WO2013009971A1 - Detection and screening method and materials useful in performance thereof - Google Patents

Detection and screening method and materials useful in performance thereof Download PDF

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Publication number
WO2013009971A1
WO2013009971A1 PCT/US2012/046437 US2012046437W WO2013009971A1 WO 2013009971 A1 WO2013009971 A1 WO 2013009971A1 US 2012046437 W US2012046437 W US 2012046437W WO 2013009971 A1 WO2013009971 A1 WO 2013009971A1
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Prior art keywords
compound
protein
oomycetes
oxysterol binding
oxysterol
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PCT/US2012/046437
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French (fr)
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John Lawrence ANDREASSI II
Steven Gutteridge
Stephen Ogden PEMBER
James A. Sweigard
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E. I. Du Pont De Nemours And Company
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Publication of WO2013009971A1 publication Critical patent/WO2013009971A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/18—Testing for antimicrobial activity of a material
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand

Definitions

  • This invention relates to polynucleotides, polypeptides, and methods of using them to screen compounds for mti-Oomycete activity and to detect resistance developed to such compounds and detecting compounds capable of overcoming such resistance.
  • Terrestrial Oomyceies the largest group of heterotrophic Stramenopiles, include a large number of plant pathogens and are believed to be among the most important plant pathogenic organisms that may be facultatively or obiigately parasitic.
  • PCT Patent Publication WO 08/013925 discloses the fungicidal azocyclic amide l-[4-[4-[5-(2,6-difluorophenyl)-4,5-dihydro-3-isoxazolyl]-2-thiazolyl]-l-piperidinyl]-
  • the present invention includes
  • a method to identify a potential anti- Oomycetes compound comprising the steps of:
  • the compound is a potential anti- Oomyceies compound if the compound binds to the Oomycetes oxysterol binding polypeptide .
  • the present invention includes a method, to identify a potential anti- Oomyceies compound wherein the oxysterol binding protein is derived from Phytophthora sp.
  • the present invention includes a method to identify a potential anti- Oomycetes compound wherein the oxysteroi binding protein is derived from Pythium sp.
  • the present invention includes a method to identify a potential anti- Oomyceies compound wherein the oxysteroi binding protein is derived from P!asmopara sp.
  • the present invention includes a method to identify a potential anti- Oomycetes compound wherein the oxysteroi binding protein is derived, from Psuedoperonospora sp.
  • the present invention includes a method to identify a potential anti- Oomyceies compound wherein the oxysteroi binding protein is selected from the group consisting of polypeptides comprising SEQ ID NOs: 1, 3, 6, 8, 1 1, 14, 17, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50 and 52.
  • the present invention includes a method to identify a potential anti- Oomyceies compound wherein the oxysteroi binding protein comprises a W at residue position 733; or 1, F, K, or Y at residue position 768; or A, 1, P, V or L at residue position 770; or I, F or Y at residue position 837; or W at residue 839; or H at residue position 861; or W or F at residue position 863;or F or Y at residue position 877 (enumeration based on SEQ ID NO: 1).
  • the present invention includes the isolated Oomycele oxysteroi binding protem which comprises a: W at residue position 733; or I, F, K, or Y at residue position 768; or A, 1, P, V or L at residue position 770; or 1, F or Y at residue position 837; or W at residue 839; or H at residue position 861 ; or W or F at residue position 863; or F or Y ⁇ at residue position 877.
  • the present invention includes a method of producing an isolated Oomycele oxysteroi binding protein which comprises: providing a host cell which comprises a recombinant construct which expresses the Oomyceies oxysteroi binding polypeptide of; and growing the host cell under conditions that are suitable for expression of the
  • the method of producing an isolated Oomycele oxysteroi binding protein may further comprise the step of purifying the Oomycele oxysteroi binding protein.
  • the invention includes all of the vectors, polypeptides, polynucleotides host cells and antibodies described herein.
  • SEQ ID NQ:4 complete cds, conserved hypothetical protein, Joint Genome Institute (JGI), Phytophthora_capsici_TOQ.
  • SEQ ID NO: 7 Sequence encoding Oxysterol Binding Domain of conserved hypothetical protein [Phytophthora infestans T3G-4], NCBl Reference Sequence: XP 002902250.1 (Derived from SEQ ID NO: l)
  • SEQ ID NO:.9 complete cds, conserved hypothetical protein, Joint Genome Institute (JGI), P. infestans supercontl.18 of Phytophthora infestans [DNA] 1385000-1389444
  • JGI Joint Genome Institute
  • SEQ ID NO: 12 complete cds, conserved hypothetical protein, Joint Genome Institute (JGI), Phytophthora ramoruml 1 scaffold 67:56451-59691
  • JGI Joint Genome Institute
  • SEQ ID NO:15 complete CDS, conserved hypothetical protein, Joint Genome Institute (JGI) Phytophthora_sojae_so 1_1 scaffold_103 :212024-214994.
  • JGI Joint Genome Institute
  • SEQ ID NO:18 complete CDS, Pythium ultimum, conserved hypothetical protein
  • SEQ ID NO: 19 genomic sequence, Pythium ultimum, conserved hypothetical protein SEQ ID NO:20 Oxysterol Birsding Domain of conserved hypothetical protein,
  • SEQ ID NO:21 coding sequence, Oxysterol Binding Domain of conserved hypothetical protein, Pseudoperonospora cubensis (cucumber downy mildew organism)
  • SEQ ID NO:29 coding sequence, Oxysterol Binding Domain of conserved hypothetical protein, Phytophihora ramorum
  • SEQ ID NO:36 Oxysterol Binding Domain of conserved hypothetical protein, Pythium ultimum
  • SEQ ID NO:44 Oxysterol Binding Domain of conserved hypothetical protein, Pythium aphanadermatum
  • SEQ ID NO:45 coding sequence Oxysterol Binding Domain of conserved hypothetical protein, Pythium aphanadermatum
  • SEQ ID NO:46 Oxysterol Binding Domain of conserv ed hypothetical protein, Pythium graminacoia
  • SEQ ID NO:47 coding sequence Oxysterol Binding Domain of conserved hypothetical protein, Pythium graminacoia
  • SEQ ID NO:48 Oxysterol Binding Domain of conserved hypothetical protein, Pythium heHcoides
  • SEQ ID NO:49 coding sequence,Oxysteroi Binding Domain of conserved hypothetical protein, Pythium helicoides
  • SEQ ID NO:51 coding sequence, Oxysterol Binding Domain of conserved hypothetical protein, Pythium deliense.
  • PCX Patent Publication WO 08/013925 discloses the fungicidal azocyciic amide l -[4-[4-[5-(2,6-difluorophenyl)-4,5-dihydro-3- isoxazolyl]-2-thiazolyi]-l-piperidm ⁇
  • compound 1 has the following structure:
  • Compuond 1 shows particularly good utility as an agent toxic to Phytophthora, P!asmopara and Pstiedoperonospora .
  • HMMER3 http://hmmer.janelia.org/) suite of programs.
  • the Pfam protein families database M. Punta, P.C. Coggiil, R.Y. Eberhardt, J. Mistry, J. Tate, C. Boursnell, N. Pang, K. Forslund, G. Ceric, J. Clements, A. Heger, L, Holm, E.L.L. Sonnhammer, S.R. Eddy, A. Bateman, R.D. Finn
  • OSBP oxysterol binding protein
  • the domain structure of the full length protein also includes a plekstrin homology domain at the N-terminal third of the protein and a START (StAR-related lipid-transfer) domain(SRPBCC (START/RHO_alpha_C/PlTP Bet_v 1 /CoxG/CalC)) ligand-bindmg domain toward the middle portion of the sequence.
  • START StAR-related lipid-transfer domain
  • the 'pleckstrin homology' (PH) domain is a domain of about 100 residues that occurs in a wide range of proteins invol ved in intracellular signaling or as constituents of the cytoskeieton. All known cases have a common structure consisting of two perpendicular anti-parallel ⁇ sheets, followed by a C-terminai amphipathic helix. The loops connecting the ⁇ -strands differ greatly in length. (Mayer B.J., Ren R, 5 Clark K.L., Baltimore D., A putative modular domain present in diverse signaling proteins, Cell 73:629-630(1993)).
  • the StAR-related lipid-transfer (START) domain is an about 200-210 residue lipid- binding motif, which is primarily involved in eukaryotic signaling mediated by lipid binding.
  • R epresentatives of the START domain family have been shown to bind different ligands such as sterols (StAR protein) and phosphatidylcholine (PC-TP).
  • Ligand binding by the START domain can also regulate the activities of other domains that co-occur with the START domain in multidomain proteins.
  • StAR protein sterols
  • PC-TP phosphatidylcholine
  • compositions comprising, “comprising,” “includes,” “including,” “has,” “having,” “contains” or “containing,” or any other variation thereof, are intended to cover a non-exclusive inclusion.
  • a composition, a mixture, process, method, article, or apparatus that comprises a list of elements is not necessarily limited, to only those elements but may include other elements not expressly listed, or inherent to such composition, mixture, process, method, article, or apparatus.
  • a polynucleotide or polypeptide comprising a reference sequence is understood to potentially include flanking sequence on either side of the reference sequence.
  • cDNA refers to a DNA that is complementary to and synthesized from a mRNA template using the enzyme reverse transcriptase. The cDNA can be single-stranded or converted, into the double-stranded form.
  • Coding sequence refers to a cDNA sequence that codes for a specific amino acid sequence.
  • This term also refer to modifications of the nucleic acid, fragments of the instant invention such as deletion or insertion or attachment to flanking sequences of one or more nucleotides that do not substantially alter the functional properties of the resulting nucleic acid fragment relative to the initial, unmodified fragment. It is therefore understood, as those skilled in the art will appreciate, that the invention described in terms of polynucleotides that consist essentially any given sequence encompasses more than such specific exemplary sequences.
  • polynucleotide that "comprising,” “includes,” “has,” or “contains” "consists of or “consists essentially of a sequence encompassed by this invention is also defined it's ability to hybridize, under moderately stringent conditions (for example, 1 X SSC, 0.1% SDS, 60°C) with the sequences exemplified herein, or to any portion of the nucleotide sequences reported herein and which are functionally equivalent to the gene or the promoter of the invention. Stringency conditions can be adjusted to screen for moderately similar fragments, such as homologous sequences from distantly related organisms, to highly similar fragments, such as genes that duplicate functional enzymes from closely related, organisms.
  • moderately similar fragments such as homologous sequences from distantly related organisms, to highly similar fragments, such as genes that duplicate functional enzymes from closely related, organisms.
  • Post- hybridization washes determine stringency conditions.
  • One set of preferred conditions involves a series of washes starting with 6X SSC, 0.5% SDS at room temperature for 15 min, then repeated with 2X SSC, 0,5% SDS at 45 °C for 30 min, and then repeated twice with 0.2X SSC, 0.5% SDS at 50°C for 30 min.
  • a more preferred set of stringent conditions involves the use of higher temperatures in which the washes are identical to those above except for the temperature of the final two 30 min washes in 0.2X SSC, 0.5% SDS was increased to 60°C.
  • Another preferred, set of highly stringent conditions involves the use of two final washes in 0.1X SSC, 0.1 % SDS at 65°C.
  • the term “consisting essentially of of a reference sequence includes flanking sequences on either side of the reference sequence but does extend to include a full length or mature polypeptide or polynucleotide encoding a mature or full length polypeptide
  • the term “contacting” means bringing together, either directly or indirectly, a compound into physical proximity to a polypeptide.
  • expression refers to the production of a functional end- product. Expression of a gene involves transcription of the gene and translation of the mRNA into a precursor or mature protein.
  • host cell refers to any cell or organism into which an isolated nucleic acid fragment has been stably or transiently introduced.
  • the host cell may be part of a larger organism, an individual in tissue culture, or a free-living organism. Examples of host cells include, but are not limited to, bacteria, fungi, insect, plant, and animal
  • Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described more fully in Sambrook, J., Fritsch, E,F. and Mania tis, T. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989 (hereinafter "Sambrook”).
  • an "intron” is an intervening sequence in a gene that does not encode a portion of the protein sequence. Thus, such sequences are transcribed into R A but are then excised and are not translated. The term is also used for the excised RNA sequences.
  • An "exon” is a portion of the sequence of a gene that is transcribed and is found in the mature messenger RNA derived from the gene, but is not necessarily a part of the sequence that encodes the final gene product.
  • isolated refers to a polynucleotide (DNA or RNA) or polypeptide that has been removed from its whole cell native environment.
  • isolated polynucleotides include, but are not limited to, recombinant DNA molecules contained in a vector, recombinant DNA molecules maintained in a heterologous host cell, partially or substantially purified nucleic acid molecules, and synthetic DNA or RNA molecules.
  • An example of an isolated polypeptide is a polypeptide which has been partially or substantially purified. Examples of describing the details of such partially purified extracts containing Oomycete oxysteroi binding polypeptides are described herein. Extracts enriched for the golgi and endoplasmic reticulum are sometimes preferred,. Specifically excluded from the definition of isolated as it applies to polynucleotides are whole chromosomes.
  • the "Oomycetes ' " are organisms classified as Stramenopiles.
  • the term "Oomycetes” as used, in this disclosure includes all genera commonly identified as plant pathogenic Oomycetes. These organisms are single-celled and produce thread-like filaments (myceiia). They resemble fungi and grow best in a moist environment. However the cell walls of the Oomycetes are built with cellulose instead of fungal chitin. Accordingly they are closely related to algae not fungi
  • the Oomycetes have individual mobile cells, with the characteristics of the Stramenopiles (for instance, two flageila of different lengths, one with forked bristles). The Oomycetes can reproduce sexually or asexualfy.
  • oospores have a single chromosome set (haploid) and are either male or female (the name Oomycetes means "egg- fungi"). These mate to produce a new thallus.
  • Phytophthora sp. are aggressive pathogens of plants.
  • Phytophthora sojae costs the soybean industry millions of dollars each year, in California and Oregon, a newly emerged Phytophthora species, P. ramorum, is responsible for a disease called sudden oak death that affects not only the live oaks that are the keystone species of the ecosystem but also a large variety of woody shrubs that inhabit the oak ecosystems, such as bay laurel and viburnum.
  • Species of Phytophthora can also destroy eucalyptus, avocado, and pineapple, as well as other tropical crop plants.
  • the Pythium sp. group is less virulent and more widespread. They are parasites in many plants, especially seedlings.
  • the downy mildews organisms cause plant diseases that look like 'mildew' growing on leaves, but are neither mildews or rusts. They cause great damage to commercial crops.
  • Plasmopara viticola nearly destroyed the entire French wine industry in the 1870s by killing the grapevines.
  • Plasmopara sp. and Psiiedoperonospora sp. are responsible for the downy mildews that affect grapes, lettuce, com, cabbage, cantaloupe, cucumber, pumpkin, squash and watermelon and other crops.
  • Oomycetes oxysterol binding polypeptide refers to a protein product of an essential gene found in Oomycetes which comprises
  • an oxysterol binding domain This definition may include among others f in combination with other embodiments listed in this specificaiton or alone) an Oomycetes oxysterol binding polypeptide; wherein the PH domain is at about between residues 28-150; and/or wherein the START domain is at about between residues 291-530; and/or wherein the oxysterol binding domain at about between residues 606-950.
  • Residue numbers in this specification are described according to the convention described below with reference to Table 1 unless otherwise noted.
  • This definition may include among others (in combination with other embodiments listed in this specificaiton or alone) an embodiment wherein the oxysterol binding domain of the Oomycetes has at least 60% 61% 62% 63%, 64%, 65%», 66%, 67%, 68%,
  • noxysterol binding domain of the invention comprises the highly conserved residues KPFNPILGET (773-777).
  • This definition may include among others (in combination with other embodiments listed in this specificaiton or alone) an embodiment wherein the oxysterol binding domain of the invention comprises the highly conserved residues CEHTSHHPPI (795-804).
  • This definition may include among others (in combination with other embodiments listed in this specificaiton or alone) an embodiment wherein the highly conserved residues KPFNPILGET (773-777) and the highly conserved residues CEHTSHHPPI (795- 804).
  • anti- Oomycetes compound is a compound which kills or inhibits the growth of Oomycetes.
  • OOSBP Olethyl oxysterol binding polypeptide
  • the oxysterol binding proteins and binding domains of the invention can be both wild-type or may contain substitutions as set forth below in Table 1. This section also describes the numbering convention used throughout this specification when describing the OOSBP of the invention.
  • Table 1 Lists the independent mutations identified, and. the changes in the protein primary sequence that results. Residue numbers are expressed relative to the Phytophthora infestans protein sequence of SEQ ID NO:l .
  • XxxxY refers to an amino acid sequence position xxx, a single amino acid X in wild type is changed to an amino acid Y.
  • the numbering system used to identify particular residues uses the sequence of SEQ ID NO: 1 for convenience. It is understand by the skilled artisan that among similar sequences the same numbering system can be used with other related, polypeptides by aligning the related sequences with SEQ ID NO: 1 utlizing for example a scoring matrix such as BLOSUM62 and perhaps introducing insertions or deletions as necessary to create the best alignment.
  • the term “L733W refers to an amino acid at sequence position 733, in which a wild type leucine is changed to tryptophan in a mutant polypeptide.
  • S768IF Y refers to an amino acid sequence position 768 in which a wild type serine is changed to isoleucine, phenyalanine, lysine or tyrosine in a mutant polypeptide.
  • G770AIPVL refers to an amino acid sequence position 770 in which a wild type glycine is changed to an alanine, isoleucine. proline, valine or leucine.
  • N837IFY refers to an amino acid sequence at position 837 in which a wild type asparagine is changed to an isoleucine, phenyalanine or tyrosine.
  • G839W refers to an amino acid sequence position 839 in which a wild type glycine is changed to a tryptophan.
  • P861H refers to an amino acid sequence at position 861 in which a wild type proline is changed to histidine.
  • L863WF refers to an ammo acid sequence at position 863 in which a wild type leucine is changed to a tryptophan or phenylalanine.
  • I877FY refers to an amino acid sequence at position 877 in which a wild type isoleucine is changed, to a phenylalanine or tyrosine.
  • polynucleotide generally refers to any polyribonucleotide or polydeoxribonucfeotide, which may be unmodified RNA or DNA or modified RNA or DNA.
  • Polynucleotides include, without limitation, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, RNA that is mixture of single- and double-stranded regions, and hybrid molecules comprising DNA and RNA that may be single -stranded or, more typically, double-stranded or a mixture of single- and. double-stranded regions.
  • Polynucleotide also embraces relatively short polynucleotides, often referred to as oligonucleotides.
  • the nucleotide sequences are presented by single strand only, in the 5' to 3' direction, from left to right.
  • polypeptide refers to any peptide or protein comprising amino acids joined to each other by peptide bonds
  • Polypeptide refers to both short chains, commonly referred to as peptides, oligopeptides or oligomers, and to longer chains, generally referred to as proteins.
  • the amino acid sequences are presented in the amino to carboxy direction, from left to right. The amino and carboxy groups are not presented in the sequence.
  • Polypeptide "similarity” as used herein is defined herein as the percentage of amino acid residues which align based on identit or similarity as determined BLASTP used with the scoring matrix BLOSUM62. Two polypeptide sequences are aligned to optimize the alignment scores using a gap opening penalty of 10, a gap extension penalty of 0.1, The BLOSUM62 scoring matrix of Henikoff and Henikoff is shown in Table 1 (amino acids are indicated by the standard one-letter codes).
  • Scores within a BLOSUM are log-odds scores that measure, in an alignment, the logarithm for the ratio of the likelihood of two amino acids appearing with a biological sense and the likelihood of the same amino acids appearing by chance
  • the matrices are based on the minimum percentage identity of the aligned protein sequence used in calculating them. Every possible identity or substitution is assigned a score based on its observed frequences in the alignment of related proteins. A positive score is given to the more likely substitutions while a negative score is given to the less likely substitutions.
  • SEQ ID NO: 6 represents a fragment which might often be found with flanking sequence of a dissimilar nature on one or both sides. When percent similarity is calculated for such a polypeptide therefore, a with respect to SEQ ID NO: 6 it is done so without regard to the unmatched amino acids not encompassed within the fragment.
  • Percent sequence "similarity" with respect to polynucleotides of the invention may be calculated as the percentage of nucleotide bases in the candidate sequence that are identical to nucleotides in the OOSBP sequence set forth in a particular polynucleotide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity.
  • similarity is identical to "identity” when used to describe a polynucleotide.
  • recombinant refers to an artificial combination of two otherwise separated, segments of sequence, e.g., by chemical synthesis or by the manipulation of isolated segments of nucleic acids by genetic engineering techniques.
  • recombinant construct refers to a functional unit of genetic material that can be inserted into the genome of a cell using standard methodology well known to one skilled in the art. Such construct may be itself or may be used in conjunction with a vector. If a vector is used then the choice of vector is dependent upon the method that will be used to transform host plants as is well known to those skilled in the art. For example, a plasmid vector can be used. The skilled artisan is well aware of the genetic elements that must be present on the vector in order to successfully transform, select and. propagate host cells comprising any of the isolated nucleic acid fragments of the invention. The skilled artisan will also recognize that different independent transformation events will result in different levels and patterns of expression (Jones et al., (1985) EMBO J.
  • wild-type refers to a gene or gene product which has the characteristics of that gene or gene product when isolated from a naturally occurring source (as the protein is found predominantly in nature among organisms which have not been exposed to selection pressure with compound 1 or similar compounds).
  • a wild-type gene is that which is most frequently observed in a population and is thus arbitrarily designated the "normal” or " wild -type” form of the gene.
  • mutant refers to a gene or gene product which displays modifications in sequence and or functional properties (i.e., altered characteristics) when compared, to the wild-type gene or gene product. It is noted that naturally-occurring mutants can be isolated; these are identified by the fact that they have altered characteristics when compared to the wild-type gene or gene product.
