Methods for identifying compounds which inhibit binding of nucleocapsid 7 protein to HIV-1 RNA Number:7,101,676 from the United States Patent and Trademark Office (PTO) owispatent

Title: Methods for identifying compounds which inhibit binding of nucleocapsid 7 protein to HIV-1 RNA

Abstract: The present invention relates to methods of identifying a molecule from a library of molecules that inhibits binding of human immunodeficiency virus nucleocapsid 7 polypeptide (NCp7) to an oligonucleotide which comprises admixing an NCp7 polypeptide with at one labeled HIV-1 psi-site oligonucleotide and an amount of the molecule to be tested under binding conditions; and determining the amount of oligonucleotide bound to the NCp7 polypeptide, wherein a decrease in the amount of oligonucleotide bound in the presence of the molecule compared with the amount of oligonucleotide bound in the absence of the molecule indicates that the molecule inhibits binding of NCp7 polypeptide to the oligonucleotide.

Patent Number: 7,101,676 Issued on 09/05/2006 to Buechter,   et al.

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Inventors:	Buechter; Douglas (Killingworth, CT), Hou; Xiaohong (Guilford, CT), Rice; William G. (Madison, CT), Marlor; Christopher W. (Bethany, CT), Yang; Wengang (Branford, CT)
Appl. No.:	10/339,217
Filed:	January 9, 2003

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Current U.S. Class:	435/7.1 ; 424/184.1; 424/207.1; 424/208.1; 435/235.1; 435/456; 435/5; 435/6; 435/69.1; 435/91.2; 530/300; 530/350
Current International Class:	G01N 33/53 (20060101)
Field of Search:	435/5,6,7.1,91.2,456,235.1,69.1 424/207.1,208.1,184.1,93.2,188.1,204.1 530/300,350

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References Cited [Referenced By]

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U.S. Patent Documents

	5652260		July 1997		Kun et al.
	5652367		July 1997		Kun et al.
	5668291		September 1997		Domagala et al.
	5670518		September 1997		Kun et al.
	5733921		March 1998		Bolton et al.
	5734081		March 1998		Domagala et al.
	5753674		May 1998		Kun et al.
	5783384		July 1998		Verdine
	5877185		March 1999		Kun et al.
	5889034		March 1999		Bolton et al.
	5929114		July 1999		Domagala et al.
	6001863		December 1999		Bolton et al.
	6004978		December 1999		Kun et al.
	6008190		December 1999		Meade et al.
	6046228		April 2000		Rice et al.
	6133270		October 2000		Bolton et al.
	6225323		May 2001		Yatscoff et al.
	6242478		June 2001		Welker et al.

Foreign Patent Documents

	WO 9609406		Mar., 1996		WO

Other References

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Sci. vol. 90, 1993, pp. 9721-9724. cited by examiner . Rice et al. Inhibitors of HIV nucleocapsid protein zinc fingers as candidates for the treatment of AIDS. Science. vol. 270, 1995, 1194-1197. cited by examiner . Tummino et al. The human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein zinc ejection activity of disulfide benzamides and benzisothiazolones: correlation with anti-HIV and virucidal activities. Antimicrobial Agents and Chemotherapy. vol. 41 (2), 1997, pp. 394-400. cited by examiner . Aldovini A, and Young RA, Mutations of RNA and Protein Sequences Involved in Human Immunodeficiency Virus Type 1 Packaging Result in Production of Noninfectious Virus, J Virol May 1990, 64(5):1920-1926. cited by other . Altschul, et al., Basic Local Alignment Search Tool, (1990) J. Mol. Biol. 215:403-410. cited by other . Amara et al., Control of a Mucosal Challenge and Prevention of AIDS by a Multiprotein DNA/MVA Vaccine, Science Apr. 6, 2001;292(5514): 69-74. cite- d by other . Amarasinghe et al., NMR Structure of the HIV-1 Nucleocapsid Protein Bound to Stem-Loop SL2 of the .PSI.-RNA Packaging Signal. Implications for Genome Recognition, J Mol Biol Aug. 11, 2000;301(2):491-511. cited by oth- er . Basrur et al., Inactivation of HIV-1 Nucleocapsid Protein P7 by Pyridinioalkanoyl Thioesters, J Biol Chem May 19, 2000;275(20):14890-14897. cited by other . Berg, Potential Metal-Binding Domains in Nucleic Acid Binding Proteins, Science Apr. 25, 1986; 232(4749):485-487. cited by other . Darlix, et al., First Glimpses at Structure-function Relationships of the Nucleocapsid Protein of Retroviruses, J Mol Biol Dec. 8, 1995; 254(4):523-537. cited by other . De Guzman et al., Structure of the HIV-1 Nucleocapsid Protein Bound to the SL3 .PSI.-RNA Recognition Element, Science Jan. 16, 1998; 279(5349):384-388. cited by other . de Rocquigny H. et al., First Large Scale Chemical Synthesis of the 72 Amino Acid HIV-1 Nucleocapsid Protein NCp7 in an Active Form, Biochem Biophys Res Commun Oct. 31, 1991;180(2):1010-8. cited by other . Dorfman et al., Mapping of Functionally Important Residues of a Cysteine-Histidine Box in the Human Immunodeficiency Virus Type 1 Nucleocapsid Protein, J Virol Oct. 1993;67(10):6159-69. cited by other . Gorelick, et al., Strict Conservation of the Retroviral Nucleocapsid Protein Zinc Finger is Strongly Influenced by its Roles in Viral Processes: Characterization of HIV-1, Particles Containing Mutant Nucleocapsid Zinc-Coordinating Sequences, Virology Mar. 30, 1999;256(1):92-104. cited by other . Guo, J, et al., Zinc Finger Structures in the Human Immunodeficiency Virus Type 1 Nucleocapsid Protein Facilitate Efficient Minus- and Plus-Strand Transfer, J Virol Oct. 2000;74(19):8980-8. cited by other . Huang M, et al., Anti-HIV Agents That Selectively Target Retroviral Nucleocapsid Protein Zinc Fingers without Affecting Cellular Zinc Finger Proteins, J Med Chem Apr. 23, 1998;41(9):1371-81. cited by other . Maynard AT, et al., Reactivity of the HIV-1 nucleocapsid protein p7 zinc finger domains from the perspective of density-functional theory, Proc Natl Acad Sci USA Sep. 29, 1998;95(20);11578-83. cited by other . Maynard AT and Covell DG, Reactivity of Zinc Finger Cores; Analysis of Protein Packing and Electrostatic Screening, J Am Chem Soc Feb. 14, 2001;123(6):1047-58. cited by other . McDonnell NB, et al., Zinc Ejection as a New Rationale for the Use of Cystamine and Related Disulfide-Containing Antiviral Agents in the Treatment of AIDS, J Med Chem Jun. 20, 1997;40(13):1969-76. cited by othe- r . Ramboarina S. et al., Structural Investigation on the Requirement of CCHH Zinc Finger Type in Nucleocapsid Protein of Human Immunodeficiency Virus 1, Biochemistry Jul. 27, 1999;38(30):9600-7. cited by other . Rice WG, et al., Inhibition of Multiple Phases of Human Immunodeficiency Virus Type 1. cited by other . South TL, et al., The Nucleocapsid Protein Isolated from HIV-1 Particles Binds Zinc and Forms Retroviral-Type Zinc Fingers, Biochemistry Aug. 28, 1990;29(34):7786-9. cited by other . South TL and Summers MF, Zinc Fingers, Adv Inorg Biochem 1990;8:199-248. cited by other . Takahashi K, et al., Two Basic Regions of NCp7 Are Sufficient for Conformational Conversion of HIV-1 Dimerization Initiation Site from Kissing-loop Dimer to Extended-duplex Dimer, Biol Chem Aug. 17, 2001;276(33):31274-8. cited by other . Turpin JA, et al., Synthesis and Biological Properties of Novel Pyridinioalkanoyl Thiolesters (PATE) as Anti-HIV-1 Agents That Target the Viral Nucleocapsid Protein Zinc Fingers, J Med Chem Jan.14, 1999;42(1):67-86. cited by other . Vuilleumier C, et al., Nucleic Acid Sequence Discrimination by the HIV-1 Nucleocapsid Protein NCp7: A Fluorescence Study, Biochemistry Dec. 21, 1999;38(51);16816-25. cited by other . Williams MC, et al., Mechanism for nucleic acid chaperone activity of HIV-1 nucleocapsid protein revealed by single molecule stretching, Proc Natl Acad Sci USA May 22, 2001;98(11):6121-6. cited by other . Rice, et al., Inhibitors of HIV Nucleocapsid Protein Zinc Fingers as Candidates for the Treatment of AIDS, Science Nov. 17, 1995;270(5239): 1194-97. cited by other . Rice, et al. The Site of Antiviral Action of 3-nitrosobenzamide on the Infectivity Process of Human Immunodeficiency Virus in Human Lymphocytes, Proc. Natl. Acad. Sci. USA Oct. 15, 1993; 90(20): 9721-24. cited by other . Tummino, et al. The vitro Ejection of Zinc From Human Immunodeficiency Virus (HIV) typ 1 Nucleocapsid Protein by Disulfide Benzamides with Cellular Anti-HIV Activity, Proc. Natl. Acad. Sci. USA Feb. 6, 1996;93(3): 969-73. cited by other . Liitsola, K. et al., "Genetic Characterization of HIV-1 Strains in the Baltic Countries and Russia," Scand J. Infect. Dis. 1996; 28: 537-541. cited by other . International Search Report for Application No. PCT/US03/00801. Mailing Date Jun. 22, 2005; Total 6 pages. cited by other.

Primary Examiner: Housel; James
Assistant Examiner: Le; Emily M.
Attorney, Agent or Firm: Wilmer Cutler Pickering Hale & Dorr LLP

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Parent Case Text

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This application claims priority of U.S. Ser. No. 60/347,369, filed Jan. 11, 2002, which is hereby incorporated by reference in its entirety.

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Claims

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What is claimed is:

1. A method for determining whether a compound inhibits formation of a complex between an HIV nucleocapsid protein 7 (NCp7) polypeptide and an HIV .PSI.-site oligonucleotide comprising the steps of: (a) admixing an NCp7 polypeptide with a compound; (b) adding an HIV .PSI.-site oligonucleotide selected from SEQ ID NO:7 and SEQ ID NO:8 to the admixture of step (a) so as to form an HIV .PSI.-site oligonucleotide-NCp7 polypeptide complex; (c) determining amounts of complex formed in step (b); and (d) comparing the amount of complex formed in step (b) in the presence of the compound with the amount of complex formed in the absence of the compound, thereby determining whether the compound inhibits complex formation, wherein a decrease in the amount of complex formed in the presence of the compound indicates that the compound inhibits complex formation.

2. The method of claim 1, wherein the NCp7 polypeptide consists the amino acid sequence set forth in any one of SEQ ID NOS: 10-18, 20, 22-32, 35, 99-101, 104-111, 121-122, 126-130, 132, and 135-144.

3. The method of claim 1, wherein the NCp7 polypeptide consists essentially of two zinc finger binding domains and an amino acid sequence which links the two zinc finger binding domains.