  • the present invention relates to molecules which comprise the gene sequences that encode the OOSBF and. constructs and recombinant host cells incorporating the gene sequences; the novel OOSBP encoded by the gene sequences; antibodies to the poHpeptides and homologs; kits employing the polynucleotides and polypeptides, and methods of making and using ail of the foregoing.
  • the present invention relates to homologs of the gene sequences and of the polypeptides and methods of making and using the same.
  • the present invention includes an isolated Oomycetes oxysterol binding polypeptide (OOSBP) which comprises/consists essentially of a polypeptide which has has 100% to 50% ( and even' integer value in between) similarity (without taking into account unmatched residues flanking the amino or carboxy terminus on either side) to the sequence set forth in SEQ ID NO: 6.
  • OOSBP Oomycetes oxysterol binding polypeptide
  • OOSBP polypeptides therefore include the amino acid sequences set out in any of SEQ ID NOs: 1 , 3 8, 11, 14, 17, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50 and 52..
  • the polypeptides may be isolated, by convention protein purificatation methods from organisms which naturally express the protein.
  • the polypeptide may be chemically synthesized, but also may be produced by recombinant procedures involving host cells of the invention. Use of eukaryotic host cells is expected to provide for such post-translational modifications (e.g., glycosylation, truncation, lipidation, and phosphorylation) as may be needed to confer optimal biological activity.
  • the invention includes isolated polypeptides which have been modified as a result of cellular expression or intentionally modified to facilitate expression,
  • OOSBP in enough quantities from any of these sources, would form the basis of assays that have utility for identifying synthetic compounds or natural products that have the same mode of action as compound 1.
  • Residues 733, 768, 770, 839, 861, 863 and 877 may substituted with another naturally occurring amino acid if the object is determine the activity of compounds against a mutant target.
  • the specific amino acids identified as substitutional variants might themselves be substituted with amino acids sharing similar characteristics (often called "conservative substitutions).
  • Insertions are provided wherein one or more amino acid residues supplement a OOSBP amino acid sequence. Insertions may be located ax either or both termini of the polypeptide, or may be positioned within internal regions of the OOSBP amino acid sequence, Insertional variants with additional residues at either or both termini can include, for example, fusion proteins and polypeptides including amino acid tags or labels.
  • Insertion variants include OOSBP wherein one or more amino acid residues are added to an OOSBP amino acid sequence, or to a biologically active fragment thereof.
  • Variant products of the invention also include mature OOSBP products, i.e., OSBP products wherein leader or signal sequences are removed. These residues might be replaced with additional amino terminal residues.
  • the additional amino terminal residues may be derived from another protein, or may include one or more residues that are not identifiable as being derived from specific proteins.
  • OOSBP products with an additional methionine residue at position -1 are contemplated.
  • variants of OOSBP with additional Met are particularly useful for enhanced recombinant protein production in bacterial host cells.
  • mutant polypeptides of the invention by mutagenisis and subsequent selection the skilled artisan recognizes that alterations we have identified of the native amino acid sequence may be accomplished by any of a number of known techniques. For example, mutations may be introduced at particular locations by procedures well known to the skilled artisan, such as oligonucleotide-directed mutagenesis, which is described by Walder et al. (Gene 42: 133 (1986)); Bauer el al.
  • Insertional variants also include fusion proteins wherein the amino terminus and/or the carboxy terminus of an oxysterol binding protein or oxysterol binding domain fragment is/are fused to another polypeptide.
  • an oxysterol binding domain fragement may be fused to the scaffold of another oxysterol binding protein from another species, such that that a "chimeric " oxysterol binding protein is created.
  • the invention therefore embraces variants having additional amino acid residues which result from use of specific expression systems.
  • either the OSBP polypeptide comprises a label or tag that facilitates its isolation.
  • An exemplary tag of this type is a poly- histidine sequence, generally around six histidine residues, that permits isolation of a compound so labeled using nickel chelation.
  • Other labels and tags such as the FLAG ⁇ tag (Eastman Kodak, Rochester, NY), well known and. routinely used in the art, are embraced by the invention..
  • the invention provides deletion variants wherein one or more ammo acid, residues in a OOSBP are removed.
  • Deletions can be effected at one or both termini of the OOSBP, or with removal of one or more non-terminal amino acid residues of an OOSBP.
  • Deletion variants therefore, include all fragments of an OOSBP.
  • polypeptides of the present invention are provided, in an isolated form, and preferably are substantially purified.
  • OOSBP may be recovered and purified, from recombinant cell cultures by well-known methods, including ammonium sulfate or ethanol precipitation, anion or cation exchange chromatography, phosphoceliulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyfapatite chromatography and lectin chromatography. Affinity chromatography is described in the examples set forth at the end of this specification.
  • the present invention provides isolated polynucleotides (e.g. , DNA sequences and R A transcripts which encode the polypeptides of the invention, both sense and.
  • isolated polynucleotides e.g. , DNA sequences and R A transcripts which encode the polypeptides of the invention, both sense and.
  • complementary aniisense strands both single- and double-stranded, including splice variants thereof) that encode OOSBP of the invention.
  • Such polynucleotides are of use for (among other uses) in the expression of and the coconstruction of hybrid OOSBP.
  • the invention therefore includes a polynucleotide which comprises/consists essentially of a polynuceltide which encodes a polypeptide which has has 100% to 50% (and every integer value in between) (without taking into account unmatched, residues flanking the amino or carboxy terminus on either side) to the sequence set forth in SEQ ID NO: 6.
  • OOSBP encoding polynucleotides therefore comprise/consist essentially of the polynucleotide sequences set out in any of SEQ ID NOs: 2, 3, 5, 7, 9, 10, 12, 13, 15, 16, 18, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51 and 53.
  • the polynucleotides of the invention comprise polynucleotides that encode polypeptides which comprise the mutant residues at 733, 768. 770, 839. 861 , 863 and 877 as described above.
  • the invention provides isolated polynucleotides (e.g., cDNA, genomic DNA, synthetic DNA, RNA, or combinations thereof, whether single- or double-stranded) that comprise a nucleotide sequ ence encoding the amino acid sequence of the polypeptides of the invention.
  • isolated polynucleotides e.g., cDNA, genomic DNA, synthetic DNA, RNA, or combinations thereof, whether single- or double-stranded
  • Such polynucleotides are useful for recombinant! ⁇ ' expressing the OOSBP and also for detecting expression of the polypeptide in cells (e.g., using Northern hybridization and in situ hybridization assays).
  • the invention also provides a purified and isolated polynucleotides
  • Genomic DNA of the invention comprises the protein-coding region for a polypeptide of the invention and is also intended to include the variants described above. It is widely understood that, for many genes, genomic DNA is transcribed into RNA transcripts that undergo one or more splicing events wherein in iron (i.e., non-coding regions) of the transcripts are removed, or "spliced out," RNA transcripts that can be spliced, by alternative mechanisms, and therefore be subject to removal of different RNA sequences but still encode a OOSBP, are referred to in the art as splice variants which are embraced by the invention. Splice variants comprehended by the invention therefore are encoded by the same original genomic DNA sequences but arise from distinct mRNA transcripts.
  • the invention also comprehends cDNA that is obtained through reverse transcription of an RNA polynucleotide encodmg OOSBP (conventionally followed by second, strand synthesis of a complementary strand to provide a double-stranded DNA).
  • Preferred DNA sequences encoding OOSBP are selected from the group consisting of 2, 4, 5, 7, 9, 10, 12, 13, 15, 16, 18, 19, 21, 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41, 43, 45, 47, 49, 51.
  • a preferred DNA of the invention comprises a double stranded molecule along with the complementary molecule (the "non-coding strand" or "complement") having a sequence unambiguously deducible from the coding strand according to Watson-Crick base- pairing rales for DNA.
  • polynucleotides encoding the OOSBP polypeptide selected from the group consisting of SEQ ID NOs: 2, 4, 5, 7, 9, 10, 12, 13, 15, 16, 1 8, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41 , 43, 45, 47, 49 and 51 which differ in sequence from the polynucleotides selected from the group consisting of SEQ ID NOs; 2, 4, 5, 7, 9, 10, 12, 13, 15, 16, 18, 19, 21 , 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51 by virtue of the well-known degeneracy of the universal nuclear genetic code.
  • Polynucleotides of the invention permit identification and isolation of
  • polynucleotides encoding related OOSBP such as allelic variants and species homologs, by well-known techniques including Southern and/or Northern hybridization, and polymerase chain reaction (PCR).
  • related polynucleotides other Oomycetes genomic sequences, including allelic variants, as well as polynucleotides encoding polypeptides having substantial similarity to SEQ ID NOs: 1 ,4, 8 , 1 1 , 14, 17, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48 and 50 and structurally related polypeptides sharing one or more biological, immunological, and/or physical properties of OOSBP.
  • Homologous polynucleotides encoding OOSBP can also be identified by Southern and/or PCR analysis. Knowledge of the sequence of any individual OOSBP also makes possible through use of Southern hybridization or polymerase chain reaction (PCR) the identification of genomic DNA sequences which include OOSBP expression control regulatory sequences such as promoters, operators, enhancers, repressors, and the like.
  • PCR polymerase chain reaction
  • Polynucleotides of the invention are also useful in hybridization assays to detect the capacity of cells to express OOSBP polynucleotides.
  • the invention may also provide a basis for diagnostic methods useful for identifying the genetic alteration(s) arising in nature (and consequent resistance to compound 1 and molecules with a mode of action similar to compound 1) in a OOSBP locus that produce the mutations outlined in Table 1.
  • the OOSBP nucleotide sequences disclosed herein may be used to identify homologs of the OOSBP, in other species. Any of the nucleotide sequences disclosed herein, or any portion thereof, can be used, for example, as probes to screen databases or nucleic acid libraries, such as, for example, genomic or cDNA libraries, to identify homologs, using screening procedures well known to those skilled, in the art. Accordingly, homologs having at least 60%, more preferably at least 70%, more preferably at least 80%. more preferably at least 90%, more preferably at least 95%, and most preferably at least 100% similarit with OOSBP sequences reported herein can be identified.
  • One preferred embodiment of the present invention provides an isolated nucleic acid molecule comprising sequences selected from the group consisting of SEQ ID NOs: 2, 4, 5, 7, 9, 10, 12, 13, 15, 16, 18, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49 and 51 and fragments thereof.
  • fragments of OOSBP encoding polynucleotides comprise at least 10, and preferably at least 12, 14, 16, 18, 20, 25, 50, or 75 consecutive nucleotides of a polynucleotide encoding an OOSBP.
  • fragment polynucleotides of the invention comprise sequences unique to the OOSBP encoding polynucleotide sequence, and therefore hybridize under highly stringent or moderately stringent conditions only (i.e., "specifically") to polynucleotides encoding OOSBP (or fragments thereof).
  • Polynucleotide fragments of genomic sequences of the invention comprise not only sequences unique to the coding region, but also include fragments of the full-length sequence derived from introns, regulatory regions, and/or other non-translated sequences. Sequences unique to polynucleotides of the invention are recognizable through sequence comparison to other known polynucleotides, and can be identified through use of alignment programs routinely utilized in the art, e.g., those made available in public sequence databases. Such sequences also are recognizable from Southern hybridization analyses to determine the number of fragments of genomic DNA to which a polynucleotide will hybridize. Polynucleotides of the invention can be labeled in a manner that permits their detection, including radioactive, fluorescent, and enzymatic labeling.
  • Fragment polynucleotides are particularly useful as probes for detection of full- length or fragments of OOSBP encoding polynucleotides.
  • One or more polynucleotides can be included in kits that are used to detect the presence of a polynucleotide encoding aqn OOSBP or used to detect variations in a polynucleotide sequence encoding an OOSBP.
  • the invention also embraces DNAs encoding an OOSBP that hybridize under high stringency conditions to the non-coding strand, or complement, of the polynucleotides set forth in sequences selected from the group consisting of SEQ ID NOs: 2, 3, 5, 7, 9, 10, 12, 13, 15, 16, 18, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41 , 43, 45, 47, 49 and 51
  • Exemplary highly stringent hybridization conditions are as follows: hybridization at 42C in a hybridization solution comprising 50% formamide, 1% SDS, 1 M NaCl, 10% Dextran sulfate, and washing twice for 30 minutes at 60C in a wash solution comprising 0.1 X SSC and 1% SDS.
  • hybridization conditions can be achieved through variation of temperature and buffer, or salt concentration as described Ausubel et al. (Eds.), Protocols in Molecular Biology, John Wiley & Sons (1994), pp. 6.0.3 to 6.4.10. Modifications in hybridization conditions can be empirically determined or precisely calculated based on the length and the percentage of guanosine/cytosine (GC) base pairing of the probe. The hybridization conditions can be calculated as described in
  • nucleotide sequence information disclosed in the present invention, one skilled in the art can identify and obtain nucleotide sequences which encode OOSBP from different sources ⁇ i.e., other Oomycetes species or fungal species) through a variety of means well known to the skilled artisan and as disclosed by, for example, Sambrook et al., "Molecular cloning: a laboratory manual", Second Edition, Cold Spring Harbor Press, Cold Spring Harbor, NY (1989), which is incorporated herein by reference in its entirety.
  • DNA that encodes an OOSBP may be obtained by screening of mRNA, cDNA, or genomic DNA with oligonucleotide probes generated from the OOSBP gene sequence information provided herein. Probes may be labeled with a detectable group, such as a fluorescent group, a radioactive atom or a chemiluminescent group in accordance with procedures known to the skilled, artisan and. used in conventional hybridization assays, as described by, for example, Sambrook et al.
  • a detectable group such as a fluorescent group, a radioactive atom or a chemiluminescent group
  • a nucleic acid, molecule comprising any of the OOSBP nucleotide sequences described above can alternatively be synthesized by use of the polymerase chain reaction (PCR) procedure, with the PGR oligonucleotide primers produced from the nucleotide sequences provided herein. See U.S. Patent Numbers 4,683, 195 to Mullis et al. and
  • the PCR reaction provides a method for selectively increasing the concentration of a particular nucleic acid, sequence even when that sequence has not been previously purified and is present only in a single copy in a particular sample.
  • the method can be used to amplify either single- or double-stranded DNA.
  • the essence of the method involves the use of two oligonucleotide probes to serve as primers for the template- dependent, polymerase mediated replication of a desired nucleic acid molecule,
  • Automated sequencing methods can be used to obtain or verify the nucleotide sequence of OOSBP.
  • the OOSBP polynucleotide sequences of the present invention are believed to be 100% accurate.
  • nucleotide sequence obtained by automated methods may contain some errors.
  • Nucleotide sequences determined by automation are typically 90 to at least about 00% identical (and e e ⁇ ' ' integer value in between) to the actual nucleotide sequence of a given nucleic acid molecule.
  • the actual sequence may be more precisely determined using manual sequencing methods, which are well known in the art.
  • An error in a sequence which results in an insertion or deletion of one or more nucleotides may result in a frame shift in translation such that the predicted amino acid sequence will differ from that which would be predicted from the actual nucleotide sequence of the nucleic acid molecule, starting at the point of the mutation.
  • nucleic acid molecules of the present invention are useful for screening for restriction fragment length polymorphism (RFLP) associated with some of which may be associated with resistance to compound 1 and compounds having a similar mode of action to compound 1.
  • RFLP restriction fragment length polymorphism
  • the polynucleotide sequence information provided by the invention makes possible large-scale expression of the encoded polypeptide by techniques well known and routinely practiced in the art.
  • vectors or recombinant expression vectors, comprising any of the nucleic acid molecules described abo ve.
  • Vectors are used herein either to amplify DNA or RN A encoding OOSBP and/or to express DNA which encodes OOSBP, Preferred vectors include, but are not limited to, piasmids. phages, cosmids, episomes, viral particles or viruses, and integratable DNA fragments (i.e., fragments integratable into the host genome by homologous recombination).
  • Preferred viral particles include, but are not limited to, adenoviruses, baculoviruses, parvoviruses, herpesviruses, poxviruses, adeno-associated viruses, Semliki Forest viruses, vaccinia viruses, and retroviruses.
  • Preferred expression vectors include, but are not limited to, pcDNA3 (mviirogen) and pSVL (Pharmacia Biotech ⁇ .
  • expression vectors include, but are not limited to, pSPORTTM vectors, pGEMTM vectors (Promega), pPROEX vectorsTM (LTI, Bethesda, MD), BluescriptTM vectors (Stratagene), pQETM vectors (Qiagen), pSE420TM (Invitrogen), and pYES2TM(lnvitrogen).
  • Expression constructs preferably comprise OOSBP encoding polynucleotides operatively linked to an endogenous or exogenous expression control DNA sequence and a transcription terminator.
  • Expression control DNA sequences include promoters, enhancers, operators, and regulatory element binding sites generally, and are typically selected based on the expression systems in which the expression construct is to be utilized. Preferred promoter and enhancer sequences are generally selected for the ability to increase gene expression, while operator sequences are generally selected for the ability to regulate gene expression.
  • Expression constructs of the invention may also include sequences encoding one or more selectable markers that permit identification of host cells bearing the construct. Expression constructs may also include sequences that facilitate, and preferably promote, homologous recombination in a host cell. Preferred constructs of the invention also include sequences necessary for replication in a host cell.
  • Expression constructs are preferably utilized for production of an encoded protein, but may also be utilized simply to amplify a OOSBP encoding polynucleotide sequence.
  • the vector is an expression vector wherein the polynucleotide of the invention is operatively linked to a polynucleotide comprising an expression control sequence.
  • Autonomously replicating recombinant expression constructs such as plasmid and viral DNA vectors incorporating polynucleotides of the invention are also provided.
  • Preferred expression vectors are replicable DNA constructs in which a DNA sequence encoding OOSBP is operably linked or connected to suitable control sequences capable of effecting the expression of the OOSBP in a suitable host.
  • DNA regions are operably linked or connected when they are functionally related to each other.
  • a promoter is operably linked or connected to a coding sequence if it controls the transcription of the sequence.
  • Amplification vectors do not require expression control domains, but rather need only the ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants.
  • the need for control sequences in the expression vector will vary depending upon the host selected and the transformation method chosen.
  • control sequences include a transcriptional promoter, an optional operator sequence to control transcription, a sequence encoding suitable mRNA ribosomal binding and sequences which control the termination of transcription and translation.
  • Preferred vectors preferably contain a promoter that is recognized by the host organism.
  • the promoter sequences of the present invention may be prokaryotic, eukaryotic or viral.
  • suitable prokaryotic sequences include the P R and P L promoters of bacteriophage lambda (The bacteriophage Lambda, Hershey, A, D., Ed., Cold Spring Harbor Press, Cold Spring Harbor, NY (1973), which is incorporated herein by reference in its entirety; Lambda 11, Hendrix, R. W., Ed., Cold. Spring Harbor Press, Cold Spring Harbor, NY (1980), which is incorporated herein by reference in its entirety); the tip, recA, heat shock, and lacZ promoters of E.
  • Additional promoters include, but are not limited to, E. coli lactose, E. coli arabinose, mouse mammary tumor virus, long terminal repeat of human immunodeficiency virus, maloney v irus, cytomegalovirus immediate early promoter, Epstein Barr virus, Rous sarcoma virus, human actin, human myosin, human hemoglobin, human muscle creatine, and. human
  • Additional regulatory sequences can also be included in preferred vectors.
  • Preferred examples of suitable regulatory sequences are represented by the S ine-Dalgamo of the replicase gene of the phage MS-2 and of the gene ell of bacteriophage lambda.
  • the Shine- Dalgarno sequence may be directly followed by DNA encoding OOSBP and result in the expression of the mature OOSBP protein.
  • suitable expression vectors can include an appropriate marker that allow r s the screening of the transformed ' transfected host cells.
  • the transformation of the selected host is carried out using any one of the various techniques well known to the expert in the art and described in Sambrook et ah, supra.
  • An origin of replication can also be provided either by construction of the vector to include an exogenous origin or may be provided by the host cell chromosomal replication mechanism. If the vector is integrated into the host cell chromosome, the latter may be sufficient.
  • one skilled in the art can transform mammalian ceils by the method of co-transformation with a selectable marker and OOSBP genomic or cDNA.
  • a suitable marker is dihydrofolate reductase (DHFR) or thymidine kinase (see, U.S. Patent No, 4,399,216).
  • Nucleotide sequences encoding OOSBP may be recombined with vector DNA in accordance with conventional techniques, including blunt-ended or staggered-ended termini for ligation, restriction enzyme digestion may provide appropriate termini, filling in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid iradesiderable joining, and ligation with, appropriate ligases. Techniques for such manipulation are disclosed, by Sambrook et al., supra and are well known in the art. Methods for construction of mammalian expression vectors are disclosed in, for example, Okayama et al, Mol. Cell Biol. , 1983, 3, 280, Cosman et al., Mol. Immunol, 1986, 23, 935, Cosman et al , Nature, 1984, 312, 768. EP-A -0367566, and WO 91/18982, each of which is incorporated herein by reference in their relevant parts.
  • host cells including prokaryotic and eukaryotic cells, comprising a polynucleotide of the invention (or v ector of the invention) in a manner that permits expression of the encoded OOSBP polynucleotides of the invention may be introduced into the host cell as part of a circular plasmid or a viral vector, or as linear DNA comprising an isolated protein coding region.
  • Methods for introducing DNA into the host cell that are well known and routinely practiced in the art include transformation, transfection, electroporation, nuclear injection, or fusion with carriers such as liposomes, micelles, ghost cells, and protoplasts.
  • Expression systems of the invention include bacterial, yeast, fungal, plant, insect, invertebrate, vertebrate, and mammalian cells systems.
  • the invention provides host cells that are transformed or transfec ed (stably or transiently) with polynucleotides of the invention or vectors of the invention. As stated, above, such host cells are useful for amplifying the polynucleotides and also for expressing the OOSBP or fragment thereof encoded by the polynucleotide.
  • the invention provides a method for producing a OOSBP (or fragment thereof) comprising the steps of growmg a host ceil of the invention in a nutrient medium and isolating the polypeptide or variant thereof from the cell or the medium.
  • OOSBP may be membrane bound, it will be appreciated that, for some applications, such as certain activity assays, the preferable isolation may involve isolation of cell membranes containing the polypeptide embedded therein, whereas for other applications a more complete isolation may be preferable.
  • transformed host cells having an expression vector comprising any of the nucleic acid molecules described, above are provided. Expression of the nucleotide sequence occurs when the expression vector is introduced into an appropriate host cell. Suitable host cells for expression of the
  • polypeptides of the invention include, but are not limited to, prokaryotes, yeast, and eukaryotes. If a prokaryotic expression vector is employed, then the appropriate host cell would be any prokaryotic cell capable of expressing the cloned sequences. Suitable prokaryotic cells include, but are not limited to, bacteria of the genera Escherichia, Bacillus, Salmonella, Pseudomonas, Streptomyces, and Staphylococcus.
  • eukaryotic cells are cells of higher eukaryotes.
  • Suitable eukaryotic cells include, but are not limited to, non-human mammalian tissue culture cells and human tissue culture cells.
  • Preferred host cells include, but are not limited to, insect cells, HeLa ceils, Chinese hamster ovary ceils (CHO cells), African green monkey kidney cells (COS cells), human 293 cells, and murine 3T3 fibroblasts. Propagation of such cells in cell culture has become a routine procedure (see, Tissue Culture, Academic Press, Kruse and Patterson, eds. (1973), which is incorporated herein by reference in its entirety).
  • yeast host may be employed as a host cell.
  • Preferred yeast cells include, but are not limited to, the genera Saccharomyces, Pichia, and. Kluveromyces.
  • Preferred yeast hosts are 5. cerevisiae and P. pastoris.
  • Preferred yeast vectors can contain an origin of replication sequence from a 2T yeast plasrnid, an autonomously replication sequence (ARS), a promoter region, sequences for polyadenylation, sequences for transcription termination, and a selectable marker gene.
  • ARS autonomously replication sequence
  • Shuttle vectors for replication in both yeast and E. coli are also included herein.
  • insect cells may be used as host cells.
  • the polypeptides of the invention are expressed using a baculovirus expression system (see, Luekow et a!. , Bio/Technology, 1988, 6, 47, Baculovirus Expression Vectors: A Laborator Manual, O'Rielfy et al. (Eds.), W.H. Freeman and Company, New York, 1992, and U.S. Patent No. 4,879,236, each of which is incorporated herein by reference in its entirety).
  • the MAXBACTM complete baculovirus expression system can, for example, be used for production in insect cells.
  • Host cells of the invention are a valuable source of immunogen for development of antibodies specifically immimoreactive with mutant and wild-type OOSBP.
  • Host cells of the invention are also useful in methods for the large-scale production of OOSBP polypeptides wherein the cells are grown in a suitable culture medium and the desired polypeptide products are isolated from the cells, or from the medium in which the cells are grown, by purification methods known in the art, e.g. , conventional chromatographic methods including immunoaffinity chromatography, receptor affinity chromatography, hydrophobic interaction chromatography, lectin affinity chromatography, size exclusion filtration, cation or anion exchange chromatography, high pressure liquid chromatography (HPLC), reverse phase HPLC, and the like.
  • HPLC high pressure liquid chromatography
  • Still other methods of purification include those methods wherein the desired protein is expressed and purified as a fusion protein having a specific tag, label, or chelating moiety that is recognized by a specific binding partner or agent.
  • the purified protein can be cleaved to yield the desired protein, or can be left as an intact fusion protein. Cleavage of the fusion component may produce a form of the desired protein having additional amino acid residues as a result of the cleavage process.
  • OOSBP DNA sequences allow r s for modification of cells to permit, or increase, expression of endogenous OOSBP.
  • Cells can be modified (e.g. , by homologous recombination) to provide increased expression by replacing, in whole or in part, the naturally occurring OOSBP promoter with all or part of a heterologous promoter so that the cells express the OOSBP at higher levels.
  • the heterologous promoter is inserted, in such a manner that it is operatively linked to endogenous OOSBP encoding sequences.
  • amplifiable marker DNA e.g., ada, dhfr, and the multifunctional CAD gene which encodes carbamoyl phosphate synthase, aspartate transcarbamylase, and dihydroorotase
  • intron DNA may be inserted, along with the heterologous promoter DNA. If linked to the OOSBPcoding sequence, amplification of the marker DNA by standard selection methods results in co-amplification of the OOSBP coding sequences in the cells.
  • the present invention therefore involves ligand binding assays to determine the avidity of a test compound for an OOSBP. Such methods are useful in identifying compounds with potential utility as anti-Oomycetes compounds.
  • the present invention includes a method for evaluating a compound for its ability to bind an Oomycete oxysterol binding polypeptide, the method comprising the steps of:
  • the invention further comprises the method stated above wherein the polypeptide has sterol/oxysierol binding activity.
  • the present invention includes a method for evaluating a compound for its ability to bind an Oomycetes oxysterol binding polypeptide wherein the oxysterol binding protein is derived from Phytophthora sp.
  • the present invention includes includes a method for evaluating a compound for its ability to bind an Oomycete oxysterol binding polypeptide wherem the oxysterol binding protein is derived from Pylhium sp.
  • the present invention includes a method for evaluating a compound for its ability to bind an Oomycete oxysterol binding polypeptide wherem the oxysterol binding protein is derived from Plasmopara sp.
  • the present invention includes a method for evaluating a compound for its ability to bind an Oomycete oxysterol binding polypeptide wherem the oxysterol binding protein is derived, from Psuedoperonospora sp.
  • the present invention includes a method for evaluating a compound for its ability to bind an Oomycete oxysterol binding polypeptide wherein the oxysterol binding protein is selected from the group consisting of polypeptides comprising SEQ ID NOs: 1 , 3, 6, 8, 1 1, 14, 17, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50 and. 52.
  • the present invention includes a method for evaluating a compound for its ability to bind, an Oomycete oxysterol binding polypeptide wherem the oxysterol binding protein comprises a W at residue position 733; or I, F, K, or Y at residue position 768; or A, I, P. V or L at residue position 770; or I, F or Y at residue position 837; or W at residue 839; or H at residue position 861 ; or W or F at residue position 863;or F or Y at residue position 877 (enumeration based on SEQ ID NO: 1).
  • a binding assay can be used to identify compounds that interact with the
  • polypeptides of the invention hi binding assays usually (but not always) one partner molecule is immobilized and the other is labeled in some fashion (e.g. using a
  • nonradioactive label such as an enzyme or fluorescent tag or by using a radioactive label
  • the amount of ligand bound is measured using an appropriate detection system.
  • Ligands showing significant binding may then be studied further by ensuring that the protein is in excess, and carrying out experiments with a dilution series of the ligand at a set of known concentrations, typically from 10 "2 to 10 ⁇ llJ M.
  • the polypeptide may be immobilized using an epitope or other affinity tag on a support material to which an appropriate antibody or binding agent for the tag is attached.
  • Suitable supports used in assays include, but are not limited to, synthetic polymer supports such as polystyrene, polypropylene, substituted, polystyrene. E.g., aminated or carboylzted polystyrene; polyacrylamides; polyamides;
  • polyvinyl chloride etc.; glass beads; agarose; nitrocellulose; nylon; polyvinyl! denedifluoride; surface-modified nylon, etc.
  • Methods to identify binding partner compounds include solution assays, in vitro assays wherein OOSBP are immobi lized, and cell-based assays. Identification of binding partner compounds of OOSBP provides novel compounds useful in the control of Oomycete sp.
  • the invention includes several assay systems for identifying OOSBP binding partners.
  • methods of the invention comprise the steps of (a) contacting a OOSBP with one or more candidate binding partner compounds and (b) identifying the compounds that bind to the OSBP polypeptide. Identification of the compounds that bind the OSBP polypeptide can be achieved by isolating the OSBP polypeptide/binding partner complex, and separating the binding partner compound from the OOSBP polypeptide. An additional step of characterizing the physical, biological, and/or biochemical properties of the binding partner compound is also comprehended in another embodiment of the invention.
  • the OOSBP polypeptide/binding partner complex is isolated using an antibody immunospecific for the OSBP polypeptide.
  • the invention provides a method comprising the steps of (a) contacting an immobilized OOSBP with a candidate binding partner compound and (b) detecting binding of the candidate compound to the OOSBP,
  • the candidate binding partner compound is immobilized and binding of OOSBP is detected.
  • Immobilization is accomplished using any of the methods well known in the art, including covalent bonding to a support, a bead, or a chromatographic resin, as well as non-covending, high affinity interactions such as antibody binding, or use of streptavidin/biotin binding wherein the immobilized compound includes a biotin moiety.
  • Detection of binding can be accomplished (i) using a radioactive label on the compound that is not immobilized, (ii) using of a fluorescent label on the non-immobilized compound, (iii) using an antibody immunospecific for the non-immobilized compound (a hapten), (iv) using a label on the non-immobilized compound that excites a fluorescent support to which the immobilized compound is attached (v) surface p!asmon resonance, as well as other techniques well known and. routinely practiced in the art.
  • the invention comprehends high-throughput screening (HTS) assays to identify compounds that interact with an OOSBP.
  • HTS assays permit screening of large numbers of compounds in an efficient manner.
  • Cell-based HTS systems are contemplated to investigate OOSBP-ligand interaction.
  • HTS assays are designed to identify "hits' " or "lead compounds” having the desired property, from which modifications can be designed to improve the desired property. Chemical modification of the "hit” or "lead compound” is often based on an identifiable structure/activity relationship between the "hit” and the OOSBP.
  • Binding can be determined by binding assays which are well known to the skilled artisan, including, but not limited to, gel-shift assays, Western blots, radiolabeled competition assay, co-fractionatio by chromatography, co-precipitation, cross linking, ELISA, and the like, which are described in, for example, Current Protocols in Molecular Biology, 1999, John Wiley & Sons, NY, which is incorporated herein by reference in its entirety.
  • the compounds to be screened include (which may include compounds which are suspected to bind an OOSBP include but are not limited to extracellular, intracellular, biologic or chemical origin.
  • the compounds of the invention exhibit a variety of chemical structures,
  • the literature is replete with, examples of the use of radiolabeled ligands i HIS binding assays for drug discovery (see Williams. Medicinal Research Reviews, 1991, 11, 147-184.; Sweetnam, el al., J. Natural Products, 1993, 56, 441 -455 for review).
  • Recombinant proteins are sometimes preferred for HTS binding assays because they allow for better specificity (higher relative purity), provide the ability to generate large amounts of binding material, and can be used in a broad variety of formats (see Hodgson,
  • methods of screening for compounds which comprise contacting test compounds with QOSBP and assaying for the presence of a complex between the compound and OOSBP.
  • the ligand is typically labeled. After suitable incubation, free ligand is separated from that present in bound, form, and the amount of free or uncomplexed label is a measure of the ability of the particular compound to bind to OOSBP.
  • an OSBP can be used for HTS binding assays in conjunction with a defined ligand (for example compound 1).
  • a defined ligand for example compound 1.
  • the compound is labeled with a suitable radioisotope, including, but not limited to, l2j l, 3 H, JJ S or j2 P, by methods that are well known to those skilled in the art.
  • the compounds may be labeled by well- known methods with a suitable fluorescent derivative (Baindur, et a!.. Drug Dev. Res. , 1994, 33, 373-398; Rogers, Drug Discovery Today, 1997, 2, 156-160).
  • Radioactive ligand specifically bound to the OOSBP can be detected in HTS assays in one of several standard ways, including filtration of the receptor-ligand complex to separate bound ligand from unbound ligand (Williams, Med. Res. Rev., 1991 , 11, 147-184.; Sweetnam, et a!., . J. Natural Products, 1993, 56, 441-455).
  • Alternative methods include a scintillation proximity assay (SPA) or a FlashPlate format in which such separation is unnecessary ( akayama, Cur. Opinion Drug Disc. Dev. , 1998, 1, 85-91 Bosse, et al., , . Biomolecular Screening, 1998, 5, 285-292.).
  • Binding of fluorescent ligands can be detected in various ways, including fluorescence energy transfer (FRET), direct spectrophotofluorometric analysis of bound ligand, or fluorescence polarization (Rogers, Drug Discovery Today, 1997, 2, 156-160; Hill, Cm: Opinion Drug Disc. Dev. , 1.998, ./, 92-97).
  • FRET fluorescence energy transfer
  • Differophotofluorometric analysis of bound ligand or fluorescence polarization
  • Candidate modulators contemplated by the invention include compounds selected from libraries. There are a number of different libraries used for the identification of small molecule modulators, including: (1 ) chemical libraries, (2) natural product libraries, and (3) combinatorial libraries. Chemical libraries consist of random chemical structures, some of which are analogs of known compounds or analogs of compounds that have been identified as “hits” or “leads” in other drug discovery screens, some of which are derived from natural products, and some of which arise from non-directed synthetic organic chemistry. Natural product libraries are collections of microorganisms, animals, plants, or marine organisms which are used to create mixtures for screening by: (1) fermentation and extraction of broths from soil, plant or marine microorganisms or (2) extraction of plants or marine organisms.
  • Natural product libraries include polyketides, non-ribosomal peptides, and variants (non- naturally occurring) thereof. For a review, see Science 282:63-68 (1998). Combinatorial libraries are composed, of large of organic compounds as a mixture. These libraries are relatively easy to prepare by traditional automated synthesis methods, PCR, cloning, or proprietary synthetic methods. For a revie of combinatorial chemistry and libraries created therefrom, see Myers, Curr. Opin. Biotechnol. 8:701-707 (1997). Identification of modulators through use of the various libraries described herein permits modification of the candidate "hit” (or “lead” ⁇ to optimize the capacity of the "hit” to modulate activity.
  • assays may be used to identify specific neuropeptide ligands of a OQ8BP including assays that identify ligands of the target protein through measuring direct binding of test ligands to the target protein, as well as assays that identify ligands of target proteins through affinity ultrafiltration with ion spray mass spectroscopy/HPLC methods or other physical and analytical methods.
  • test ligands may be used to search for agents that bind to the target protein.
  • One such screening method to identify direct binding of test ligands to a target protein is described in U.S. Patent No. 5,585,277, incorporated herein by reference. This method relies on the principle that proteins generally exist as a mixture of folded and unfolded states, and continually alternate between the two states.
  • the target protein molecule bound by the ligand remains in its folded, state.
  • the folded, target protein is present to a greater extent in the presence of a test ligand which binds the target protein, than in the absence of a ligand. Binding of the ligand to the target protein can be determined by any method which distinguishes between the folded and unfolded states of the target protein. The function of the target protein need not be known in order for this assay to be performed. Virtually any agent can be assessed by this method as a test ligand, including, but not limited to, metals, polypeptides, proteins, lipids, polysaccharides, polynucleotides and small organic molecules. Another method for identifying ligands of a target protein is described in Wieboldt et aL, Anal.
  • antibodies e.g., monoclonal and polyclonal antibodies, single chain antibodies, chimeric antibodies, bifimctional/bispecific antibodies, humanized antibodies, human antibodies, and complementary determining region (CDR)-grafted antibodies, including compounds which include CDR sequences which specifically recognize a polypeptide of the invention) specific for OSBP or fragments thereof.
  • CDR complementary determining region
  • variable regions of the antibodies of the invention recognize and bind OSBP polypeptides exclusively (i.e. , are able to distinguish OSBP polypeptides from other polypeptides by virtue of measurable differences in binding affinity, despite the possible existence of localized sequence identity, homology, or similarity between an OOSBP and such polypeptides.
  • specific antibodies may also interact with other proteins (for example, S. aureus protein A or other antibodies in EL1SA techniques) through interactions with sequences outside the variable region of the antibodies, and, in particular, in the constant region of the molecule. Screening assays to determine binding specificity of an antibody of the invention are well known and routinely practiced in the art.
  • Antibodies that recognize and bind fragments of the OOSBPs of the invention are also contemplated, provided that the antibodies are specific for an OOSBP.
  • Antibodies of the invention can be produced using any method well known and routinely practiced in the art.
  • Another aspect of the present invention is directed to methods of inducing an immune response in a mammal against a polypeptide of the invention by administering to the mammal an amount of the polypeptide sufficient to induce an immune response.
  • the amount will be dependent on the animal species, size of the animal, and the like but can be determined by those skilled in the art.
  • binding molecules including natural ligands and synthetic compounds, can be identified or developed, using isolated or recombinant OSBP products, OSBP variants, or preferably, ceils expressing such products. Binding partners are useful for purifying OSBP products and detection or quantification of OSBP products in fluid amples using known immunological procedures. Binding molecules are also manifestly useful in modulating (i.e., blocking, inhibiting or stimulating) the biological activities of OOSBP.
  • Mutations such as those described in Table 1 (and others) may be identified at the nucleic acid level methods well known in the art. These may include nucleic acid sequencing, hybridization and extension of primers comprising a particular mutation.
  • the sequence of the protein is valuable for designing antibodies that are either specific for a particular variant of OSBP or alternatively using those areas of homology, generally to cross react with many forms of the protein.
  • An additional use of the polypeptide sequence is to identify regions that are susceptible to digestion by specific proteases. Digestion of the protein into subfragments with or without bound molecules would allow the sites where these molecules bind to be mapped. Understanding the nature of the binding of small organic molecules to the oxysterol binding site could, be used to apply a number of different computational modeling approaches to design other small molecules that target the same site and may have other desired biological and ecotoxicological activities and attributes.
  • Phytophthora. capsici or P.infestans were grown in Malt+ media ( ) at 27 °C.
  • Mycelia were harvested, from culture by filtration, flash frozen in liq N 2 , ground into a fine white powder and then resuspended in extraction buffer (50 mM Hepes (7.5), 2 mM DTT, complete protease inhibitor tablets (Roche) supplemented with 3 uM pepstatin (Sigma), 5 mM EDTA, 5% Glycerol (v/v) and 0.4 M aCl).
  • the suspension was subjected to sonication and then made 0.1 % in Brij-35 , allowed, to stir at 4 °C for one hour and then centrifuged at 1300 x g for 25 min (4 °C) to remove cell debris.
  • the solution was centrifuged at 30,000 x g for 3 min to remove any remaining particulates and the supernatant subjected to centrifugation at 100,000 x g for one hour at 4 *C.
  • the soluble fraction was aspirated, quick frozen in liq 2 and stored at - 80 °C.
  • the sample was separated by S400 chromatography by pumping Buffer A at a flow rate of 1.2 mlJ min and 3.6 niL fractions were collected.
  • the "target” protein was determined by performing a binding assay on column fractions (see Example 3). Fractions containing highest specific binding activity were pooled to give a protein solution enriched in the target protein.
  • a filter binding assay was used for saturation binding studies. Three hundred micrograms of V.infestans or V.capsici soluble protein (prepared as in Example 2) was incubated with various conce trations of tritiated compound in binding buffer (50 mM NaCl , 50 mM sodium phosphate, pH7.2) for 55 min at room temperature in a total volume of 200 fiL, Non-specific background controls included the tritiated compound 1 plus unlabeled compound at a concentration 550 times more than the labeled material A displacement assay was also developed that used a fixed amount of tritiated probe which was then displaced with increasing concentrations of an unlabelled compound.1.
  • the method was adapted for 96-well plates.
  • the tritiated ligand and protein were incubated in polypropylene plates for 55 min at room temperature using the same reaction conditions as the single filter assay.
  • the binding reactions were then transferred to a GF/B filter plate (Packard) via a Packard Filterrnate harvester followed by several rinses with cold buffer. Filter plates were allowed to dry at Room Temperature for 30 mm and then 50 uL of MicroScint 20 scintillation fluid (Perkin Elmer) was added, to each well.
  • GF/B filter plate Packard
  • MicroScint 20 scintillation fluid Perkin Elmer
  • Example 4 Membrane isolation and Protein extraction.
  • Target protein was also enriched further by isolation of golgi/endosomal membranes.
  • Phytophthora mycefia were harvested and ground under liquid N2 as described above in the absence of detergent. The viscosity of the extract was reduced by homogenizing with a polytron briefly. The extract was then centrifuged at 600 x g to remove cell debris (pellet P2) and the supernatant (S I ) was then transferred to a new tube and subjected, to a 20,000 x g spin to give a supernatant (S2) depleted of heavier plasma membrane fragments and.
  • membrane pellet P3 consisting of primarily golgi and endosomal vesicles.
  • Pellet P3 was homogenized in extraction buffer by applying ten strokes in a d ounce homogenizer. Soluble target protein was then extracted by sonicating the homogenized P3 followed by incubation with 0.2 % Brij-35. The solution was clarified by centrifugation at 100.000 x g resulting in a membrane pellet (P4) and. a supernatant containing soluble target (S4).
  • Compound 2 has a carboxyl group appendage to the phenyl ring to facilitate linkage to the resin.
  • the methy ester of compound 2 was also synthesized and screened for biological activity with the assumption that the ester form would resemble a ligand that was tethered through a carboxyl moiety. We verified that both compound 2 and and its methyl ester showed activity.
  • Affinity resins were produced by coupling compound 2 to Uliralink® Biosupport (Pierce) though a suitable spacer.
  • the N-hydroxysuccinamide (NHS) ester of compound 2 was first prepared by coupling with 1.
  • Dicyclohexylurea was removed by filtration, and the product recovered by preparative TLC.
  • 4, 9-dioxa-diaminododecane (DDD) was directly coupled to Ultralink® resin by reacting the amine with azlactone functionalities on the resin. Briefly, 30 ml. of a 2M solution of DDD in water was adjusted to pH 1 1 with HC1, To this solution 0.26 gm of resin was added, and the slurr gently stirred for 4 hrs. Excess DDD was removed by filtration, and the resin washed thoroughly with water. The resin was suspended in 1 M NaCl and stirred, for 15 mm.