4. A method for determining whether a compound inhibits binding of human immunodeficiency virus (HIV) nucleocapsid 7 polypeptide (NCp7) to an oligonucleotide which comprises: (a) attaching an NCp7 polypeptide to a solid support; (b) incubating the solid support with the NCp7 polypeptide linked thereto with a blocking agent; (c) incubating the solid support with the NCp7 polypeptide linked thereto with: (i) at least one labeled oligonucleotide selected from SEQ ID NO:7 and SEQ ID NO:8, and (ii) an amount of the compound; and (d) determining the amount of labeled oligonucleotide bound to the NCp7 polypeptide, wherein a decrease in the amount of oligonucleotide bound to the NCp7 polypeptide in the presence of the compound compared with the amount of oligonucleotide bound to the NCp7 polypeptide in the absence of the compound indicates that the compound inhibits binding of NCp7 polypeptide to the oligonucleotide.

5. The method of claim 4, wherein the NCp7 polypeptide consists of the amino acid sequence set forth in any one of SEQ ID NOS:10-18, 20, 22-32, 35, 99-101, 104-111, 121-122, 126-130, 132, and 135-144.

6. The method of claim 4, wherein the NCp7 polypeptide consists essentially of two zinc finger binding domains and an amino acid sequence which links the two zinc finger binding domains.

7. The method of claim 4, wherein the blocking agent is selected from the group consisting of: bovine serum albumin (BSA), poly-L-lysine, poly-DL-lysine, poly-L-glutamic acid, poly-DL-glutamic acid polyethyleneimine, poly-4-vinylpyridine, poly-2-vinylpyridine, poly-3-vinylpyridine, polylethylene oxide, bacterial tRNA, yeast tRNA, casein, ovalbumin gamma-globulin, heparin, polybrene, polyacrylic acid, polymethacrylic acid, ampholytic copolymers of acrylic acid with acrylamide, poly-N-carboxyethylacrylamide, poly-N-carboxymethylacrylamide poly-N-carboxypropylacrylamide, poly(glycolic acid), a copolymer of polyacrylic acid, poly(glycolic acid), polylactic acid oligomers and any combination thereof.

8. A kit for detection of agents that inhibit binding of an HIV nucleocapsid polypeptide to an HIV .PSI.-site oligonucleotide, comprising: (a) an NCp7 polypeptide; (b) an isolated HIV .PSI.-site oligonucleotide selected from SEQ ID NO:7 and SEQ ID NO:8; (c) instructions describing how to create the appropriate binding conditions.

9. The kit of claim 8, wherein the NCp7 polypeptide is affixed to a solid support.

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Description

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FIELD OF THE INVENTION

The present invention relates to the field of compound screening assays. This invention relates to biopolymer binding assays, and more particularly to methods for assaying binding between nucleic acids and the human immunodeficiency virus (HIV) nucleocapsid 7 protein (NCp7) and related protein and peptide sequences.

BACKGROUND OF THE INVENTION

It is estimated that there are greater than 30 million cases of HIV infection and AIDS worldwide. There are steep rises of new infections in Eastern Europe and Latin America. In the Caribbean region, AIDS is the primary cause of death among young men and women. In Asia, some 7 million people are living with HIV. In industrialized countries, AIDS continues to have a significant impact in minority communities where complacency in the face of a major health risk is a growing problem (Report of the Executive Director of the Joint United Nations Program on HIV/AIDS (UNAIDS)).

HIV-1 is a retrovirus and thus utilizes RNA as its genomic message. Genome packaging is directed by a gag polyprotein produced in the host cell during late stages of the infectious cycle. An element of gag that is essential for genome recognition and the packaging of infectious RNA is a 55 amino acid nucleocapsid protein, NCp7. NC proteins of all known classes of retrovirus (except spumavirus) contain one or two copies of a retroviral zinc finger (ZF) motif, Cys(X).sub.2Cys(X).sub.4His(X).sub.4Cys, where X is a variable amino acid and Zn.sup.2+ is coordinated to the invariant cysteine and histidine residues. As part of gag, NCp7 initiates genomic RNA encapsidation by recognition of a ca. 120 nucleotide sequence (psi-site or .PSI.-site) of the RNA genome that contains four stem-loop (SL) sequences in its secondary structure (e.g., SL1, SL2, SL3, and SL4). Although multi-drug therapy of AIDS with inhibitors of HIV-1 reverse transcriptase and HIV-1 protease has dramatically delayed the onset of clinical disease and death due to AIDS, problems with this therapy are of increasing concern.

Currently available drugs for the treatment of HIV include six nucleoside reverse transcriptase (RT) inhibitors (zidovudine, didanosine, stavudine, lamivudine, zalcitabine and abacavir), three non-nucleoside reverse transcriptase inhibitors (nevirapine, delavirdine and efavirenz), and five peptidomimetic protease inhibitors (saquinavir, indinavir, ritonavir, nelfinavir and amprenavir). Each of these drugs can only transiently restrain viral replication if used alone. However, when used in combination, these drugs have a profound effect on viremia and disease progression. In fact, significant reductions in death rates among AIDS patients have been recently documented as a consequence of the widespread application of combination therapy. However, despite these impressive results, 30 to 50% of patients ultimately fail combination drug therapies. Insufficient drug potency, non-compliance, restricted tissue penetration and drug-specific limitations within certain cell types (e.g. many nucleoside analogs cannot be phosphorylated in resting cells, which is required for biological activity) may account for the incomplete suppression of sensitive viruses. Furthermore, the high replication rate and rapid turnover of HIV-1 combined with the frequent incorporation of mutations, leads to the appearance of drug-resistant variants and treatment failures when sub-optimal drug concentrations are present Therefore, novel anti-HIV agents exhibiting distinct resistance patterns, and favorable pharmacokinetic as well as safety profiles are needed to provide more treatment options.