  • the resin was recovered by low speed centrifugation (lOOOg), and subsequently washed with water. Substitution of the resin was confirmed by reaction of small amounts of resin with ninhydrin.
  • linker substituted resin was exchanged into DMSO (3 mL), and the N-Hydroxysuccinimide (NHS) ester of compound 2 was added to about 2x molar excess over resin bound amine. Triethylamine was then added (about 36 umoles), and the suspension stirred gently under nitrogen for 3 hrs.
  • Ligand substituted resin was isolated by filtration, washed with DMSO, and then thoroughly with water. Resin was suspended in water containg 10% ethanol, and stored at 4°C. Ligand substitution of the resin was confirmed by 19F NMR (proton decoupled). Ligand density was estimated by IJV/Vis spectroscopy to be about 0.2 mM.
  • the structure of compound 3 is set forth below.
  • affinity resin Fifty microliters of affinity resin, equilibrated in Buffer C (extraction buffer with 0.1 M NaCl), was added, to the 20 mL protem solution and allowed to incubate at room temperature for one hour. The resin was collected by centrifugation at 1500 x g and washed three times with 1.0 mL of Buffer C. The resin was then washed twice with 0.5 mL of a 10 uM solution of compound 3. Finally, the resin was incubated with 0.5 mL of a solution of compound 2 (at lOOx the 1C50 of the tritiated probe) to displace protein species interacting specifically with the affinity matrix.
  • Buffer C extraction buffer with 0.1 M NaCl
  • the individual protein fractions collected from the affinity chromatography protocol were concentrated by TCA precipitation and the pellets were redissolved in 75 uL of LDS buffer. Each sample was separated by SDS-PAGE on a 6- 12% gradient gel (Invitrogen) using a MES buffer system and bands were visualized with colloidal blue stain . Proteins eluted using "inactive" compound washes and active compound elution were compared.. Those unique to the active compound elution lane were excised for tryptic fragment analysis. Amino acid sequences of those fragments were determined using MALDI-ToF mass spectroscopy and. then matched to those masses inferred from a Phytophthora infestans database of known protein sequences available in a public database.
  • Phytophthora capsici mutants were generated using TJV mutagenesis.
  • P. capsici cultures were grown for 10 days on V8 agar medium and zoospore formation induced at 4 C. After 30 minutes, the culture plates were moved to room temperature and each culture flooded with water. The motile zoospores were induced to encyst by addition of 2X encystment medium. The encysted, zoospores were collected by centrifugation and resuspended in water. The number of zoospores was determined with a hemacytometer and adjusted to a count of ⁇ 1 X 107ml with water.
  • the encysted zoospore suspension was placed in a sterile glass petri dish and irradiated with UV light as the zoospore suspension was gently stirred. After irradiation the cysts were transferred, to liquid nutrient medium and allowed to grow at 25 C for 18-24 hours. Enough compound was added at a concentration to completely inhibit hyphal growth. Six- 12 days after addition of compound selection, those colonies able to grow in the presence of the selective agent were picked and single zoospore cultures were obtained by microscopic dissection.
  • Example 8 Preparation of total RNA from P eapsiei and production of transcriptome seque ce
  • RNA was prepared from P. capsici wild type and. resistant germlings and involved inoculating encysted zoospores into a flask with nutrient medium and grown overnight with shaking at 25 C. Mycelia were then harvested by filtration and ⁇ 2 g ground to a fine powder in liquid nitrogen. The ground powder was treated with Triazol reagent (Sigma, St. Louis, MO) to isolate the total RNA using the steps recommended by the manufacturer The RNA was used to prepare a normalized cDNA library suitable for sequencing using an Illumina Selexa parallel sequencer
  • Example 9 Identification of single nucleotide differences between wild-type and Compound resistant spores
  • Phytophthora capsici is diploid and. thus it was predicted, that resistant mutants would differ from wild-type sensitive strains by a single base change in one of two alleles (ie be heterozygous). Furthermore, it was surmised that the base pair difference must result in an amino acid change in some protein encompassed by this region of sequence. Therefore, each possible nucleotide difference between the wild-type and the resistant mutant was given a quality score. For wild-type the score evaluated the probability that the nucleotide in question was homozygous and for the resistant isolate the score e valuated the probability that the nucleotide in question was heterozygous. The possible base pair differences between the two tran scrip tomes were therefore sorted based on these probabilities .
  • Example 10 identification of the target of the chemistry Proteins identified from gel fragments taken from poiyacrylamide gels of samples eluted from inactive affinity resins and. active resins (Example 5) were compared. The major difference in the proteins populating that region of the gel showing most variability in band pattern was the presence of an oxysterol binding polypeptide (OOSBP) of unknown function. This information was used to search for single nucleotide polymorphisms in the sequences of the many independent resistant Phytophthora capsici mutations generated by UV irradiation. Indeed, in all 353 mutant populations, the OOSBP sequence was found to harbor one of eight different amino acid changes (see Table 1)
  • Example 11 Confirmation of single nucleotide polymorphisms by Sanger sequencing Each of the single nucleotide polymorphisms that mapped to the OSBP sequence were confirmed by Sanger sequencing.
  • the OSB region was amplified with primers SI 510 (5' ⁇ CTCGTACGAATCTGGAAC- 3;) SEQ ID NO: 54 and S1380 (5'-
  • the full coding sequence corresponding to amino acids i- 948 of the identified target protein was amplified, by PGR from both P.infestans and P, capsici genomic DNA.
  • PGR products were restricted with BamHl/EcoRl and ligated into a modified pGEM-T vector containing DNA sequence encoding E.coii maltose binding protein (MBP) containing an N-terminal 6x His-tag.
  • MBP E.coii maltose binding protein
  • the 6x-His-MBP tag was linked to the N- terminus of target protein by sequence encoding either a thrombin or HRV3C protease recognition site.
  • a truncated form of gene encoding 350 amino acids of the C -terminus of the protein encompassing the oxysterol domain was also produced in the same manner described above for the full length gene.
  • Expression plasmids containing the desired coding sequences were used to transform BL21-DE3 E.coii cells. When liquid cultures reached an O.D. of 0.5 - 0.6, cells were induced to express fusion protein by the addition of 0.2 mM
  • Phytopihora capsici and P. injestans are signficantly (>100x) more sensitive to Compound 1 and its analogs than Pythium ultimum in radial growth assays.
  • HZQ10 analogs provide exceptional plant disease control of Phytopihora capsici, P injestans and downy mildews (which have very conserved oxysterol-binding domains) but poor or no control of diseases caused by Pythium spp (the oxysterol-binding domain is much less similar between Phytophthora and Pythium spp.).
  • the reduced sensitivity of Pythium spp. to Compound 1 and its analogs could be due to one of several mechanisms including reduced binding to the target, compound efflux, or compound metabolism.
  • the "reduced binding" explanation seemed plausible because the Pythium ultimum oxysterol-binding domain contains 3 amino acid changes at positions known to change in resistant Phytophthora capsici (first alignment).
  • a chimeric OSBP gene was constructed where the P. capsici oxysterol-binding domain was replaced with the P. ultimum oxysterol-binding domain.
  • the sequence of the chimeric protein is set forth in SEQ IB NO: 52.
  • the encoding nucleic acid is set forth in SEQ ID NO:53
  • the chimeric OSBP gene and control plasmids were transformed into Phytophthor capsici zoospores by electroporation.
  • the native Pc OSBP did not transform P. capsici to Compound 1 resistance.
  • Compound. 1 has been tested for control of grape downy mildew in a vineyard in Freiburg. Germany for roughly 4 years. In 2010, in trials of Compound 1, complete control was not provided by the compound. Sporulating lesions were picked from vine leaves, DNA prepared, and the sequence of the oxysterof-binding domain determined. Lesions taken from the Compound 1 -treated vines show two amino acid changes in the OSBP domain (N837I and L863W). Both of these mutations, individually, have been found to confer resistance to HZQ10 analogs in mutated labora tory strains oi Phytophthora capsici.
  • Polyclonal antibodies were produced by GenScript (Piseataway, NJ) using the follow synthetic peptides as antigen:
  • a-OOSBP2 CYPEDR1LASDSRYR (SEQ ID NO:58)
  • a-OOSBP3 HHQHRPHRTRTQRLC (SEQ ID NO:59)
  • a fourth polyclonal antibody (a-OOSBP4) was produced by GenScript using a P.capsici OOSBP peptide sequence corresponding to the terminal 350 amino acids of the protein.
  • the P.capsici OOSBP C-terminal domain was expressed in E.coli and purified to near homogeneity as described above. All polyclonal IgG antibodies were purified to >95% from immune serum by octanoic acid precipitation followed by chromatography on Protein A Sepharose (GE lifesciences).
  • Example 16 isolation of OOSBP by density gradient fractionation.
  • Example 4 An extract was prepared as described in Example 4: "Membrane Isolation and Protein extraction with modifications". The extract was centrifuged at 600 x g to remove cell debris and the supernatant was then transferred to a new tube and subjected to a brief spin at 10,000 x g to further remove particulate, followed by centrifugation at 100,000 x g to collect a total membrane pellet. The pellet was homogenized in Buffer D (50 mM Hepes (7.5), 0.1 M mannitol, 2x Complete protease inhibitor tablets (Roche), 5 mM EDTA,
  • the fractions are enriched in golgi and endoplasmic reticulum membranes.
  • a linearized DNA sequence containing a FLAG tripeptide fused to the N-terrainus of OSBP and a Geneticin resistance marker was used to transform Phytophthora.capsici (Pc) cells.
  • Geneticin resistant clones were screened by Western analysis using a monoclonal anti- Flag antibody (Sigma) and a polyclonal anti-OSBP C-terminal domain Ab.
  • FLAG-OSBP was extracted from membrane fractions as described above (Example 1 ) and exposed to anti- FLAG affinity resin (Sigma).
  • wild type Pc extracted membranes were also incubated with anti-FLAG affinity resin as a negative control. Native elution from anti-Flag affinity resin was achieved using a synthetic FLAG peptide (Sigma).

Abstract

This invention relates to polynucleotides, polypeptides, and methods of using them to screen compounds for anti-Oomycetes activity and to to detect resistance developed to such compounds and detecting compounds capable of overcoming such resistance.

Description

This application claims the benefit of US Provisional Application Serial No 61/506,942 which is hereby incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
This invention relates to polynucleotides, polypeptides, and methods of using them to screen compounds for mti-Oomycete activity and to detect resistance developed to such compounds and detecting compounds capable of overcoming such resistance.
Terrestrial Oomyceies, the largest group of heterotrophic Stramenopiles, include a large number of plant pathogens and are believed to be among the most important plant pathogenic organisms that may be facultatively or obiigately parasitic.
PCT Patent Publication WO 08/013925 discloses the fungicidal azocyclic amide l-[4-[4-[5-(2,6-difluorophenyl)-4,5-dihydro-3-isoxazolyl]-2-thiazolyl]-l-piperidinyl]-
2-[5-methyl-3-(trifluoi methyl)-lH-pyrazol-l-yl]ethanone and methods for its preparation, as well as the utility of this compound, as a fungicide. This compound shows particularly good utility as an agent toxic to Oomyceies of particular economic importance. We have discovered the previously unknown molecular target of this compound. This insight led us to the conception of the invention described below.
The present invention includes
A method to identify a potential anti- Oomycetes compound , the method comprising the steps of:
(a) contacting an isolated Oomycetes oxysterol binding polypeptide with a compound to be tested; and
(b) evaluating whether the compound to be tested is bound by said Oomycetes oxysterol binding polypeptide:
wherein the compound is a potential anti- Oomyceies compound if the compound binds to the Oomycetes oxysterol binding polypeptide ..
The present invention includes a method, to identify a potential anti- Oomyceies compound wherein the oxysterol binding protein is derived from Phytophthora sp. The present invention includes a method to identify a potential anti- Oomycetes compound wherein the oxysteroi binding protein is derived from Pythium sp.
The present invention includes a method to identify a potential anti- Oomyceies compound wherein the oxysteroi binding protein is derived from P!asmopara sp.
The present invention includes a method to identify a potential anti- Oomycetes compound wherein the oxysteroi binding protein is derived, from Psuedoperonospora sp. The present invention includes a method to identify a potential anti- Oomyceies compound wherein the oxysteroi binding protein is selected from the group consisting of polypeptides comprising SEQ ID NOs: 1, 3, 6, 8, 1 1, 14, 17, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50 and 52.
The present invention includes a method to identify a potential anti- Oomyceies compound wherein the oxysteroi binding protein comprises a W at residue position 733; or 1, F, K, or Y at residue position 768; or A, 1, P, V or L at residue position 770; or I, F or Y at residue position 837; or W at residue 839; or H at residue position 861; or W or F at residue position 863;or F or Y at residue position 877 (enumeration based on SEQ ID NO: 1).
The present invention includes the isolated Oomycele oxysteroi binding protem which comprises a: W at residue position 733; or I, F, K, or Y at residue position 768; or A, 1, P, V or L at residue position 770; or 1, F or Y at residue position 837; or W at residue 839; or H at residue position 861 ; or W or F at residue position 863; or F or Y~ at residue position 877.
The present invention includes a method of producing an isolated Oomycele oxysteroi binding protein which comprises: providing a host cell which comprises a recombinant construct which expresses the Oomyceies oxysteroi binding polypeptide of; and growing the host cell under conditions that are suitable for expression of the
recombinant construct. Optionally the method of producing an isolated Oomycele oxysteroi binding protein may further comprise the step of purifying the Oomycele oxysteroi binding protein.
The invention includes all of the vectors, polypeptides, polynucleotides host cells and antibodies described herein. SEQ ID NO:l conserved hypothetical protein [Phytophthora infestans T30-4], CBl Reference Sequence: XP_002902250.1
SEQ ID N():2 complete cds conserved hypothetical protein, Phytophthora infestans T30-4 (PJTG_10462) , NCBl Reference Sequence: XM__Q02902204.1
SEQ ID NO:3 conserved hypothetical protein, Joint Genome Institute (JGI)
Phytophthora capsici TOQ1.
SEQ ID NQ:4 complete cds, conserved hypothetical protein, Joint Genome Institute (JGI), Phytophthora_capsici_TOQ.
SEQ ID O:5 genomic sequence, consented hypothetical protein, Joint Genome Institute (JGI), Phytophthora_capsici_TOQ.
SEQ ID NO:6 Oxysterol Binding Domain of conserved hypothetical protein
[Phytophthora infestans T30-4], NCBl Reference Sequence: XP J3G2902250.1 (residues 733-877)
SEQ ID NO: 7 Sequence encoding Oxysterol Binding Domain of conserved hypothetical protein [Phytophthora infestans T3G-4], NCBl Reference Sequence: XP 002902250.1 (Derived from SEQ ID NO: l)
SEQ ID NO:8 conserved hypothetical protein, Joint Genome Institute (JGI) P. infestans supercontl .18 of Phytophthora infestans 1385000- 1389444.
SEQ ID NO:.9 complete cds, conserved hypothetical protein, Joint Genome Institute (JGI), P. infestans supercontl.18 of Phytophthora infestans [DNA] 1385000-1389444
SEQ ID NO:10 genomic sequence, conserved hypothetical protein, Joint Genome Institute (JGI), P. infestans supercontl.18 of Phytophthora infestans [DNA] 1385000-1389444 SEQ ID NO: 11 conserved hypothetical protein. Joint Genome Institute (JGI)
Phytophthora_ramorum l_l scaffold_67:56451-59691,
SEQ ID NO: 12 complete cds, conserved hypothetical protein, Joint Genome Institute (JGI), Phytophthora ramoruml 1 scaffold 67:56451-59691
SEQ ID NO:13 genomic sequence, conserved hypothetical protein, Joint Genome Institute (JGI), Phytophthora ramorum 1 1 scaffold 67 : 56451-59691
SEQ ID NO: 14 conserved hypothetical protein. Joint Genome Institute (JGI)
Phytophthora_sojae_so 1 J scaffold J 03 :212024-214994.
SEQ ID NO:15 complete CDS, conserved hypothetical protein, Joint Genome Institute (JGI) Phytophthora_sojae_so 1_1 scaffold_103 :212024-214994.
SEQ ID NO:1 genomic DNA, conserved hypothetical protein, Joint Genome Institute (JGI) Phytophthora. sojae_._sol.__ l scaffold , .103:212024-214994.
SEQ ID NO:17 Pytbium_ultimurn, conserved hypothetical protein
SEQ ID NO:18 complete CDS, Pythium ultimum, conserved hypothetical protein
SEQ ID NO: 19 genomic sequence, Pythium ultimum, conserved hypothetical protein SEQ ID NO:20 Oxysterol Birsding Domain of conserved hypothetical protein,
Pseudoperonospora cubensis (cucumber downy mildew organism)
SEQ ID NO:21 coding sequence, Oxysterol Binding Domain of conserved hypothetical protein, Pseudoperonospora cubensis (cucumber downy mildew organism)
SEQ ID NO:22 Oxysterol Binding Domain of conserved hypothetical protein, Plasmopora viticola (grape downy mildew organism)
SEQ ID NO:23 coding sequence, Oxysterol Binding Domain of conserved hypothetical protein, Plasmopora viticola (grape downy mildew organism)
SEQ ID O:24 Oxysterol Binding Domain of conserved hypothetical protein Phytophihora pseudotsugae.
SEQ ID NO:25 coding sequence, Oxysterol Binding Domain of conserved hypothetical protein Phytophihora pseudotsugae
SEQ ID NO:26 Oxysterol Binding Domain of conserved hypothetical protein, Phytophihora capsici
SEQ ID ():27 coding sequence, Oxysterol Binding Domain of conserved hypothetical protein Phytopathora capsici
SEQ ID NO:28 Oxysterol Binding Domain of conserved hypothetical protein, Phytophihora ramorum
SEQ ID NO:29 coding sequence, Oxysterol Binding Domain of conserved hypothetical protein, Phytophihora ramorum
SEQ ID ():30 Oxysterol Binding Domain of conserved hypothetical protein, Phytophihora erythriseptica
SEQ ID NO:31 coding sequence, Oxysterol Binding Domain of conserved hypothetical protein, Phytophihora erythriseptica
SEQ ID NQ:32 Oxysterol Binding Domain of conserved hypothetical protein, Phytophihora sojae
SEQ ID NO:33 coding sequence, Oxysterol Binding Domain of conserved hypothetical protein, Phytophihora sojae
SEQ ID O:34 Oxysterol Binding Domain of conserved hypothetical protein,
Phytophthorum alni
SEQ ID NO:35 coding sequence Oxysterol Binding Domain of conserved hypothetical protein, alni
SEQ ID NO:36 Oxysterol Binding Domain of conserved hypothetical protein, Pythium ultimum
SEQ ID NG:37 coding sequence, Oxysterol Binding Domain of conserved hypothetical protein, Pythium ultimum
SEQ ID ():38 Oxysterol Binding Domain of conserved hypothetical protein, Pythium plendens SEQ ID NO:39 coding sequence, Oxysterol Binding Domain of conserved hypothetical protein, Pythium plendens
SEQ ID NO:40 Oxysterol Binding Domain of conserved hypothetical protein, Pythium sylvaticum
SEQ ID NO:41 coding sequence Oxysterol Binding Domain of conserved hypothetical protein, Pythium sylvaticum
SEQ ID NO: 42 Oxysterol Binding Domain of conserved hypothetical protein, Pythium arrhenomanes
SEQ ID NO:43 coding sequence, Oxysterol Binding Domain of conserved hypothetical protein, Pythium arrhenomanes
SEQ ID NO:44 Oxysterol Binding Domain of conserved hypothetical protein, Pythium aphanadermatum
SEQ ID NO:45 coding sequence Oxysterol Binding Domain of conserved hypothetical protein, Pythium aphanadermatum
SEQ ID NO:46 Oxysterol Binding Domain of conserv ed hypothetical protein, Pythium graminacoia
SEQ ID NO:47 coding sequence Oxysterol Binding Domain of conserved hypothetical protein, Pythium graminacoia
SEQ ID NO:48 Oxysterol Binding Domain of conserved hypothetical protein, Pythium heHcoides
SEQ ID NO:49 coding sequence,Oxysteroi Binding Domain of conserved hypothetical protein, Pythium helicoides
SEQ ID NO: 50 Oxysterol Binding Domain of conserved hypothetical protein, Pythium deliense
SEQ ID NO:51 coding sequence, Oxysterol Binding Domain of conserved hypothetical protein, Pythium deliense.
SEQ ID NO: 52 chimeric protein described in Example 13
SEQ ID NO:53 coding sequence for SEQ ID NO:52
SEQ ID NO:54 primer Example 1 1
SEQ ID NO:55 primer Example 1 1
SEQ ID NO: 56 primer Example 1 1
SEQ ID NO: 57 peptide Example 15
SEQ ID NO: 58 peptide Example 15
SEQ ID NO:59 peptide Example 15
DETAILS OE THE INVENTION
As noted in the background section, PCX Patent Publication WO 08/013925 discloses the fungicidal azocyciic amide l -[4-[4-[5-(2,6-difluorophenyl)-4,5-dihydro-3- isoxazolyl]-2-thiazolyi]-l-piperidm^
yijethanone and methods for its preparation, as well as the utility of this compound, as a fungicide. This compound (hereinafter designated "compound 1 ") has the following structure:
Figure imgf000007_0001
Compuond 1 shows particularly good utility as an agent toxic to Phytophthora, P!asmopara and Pstiedoperonospora .
We used an approach of sequential enrichment of cellular extracts of Phytophthora capsici or Phytophthora infeslans, binding assays and affinity chromatography to identify proteins which bound compound i but not an inactive analogue. The protein with the most avidity for compound 1 was identified as a protein of unknown function, of approximately 1000 residues in length the sequence of which is found in Genbank and is designated with accession number XP_002902250. . The identification of domains that occur within this protein can provide insight into its physiologic function. Utilizing matching against the the Pfam protein families database. Pfam is a database of protein families, where families are sets of protein regions that share a significant degree of sequence similarity, thereby suggesting homology. Similarity is detected using the
HMMER3 (http://hmmer.janelia.org/) suite of programs. (The Pfam protein families database: M. Punta, P.C. Coggiil, R.Y. Eberhardt, J. Mistry, J. Tate, C. Boursnell, N. Pang, K. Forslund, G. Ceric, J. Clements, A. Heger, L, Holm, E.L.L. Sonnhammer, S.R. Eddy, A. Bateman, R.D. Finn
Nucleic Acids Research (2012) 40 (Dl): D290-D301).
Although its physiologic function in the cell (other than its essential nature) is not completely elucidated we have designated the full-length protein as an oxysterol binding protein (OSBP) due to the identification of an OSBP domain defined by the C -terminal portion of the sequenceOxysterol binding domains generally have a consensus sequence which can be represented as:
E-[KQ]-x-[SC]-H-[HR]-[PG]-[PL]-x(l ,2)-[STACFI]-[ACGY] (although the C terminal residues are not as conserved in the Oomycetes.
The domain structure of the full length protein also includes a plekstrin homology domain at the N-terminal third of the protein and a START (StAR-related lipid-transfer) domain(SRPBCC (START/RHO_alpha_C/PlTP Bet_v 1 /CoxG/CalC)) ligand-bindmg domain toward the middle portion of the sequence.
The 'pleckstrin homology' (PH) domain is a domain of about 100 residues that occurs in a wide range of proteins invol ved in intracellular signaling or as constituents of the cytoskeieton. All known cases have a common structure consisting of two perpendicular anti-parallel β sheets, followed by a C-terminai amphipathic helix. The loops connecting the β-strands differ greatly in length. (Mayer B.J., Ren R,5 Clark K.L., Baltimore D., A putative modular domain present in diverse signaling proteins, Cell 73:629-630(1993)).
There are no invariant residues in the PH domain however the PH domain is easily detectable in the Oomycetes.
The StAR-related lipid-transfer (START) domain is an about 200-210 residue lipid- binding motif, which is primarily involved in eukaryotic signaling mediated by lipid binding. R epresentatives of the START domain family have been shown to bind different ligands such as sterols (StAR protein) and phosphatidylcholine (PC-TP). Ligand binding by the START domain can also regulate the activities of other domains that co-occur with the START domain in multidomain proteins. (Ponting CP and Aravind L. START: a lipid- binding domain in StAR, HD-Z1P and signalling proteins Trends Biochem. Sci. 24: 130- 132(1999)).
We confirmed our binding study results by mutation analysis. Mutagenized live organisms subjected to selection with compound 1 show a consistent pattern of
substitutions at the following residues within the oxysteroi binding domain as shown in Table 1 . A model of the oxysteroi portion based on the three-dimensional structure of a yeast oxysteroi binding protein, shows that the mutations are located, spatially to a small region of this domain.
By virtue of our discovery we have identified an essential Oomycete sp. gene product. The skilled artisan recognizes that such a discovery provides a means of screening new compounds active against Oomycete sp. Additionally our identification of resistant mutants provides the skilled, artisan a means of screening compounds capable of controlling Oomycete sp, which have become naturally resistant to compounds with a similar mode of action as compound 1.
Our identification of the residues forming important contacts with synthetic molecules provides a means of the skilled artisan designing compounds capable of controlling a broader range of Oomycete sp, and other fungi dependant on this protein for growth and survival.
DEFINITIONS
Various definitions are made throughout this document. Most words have the meaning that would be aitributed to those words by one skilled in the art. Words specifically defined either below or elsewhere in this document have the meaning provided in the context of the present invention as a whole.
The indefinite articles "a" and "an" preceding an element or component of the invention are intended, to be nonrestrictive regarding the number of instances (i.e. occurrences) of the element or component. Therefore "a" or "an" should be read to include one or at least one. and the singular word form of the element or component also includes the plural unless the number is obviously meant to be singular.
As used herein, the terms "comprises," "comprising," "includes," "including," "has," "having," "contains" or "containing," or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a composition, a mixture, process, method, article, or apparatus that comprises a list of elements is not necessarily limited, to only those elements but may include other elements not expressly listed, or inherent to such composition, mixture, process, method, article, or apparatus. By way of example, a polynucleotide or polypeptide comprising a reference sequence is understood to potentially include flanking sequence on either side of the reference sequence.
Further, unless expressly stated to the contrary, "or" refers to an inclusive or and not to an exclusive or. For example, a condition A or B is satisfied by any one of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and. both A and B are true (or present). A nucleic acid comprising a reference sequence is understood to potentially include flanking sequence on either side of the reference sequence. "cDNA" refers to a DNA that is complementary to and synthesized from a mRNA template using the enzyme reverse transcriptase. The cDNA can be single-stranded or converted, into the double-stranded form.
"Coding sequence" refers to a cDNA sequence that codes for a specific amino acid sequence.
The term "consisting essentially of with respect to a polynucleotide contemplates changes to the nucleic acid wherein the changes in one or more nucleotide bases does not affect the ability of the nucleic acid fragment to mediate gene expression or produce a certain phenotype. This term also refer to modifications of the nucleic acid, fragments of the instant invention such as deletion or insertion or attachment to flanking sequences of one or more nucleotides that do not substantially alter the functional properties of the resulting nucleic acid fragment relative to the initial, unmodified fragment. It is therefore understood, as those skilled in the art will appreciate, that the invention described in terms of polynucleotides that consist essentially any given sequence encompasses more than such specific exemplary sequences. The skilled artisan recognizes a polynucleotide that "comprising," "includes," "has," or "contains" "consists of or "consists essentially of a sequence encompassed by this invention is also defined it's ability to hybridize, under moderately stringent conditions (for example, 1 X SSC, 0.1% SDS, 60°C) with the sequences exemplified herein, or to any portion of the nucleotide sequences reported herein and which are functionally equivalent to the gene or the promoter of the invention. Stringency conditions can be adjusted to screen for moderately similar fragments, such as homologous sequences from distantly related organisms, to highly similar fragments, such as genes that duplicate functional enzymes from closely related, organisms. Post- hybridization washes determine stringency conditions. One set of preferred conditions involves a series of washes starting with 6X SSC, 0.5% SDS at room temperature for 15 min, then repeated with 2X SSC, 0,5% SDS at 45 °C for 30 min, and then repeated twice with 0.2X SSC, 0.5% SDS at 50°C for 30 min. A more preferred set of stringent conditions involves the use of higher temperatures in which the washes are identical to those above except for the temperature of the final two 30 min washes in 0.2X SSC, 0.5% SDS was increased to 60°C. Another preferred, set of highly stringent conditions involves the use of two final washes in 0.1X SSC, 0.1 % SDS at 65°C. In the context of this invention the term "consisting essentially of of a reference sequence (polynucleotide or polypeptide) includes flanking sequences on either side of the reference sequence but does extend to include a full length or mature polypeptide or polynucleotide encoding a mature or full length polypeptide As used herein, the term "contacting" means bringing together, either directly or indirectly, a compound into physical proximity to a polypeptide.
The term "expression", as used herein, refers to the production of a functional end- product. Expression of a gene involves transcription of the gene and translation of the mRNA into a precursor or mature protein.
The term "host cell" refers to any cell or organism into which an isolated nucleic acid fragment has been stably or transiently introduced. The host cell may be part of a larger organism, an individual in tissue culture, or a free-living organism. Examples of host cells include, but are not limited to, bacteria, fungi, insect, plant, and animal
Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described more fully in Sambrook, J., Fritsch, E,F. and Mania tis, T. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989 (hereinafter "Sambrook").
An "intron" is an intervening sequence in a gene that does not encode a portion of the protein sequence. Thus, such sequences are transcribed into R A but are then excised and are not translated. The term is also used for the excised RNA sequences. An "exon" is a portion of the sequence of a gene that is transcribed and is found in the mature messenger RNA derived from the gene, but is not necessarily a part of the sequence that encodes the final gene product.
As used herein, the term "isolated" with respect to a polynucleotide or polypeptide refers to a polynucleotide (DNA or RNA) or polypeptide that has been removed from its whole cell native environment. Examples of isolated polynucleotides include, but are not limited to, recombinant DNA molecules contained in a vector, recombinant DNA molecules maintained in a heterologous host cell, partially or substantially purified nucleic acid molecules, and synthetic DNA or RNA molecules. An example of an isolated polypeptide is a polypeptide which has been partially or substantially purified. Examples of describing the details of such partially purified extracts containing Oomycete oxysteroi binding polypeptides are described herein. Extracts enriched for the golgi and endoplasmic reticulum are sometimes preferred,. Specifically excluded from the definition of isolated as it applies to polynucleotides are whole chromosomes.
The "Oomycetes'" are organisms classified as Stramenopiles. The term "Oomycetes" as used, in this disclosure includes all genera commonly identified as plant pathogenic Oomycetes. These organisms are single-celled and produce thread-like filaments (myceiia). They resemble fungi and grow best in a moist environment. However the cell walls of the Oomycetes are built with cellulose instead of fungal chitin. Accordingly they are closely related to algae not fungi The Oomycetes have individual mobile cells, with the characteristics of the Stramenopiles (for instance, two flageila of different lengths, one with forked bristles). The Oomycetes can reproduce sexually or asexualfy. They sometimes produce asexual spores that use surface water (on soil or plants) to move and. then divide. They also produce thick-shelled sexual spores called oospores. The oospores have a single chromosome set (haploid) and are either male or female (the name Oomycetes means "egg- fungi"). These mate to produce a new thallus.
There are many groups of organisms within the Oomycetes but the most ecomically important genera are: Phytophlhora sp. Pythium sp. and downy mildew organisms.
Phytophthora sp. are aggressive pathogens of plants. One species, Phytophthora infestans, caused the Irish potato famine of 1846 which starved and dislocated millions of people. Phytophthora sojae costs the soybean industry millions of dollars each year, in California and Oregon, a newly emerged Phytophthora species, P. ramorum, is responsible for a disease called sudden oak death that affects not only the live oaks that are the keystone species of the ecosystem but also a large variety of woody shrubs that inhabit the oak ecosystems, such as bay laurel and viburnum. Species of Phytophthora can also destroy eucalyptus, avocado, and pineapple, as well as other tropical crop plants.
The Pythium sp. group is less virulent and more widespread. They are parasites in many plants, especially seedlings.
The downy mildews organisms cause plant diseases that look like 'mildew' growing on leaves, but are neither mildews or rusts. They cause great damage to commercial crops.
One species, Plasmopara viticola, nearly destroyed the entire French wine industry in the 1870s by killing the grapevines. Plasmopara sp. and Psiiedoperonospora sp. are responsible for the downy mildews that affect grapes, lettuce, com, cabbage, cantaloupe, cucumber, pumpkin, squash and watermelon and other crops.
The term "Oomycetes oxysterol binding polypeptide" as used herein refers to a protein product of an essential gene found in Oomycetes which comprises
a) a plekstrin homology domain;
b) a linking sequence of about .140 amino acids;
c) START domain;
d) a linking sequence of about 75 ami no acids; and
e) an oxysterol binding domain. This definition may include among others f in combination with other embodiments listed in this specificaiton or alone) an Oomycetes oxysterol binding polypeptide; wherein the PH domain is at about between residues 28-150; and/or wherein the START domain is at about between residues 291-530; and/or wherein the oxysterol binding domain at about between residues 606-950.
Residue numbers in this specification are described according to the convention described below with reference to Table 1 unless otherwise noted.
This definition may include among others (in combination with other embodiments listed in this specificaiton or alone) an embodiment wherein the the oxysterol binding domain of the Oomycetes has at least 60% 61% 62% 63%, 64%, 65%», 66%, 67%, 68%,
69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% percent similarity to the protein sequence set forth in SEQ ID NO: 6 without taking into account any unmatched, sequence on either side,
This definition may include among others (in combination with other embodiments listed in this specificaiton or alone) an embodiment wherein the noxysterol binding domain of the invention comprises the highly conserved residues KPFNPILGET (773-777).
This definition may include among others (in combination with other embodiments listed in this specificaiton or alone) an embodiment wherein the oxysterol binding domain of the invention comprises the highly conserved residues CEHTSHHPPI (795-804).
This definition may include among others (in combination with other embodiments listed in this specificaiton or alone) an embodiment wherein the the highly conserved residues KPFNPILGET (773-777) and the highly conserved residues CEHTSHHPPI (795- 804).
The term "anti- Oomycetes compound" is a compound which kills or inhibits the growth of Oomycetes.
For the sake of convenience "Oomycete oxysterol binding polypeptide" is abbreviated "OOSBP" in this specification.
The oxysterol binding proteins and binding domains of the invention can be both wild-type or may contain substitutions as set forth below in Table 1. This section also describes the numbering convention used throughout this specification when describing the OOSBP of the invention.
Table 1. Lists the independent mutations identified, and. the changes in the protein primary sequence that results. Residue numbers are expressed relative to the Phytophthora infestans protein sequence of SEQ ID NO:l .
L 733 W G 770 A I P V L
N 837 I F Ϊ
G 839 W
P 861 H
L 863 W F
I 877 F Y
The term "XxxxY" as it is used in Table 1, refers to an amino acid sequence position xxx, a single amino acid X in wild type is changed to an amino acid Y. The numbering system used to identify particular residues uses the sequence of SEQ ID NO: 1 for convenience. It is understand by the skilled artisan that among similar sequences the same numbering system can be used with other related, polypeptides by aligning the related sequences with SEQ ID NO: 1 utlizing for example a scoring matrix such as BLOSUM62 and perhaps introducing insertions or deletions as necessary to create the best alignment. For example, the term "L733W refers to an amino acid at sequence position 733, in which a wild type leucine is changed to tryptophan in a mutant polypeptide. The term
"S768IF Y" refers to an amino acid sequence position 768 in which a wild type serine is changed to isoleucine, phenyalanine, lysine or tyrosine in a mutant polypeptide. The term G770AIPVL refers to an amino acid sequence position 770 in which a wild type glycine is changed to an alanine, isoleucine. proline, valine or leucine. The term N837IFY refers to an amino acid sequence at position 837 in which a wild type asparagine is changed to an isoleucine, phenyalanine or tyrosine. The term G839W refers to an amino acid sequence position 839 in which a wild type glycine is changed to a tryptophan. The term P861H refers to an amino acid sequence at position 861 in which a wild type proline is changed to histidine. The term L863WF refers to an ammo acid sequence at position 863 in which a wild type leucine is changed to a tryptophan or phenylalanine. The term I877FY refers to an amino acid sequence at position 877 in which a wild type isoleucine is changed, to a phenylalanine or tyrosine.
As used herein, "polynucleotide" generally refers to any polyribonucleotide or polydeoxribonucfeotide, which may be unmodified RNA or DNA or modified RNA or DNA. "Polynucleotides" include, without limitation, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, RNA that is mixture of single- and double-stranded regions, and hybrid molecules comprising DNA and RNA that may be single -stranded or, more typically, double-stranded or a mixture of single- and. double-stranded regions. "Polynucleotide" also embraces relatively short polynucleotides, often referred to as oligonucleotides. The nucleotide sequences are presented by single strand only, in the 5' to 3' direction, from left to right.
As used herein, "polypeptide" refers to any peptide or protein comprising amino acids joined to each other by peptide bonds, "Polypeptide" refers to both short chains, commonly referred to as peptides, oligopeptides or oligomers, and to longer chains, generally referred to as proteins. The amino acid sequences are presented in the amino to carboxy direction, from left to right. The amino and carboxy groups are not presented in the sequence.
Polypeptide "similarity" as used herein is defined herein as the percentage of amino acid residues which align based on identit or similarity as determined BLASTP used with the scoring matrix BLOSUM62. Two polypeptide sequences are aligned to optimize the alignment scores using a gap opening penalty of 10, a gap extension penalty of 0.1, The BLOSUM62 scoring matrix of Henikoff and Henikoff is shown in Table 1 (amino acids are indicated by the standard one-letter codes).
A c D E F G H I K L M N P Q R S T V W
A 8
C 0 18
D -4 -6 12
E -2 -8 4 10
F -4 -4 -6 - 6 12
G 0 -6 - 4 -6 12
H -- 4 --6 — 9 0 -2 - 4 16
I 2 2 0 -- o --6
K 2 --6 -2 2 -6 -4 2 -6 10
L -2 -2 -8 -6 - o -6 4 -4 8
M -2 -2 -6 -4 - 6 -4 2 -2 4 10
N -4 - 6 2 0 — 6 0 2 -6 0 -6 -4 12
P -2 - 6 -2 -- 2 -- -4 -- 2 --6 14
Q -2 - 6 o 4 --6 o -- 6 2 -4 0 0 -2 10
R -2 - 6 -4 0 --6 o -- 6 4 -4 -2 0 -4 2 10
S 2 -2 Q 0 -4 0 -2 -4 0 -4 -2 2 -2 0 -2 8
T 0 - 2 -2 -4 -4 -4 -2 -2 -2 -2 0 - 2 -2 -2 2 10
V 0 - 2 -6 -4 -2 -6 -6 6 -4 2 2 - 6 -4 -4 -6 -4 0 8
w -- 6 -4 2 - 4 -4 -6 - 6 -4 --2 --8 --8 - 4 - 6 - 6 - 4 -- 6
Y - 4 -4 - 4 6 -- 6 4 - 4 ...2 --2 - 4 --6 -2 -4 -4 -4 -2
Scores within a BLOSUM are log-odds scores that measure, in an alignment, the logarithm for the ratio of the likelihood of two amino acids appearing with a biological sense and the likelihood of the same amino acids appearing by chance The matrices are based on the minimum percentage identity of the aligned protein sequence used in calculating them. Every possible identity or substitution is assigned a score based on its observed frequences in the alignment of related proteins. A positive score is given to the more likely substitutions while a negative score is given to the less likely substitutions.
This specification frequently makes reference to percent similarity with respect to a polypeptide comprising SEQ ID NO: 6. The skilled artisan understands that SEQ ID NO: 6 represents a fragment which might often be found with flanking sequence of a dissimilar nature on one or both sides. When percent similarity is calculated for such a polypeptide therefore, a with respect to SEQ ID NO: 6 it is done so without regard to the unmatched amino acids not encompassed within the fragment.
Percent sequence "similarity" with respect to polynucleotides of the invention may be calculated as the percentage of nucleotide bases in the candidate sequence that are identical to nucleotides in the OOSBP sequence set forth in a particular polynucleotide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. In eonstrast to polypeptides, "similarity" is identical to "identity" when used to describe a polynucleotide.
The term "recombinant" refers to an artificial combination of two otherwise separated, segments of sequence, e.g., by chemical synthesis or by the manipulation of isolated segments of nucleic acids by genetic engineering techniques.
The terms "recombinant construct", "expression construct" and "recombinant expression construct" are used, interchangeably herein. These terms refer to a functional unit of genetic material that can be inserted into the genome of a cell using standard methodology well known to one skilled in the art. Such construct may be itself or may be used in conjunction with a vector. If a vector is used then the choice of vector is dependent upon the method that will be used to transform host plants as is well known to those skilled in the art. For example, a plasmid vector can be used. The skilled artisan is well aware of the genetic elements that must be present on the vector in order to successfully transform, select and. propagate host cells comprising any of the isolated nucleic acid fragments of the invention. The skilled artisan will also recognize that different independent transformation events will result in different levels and patterns of expression (Jones et al., (1985) EMBO J.
4:2411-2418; De Almeida et al, (1989) Moi. Gen. Genetics 2/5:78-86), and thus that multiple events must be screened in order to obtain lines displaying the desired expression level and pattern. Such screening may be accomplished by Southern analysis of DNA, Northern analysis of mRNA expression, Western analysis of protein expression or phenotypic analysis. The term " wild-type" refers to a gene or gene product which has the characteristics of that gene or gene product when isolated from a naturally occurring source (as the protein is found predominantly in nature among organisms which have not been exposed to selection pressure with compound 1 or similar compounds). A wild-type gene is that which is most frequently observed in a population and is thus arbitrarily designated the "normal" or " wild -type" form of the gene.
In contrast, the term "mutant" refers to a gene or gene product which displays modifications in sequence and or functional properties (i.e., altered characteristics) when compared, to the wild-type gene or gene product. It is noted that naturally-occurring mutants can be isolated; these are identified by the fact that they have altered characteristics when compared to the wild-type gene or gene product.
The present invention relates to molecules which comprise the gene sequences that encode the OOSBF and. constructs and recombinant host cells incorporating the gene sequences; the novel OOSBP encoded by the gene sequences; antibodies to the poHpeptides and homologs; kits employing the polynucleotides and polypeptides, and methods of making and using ail of the foregoing. In add ition, the present invention relates to homologs of the gene sequences and of the polypeptides and methods of making and using the same.
Polypeptides
The present invention includes an isolated Oomycetes oxysterol binding polypeptide (OOSBP) which comprises/consists essentially of a polypeptide which has has 100% to 50% ( and even' integer value in between) similarity (without taking into account unmatched residues flanking the amino or carboxy terminus on either side) to the sequence set forth in SEQ ID NO: 6.
OOSBP polypeptides therefore include the amino acid sequences set out in any of SEQ ID NOs: 1 , 3 8, 11, 14, 17, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50 and 52..