Currently marketed HIV-1 drugs are dominated by either nucleoside reverse transcriptase inhibitors or peptidomimetic protease inhibitors. Non-nucleoside reverse transcriptase inhibitors (NNRTIs) have recently gained an increasingly important role in the therapy of HIV infections At least 30 different classes of NNRTI have been described in the literature (De Clercq) and several NNRTIs have been evaluated in clinical trials. Dipyridodiazepinone (nevirapine), benzoxazinone (efavirenz) and bis(heteroaryl) piperazine derivatives (delavirdine) have been approved for clinical use. However, the major drawback to the development and application of NNRTIs is the propensity for rapid emergence of drug resistant strains, both in tissue cell culture and in treated individuals, particularly those subject to monotherapy.

Furthermore, although work continues to advance in the development of vaccines against HIV-1, there is currently no vaccine available with proven effectiveness in humans (Amara et al, Science 2001 Apr. 6; 292(5514): 69-74). In addition, it is clear that there is a need for anti-HIV drugs targeted against novel viral targets that are less prone to the development of resistant virus. These facts stress the importance of methods for the identification of new anti-HIV molecules or compounds and HIV targets that possess the following properties: 1) anti-HIV molecules or compounds against the new targets would not exert cross-resistance with current anti-HIV drugs that affect other targets, 2) structural distinctiveness of the target compared to mammalian counterparts such that new molecule or compound selectivity can be achieved toward the HIV target, and 3) target structural and functional conservation so that mutational escape toward drug-resistance is minimized.

SUMMARY OF THE INVENTION

The present invention is directed to a method for determining whether a compound inhibits formation of a complex between an HIV nucleocapsid protein 7 (NCp7) polypeptide and an HIV .PSI.-site oligonucleotide comprising the steps of: (a) admixing an NCp7 polypeptide with a compound; (b) adding an HIV 1-site oligonucleotide to the admixture of step (a) under appropriate binding conditions so as to form an HIV .PSI.-site oligonucleotide-NCp7 polypeptide complex; and (c) comparing the amount of complex formed in step (b) with the amount of complex formed in the absence of the compound, thereby determining whether the compound inhibits complex formation, wherein a decrease in the amount of complex formed in the presence of the compound indicates that the compound inhibits complex formation.

The present invention is directed to a method for determining whether a molecule inhibits binding of NCp7 to an oligonucleotide which comprises: (a) attaching a NCp7 polypeptide to a solid support, wherein the NCp7 polypeptide is selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4; SEQ ID NOS:10-17, or SEQ ID NOS:18-144 (but not limited thereto); (b) incubating the solid support with the NCp7 polypeptide linked thereto with a blocking agent; (c) incubating the solid support with the NCp7 polypeptide linked thereto with: (i) at least one labeled oligonucleotide selected from the group consisting of: SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, a portion of a HIV-1 .PSI.-site RNA which binds to NCp7 polypeptide, an oligonucleotide which binds to the NCp7 polypeptide under physiological conditions, and any combination thereof, and (ii) an amount of the molecule to be tested under binding conditions; and (d) determining the amount of oligonucleotide bound to the NCp7 polypeptide, wherein a decrease in the amount of oligonucleotide bound in the presence of the molecule compared with the amount of oligonucleotide bound in the absence of the molecule indicates that the molecule inhibits binding of NCp7 polypeptide to the oligonucleotide.

The present invention is further directed to a method for determining whether a test molecule has an ability to inhibit binding of NCp7 to an oligonucleotide. The method comprises: (a) attaching to a solid support at least one labeled oligonucleotide selected from the group consisting of: SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, a portion of a HIV-1 .PSI.-site RNA which binds to NCp7 polypeptide, an oligonucleotide which binds to the NCp7 polypeptide under physiological conditions, and any combination thereof, (b) incubating the solid support with the oligonucleotide linked thereto with a blocking agent; (c) incubating the solid support with the oligonucleotide linked thereto with: (i) NCp7 polypeptide wherein the NCp7 polypeptide is selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3; SEQ ID NO:4; SEQ ID NOS:10-17, or SEQ ID NOS:18-144 (but not limited thereto); and (ii) an amount of the molecule to be tested under binding conditions; and (iii) determining the amount of oligonucleotide bound to the NCp7 polypeptide, wherein a decrease in the amount of oligonucleotide bound in the presence of the molecule compared with the amount of oligonucleotide bound in the absence of the molecule indicates that the molecule inhibits binding of NCp7 polypeptide to the oligonucleotide.

The present invention is further directed to screening methods for identifying a molecule from a library of molecules that inhibits binding of NCp7 polypeptide to an oligonucleotide, the method comprising: (a) attaching an NCp7 polypeptide to a solid support, such as an NCp7 zinc finger (ZF) motif having the polypeptide sequence as follows: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3; SEQ ID NO:4; SEQ ID NOS:10-17, or SEQ ID NOS:18-144 (but not limited thereto); (b) incubating the solid support with a blocking agent; (c) washing the solid support so as to remove unbound blocking agent; (d) incubating the solid support with at least one labeled oligonucleotide selected from the group consisting of: SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, a portion of a HIV-1 .PSI.-site RNA which binds to NCp7 polypeptide, an oligonucleotide which binds to the NCp7 polypeptide under physiological conditions, and any combination thereof, and a sufficient amount of a test molecule under appropriate binding conditions; (e) washing the solid support to remove unbound oligonucleotide and test molecules; and (f) determining the amount of oligonucleotide bound to the NCp7 polypeptide, wherein a decrease in the amount of oligonucleotide bound in the presence of the test molecule compared with the amount of oligonucleotide bound in the absence of the test molecule indicates that the molecule inhibits binding of NCp7 polypeptide to the oligonucleotide.