The polypeptides may be isolated, by convention protein purificatation methods from organisms which naturally express the protein. The polypeptide may be chemically synthesized, but also may be produced by recombinant procedures involving host cells of the invention. Use of eukaryotic host cells is expected to provide for such post-translational modifications (e.g., glycosylation, truncation, lipidation, and phosphorylation) as may be needed to confer optimal biological activity. The invention includes isolated polypeptides which have been modified as a result of cellular expression or intentionally modified to facilitate expression,
OOSBP in enough quantities from any of these sources, would form the basis of assays that have utility for identifying synthetic compounds or natural products that have the same mode of action as compound 1.
Residues 733, 768, 770, 839, 861, 863 and 877 may substituted with another naturally occurring amino acid if the object is determine the activity of compounds against a mutant target. We have identified mutations set forth belo in Table 1 above which confer tolerance to compound 1. Although the invention contemplates the specific substitution at the positions indicated in Table 1. The specific amino acids identified as substitutional variants might themselves be substituted with amino acids sharing similar characteristics (often called "conservative substitutions).
For example conservative amino acids can be grouped as described in Lehninger, (Biochemistry, Second Edition; Worth Publishers, Inc. NY, NY (1975), pp.71-77} as set out in Table 2, immediately below.
Table 2
Conservative Substitutions ii
SIDE CHAIN CHARACTERISTIC AMINO ACID
Non-polar (hydrophobic)
A. Aliphatic: A L I V P
B. Aromatic: F W
C. Sulfur-containing: M
D. Borderline: G
Uncharged-polar
A. Hydroxy!: S T Y
B. Amides: N Q
C. Sulfhydryl: C
D. Borderline: G
Positively Charged (Basic): K R H
Negatively Charged (Acidic): D E
Other variants are also contemplated, insertion variants are provided wherein one or more amino acid residues supplement a OOSBP amino acid sequence. Insertions may be located ax either or both termini of the polypeptide, or may be positioned within internal regions of the OOSBP amino acid sequence, Insertional variants with additional residues at either or both termini can include, for example, fusion proteins and polypeptides including amino acid tags or labels.
Insertion variants include OOSBP wherein one or more amino acid residues are added to an OOSBP amino acid sequence, or to a biologically active fragment thereof.
Variant products of the invention also include mature OOSBP products, i.e., OSBP products wherein leader or signal sequences are removed. These residues might be replaced with additional amino terminal residues. The additional amino terminal residues may be derived from another protein, or may include one or more residues that are not identifiable as being derived from specific proteins. OOSBP products with an additional methionine residue at position -1 (Met'-OSBP) are contemplated.
Variants of OOSBP with additional Met. Met-Lys, Lys residues (or one or more basic residues in general} are particularly useful for enhanced recombinant protein production in bacterial host cells. Although we have discovered the mutant polypeptides of the invention by mutagenisis and subsequent selection the skilled artisan recognizes that alterations we have identified of the native amino acid sequence may be accomplished by any of a number of known techniques. For example, mutations may be introduced at particular locations by procedures well known to the skilled artisan, such as oligonucleotide-directed mutagenesis, which is described by Walder et al. (Gene 42: 133 (1986)); Bauer el al. [Gene 37:73 (1985)); Craik {BioTechniques, January 1985, pp. 12-19); Smith el al. (Genetic Engineering: Principles and Methods, Plenum Press (1981)); and U.S. Patent Nos. 4,518,584 and 4,737,462.
Insertional variants also include fusion proteins wherein the amino terminus and/or the carboxy terminus of an oxysterol binding protein or oxysterol binding domain fragment is/are fused to another polypeptide. Also, an oxysterol binding domain fragement may be fused to the scaffold of another oxysterol binding protein from another species, such that that a "chimeric " oxysterol binding protein is created. The invention therefore embraces variants having additional amino acid residues which result from use of specific expression systems. For example, use of commercially available vectors that express a desired polypeptide as part of a glutathione-S-transferase (GST) tusion product provides the desired polypeptide having an additional glycine residue at position -1 after cleavage of the GST component from the desired polypeptide. Variants which result from expression in other vector systems are also contemplated. In one embodiment, either the OSBP polypeptide comprises a label or tag that facilitates its isolation. An exemplary tag of this type is a poly- histidine sequence, generally around six histidine residues, that permits isolation of a compound so labeled using nickel chelation. Other labels and tags, such as the FLAG© tag (Eastman Kodak, Rochester, NY), well known and. routinely used in the art, are embraced by the invention..
In another aspect, the invention provides deletion variants wherein one or more ammo acid, residues in a OOSBP are removed. Deletions can be effected at one or both termini of the OOSBP, or with removal of one or more non-terminal amino acid residues of an OOSBP. Deletion variants, therefore, include all fragments of an OOSBP.
The polypeptides of the present invention are provided, in an isolated form, and preferably are substantially purified. OOSBP may be recovered and purified, from recombinant cell cultures by well-known methods, including ammonium sulfate or ethanol precipitation, anion or cation exchange chromatography, phosphoceliulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyfapatite chromatography and lectin chromatography. Affinity chromatography is described in the examples set forth at the end of this specification.
Polynucleotides
The present invention provides isolated polynucleotides (e.g. , DNA sequences and R A transcripts which encode the polypeptides of the invention, both sense and.
complementary aniisense strands, both single- and double-stranded, including splice variants thereof) that encode OOSBP of the invention. Such polynucleotides are of use for (among other uses) in the expression of and the coconstruction of hybrid OOSBP.
The invention therefore includes a polynucleotide which comprises/consists essentially of a polynuceltide which encodes a polypeptide which has has 100% to 50% (and every integer value in between) (without taking into account unmatched, residues flanking the amino or carboxy terminus on either side) to the sequence set forth in SEQ ID NO: 6.
OOSBP encoding polynucleotides therefore comprise/consist essentially of the polynucleotide sequences set out in any of SEQ ID NOs: 2, 3, 5, 7, 9, 10, 12, 13, 15, 16, 18, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51 and 53. The polynucleotides of the invention comprise polynucleotides that encode polypeptides which comprise the mutant residues at 733, 768. 770, 839. 861 , 863 and 877 as described above.
The invention provides isolated polynucleotides (e.g., cDNA, genomic DNA, synthetic DNA, RNA, or combinations thereof, whether single- or double-stranded) that comprise a nucleotide sequ ence encoding the amino acid sequence of the polypeptides of the invention. Such polynucleotides are useful for recombinant!}' expressing the OOSBP and also for detecting expression of the polypeptide in cells (e.g., using Northern hybridization and in situ hybridization assays). It will be appreciated that numerous other polynucleotides sequences exist that also encode OOSBP having the sequence selected from the group consisting of SEQ ID NOs: 1 ,3, 8, 1 1, 14, 1 7, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48 and 50 due to the well-known degeneracy of the universal genetic code.
The invention also provides a purified and isolated polynucleotides
comprising/consisting essentially of a nucleotide sequence that encodes a OOSBP, wherein the polynucleotide hybridizes to a polynucleotide having the sequence selected from the group consisting of those set forth in SEQ ID NOs: 2, 4, 5, 7, 9, 10, 12, 13, 15, 16, 18, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41 , 43, 45, 47, 49, 51 or the non-coding strand complementary thereto, under the following hybridization conditions: (a) hybridization for 6 hours at 42C in a hybridization solution comprising 50% formamide, 1% SDS, 1 M NaCl, 10% dextran sulfate; and. (b) washing 2 times for 30 minutes each at 60C in a wash solution comprising 0.1% SSC, 1% SDS.
Genomic DNA of the invention comprises the protein-coding region for a polypeptide of the invention and is also intended to include the variants described above. It is widely understood that, for many genes, genomic DNA is transcribed into RNA transcripts that undergo one or more splicing events wherein in iron (i.e., non-coding regions) of the transcripts are removed, or "spliced out," RNA transcripts that can be spliced, by alternative mechanisms, and therefore be subject to removal of different RNA sequences but still encode a OOSBP, are referred to in the art as splice variants which are embraced by the invention. Splice variants comprehended by the invention therefore are encoded by the same original genomic DNA sequences but arise from distinct mRNA transcripts.
The invention also comprehends cDNA that is obtained through reverse transcription of an RNA polynucleotide encodmg OOSBP (conventionally followed by second, strand synthesis of a complementary strand to provide a double-stranded DNA). Preferred DNA sequences encoding OOSBP are selected from the group consisting of 2, 4, 5, 7, 9, 10, 12, 13, 15, 16, 18, 19, 21, 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41, 43, 45, 47, 49, 51. A preferred DNA of the invention comprises a double stranded molecule along with the complementary molecule (the "non-coding strand" or "complement") having a sequence unambiguously deducible from the coding strand according to Watson-Crick base- pairing rales for DNA. Also preferred are other polynucleotides encoding the OOSBP polypeptide selected from the group consisting of SEQ ID NOs: 2, 4, 5, 7, 9, 10, 12, 13, 15, 16, 1 8, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41 , 43, 45, 47, 49 and 51, which differ in sequence from the polynucleotides selected from the group consisting of SEQ ID NOs; 2, 4, 5, 7, 9, 10, 12, 13, 15, 16, 18, 19, 21 , 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51 by virtue of the well-known degeneracy of the universal nuclear genetic code.
Polynucleotides of the invention permit identification and isolation of
polynucleotides encoding related OOSBP, such as allelic variants and species homologs, by well-known techniques including Southern and/or Northern hybridization, and polymerase chain reaction (PCR). Examples of related polynucleotides other Oomycetes genomic sequences, including allelic variants, as well as polynucleotides encoding polypeptides having substantial similarity to SEQ ID NOs: 1 ,4, 8 , 1 1 , 14, 17, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48 and 50 and structurally related polypeptides sharing one or more biological, immunological, and/or physical properties of OOSBP. Homologous polynucleotides encoding OOSBP can also be identified by Southern and/or PCR analysis. Knowledge of the sequence of any individual OOSBP also makes possible through use of Southern hybridization or polymerase chain reaction (PCR) the identification of genomic DNA sequences which include OOSBP expression control regulatory sequences such as promoters, operators, enhancers, repressors, and the like.
Polynucleotides of the invention are also useful in hybridization assays to detect the capacity of cells to express OOSBP polynucleotides. The invention may also provide a basis for diagnostic methods useful for identifying the genetic alteration(s) arising in nature (and consequent resistance to compound 1 and molecules with a mode of action similar to compound 1) in a OOSBP locus that produce the mutations outlined in Table 1.
According to the present invention, the OOSBP nucleotide sequences disclosed herein may be used to identify homologs of the OOSBP, in other species. Any of the nucleotide sequences disclosed herein, or any portion thereof, can be used, for example, as probes to screen databases or nucleic acid libraries, such as, for example, genomic or cDNA libraries, to identify homologs, using screening procedures well known to those skilled, in the art. Accordingly, homologs having at least 60%, more preferably at least 70%, more preferably at least 80%. more preferably at least 90%, more preferably at least 95%, and most preferably at least 100% similarit with OOSBP sequences reported herein can be identified.
One preferred embodiment of the present invention provides an isolated nucleic acid molecule comprising sequences selected from the group consisting of SEQ ID NOs: 2, 4, 5, 7, 9, 10, 12, 13, 15, 16, 18, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49 and 51 and fragments thereof.
As used in the present invention, fragments of OOSBP encoding polynucleotides comprise at least 10, and preferably at least 12, 14, 16, 18, 20, 25, 50, or 75 consecutive nucleotides of a polynucleotide encoding an OOSBP. Preferably, fragment polynucleotides of the invention comprise sequences unique to the OOSBP encoding polynucleotide sequence, and therefore hybridize under highly stringent or moderately stringent conditions only (i.e., "specifically") to polynucleotides encoding OOSBP (or fragments thereof).
Polynucleotide fragments of genomic sequences of the invention comprise not only sequences unique to the coding region, but also include fragments of the full-length sequence derived from introns, regulatory regions, and/or other non-translated sequences. Sequences unique to polynucleotides of the invention are recognizable through sequence comparison to other known polynucleotides, and can be identified through use of alignment programs routinely utilized in the art, e.g., those made available in public sequence databases. Such sequences also are recognizable from Southern hybridization analyses to determine the number of fragments of genomic DNA to which a polynucleotide will hybridize. Polynucleotides of the invention can be labeled in a manner that permits their detection, including radioactive, fluorescent, and enzymatic labeling.
Fragment polynucleotides are particularly useful as probes for detection of full- length or fragments of OOSBP encoding polynucleotides. One or more polynucleotides can be included in kits that are used to detect the presence of a polynucleotide encoding aqn OOSBP or used to detect variations in a polynucleotide sequence encoding an OOSBP.
The invention also embraces DNAs encoding an OOSBP that hybridize under high stringency conditions to the non-coding strand, or complement, of the polynucleotides set forth in sequences selected from the group consisting of SEQ ID NOs: 2, 3, 5, 7, 9, 10, 12, 13, 15, 16, 18, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41 , 43, 45, 47, 49 and 51 Exemplary highly stringent hybridization conditions are as follows: hybridization at 42C in a hybridization solution comprising 50% formamide, 1% SDS, 1 M NaCl, 10% Dextran sulfate, and washing twice for 30 minutes at 60C in a wash solution comprising 0.1 X SSC and 1% SDS. It is understood in the art thai conditions of equivalent stringency can be achieved through variation of temperature and buffer, or salt concentration as described Ausubel et al. (Eds.), Protocols in Molecular Biology, John Wiley & Sons (1994), pp. 6.0.3 to 6.4.10. Modifications in hybridization conditions can be empirically determined or precisely calculated based on the length and the percentage of guanosine/cytosine (GC) base pairing of the probe. The hybridization conditions can be calculated as described in
Sambrook, et ah, (Eds.), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York (1989), pp. 9.47 to 9.51 .
With the knowledge of the nucleotide sequence information disclosed in the present invention, one skilled in the art can identify and obtain nucleotide sequences which encode OOSBP from different sources {i.e., other Oomycetes species or fungal species) through a variety of means well known to the skilled artisan and as disclosed by, for example, Sambrook et al., "Molecular cloning: a laboratory manual", Second Edition, Cold Spring Harbor Press, Cold Spring Harbor, NY (1989), which is incorporated herein by reference in its entirety.
For example, DNA that encodes an OOSBP may be obtained by screening of mRNA, cDNA, or genomic DNA with oligonucleotide probes generated from the OOSBP gene sequence information provided herein. Probes may be labeled with a detectable group, such as a fluorescent group, a radioactive atom or a chemiluminescent group in accordance with procedures known to the skilled, artisan and. used in conventional hybridization assays, as described by, for example, Sambrook et al.
A nucleic acid, molecule comprising any of the OOSBP nucleotide sequences described above can alternatively be synthesized by use of the polymerase chain reaction (PCR) procedure, with the PGR oligonucleotide primers produced from the nucleotide sequences provided herein. See U.S. Patent Numbers 4,683, 195 to Mullis et al. and
4,683,202 to Mullis. The PCR reaction provides a method for selectively increasing the concentration of a particular nucleic acid, sequence even when that sequence has not been previously purified and is present only in a single copy in a particular sample. The method can be used to amplify either single- or double-stranded DNA. The essence of the method involves the use of two oligonucleotide probes to serve as primers for the template- dependent, polymerase mediated replication of a desired nucleic acid molecule,
A wide variety of alternative cloning and in vitro amplification methodologies are well known to those skilled in the art. Examples of these techniques are found in, for example, Berger et ah, Guide to Molecular Cloning Techniques, Methods in Enzymology 152. Academic Press, Inc., San Diego, CA (Berger), which is incorporated herein by reference in its entirety.
Automated sequencing methods can be used to obtain or verify the nucleotide sequence of OOSBP. The OOSBP polynucleotide sequences of the present invention are believed to be 100% accurate. However, as is known in the art, nucleotide sequence obtained by automated methods may contain some errors. Nucleotide sequences determined by automation are typically 90 to at least about 00% identical (and e e }'' integer value in between) to the actual nucleotide sequence of a given nucleic acid molecule. The actual sequence may be more precisely determined using manual sequencing methods, which are well known in the art. An error in a sequence which results in an insertion or deletion of one or more nucleotides may result in a frame shift in translation such that the predicted amino acid sequence will differ from that which would be predicted from the actual nucleotide sequence of the nucleic acid molecule, starting at the point of the mutation.
The nucleic acid molecules of the present invention, and fragments derived therefrom, are useful for screening for restriction fragment length polymorphism (RFLP) associated with some of which may be associated with resistance to compound 1 and compounds having a similar mode of action to compound 1.
The polynucleotide sequence information provided by the invention makes possible large-scale expression of the encoded polypeptide by techniques well known and routinely practiced in the art.
Vectors
Another aspect of the present invention is directed to vectors, or recombinant expression vectors, comprising any of the nucleic acid molecules described abo ve. Vectors are used herein either to amplify DNA or RN A encoding OOSBP and/or to express DNA which encodes OOSBP, Preferred vectors include, but are not limited to, piasmids. phages, cosmids, episomes, viral particles or viruses, and integratable DNA fragments (i.e., fragments integratable into the host genome by homologous recombination). Preferred viral particles include, but are not limited to, adenoviruses, baculoviruses, parvoviruses, herpesviruses, poxviruses, adeno-associated viruses, Semliki Forest viruses, vaccinia viruses, and retroviruses. Preferred expression vectors include, but are not limited to, pcDNA3 (mviirogen) and pSVL (Pharmacia Biotech}. Other expression vectors include, but are not limited to, pSPORT™ vectors, pGEM™ vectors (Promega), pPROEX vectors™ (LTI, Bethesda, MD), Bluescript™ vectors (Stratagene), pQE™ vectors (Qiagen), pSE420™ (Invitrogen), and pYES2™(lnvitrogen).
Expression constructs preferably comprise OOSBP encoding polynucleotides operatively linked to an endogenous or exogenous expression control DNA sequence and a transcription terminator. Expression control DNA sequences include promoters, enhancers, operators, and regulatory element binding sites generally, and are typically selected based on the expression systems in which the expression construct is to be utilized. Preferred promoter and enhancer sequences are generally selected for the ability to increase gene expression, while operator sequences are generally selected for the ability to regulate gene expression. Expression constructs of the invention may also include sequences encoding one or more selectable markers that permit identification of host cells bearing the construct. Expression constructs may also include sequences that facilitate, and preferably promote, homologous recombination in a host cell. Preferred constructs of the invention also include sequences necessary for replication in a host cell.
Expression constructs are preferably utilized for production of an encoded protein, but may also be utilized simply to amplify a OOSBP encoding polynucleotide sequence. In preferred embodiments, the vector is an expression vector wherein the polynucleotide of the invention is operatively linked to a polynucleotide comprising an expression control sequence. Autonomously replicating recombinant expression constructs such as plasmid and viral DNA vectors incorporating polynucleotides of the invention are also provided.
Preferred expression vectors are replicable DNA constructs in which a DNA sequence encoding OOSBP is operably linked or connected to suitable control sequences capable of effecting the expression of the OOSBP in a suitable host. DNA regions are operably linked or connected when they are functionally related to each other. For example, a promoter is operably linked or connected to a coding sequence if it controls the transcription of the sequence. Amplification vectors do not require expression control domains, but rather need only the ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants. The need for control sequences in the expression vector will vary depending upon the host selected and the transformation method chosen. Generally, control sequences include a transcriptional promoter, an optional operator sequence to control transcription, a sequence encoding suitable mRNA ribosomal binding and sequences which control the termination of transcription and translation.
Preferred vectors preferably contain a promoter that is recognized by the host organism. The promoter sequences of the present invention may be prokaryotic, eukaryotic or viral. Examples of suitable prokaryotic sequences include the PR and PL promoters of bacteriophage lambda (The bacteriophage Lambda, Hershey, A, D., Ed., Cold Spring Harbor Press, Cold Spring Harbor, NY (1973), which is incorporated herein by reference in its entirety; Lambda 11, Hendrix, R. W., Ed., Cold. Spring Harbor Press, Cold Spring Harbor, NY (1980), which is incorporated herein by reference in its entirety); the tip, recA, heat shock, and lacZ promoters of E. coli and the SV40 early promoter (Benoist et ah Nature, 1981 , 290, 304-310, wrhich is incorporated herein by reference in. its entirety). Additional promoters include, but are not limited to, E. coli lactose, E. coli arabinose, mouse mammary tumor virus, long terminal repeat of human immunodeficiency virus, maloney v irus, cytomegalovirus immediate early promoter, Epstein Barr virus, Rous sarcoma virus, human actin, human myosin, human hemoglobin, human muscle creatine, and. human
metalothionein.
Additional regulatory sequences can also be included in preferred vectors. Preferred examples of suitable regulatory sequences are represented by the S ine-Dalgamo of the replicase gene of the phage MS-2 and of the gene ell of bacteriophage lambda. The Shine- Dalgarno sequence may be directly followed by DNA encoding OOSBP and result in the expression of the mature OOSBP protein.
Moreover, suitable expression vectors can include an appropriate marker that allowrs the screening of the transformed'transfected host cells. The transformation of the selected host is carried out using any one of the various techniques well known to the expert in the art and described in Sambrook et ah, supra.
An origin of replication can also be provided either by construction of the vector to include an exogenous origin or may be provided by the host cell chromosomal replication mechanism. If the vector is integrated into the host cell chromosome, the latter may be sufficient. Alternatively, rather than using vectors which contain viral origins of replication, one skilled in the art can transform mammalian ceils by the method of co-transformation with a selectable marker and OOSBP genomic or cDNA. An example of a suitable marker is dihydrofolate reductase (DHFR) or thymidine kinase (see, U.S. Patent No, 4,399,216). Nucleotide sequences encoding OOSBP may be recombined with vector DNA in accordance with conventional techniques, including blunt-ended or staggered-ended termini for ligation, restriction enzyme digestion may provide appropriate termini, filling in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid iradesiderable joining, and ligation with, appropriate ligases. Techniques for such manipulation are disclosed, by Sambrook et al., supra and are well known in the art. Methods for construction of mammalian expression vectors are disclosed in, for example, Okayama et al, Mol. Cell Biol. , 1983, 3, 280, Cosman et al., Mol. Immunol, 1986, 23, 935, Cosman et al , Nature, 1984, 312, 768. EP-A -0367566, and WO 91/18982, each of which is incorporated herein by reference in their relevant parts.
Host cells
According to another aspect of the invention, host cells are provided, including prokaryotic and eukaryotic cells, comprising a polynucleotide of the invention (or v ector of the invention) in a manner that permits expression of the encoded OOSBP polynucleotides of the invention may be introduced into the host cell as part of a circular plasmid or a viral vector, or as linear DNA comprising an isolated protein coding region. Methods for introducing DNA into the host cell that are well known and routinely practiced in the art include transformation, transfection, electroporation, nuclear injection, or fusion with carriers such as liposomes, micelles, ghost cells, and protoplasts. Expression systems of the invention include bacterial, yeast, fungal, plant, insect, invertebrate, vertebrate, and mammalian cells systems.
The invention provides host cells that are transformed or transfec ed (stably or transiently) with polynucleotides of the invention or vectors of the invention. As stated, above, such host cells are useful for amplifying the polynucleotides and also for expressing the OOSBP or fragment thereof encoded by the polynucleotide.
In still another related embodiment, the invention provides a method for producing a OOSBP (or fragment thereof) comprising the steps of growmg a host ceil of the invention in a nutrient medium and isolating the polypeptide or variant thereof from the cell or the medium. Because the OOSBP may be membrane bound, it will be appreciated that, for some applications, such as certain activity assays, the preferable isolation may involve isolation of cell membranes containing the polypeptide embedded therein, whereas for other applications a more complete isolation may be preferable. According to some aspects of the present invention, transformed host cells having an expression vector comprising any of the nucleic acid molecules described, above are provided. Expression of the nucleotide sequence occurs when the expression vector is introduced into an appropriate host cell. Suitable host cells for expression of the
polypeptides of the invention include, but are not limited to, prokaryotes, yeast, and eukaryotes. If a prokaryotic expression vector is employed, then the appropriate host cell would be any prokaryotic cell capable of expressing the cloned sequences. Suitable prokaryotic cells include, but are not limited to, bacteria of the genera Escherichia, Bacillus, Salmonella, Pseudomonas, Streptomyces, and Staphylococcus.
If an eukaryotic expression vector is employed, then the appropriate host cell would be any eukaryotic cell capable of expressing the cloned sequence. Preferably, eukaryotic cells are cells of higher eukaryotes. Suitable eukaryotic cells include, but are not limited to, non-human mammalian tissue culture cells and human tissue culture cells. Preferred host cells include, but are not limited to, insect cells, HeLa ceils, Chinese hamster ovary ceils (CHO cells), African green monkey kidney cells (COS cells), human 293 cells, and murine 3T3 fibroblasts. Propagation of such cells in cell culture has become a routine procedure (see, Tissue Culture, Academic Press, Kruse and Patterson, eds. (1973), which is incorporated herein by reference in its entirety).
In addition, a yeast host may be employed as a host cell. Preferred yeast cells include, but are not limited to, the genera Saccharomyces, Pichia, and. Kluveromyces.
Preferred yeast hosts are 5. cerevisiae and P. pastoris. Preferred yeast vectors can contain an origin of replication sequence from a 2T yeast plasrnid, an autonomously replication sequence (ARS), a promoter region, sequences for polyadenylation, sequences for transcription termination, and a selectable marker gene. Shuttle vectors for replication in both yeast and E. coli are also included herein.
Alternatively, insect cells may be used as host cells. In a preferred embodiment, the polypeptides of the invention are expressed using a baculovirus expression system (see, Luekow et a!. , Bio/Technology, 1988, 6, 47, Baculovirus Expression Vectors: A Laborator Manual, O'Rielfy et al. (Eds.), W.H. Freeman and Company, New York, 1992, and U.S. Patent No. 4,879,236, each of which is incorporated herein by reference in its entirety). In addition, the MAXBAC™ complete baculovirus expression system (Invitrogen) can, for example, be used for production in insect cells. Host cells of the invention are a valuable source of immunogen for development of antibodies specifically immimoreactive with mutant and wild-type OOSBP. Host cells of the invention are also useful in methods for the large-scale production of OOSBP polypeptides wherein the cells are grown in a suitable culture medium and the desired polypeptide products are isolated from the cells, or from the medium in which the cells are grown, by purification methods known in the art, e.g. , conventional chromatographic methods including immunoaffinity chromatography, receptor affinity chromatography, hydrophobic interaction chromatography, lectin affinity chromatography, size exclusion filtration, cation or anion exchange chromatography, high pressure liquid chromatography (HPLC), reverse phase HPLC, and the like. Still other methods of purification include those methods wherein the desired protein is expressed and purified as a fusion protein having a specific tag, label, or chelating moiety that is recognized by a specific binding partner or agent. The purified protein can be cleaved to yield the desired protein, or can be left as an intact fusion protein. Cleavage of the fusion component may produce a form of the desired protein having additional amino acid residues as a result of the cleavage process.
Knowledge of OOSBP DNA sequences allowrs for modification of cells to permit, or increase, expression of endogenous OOSBP. Cells can be modified (e.g. , by homologous recombination) to provide increased expression by replacing, in whole or in part, the naturally occurring OOSBP promoter with all or part of a heterologous promoter so that the cells express the OOSBP at higher levels. The heterologous promoter is inserted, in such a manner that it is operatively linked to endogenous OOSBP encoding sequences. (See, for example, PCT International Publication No. WO 94/12650, PCT International Publication No, WO 92/20808, and PCT International Publication No. WO 91/09955.) It is also contemplated that, in addition to heterologous promoter DNA, amplifiable marker DNA (e.g., ada, dhfr, and the multifunctional CAD gene which encodes carbamoyl phosphate synthase, aspartate transcarbamylase, and dihydroorotase) and/or intron DNA may be inserted, along with the heterologous promoter DNA. If linked to the OOSBPcoding sequence, amplification of the marker DNA by standard selection methods results in co-amplification of the OOSBP coding sequences in the cells.
Assays
The initial goal of our research was to determine the molecular target responsible for the toxicity of compound i to Oomycet.es particularly Phytophlhora sp. and downy mildew organisms. In doing so we have also identified a previously unidentified molecular target present in all Oomycetes which will prove useful in the identification of other classes of chemistry useful against many economically important plant pathogens within the group
The present invention therefore involves ligand binding assays to determine the avidity of a test compound for an OOSBP. Such methods are useful in identifying compounds with potential utility as anti-Oomycetes compounds.
The present invention includes a method for evaluating a compound for its ability to bind an Oomycete oxysterol binding polypeptide, the method comprising the steps of:
(a) contacting an isolated oxysterol binding polypeptide with a compound to be tested; and
(b) evaluating whether the compound, to be tested is bound to a polypeptide
comprising/consisting essentially of a sequence having at least 60% 61% 62% 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 9 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% similaritywit SEQ ID NG:6.
The invention further comprises the method stated above wherein the polypeptide has sterol/oxysierol binding activity.
The present invention includes a method for evaluating a compound for its ability to bind an Oomycetes oxysterol binding polypeptide wherein the oxysterol binding protein is derived from Phytophthora sp.
The present invention includes includes a method for evaluating a compound for its ability to bind an Oomycete oxysterol binding polypeptide wherem the oxysterol binding protein is derived from Pylhium sp.
The present invention includes a method for evaluating a compound for its ability to bind an Oomycete oxysterol binding polypeptide wherem the oxysterol binding protein is derived from Plasmopara sp.
The present invention includes a method for evaluating a compound for its ability to bind an Oomycete oxysterol binding polypeptide wherem the oxysterol binding protein is derived, from Psuedoperonospora sp.
The present invention includes a method for evaluating a compound for its ability to bind an Oomycete oxysterol binding polypeptide wherein the oxysterol binding protein is selected from the group consisting of polypeptides comprising SEQ ID NOs: 1 , 3, 6, 8, 1 1, 14, 17, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50 and. 52.
The present invention includes a method for evaluating a compound for its ability to bind, an Oomycete oxysterol binding polypeptide wherem the oxysterol binding protein comprises a W at residue position 733; or I, F, K, or Y at residue position 768; or A, I, P. V or L at residue position 770; or I, F or Y at residue position 837; or W at residue 839; or H at residue position 861 ; or W or F at residue position 863;or F or Y at residue position 877 (enumeration based on SEQ ID NO: 1).
A binding assay can be used to identify compounds that interact with the
polypeptides of the invention, hi binding assays usually (but not always) one partner molecule is immobilized and the other is labeled in some fashion (e.g. using a
nonradioactive label such as an enzyme or fluorescent tag or by using a radioactive label) and added free in solution. After mcubation to allow molecular interaction, and a wash step, the amount of ligand bound is measured using an appropriate detection system. Ligands showing significant binding may then be studied further by ensuring that the protein is in excess, and carrying out experiments with a dilution series of the ligand at a set of known concentrations, typically from 10"2 to 10 ~llJM. The polypeptide may be immobilized using an epitope or other affinity tag on a support material to which an appropriate antibody or binding agent for the tag is attached. Suitable supports used in assays include, but are not limited to, synthetic polymer supports such as polystyrene, polypropylene, substituted, polystyrene. E.g., aminated or carboylzted polystyrene; polyacrylamides; polyamides;
polyvinyl chloride, etc.; glass beads; agarose; nitrocellulose; nylon; polyvinyl! denedifluoride; surface-modified nylon, etc.
Methods to identify binding partner compounds include solution assays, in vitro assays wherein OOSBP are immobi lized, and cell-based assays. Identification of binding partner compounds of OOSBP provides novel compounds useful in the control of Oomycete sp.
The invention includes several assay systems for identifying OOSBP binding partners. In solution assays, methods of the invention comprise the steps of (a) contacting a OOSBP with one or more candidate binding partner compounds and (b) identifying the compounds that bind to the OSBP polypeptide. Identification of the compounds that bind the OSBP polypeptide can be achieved by isolating the OSBP polypeptide/binding partner complex, and separating the binding partner compound from the OOSBP polypeptide. An additional step of characterizing the physical, biological, and/or biochemical properties of the binding partner compound is also comprehended in another embodiment of the invention. In one aspect, the OOSBP polypeptide/binding partner complex is isolated using an antibody immunospecific for the OSBP polypeptide.
In one variation of an in vitro assay, the invention provides a method comprising the steps of (a) contacting an immobilized OOSBP with a candidate binding partner compound and (b) detecting binding of the candidate compound to the OOSBP, In an alternative embodiment, the candidate binding partner compound is immobilized and binding of OOSBP is detected. Immobilization is accomplished using any of the methods well known in the art, including covalent bonding to a support, a bead, or a chromatographic resin, as well as non-covaient, high affinity interactions such as antibody binding, or use of streptavidin/biotin binding wherein the immobilized compound includes a biotin moiety.
Detection of binding can be accomplished (i) using a radioactive label on the compound that is not immobilized, (ii) using of a fluorescent label on the non-immobilized compound, (iii) using an antibody immunospecific for the non-immobilized compound (a hapten), (iv) using a label on the non-immobilized compound that excites a fluorescent support to which the immobilized compound is attached (v) surface p!asmon resonance, as well as other techniques well known and. routinely practiced in the art.
The invention comprehends high-throughput screening (HTS) assays to identify compounds that interact with an OOSBP. HTS assays permit screening of large numbers of compounds in an efficient manner. Cell-based HTS systems are contemplated to investigate OOSBP-ligand interaction. HTS assays are designed to identify "hits'" or "lead compounds" having the desired property, from which modifications can be designed to improve the desired property. Chemical modification of the "hit" or "lead compound" is often based on an identifiable structure/activity relationship between the "hit" and the OOSBP.
Binding can be determined by binding assays which are well known to the skilled artisan, including, but not limited to, gel-shift assays, Western blots, radiolabeled competition assay, co-fractionatio by chromatography, co-precipitation, cross linking, ELISA, and the like, which are described in, for example, Current Protocols in Molecular Biology, 1999, John Wiley & Sons, NY, which is incorporated herein by reference in its entirety. The compounds to be screened include (which may include compounds which are suspected to bind an OOSBP include but are not limited to extracellular, intracellular, biologic or chemical origin.
The compounds of the invention exhibit a variety of chemical structures, The literature is replete with, examples of the use of radiolabeled ligands i HIS binding assays for drug discovery (see Williams. Medicinal Research Reviews, 1991, 11, 147-184.; Sweetnam, el al., J. Natural Products, 1993, 56, 441 -455 for review).
Recombinant proteins are sometimes preferred for HTS binding assays because they allow for better specificity (higher relative purity), provide the ability to generate large amounts of binding material, and can be used in a broad variety of formats (see Hodgson,
Bio/Technology, 1992, 10, 973-980.
In preferred embodiments of the invention, methods of screening for compounds which comprise contacting test compounds with QOSBP and assaying for the presence of a complex between the compound and OOSBP. In such assays, the ligand is typically labeled. After suitable incubation, free ligand is separated from that present in bound, form, and the amount of free or uncomplexed label is a measure of the ability of the particular compound to bind to OOSBP.
Generally an OSBP can be used for HTS binding assays in conjunction with a defined ligand (for example compound 1). The compound is labeled with a suitable radioisotope, including, but not limited to, l2jl, 3H, JJS or j2P, by methods that are well known to those skilled in the art. Alternatively, the compounds may be labeled by well- known methods with a suitable fluorescent derivative (Baindur, et a!.. Drug Dev. Res. , 1994, 33, 373-398; Rogers, Drug Discovery Today, 1997, 2, 156-160). Radioactive ligand specifically bound to the OOSBP can be detected in HTS assays in one of several standard ways, including filtration of the receptor-ligand complex to separate bound ligand from unbound ligand (Williams, Med. Res. Rev., 1991 , 11, 147-184.; Sweetnam, et a!., . J. Natural Products, 1993, 56, 441-455). Alternative methods include a scintillation proximity assay (SPA) or a FlashPlate format in which such separation is unnecessary ( akayama, Cur. Opinion Drug Disc. Dev. , 1998, 1, 85-91 Bosse, et al., , . Biomolecular Screening, 1998, 5, 285-292.). Binding of fluorescent ligands can be detected in various ways, including fluorescence energy transfer (FRET), direct spectrophotofluorometric analysis of bound ligand, or fluorescence polarization (Rogers, Drug Discovery Today, 1997, 2, 156-160; Hill, Cm: Opinion Drug Disc. Dev. , 1.998, ./, 92-97).
Candidate modulators contemplated by the invention include compounds selected from libraries. There are a number of different libraries used for the identification of small molecule modulators, including: (1 ) chemical libraries, (2) natural product libraries, and (3) combinatorial libraries. Chemical libraries consist of random chemical structures, some of which are analogs of known compounds or analogs of compounds that have been identified as "hits" or "leads" in other drug discovery screens, some of which are derived from natural products, and some of which arise from non-directed synthetic organic chemistry. Natural product libraries are collections of microorganisms, animals, plants, or marine organisms which are used to create mixtures for screening by: (1) fermentation and extraction of broths from soil, plant or marine microorganisms or (2) extraction of plants or marine organisms. Natural product libraries include polyketides, non-ribosomal peptides, and variants (non- naturally occurring) thereof. For a review, see Science 282:63-68 (1998). Combinatorial libraries are composed, of large of organic compounds as a mixture. These libraries are relatively easy to prepare by traditional automated synthesis methods, PCR, cloning, or proprietary synthetic methods. For a revie of combinatorial chemistry and libraries created therefrom, see Myers, Curr. Opin. Biotechnol. 8:701-707 (1997). Identification of modulators through use of the various libraries described herein permits modification of the candidate "hit" (or "lead"} to optimize the capacity of the "hit" to modulate activity.
Other assays may be used to identify specific neuropeptide ligands of a OQ8BP including assays that identify ligands of the target protein through measuring direct binding of test ligands to the target protein, as well as assays that identify ligands of target proteins through affinity ultrafiltration with ion spray mass spectroscopy/HPLC methods or other physical and analytical methods.
Other assays may be used to search for agents that bind to the target protein. One such screening method to identify direct binding of test ligands to a target protein is described in U.S. Patent No. 5,585,277, incorporated herein by reference. This method relies on the principle that proteins generally exist as a mixture of folded and unfolded states, and continually alternate between the two states. When a test ligand binds to the folded form of a target protein (i.e., when the test ligand is a ligand of the target protein), the target protein molecule bound by the ligand remains in its folded, state. Thus, the folded, target protein is present to a greater extent in the presence of a test ligand which binds the target protein, than in the absence of a ligand. Binding of the ligand to the target protein can be determined by any method which distinguishes between the folded and unfolded states of the target protein. The function of the target protein need not be known in order for this assay to be performed. Virtually any agent can be assessed by this method as a test ligand, including, but not limited to, metals, polypeptides, proteins, lipids, polysaccharides, polynucleotides and small organic molecules. Another method for identifying ligands of a target protein is described in Wieboldt et aL, Anal. Chem., 69: 1683-1691 (1997), incorporated herein by reference. This technique screens combinatorial libraries of 20-30 agents at a time in solution phase for binding to the target protein. Agents that bind to the target protein are separated from other library components by simple membrane washing. The specifically selected molecules that are retained on the filter are subsequently liberated, from the target protein and analyzed by HPLC and pneumatically assisted electrospray (ion spray) ionization mass spectroscopy. This procedure selects library components with the greatest affinity for the target protein, and is particularly useful for small molecule libraries.
Antibodies
Also comprehended by the present invention are antibodies (e.g., monoclonal and polyclonal antibodies, single chain antibodies, chimeric antibodies, bifimctional/bispecific antibodies, humanized antibodies, human antibodies, and complementary determining region (CDR)-grafted antibodies, including compounds which include CDR sequences which specifically recognize a polypeptide of the invention) specific for OSBP or fragments thereof.
The term "specific for," when used to describe antibodies of the invention, indicates that the variable regions of the antibodies of the invention recognize and bind OSBP polypeptides exclusively (i.e. , are able to distinguish OSBP polypeptides from other polypeptides by virtue of measurable differences in binding affinity, despite the possible existence of localized sequence identity, homology, or similarity between an OOSBP and such polypeptides. It will be understood that specific antibodies may also interact with other proteins (for example, S. aureus protein A or other antibodies in EL1SA techniques) through interactions with sequences outside the variable region of the antibodies, and, in particular, in the constant region of the molecule. Screening assays to determine binding specificity of an antibody of the invention are well known and routinely practiced in the art. For a comprehensive discussion of such assays, see Harlow et al. (Eds.), Antibodies A Laboratory Manual: Cold Spring Harbor Laboratory; Cold Spring Harbor , NY (1988), Chapter 6. Antibodies that recognize and bind fragments of the OOSBPs of the invention are also contemplated, provided that the antibodies are specific for an OOSBP. Antibodies of the invention can be produced using any method well known and routinely practiced in the art.
Another aspect of the present invention is directed to methods of inducing an immune response in a mammal against a polypeptide of the invention by administering to the mammal an amount of the polypeptide sufficient to induce an immune response. The amount will be dependent on the animal species, size of the animal, and the like but can be determined by those skilled in the art.
Specific binding molecules, including natural ligands and synthetic compounds, can be identified or developed, using isolated or recombinant OSBP products, OSBP variants, or preferably, ceils expressing such products. Binding partners are useful for purifying OSBP products and detection or quantification of OSBP products in fluid amples using known immunological procedures. Binding molecules are also manifestly useful in modulating (i.e., blocking, inhibiting or stimulating) the biological activities of OOSBP.
Detection of Mutations
Mutations, such as those described in Table 1 (and others) may be identified at the nucleic acid level methods well known in the art. These may include nucleic acid sequencing, hybridization and extension of primers comprising a particular mutation. The sequence of the protein is valuable for designing antibodies that are either specific for a particular variant of OSBP or alternatively using those areas of homology, generally to cross react with many forms of the protein. An additional use of the polypeptide sequence is to identify regions that are susceptible to digestion by specific proteases. Digestion of the protein into subfragments with or without bound molecules would allow the sites where these molecules bind to be mapped. Understanding the nature of the binding of small organic molecules to the oxysterol binding site could, be used to apply a number of different computational modeling approaches to design other small molecules that target the same site and may have other desired biological and ecotoxicological activities and attributes.
Figure imgf000037_0001
Example 1: Preparation of Phytophthora soluble protein extracts
Phytophthora. capsici or P.infestans were grown in Malt+ media ( ) at 27 °C. Mycelia were harvested, from culture by filtration, flash frozen in liq N2, ground into a fine white powder and then resuspended in extraction buffer (50 mM Hepes (7.5), 2 mM DTT, complete protease inhibitor tablets (Roche) supplemented with 3 uM pepstatin (Sigma), 5 mM EDTA, 5% Glycerol (v/v) and 0.4 M aCl). The suspension was subjected to sonication and then made 0.1 % in Brij-35 , allowed, to stir at 4 °C for one hour and then centrifuged at 1300 x g for 25 min (4 °C) to remove cell debris. The solution was centrifuged at 30,000 x g for 3 min to remove any remaining particulates and the supernatant subjected to centrifugation at 100,000 x g for one hour at 4 *C. The soluble fraction was aspirated, quick frozen in liq 2 and stored at - 80 °C.
Example 2: Enrichment of binding activity
Solid ammonium sulfate was added to Phytophthora protein extracts (see Example 1) to give a final saturation of 30% at 4 CC. The precipitated protein was collected by centrifugation at 30,000 x g and resolubilized in extraction buffer. Approximately 100 rng of material from the ammonium sulfate fractionation step was loaded onto a 2,6 x 60 crn Sephacryl 400 column equilibrated in Buffer A (25 111M Hepes (7.5), 150 mM NaCl, Ix Complete protease inhibitor tablets (Roche), 1 mM DTT, 2 mM EDTA and 0.02% Brij-35). The sample was separated by S400 chromatography by pumping Buffer A at a flow rate of 1.2 mlJ min and 3.6 niL fractions were collected. The "target" protein was determined by performing a binding assay on column fractions (see Example 3). Fractions containing highest specific binding activity were pooled to give a protein solution enriched in the target protein.