The present invention is still further directed towards methods for identifying a molecule from a library of molecules by means of screening said library, wherein a particular compound which is a member of said library is found to inhibit association of NCp7 to an oligonucleotide. Such methods are comprised of (a) attaching to a solid support at least one labeled oligonucleotide selected from the group consisting of: SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, a portion of a HIV-1 .PSI.-site RNA which binds to NCp7 polypeptide, an oligonucleotide which binds to the NCp7 polypeptide under physiological conditions, and any combination thereof, (b) incubating the solid support with the oligonucleotide linked thereto with a blocking agent; (c) incubating the solid support with the oligonucleotide linked thereto with: (i) NCp7 polypeptide wherein the NCp7 polypeptide is selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3; SEQ ID NO:4; SEQ ID NOS:10-17, or SEQ ID NOS:18-144 (but not limited thereto); and (ii) an amount of the molecule to be tested under binding conditions; and (d) determining the amount of oligonucleotide bound to the NCp7 polypeptide, wherein a decrease in the amount of oligonucleotide bound in the presence of the molecule compared with the amount of oligonucleotide bound in the absence of the molecule indicates that the molecule inhibits binding of NCp7 polypeptide to the oligonucleotide.

Aspects of the present invention are directed to methods of treating a subject suffering from HIV infection which comprises administering to the subject an effective amount of a composition comprising the molecule identified by the above screening method.

In the present embodiments of the assay, the blocking agent is selected from the group consisting of: bovine serum albumin (BSA), poly-L-lysine, poly-DL-lysine, poly-L-glutamic acid, poly-DL-glutamic acid, polyethyleneimine, poly-4-vinylpyridine, poly-2-vinylpyridine, poly-3-vinylpyridine, polylethylene oxide, bacterial tRNA, yeast tRNA, casein, ovalbumin, gamma-globulin, heparin, polybrene, polyacrylic acid, polymethacrylic acid, ampholytic copolymers of acrylic acid with acrylamide, poly-N-carboxyethylacrylamide, poly-N-carboxymethylacrylamide, poly-N-carboxypropylacrylamide, poly(glycolic acid), copolymers of polyacrylic acid and poyl(glycolic acid), polylactic acid oligomers and any combination thereof.

In one embodiment, the amount of BSA comprises from about 0.25% to about 10%. In another embodiment, the amount of BSA comprises from about 0.5% to about 5%. In still another embodiment, the amount of BSA comprises from about 1% to about 3%.

In the present embodiment of the invention, the solid support is selected from the group consisting of: a solid phase column support; a silica support; a magnetic support; a gel support; a glass support; a polystyrene support; a polypropylene support; a polycarbonate surface derivatized with tetraethoxysilane; a polycarbonate surface derivatized with dimethyldiethoxysilane; a polycarbonate surface derivatized with silicon tetraacetate; a polycarbonate surface derivatized with methyltriacetoxysilane; a polycarbonate support derivatized with any di- or tri-alkoxysilane; an synthetic alumina surface, whereby hydroxyl groups upon the said surface may be optionally derivatized with tetraethoxysilane, dimethyldiethoxysilane; methyltriacetoxysilane; or with any di- or tri-alkoxysilane; a silicon monoxide surface, whereby hydroxyl groups upon the said surface may be optionally derivatized with tetraethoxysilane, dimethyldiethoxysilane; methyltriacetoxysilane; or with any di- or tri-alkoxysilane; a silicon monoxide surface upon a silicon substrate, whereby hydroxyl groups upon the said surface may be optionally derivatized with tetraethoxysilane, dimethyldiethoxysilane; methyltriacetoxysilane; or with any di- or tri-alkoxysilane , a titanium dioxide surface whereby hydroxyl groups upon the said surface may be optionally derivatized with tetraethoxysilane, dimethyldiethoxysilane; methyltriacetoxysilane; or with any di- or tri-alkoxysilane, a zirconium dioxide surface whereby hydroxyl groups upon the said surface may be optionally derivatized with tetraethoxysilane, dimethyldiethoxysilane; methyltriacetoxysilane; or with any di- or tri-alkoxysilane, a tin oxide surface of electrically conductive nature, whereby hydroxyl groups upon the said surface may be optionally derivatized with tetraethoxysilane, dimethyldiethoxysilane; methyltriacetoxysilane; or with any di- or tri-alkoxysilane , a polycarbonate, polystyrene, or polypropylene support derivatized with streptavidin; and any combination thereof.

In one embodiment of the invention, the oligonucleotide is labeled with biotin, a fluorescent label, a radioactive label, a chemiluminescent label, a protein detectable by an antibody, an avidin, a horseradish peroxidase, a green fluorescent protein or any combination thereof.

In one embodiment of the invention, the oligonucleotide is attached to the solid support covalently or non-covalently.

In another embodiment, the molecule comprises an azodicarbonamide (ADA) or a derivative thereof. In a further aspect of the invention, the molecule comprises a 2,2'-dithiobisbenzamide (DIBA-1) or a derivative thereof.

In another aspect of the invention, the molecule has the structure ##STR00001##

In another aspect of the invention, the molecule has the structure ##STR00002##

In another aspect of the invention, the molecule has the structure ##STR00003##

In another aspect of the invention, the molecule has the structure ##STR00004##

In another aspect of the invention, the molecule has the structure ##STR00005##

In another aspect of the invention, the molecule has the structure ##STR00006##

In another aspect of the invention, the molecule has the structure ##STR00007##

In another aspect of the invention, the molecule has the structure ##STR00008##

In another aspect of the invention, the molecule has the structure ##STR00009##

wherein R1 and R2 are independently alkyl, trifluoroalkyl, dialkylamino, nitro, trifluoroalkoxy, or any combination thereof.

In another aspect of the invention, the molecule has the structure ##STR00010##

In another aspect of the invention, the molecule has the structure ##STR00011##

In another aspect of the invention, the molecule has the structure ##STR00012##

In another aspect of the invention, the molecule has the structure ##STR00013##

In another aspect of the invention, the molecule has the structure ##STR00014##

In another aspect of the invention, the binding conditions comprise from about 5 mM KCl to about 100 mM KCl and from about 5 mM MgCl.sub.2 to about 100 mM MgCl.sub.2.

In another aspect of the invention, the compound comprises a molecule selected from the group consisting of: tetramethylthiuram disulfide, tetraethylthiuram disulfide, tetraisopropylthiuram disulfide, tetrabutylthiuram disulfide, dicyclopentamethylenethiuram disulfide, isopropylxanthic disulfide, O,O-diethyl dithiobis-(thioformate), benzoyl disulfide, benzoylmethyl disulfide, formamidine disulfide 2HCl, 2-(diethylamino)ethyl disulfide, aldrithiol-2, aldrithiol-4,2,2-dithiobis(pyridine N-oxide), 6,6-dithiodinicotinic acid, 4-methyl-2-quinolyl disulfide, 2-quinolyl disulfide, 2,2 dithiobis(benzothiazole), 2,2-dithiobis(4-tert-butyl-1-isopropyl)-imidazole, 4-(dimethylamino)phenyl disulfide, 2-acetamidophenyl disulfide, 2,3-dimethoxyphenyl disulfide, 4-acetamidophenyl disulfide, 2-(ethoxycarboxamido)phenyl disulfide, 3-nitrophenyl disulfide, 4-nitrophenyl disulfide, 2-aminophenyl disulfide, 2,2 dithiobis(benzonitrile), p-tolyl disulfoxide, 2,4,5-trichlorophenyl disulfide, 4-methylsulfonyl-2-nitrophenyl disulfide, 4-methylsulfonyl-2-nitrophenyl disulfide, 3,3-dithiodipropionic acid, N,N-diformyl-1-cystine, trans-1,2-dithiane-4,5-diol, 2-chloro-5-nitrophenyl disulfide, 2-amino-4-chlorophenyl disulfide, 5,5-dithiobis(2-nitrobenzoic acid), 2,2-dithiobis(1-naphtylamine), 2,4-dinitrophenyl p-tolyl disulfide, 4-nitrophenyl p-tolyl disulfide, 4-chloro-3-nitrophenyl disulfideformamidine disulfide dihydrochloride and any combination thereof.

In one aspect of the invention, the molecule is in a library of test molecules.

The invention is also directed to a method for treating a subject infected with a HIV which comprises administering to the subject an effective amount of the molecule or compound identified by methods described herein. In one aspect of the invention, the administration comprises intralesional, intraperitoneal, intramuscular or intravenous injection; infusion; liposome-mediated delivery; or topical, nasal, oral, anal, ocular or otic delivery.

The invention is also directed to a method for inhibiting HIV viral replication in a subject which comprises administering to the subject an effective amount of a composition comprising the molecule identified by the methods disclosed herein, wherein the composition inhibits HIV viral replication in the subject.

The invention is also directed to a composition for inhibiting viral replication which comprises a molecule identified by the screening methods disclosed herein or a derivative thereof and a carrier. In one aspect of the invention, the carrier is an aqueous carrier, a liposome, or a lipid carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates results showing binding of biotinylated-SL3 to plate immobilized NCp7 proteins. The X-axis shows the conditions of the specific binding assay. The Y-axis shows the OD.sub.450.

FIG. 2 illustrates percent binding of biotinylated SL3 to plate immobilized NCp7 proteins vs. azodicarbonamide (ADA) concentration.

FIG. 3 illustrates percent binding of biotinylated SL3 to plate immobilized NCp7 proteins vs. 2,2'-dithiobisbenzamide (DIBA-1) concentration.

FIG. 4 illustrates the effect of ADA SL3 binding to NCp7 in Tris buffer.

FIG. 5 illustrates ADA sensitive and non-sensitive SL3 binding to NCp7 in Tris buffer.

FIG. 6 illustrates a binding competition with three different SL3 oligonucleotides, i.e., SL3 DNA (SEQ ID NO:8), SL3 RNA (SEQ ID NO:7) and SL3 A4 DNA (SEQ ID NO:9).

FIG. 7 illustrates competition with SL3 oligonucleotides ADA-sensitive binding.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

By "nucleic acid" or "oligonucleotide" or grammatical equivalents, herein means at least two nucleotides covalently linked together. A nucleic acid of the present invention will generally contain phosphodiester bonds, although in some cases, as outlined below, nucleic acid analogs are included that may have alternate backbones, comprising, for example, phosphoramide (Beaucage et al., Tetrahedron 49(10):1925 (1993) and references therein; Letsinger, J. Org. Chem. 35:3800 (1970); Sprinzl et al., Eur. J. Biochem. 81:579 (1977); Letsinger et al., Nucl. Acids Res. 14:3487 (1986); Sawai et al, Chem. Lett. 805 (1984), Letsinger et al., J. Am. Chem. Soc. 110:4470 (1988); and Pauwels et al., Chemica Scripta 26:141 91986)), phosphorothioate (Mag et al., Nucleic Acids Res. 19:1437 (1991); and U.S. Pat. No. 5,644,048), phosphorodithioate (Briu et al., J. Am. Chem. Soc. 111:2321 (1989), O-methylphophoroamidite linkages (see Eckstein, Oligonucleotides and Analogues: A Practical Approach, Oxford University Press), and peptide nucleic acid backbones and linkages (see Egholm, J. Am. Chem. Soc. 114:1895 (1992); Meier et al., Chem. Int. Ed. Engl. 31:1008 (1992); Nielsen, Nature, 365:566 (1993); Carlsson et al., Nature 380:207 (1996), all of which are incorporated by reference). Other analog nucleic acids include those with positive backbones (Denpcy et al., Proc. Natl. Acad. Sci. USA 92:6097 (1995); non-ionic backbones (U.S. Pat. Nos. 5,386,023, 5,637,684, 5,602,240, 5,216,141 and 4,469,863; Kiedrowshi et al., Angew. Chem. Intl. Ed. English 30:423 (1991); Letsinger et al., J. Am. Chem. Soc. 110:4470 (1988); Letsinger et al., Nucleoside & Nucleotide 13:1597 (1994); Chapters 2 and 3, ASC Symposium Series 580, "Carbohydrate Modifications in Antisense Research", Ed. Y. S. Sanghui and P. Dan Cook; Mesmaeker et al., Bioorganic & Medicinal Chem. Lett. 4:395 (1994); Jeffs et al., J. Biomolecular NMR 34:17 (1994); Tetrahedron Lett. 37:743 (1996)) and non-ribose backbones, including those described in U.S. Pat. Nos. 5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, "Carbohydrate Modifications in Antisense Research", Ed. Y. S. Sanghui and P. Dan Cook. Nucleic acids containing one or more carbocyclic sugars are also included within the definition of nucleic acids (see Jenkins et al., Chem. Soc. Rev. (1995) pp169-176). Several nucleic acid analogs are described in Rawls, C & E News Jun. 2, 1997 page 35. All of these references are hereby expressly incorporated by reference. These modifications of the ribose-phosphate backbone may be done to facilitate the addition of additional moieties such as labels, or to increase the stability and half-life of such molecules in physiological environments.