Example 3: Ligand Binding Assay
A filter binding assay was used for saturation binding studies. Three hundred micrograms of V.infestans or V.capsici soluble protein (prepared as in Example 2) was incubated with various conce trations of tritiated compound in binding buffer (50 mM NaCl , 50 mM sodium phosphate, pH7.2) for 55 min at room temperature in a total volume of 200 fiL, Non-specific background controls included the tritiated compound 1 plus unlabeled compound at a concentration 550 times more than the labeled material A displacement assay was also developed that used a fixed amount of tritiated probe which was then displaced with increasing concentrations of an unlabelled compound.1. After the incubation period, reactions were quenched, with 5 niL of binding buffer and poured into a vacuum filtration apparatus housing Whatman GF/F filters presoaked in 1 % PEL Filters were then dried at RT and scintillation counting was performed in a Packard instrument
The method was adapted for 96-well plates. In this case the tritiated ligand and protein were incubated in polypropylene plates for 55 min at room temperature using the same reaction conditions as the single filter assay. The binding reactions were then transferred to a GF/B filter plate (Packard) via a Packard Filterrnate harvester followed by several rinses with cold buffer. Filter plates were allowed to dry at Room Temperature for 30 mm and then 50 uL of MicroScint 20 scintillation fluid (Perkin Elmer) was added, to each well.
An alternative approach utilized gel filtration to separate bound from free ligand. Twenty-five microliters of each binding reaction was added, to the top of individual spin columns or 96 well filter plates containing 100 μί,, of G-25 sepb.ad.ex (GE Life Sciences) per column or well, equilibrated in binding buffer. Columns or plates were centrifuged at 1000 x g for 3 minutes and. eluates were analyzed by scintillation counting and specific binding determined as described above.
Example 4: Membrane isolation and Protein extraction.
Target protein was also enriched further by isolation of golgi/endosomal membranes. Phytophthora mycefia were harvested and ground under liquid N2 as described above in the absence of detergent. The viscosity of the extract was reduced by homogenizing with a polytron briefly. The extract was then centrifuged at 600 x g to remove cell debris (pellet P2) and the supernatant (S I ) was then transferred to a new tube and subjected, to a 20,000 x g spin to give a supernatant (S2) depleted of heavier plasma membrane fragments and.
mitochondria . The S2 fraction was then centrifuged at 100,000 x g to give membrane pellet P3 consisting of primarily golgi and endosomal vesicles. Pellet P3 was homogenized in extraction buffer by applying ten strokes in a d ounce homogenizer. Soluble target protein was then extracted by sonicating the homogenized P3 followed by incubation with 0.2 % Brij-35. The solution was clarified by centrifugation at 100.000 x g resulting in a membrane pellet (P4) and. a supernatant containing soluble target (S4).
Example 5: Affinity Resin Synthesis
A close analog of compound i designated compound. 2 (2-[4,5-dihydro-3-[2-[ l -[2- [5-methyl-3-(trifluorom.ethyl)- l H-^
isoxazolyl] benzoic acid was synthesized.. The structure of compound 2 is set forth below.
Figure imgf000039_0001
Compound 2 Compound. 2 has a carboxyl group appendage to the phenyl ring to facilitate linkage to the resin. The methy ester of compound 2 was also synthesized and screened for biological activity with the assumption that the ester form would resemble a ligand that was tethered through a carboxyl moiety. We verified that both compound 2 and and its methyl ester showed activity.
Affinity resins were produced by coupling compound 2 to Uliralink® Biosupport (Pierce) though a suitable spacer. The N-hydroxysuccinamide (NHS) ester of compound 2 was first prepared by coupling with 1. 3-dicyclohexylcarbodiimide (DCC). Briefly, 100 fimoles of compound 2 was combined with equal mole equivalents of N- hydroxysuccinamide and DCC in 3 mL of THF and stirred at room temperature overnight under nitrogen.
Dicyclohexylurea was removed by filtration, and the product recovered by preparative TLC. 4, 9-dioxa-diaminododecane (DDD) was directly coupled to Ultralink® resin by reacting the amine with azlactone functionalities on the resin. Briefly, 30 ml. of a 2M solution of DDD in water was adjusted to pH 1 1 with HC1, To this solution 0.26 gm of resin was added, and the slurr gently stirred for 4 hrs. Excess DDD was removed by filtration, and the resin washed thoroughly with water. The resin was suspended in 1 M NaCl and stirred, for 15 mm. The resin was recovered by low speed centrifugation (lOOOg), and subsequently washed with water. Substitution of the resin was confirmed by reaction of small amounts of resin with ninhydrin. For coupling of the ligand, linker substituted resin was exchanged into DMSO (3 mL), and the N-Hydroxysuccinimide (NHS) ester of compound 2 was added to about 2x molar excess over resin bound amine. Triethylamine was then added (about 36 umoles), and the suspension stirred gently under nitrogen for 3 hrs. Ligand substituted resin was isolated by filtration, washed with DMSO, and then thoroughly with water. Resin was suspended in water containg 10% ethanol, and stored at 4°C. Ligand substitution of the resin was confirmed by 19F NMR (proton decoupled). Ligand density was estimated by IJV/Vis spectroscopy to be about 0.2 mM.
Example 6: Affinity Chromatography and Target isolation
Target isolation using affinity matrices invol ved taking 5 mL of target enriched protein extract (8 mg/mL) was diluted four-fold in buffer B (extraction buffer with no NaCl). The protein solution was then made 5 uM in an "inactive'" analogue designated compound 3 (N- methyl-N-(2^yridinylmethyl)-2-[ l -[2-[3-(trifluoroniethyl)phenyl] acetyl]-4-piperidinyl]-4- thiazoiecarhoxamide) or compound 3 and allowed to incubate at room temperature for 30 min. The structure of compound 3 is set forth below.
Figure imgf000041_0001
Compound. 3
Fifty microliters of affinity resin, equilibrated in Buffer C (extraction buffer with 0.1 M NaCl), was added, to the 20 mL protem solution and allowed to incubate at room temperature for one hour. The resin was collected by centrifugation at 1500 x g and washed three times with 1.0 mL of Buffer C. The resin was then washed twice with 0.5 mL of a 10 uM solution of compound 3. Finally, the resin was incubated with 0.5 mL of a solution of compound 2 (at lOOx the 1C50 of the tritiated probe) to displace protein species interacting specifically with the affinity matrix. The individual protein fractions collected from the affinity chromatography protocol were concentrated by TCA precipitation and the pellets were redissolved in 75 uL of LDS buffer. Each sample was separated by SDS-PAGE on a 6- 12% gradient gel (Invitrogen) using a MES buffer system and bands were visualized with colloidal blue stain . Proteins eluted using "inactive" compound washes and active compound elution were compared.. Those unique to the active compound elution lane were excised for tryptic fragment analysis. Amino acid sequences of those fragments were determined using MALDI-ToF mass spectroscopy and. then matched to those masses inferred from a Phytophthora infestans database of known protein sequences available in a public database.
Example 7 Generation of Phytophthora capsici mutants resistant to Compound 1
Phytophthora capsici mutants were generated using TJV mutagenesis. P. capsici cultures were grown for 10 days on V8 agar medium and zoospore formation induced at 4 C. After 30 minutes, the culture plates were moved to room temperature and each culture flooded with water. The motile zoospores were induced to encyst by addition of 2X encystment medium. The encysted, zoospores were collected by centrifugation and resuspended in water. The number of zoospores was determined with a hemacytometer and adjusted to a count of ~1 X 107ml with water. The encysted zoospore suspension was placed in a sterile glass petri dish and irradiated with UV light as the zoospore suspension was gently stirred. After irradiation the cysts were transferred, to liquid nutrient medium and allowed to grow at 25 C for 18-24 hours. Enough compound was added at a concentration to completely inhibit hyphal growth. Six- 12 days after addition of compound selection, those colonies able to grow in the presence of the selective agent were picked and single zoospore cultures were obtained by microscopic dissection.
Example 8: Preparation of total RNA from P eapsiei and production of transcriptome seque ce
Total RNA was prepared from P. capsici wild type and. resistant germlings and involved inoculating encysted zoospores into a flask with nutrient medium and grown overnight with shaking at 25 C. Mycelia were then harvested by filtration and ~2 g ground to a fine powder in liquid nitrogen. The ground powder was treated with Triazol reagent (Sigma, St. Louis, MO) to isolate the total RNA using the steps recommended by the manufacturer The RNA was used to prepare a normalized cDNA library suitable for sequencing using an Illumina Selexa parallel sequencer
Example 9: Identification of single nucleotide differences between wild-type and Compound resistant spores
Phytophthora capsici is diploid and. thus it was predicted, that resistant mutants would differ from wild-type sensitive strains by a single base change in one of two alleles (ie be heterozygous). Furthermore, it was surmised that the base pair difference must result in an amino acid change in some protein encompassed by this region of sequence. Therefore, each possible nucleotide difference between the wild-type and the resistant mutant was given a quality score. For wild-type the score evaluated the probability that the nucleotide in question was homozygous and for the resistant isolate the score e valuated the probability that the nucleotide in question was heterozygous. The possible base pair differences between the two tran scrip tomes were therefore sorted based on these probabilities .
Example 10: identification of the target of the chemistry Proteins identified from gel fragments taken from poiyacrylamide gels of samples eluted from inactive affinity resins and. active resins (Example 5) were compared.. The major difference in the proteins populating that region of the gel showing most variability in band pattern was the presence of an oxysterol binding polypeptide (OOSBP) of unknown function. This information was used to search for single nucleotide polymorphisms in the sequences of the many independent resistant Phytophthora capsici mutations generated by UV irradiation. Indeed, in all 353 mutant populations, the OOSBP sequence was found to harbor one of eight different amino acid changes (see Table 1)
Example 11: Confirmation of single nucleotide polymorphisms by Sanger sequencing Each of the single nucleotide polymorphisms that mapped to the OSBP sequence were confirmed by Sanger sequencing. The OSB region was amplified with primers SI 510 (5'~ CTCGTACGAATCTGGAAC- 3;) SEQ ID NO: 54 and S1380 (5'-
GGGTCTTGGAGCTCGAA -3') SEQ ID NO: 55. The product was sequenced with primer S1377 (5'- GTGTATGCGACTTGATGC -3') SEQ ID NO: 56. Mutations were detected by electronic analysis of the sequence and by visual inspection of the sequence
chromatogram trace.
The independent mutations thus identified and the changes in the protein primary sequence that results are described above and set forth in Table 1. Example 12: Heterologous Expression and Purification of Target protein
The full coding sequence corresponding to amino acids i- 948 of the identified target protein was amplified, by PGR from both P.infestans and P, capsici genomic DNA. PGR products were restricted with BamHl/EcoRl and ligated into a modified pGEM-T vector containing DNA sequence encoding E.coii maltose binding protein (MBP) containing an N-terminal 6x His-tag. The 6x-His-MBP tag was linked to the N- terminus of target protein by sequence encoding either a thrombin or HRV3C protease recognition site. A truncated form of gene encoding 350 amino acids of the C -terminus of the protein encompassing the oxysterol domain was also produced in the same manner described above for the full length gene. Expression plasmids containing the desired coding sequences were used to transform BL21-DE3 E.coii cells. When liquid cultures reached an O.D. of 0.5 - 0.6, cells were induced to express fusion protein by the addition of 0.2 mM
IPTG and further incubation for 3 - 4 hours. Cells were harvested by centrifugation and disrupted by sonication in extraction buffer. Target protein was purified by c chhrroommaattooggrraapphhyy oonn A Ammyylloossee rreessiinn ((NNEEBB)) ffoolllloowweedd bbyy cchhrroommaattooggrraapphhyy oonn NNii22++--sseepphhaarroossee ((GGEE lliiffeesscciieenncceess)).. FFrraaccttiioonnss ccoonnttaaiinniinngg ppuurriiffiieedd ffuussiioonn pprrootteeiinn wweerree ppoooolleedd aanndd tthheenn cclleeaavveedd wwiitthh eeiitthheerr tthhrroommbbiinn oorr HHRRVV33CC pprrootteeaassee iinn aann oovveerrnniigghhtt ddiiggeesstt aatt 44 aaCC..
Figure imgf000044_0001
domain confers resistance to Compound 1
Phytopihora capsici and P. injestans are signficantly (>100x) more sensitive to Compound 1 and its analogs than Pythium ultimum in radial growth assays. Also, HZQ10 analogs provide exceptional plant disease control of Phytopihora capsici, P injestans and downy mildews (which have very conserved oxysterol-binding domains) but poor or no control of diseases caused by Pythium spp (the oxysterol-binding domain is much less similar between Phytophthora and Pythium spp.). The reduced sensitivity of Pythium spp. to Compound 1 and its analogs could be due to one of several mechanisms including reduced binding to the target, compound efflux, or compound metabolism. The "reduced binding" explanation seemed plausible because the Pythium ultimum oxysterol-binding domain contains 3 amino acid changes at positions known to change in resistant Phytophthora capsici (first alignment).
To evaluate the capacity of the P. ultimum oxysterol-binding domain to confer resistance to Compound 1 a chimeric OSBP gene was constructed where the P. capsici oxysterol-binding domain was replaced with the P. ultimum oxysterol-binding domain. The sequence of the chimeric protein is set forth in SEQ IB NO: 52. The encoding nucleic acid is set forth in SEQ ID NO:53 The chimeric OSBP gene and control plasmids were transformed into Phytophthor capsici zoospores by electroporation. The native Pc OSBP did not transform P. capsici to Compound 1 resistance. Transformation of wild-type Phytophthora capsici with PcOSBP G770V or G839W confers resistance to QGU42 and the chimeric Phytopihora capsici and P. injestans form of the gene did transform P capsici zoospores to resistance, suggesting that Pythium spp. is less sensitive to Compound 1 and its analogs due to reduced, binding of the compound to the target site. Example held isolate i grape dowi mildew ressstant to contains a mutation found in laboratory-generated Phytophthora capsici resistant mutants
Compound. 1 has been tested for control of grape downy mildew in a vineyard in Freiburg. Germany for roughly 4 years. In 2010, in trials of Compound 1, complete control was not provided by the compound. Sporulating lesions were picked from vine leaves, DNA prepared, and the sequence of the oxysterof-binding domain determined. Lesions taken from the Compound 1 -treated vines show two amino acid changes in the OSBP domain (N837I and L863W). Both of these mutations, individually, have been found to confer resistance to HZQ10 analogs in mutated labora tory strains oi Phytophthora capsici.
Example 15 : Production and purification of anti-OOSBP antibodies
Polyclonal antibodies were produced by GenScript (Piseataway, NJ) using the follow synthetic peptides as antigen:
1. -OOSBPl = CSAAPDSQDSSDDKS (SEQ ID NO:57)
2. a-OOSBP2 = CYPEDR1LASDSRYR (SEQ ID NO:58)
3. a-OOSBP3 = HHQHRPHRTRTQRLC (SEQ ID NO:59)
These peptides correspond to amino acid sequence in different regions of P. infestans OOSBP. A fourth polyclonal antibody (a-OOSBP4) was produced by GenScript using a P.capsici OOSBP peptide sequence corresponding to the terminal 350 amino acids of the protein. The P.capsici OOSBP C-terminal domain was expressed in E.coli and purified to near homogeneity as described above. All polyclonal IgG antibodies were purified to >95% from immune serum by octanoic acid precipitation followed by chromatography on Protein A Sepharose (GE lifesciences).
Example 16 : isolation of OOSBP by density gradient fractionation.
An extract was prepared as described in Example 4: "Membrane Isolation and Protein extraction with modifications". The extract was centrifuged at 600 x g to remove cell debris and the supernatant was then transferred to a new tube and subjected to a brief spin at 10,000 x g to further remove particulate, followed by centrifugation at 100,000 x g to collect a total membrane pellet. The pellet was homogenized in Buffer D (50 mM Hepes (7.5), 0.1 M mannitol, 2x Complete protease inhibitor tablets (Roche), 5 mM EDTA,
6 uM Pepstatin A and 0.1 mM Benzamidine). One milliliter of homogenate was then layered on the top of a pre-formed 5-25% lodixanol (OptiPrep, Axis-Shield, Oslo, Norway) gradient and centrifuged at 200,000 xg in a Beckman uftracentrifuge. Fractions were pipetted from the top of the meniscus and samples were analyzed for binding activity as well as the presence of immunoreactive bands after incubation with a-OOSBP4 by Western analysis. Ligand binding activity and -OOSBP4 antigen positive bands exactly overlapped with peak activities migrating to a density of 1.13-1.17 or approximately 22% to 29% lodixanol.
The fractions are enriched in golgi and endoplasmic reticulum membranes.
Example 17 Expression of tagged, full length PC-OSBP in P ytophthora cells
A linearized DNA sequence containing a FLAG tripeptide fused to the N-terrainus of OSBP and a Geneticin resistance marker was used to transform Phytophthora.capsici (Pc) cells. Geneticin resistant clones were screened by Western analysis using a monoclonal anti- Flag antibody (Sigma) and a polyclonal anti-OSBP C-terminal domain Ab. FLAG-OSBP was extracted from membrane fractions as described above (Example 1 ) and exposed to anti- FLAG affinity resin (Sigma). In addition wild type Pc extracted membranes were also incubated with anti-FLAG affinity resin as a negative control. Native elution from anti-Flag affinity resin was achieved using a synthetic FLAG peptide (Sigma). A sample of the control and experimental elution fractions from anti-flag affinity chromatography were separated by SDS-PAGE and all proteins from each condition were identified by mass spectrometry (Harvard Microchemistry). The major species eluted from the affinity resin exposed to FLAG-OSBP containing extract was determined to be OSBP. The purity was approximately 40% (as determined by MS data) and the tagged, species actively bound the 3H-QCX03 ligand with a Kd of 0.3 uM. All protein components of the "purified" FLAG- OSBP traction were identified and approximately 60 proteins were found to be unique to FLAG-expressing cells suggesting a physical interaction/localization with OSBP. The most abundant proteins were those localized to the endoplasmic reticulum, and to a lesser extent mitochondrial and nuclear proteins. Although the present invention and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
Figure imgf000048_0001

Claims

BA9498PWO 2013/009971 PCT/US2012/046437 CLAIMS What is claimed is:
1. A method to identify a potential anti- Oomycetes compound . the method comprising the steps of:
5 (a) contacting an isoiated Oomycetes oxysterol binding polypeptide with a compound to be tested; and
(b) evaluating whether the compound to be tested is bound by said Oomycetes oxysterol binding polypeptide:
wherein the compound is a potential anti- Oomycetes compound if the compound binds to 10 the Oomycetes oxysterol binding polypeptide .
2. The method, of claim 1 w herein the oxysterol binding protein is derived from Phytophthora sp.
15 3. The method of claim 1 wherein the oxysterol binding protein is derived from Pythium sp.
4. The method, of claim 1 wherein the oxysterol binding protein is derived from Plasmopara sp.
20 5. The method of claim 1 wherein the oxysterol binding protein is derived from
Psuedoperonospora sp.
6. The method, of claim 1 w herein the oxysterol binding protein is selected, from the group consisting of a polypeptides comprising SEQ ID NOs: 1 , 3, 6, 8, 1 1 , 14, 17, 20, 22, 24, 26,
25 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50 and 52,
7. The method of claim 1 wherein the Oomycetes oxysterol binding polypeptide comprises or is modified to comprise a:
a) W at residue position 733; or
30 b) I, F, K, or Y at residue position 768; or
c) A, 1, P, V or L at residue position 770; or
d.) I, F or Y at residue position 837; or
e) W at residue 839; or
f) H at residue position 861; or
35 g) W or F at residue position 863 ;or h) F or Y at residue position 877.
8 The method of any of claims 1 -7 wherein the Oomvceie oxysterol binding polypeptide is in the form of an endoplasmic reticulum-golgi enriched extract.
9. A method of producing an isolated Oomycetes oxysterol binding protein which comprises
(a) providing a host cell which comprises a recombinant expression vector which expresses mOomycetes oxysterol binding polypeptide; and
(b) growing the host cell under conditions that are suitable for expression of the
Oomycetes oxysterol binding polypeptide.
10. The method of claim 9 which further comprises the step of purifying the Oomycetes oxysterol binding protein.
11. The method of claim 10 wherem the Oomycete oxysterol binding polypeptide is in the form of the an endoplasmic reticulum-golgi enriched extract.
12 A recombinant expression vector which comprises a polynucleotide which expresses an Oomycete oxysterol binding polypeptide .
13. A host cell which comprises the expression vector of claim 12 .
14 The isolated Oomycetes oxysterol binding protein wherein the Oomycete oxysterol binding polypeptide is in the form of the an endoplasmic reticulum-golgi enriched extract.
15. The isolated Oomycetes oxysterol binding protein of claim 14 which comprises a: a) W at residue position 733; or
b) I, F, K, or Y at residue position 768; or
c) A, I, P, V or L at residue position 770; or
d) 1, F or Y at residue position 837; or
e) W at residue 839; or
f) H at residue position 861 ; or g) W or F at residue position 863;or
h) F or Y at residue position 877.
16. An isolated Oomycetes ox ysterol binding polypeptide of claim 14 wherein the polypeptide is operably linked to a peptide purification tag,
17 . The isolated peptide of claims 16 wherein the peptide purification tag is selected from the group consisting of: polyhistidine, maltose binding peptide, calmodulin binding peptide, , streptavidin binding peptide, and an epitope tag.
18 An isolated antibody that binds specifically to the Oomycete oxysterol binding polypeptide of claims 14-17.
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