As will be appreciated by those in the art, all of these nucleic acid analogs may find use in the present invention. In addition, mixtures of naturally occurring nucleic acids and analogs can be made. Alternatively, mixtures of different nucleic acid analogs, and mixtures of naturally occurring nucleic acids and analogs may be made.

The nucleic acids may be single stranded or double stranded, as specified, or contain portions of both double stranded or single stranded sequence. The nucleic acid may be DNA, both genomic and cDNA, RNA or a hybrid, where the nucleic acid contains any combination of deoxyribo- and ribo-nucleotides, and any combination of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xathanine hypoxathanine, isocytosine, isoguanine, etc.

As used herein, the term "nucleoside" includes nucleotides and nucleoside and nucleotide analogs, and modified nucleosides such as amino modified nucleosides. In addition, "nucleoside" includes non-naturally occurring analog structures. Thus for example the individual units of a peptide nucleic acid, each containing a base, are referred to herein as a nucleoside.

The term "about" is used herein to mean approximately, roughly, around, or in the region of. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term "about" is used herein to modify a numerical value above and below the stated value by a variance of 20 percent up or down (higher or lower).

As used herein, the word "or" means any one member of a particular list and also includes any combination of members of that list.

Two nucleic acid or polypeptide sequences are "substantially homologous" or "substantially identical" when at least about 80% (preferably at least about 90%, and most preferably at least about 95%) of the nucleotides or amino acids match over a defined length of the molecule. Nucleic acid sequences that are substantially identical can be identified in a Southern hybridization, experiment under, for example, stringent conditions, as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Sambrook et al., supra; DNA Cloning, vols I & II, supra; Nucleic Acid Hybridization, supra. Stringent hybridization conditions are used to permit selective hybridization of nucleic acids which are from about 95% identical to about 99.9% identical. Slightly less stringent hybridization conditions can be employed to allow hybridization of nucleic acid molecules which are from about 85% identical to about 95% identical. As used herein, "homology" has the same meaning as "identity" in the context of nucleotide sequences. However, it will be appreciated that amino acid sequence "homology" includes conservative amino acid substitutions.

As used herein, the abbreviation "NCp7" refers to the nucleocapsid protein 7 of HIV-1 including the protein in the form of a polyprotein as part of the gag gene product and in the form of a mature protein, i.e., spliced away from the polyprotein and existing as an independent protein.

As used herein, "NCp7 polypeptide" encompasses any polypeptide derived from the full length and naturally occurring HIV-1 NCp7 polypeptide. This includes the polypeptides listed herein as SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4. An NCp7 polypeptide also includes any other polypeptide or fragment derived from these polypeptides, which retains the ability to bind the psi-site of HIV-1 RNA. For example, an NCp7 polypeptide includes, and is not limited to, a truncated peptide wherein the C-terminal or N-terminal portion of the full-length NCp7 polypeptide is deleted; a peptide which includes amino acid residue substitutions or additions or deletions which do not affect the ability of the peptide to bind to the psi-site RNA, to which it would bind under normal, viral physiological conditions. The NCp7 polypeptides exemplified in this application are provided only as examples and do not limit the intent of the invention to include other polypeptides. Of course, replacement of amino acid residues which do not affect the function of the zinc-finger binding domains of the NCp7 polypeptide are considered other polypeptides encompassed by the invention.

As used herein, the term "compound" encompasses molecules, such as small organic molecules, nucleic acid molecules and polypeptides or any combination thereof. The terms "compound" and "molecule" are used interchangeably herein. The molecule of the invention can exist as a single, isolated molecule, or as a member of a population of molecules, as in a library of molecules. One example of a polypeptide is an antibody or a fragment thereof which retains binding specificity. The invention includes derivatives of such compounds and salts thereof which would be useful for preparation of a pharmaceutical composition. The invention also includes methods for using such pharmaceutical compositions or molecules or compounds in the treatment of subjects suffering from HIV infection. The present invention encompasses methods to test for the ability of a compound to inhibit the interaction between NCp7 polypeptide and an oligonucleotide which contains all or part of the psi (.PSI.)-site of HIV-1 RNA. The genome of the HIV-1 contains a stretch of approximately 120 nucleotides known as the psi-site that is essential for RNA packaging during virus assembly. The nucleotides have been proposed to form four stem-loops (SL1-SL4) that have both independent and overlapping functions. The stem loop number 3 (SL3) within the psi-site RNA has been mapped as a major determinant for specific packaging.

"Small organic molecules," as used herein, are organic molecules which are of a molecular weight no larger than 10,000 Daltons, preferably no larger than 5,000 Daltons and most preferably no larger than 1,000 Daltons. Such small organic molecules are capable of interfering, either competitively or non-competitively with the binding interaction of NCp7 and the oligonucleotide sequence to which it normally binds. The small organic molecule which could interfere with such binding would be identifiable by the screening assays which are described herein. The small organic molecules interact by covalent or non-covalent means with the NCp protein.

As used herein, "inhibits" means reducing, slowing or interfering with a process. The term "inhibits" does not require complete reversal, but rather encompasses any detectable level of slowing or reducing a process.

As used herein, the term "alkyl" is, for example, a C1-C6 straight or branched chain alkyl group, which includes methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, tert-pentyl, n-hexyl, isohexyl, and the like, n-butyl, isobutyl, and tert-butyl. The term "alkyl" here includes cycloalkyl lower alkyl. The term "cycloalkyl lower alkyl" is, for example, the above-mentioned lower alkyl group substituted with C3-C6 cycloalkyl, which includes cyclopropyl methyl, 2-cyclopropyl ethyl, 4-cyclopropyl butyl, cyclopentyl methyl, 3-cyclopentyl propyl, cyclohexyl methyl, 2-cyclohexyl ethyl, and the like.

As used herein, "alkoxy" includes O-methyl, O-ethyl, O-n-propyl, O-2-propyl, O-n-butyl, O-sec-butyl, or O-t-butyl, wherein the Si atom is optionally substituted with an additional one or two alkoxy groups, a halo group, an alkylthio group (wherein alkyl is C1 through C8), an alkyl group, a phenoxy group, a p-chlorophenoxy group, an amino group, or a straight chain or branched alkylamino group, wherein the amino group may be optionally substituted with one or two alkyl groups.

The term trifluoroalkyl refers to any of the above alkyl or alkoxy compounds in which most preferably one, but optionally more than one carbon has its hydrogens completely replaced by fluorine.

As used herein, a fluorescent label includes a composition or compound which includes any organic dye or protein which when excited by light from wavelength of 200 nm to 1000 nm efficiently emits light by virtue of decay from the singlet excited state; as typified by those reagents described in the Handbook of Fluorescent Probes, Richard Haughland, 1999. For example, a fluorescent label includes green fluorescent protein. Specific fluorescent dyes of interest include: xanthene dyes, e.g. fluorescein and rhodamine dyes, such as fluorescein isothiocyanate (FITC), 6-carboxyfluorescein (commonly known by the abbreviations FAM and F), 6-carboxy-2',4',7',4,7-hexachlorofluorescein (HEX), 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein (JOE or J), N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA or T), 6-carboxy-X-rhodamine (ROX or R), 5-carboxyrhodamine-6G (R6G.sup.5 or G.sup.5), 6-carboxyrhodamine-6G (R.sup.6G.sup.6 or G.sup.6), and rhodamine 110; cyanine dyes, e.g. Cy3, Cy5 and Cy7 dyes; coumarins, e.g umbelliferone; benzimide dyes, e.g. Hoechst 33258; phenanthridine dyes, e.g. Texas Red; ethidium dyes; acridine dyes; carbazole dyes; phenoxazine dyes; porphyrin dyes; polymethine dyes, e.g. cyanine dyes such as Cy3, Cy5, etc; BODIPY dyes and quinoline dyes. Specific fluorophores of interest that are commonly used in microbiology applications include: Pyrene, Coumarin, Diethylaminocoumarin, FAM, Fluorescein Chlorotriazinyl, Fluorescein, R110, Eosin, JOE, R6G, Tetramethylrhodamine, TAMRA, Lissamine, ROX, Napthofluorescein, Texas Red, Napthofluorescein, Cy3, and Cy5, etc. Some preferred labels include a fluorescein nucleus, an eosin nucleus, a cyanine nucleus, a pyrene nucleus, all of which are described in Haughland, supra, various chelates of europium and gadolinium as are described in Anal. Chem. [2001 Sep. 1; 73(17):4277-85; Long-wavelength long-lifetime luminophores. Maliwal B P, Gryczynski Z, Lakowicz J R].

As used herein, a radioactive label includes, but is not limited to .sup.3H, .sup.14C, .sup.11C, .sup.35S, .sup.31P, .sup.32P, .sup.45Ca, .sup.44mSc, .sup.46Sc, .sup.51Cr, .sup.49V, .sup.55Fe, .sup.59Fe, .sup.57Co, .sup.58Co, .sup.60Co, .sup.65Zn, .sup.67Ga, .sup.90Sr, .sup.93mNb, .sup.97mTC, .sup.99mTc, .sup.114mIn, .sup.123I, .sup.125I, .sup.131I, .sup.137Cs, .sup.145Sm, .sup.184Rh, or any combination thereof. Of these, most preferable are .sup.3H, .sup.14C, .sup.35S, and .sup.32P.

As used herein, binding conditions include, but are not limited to Tris, phosphate, or HEPES buffer containing between 0 mM and 100 mM KCl and from about 0 mM MgCl.sub.2 to about 100 mM MgCl.sub.2. In addition, binding conditions include a temperature within a temperature range permissive for binding, such as about 37.degree. C. Binding conditions also include an incubation time from about 10 minutes to about five (5) hours during which time the polypeptide and the oligonucleotide bind to each other to form a complex. In one example, the binding conditions include a time of two (2) hours. Example of buffers which are useful in the assay shown in the Table below:

TABLE-US-00001 pH BUFFER RANGE MES(2-[N-Morpholino]ethanesulfonic acid) 5.5-6.7 BIS-TIS(bis[2-Hydroxyethyl]imino-tris-[hydroxymethyl]- 5.8-7.2 methane) ADA(N-[2-Acetamido]-2-iminodiacetic acid) 6.0-7.2 PIPES(Piperazine-N,N'-bis[2-ethanesulfonic acid)) 6.1-7.5 ACES(2-[(2-Amino-2-oxoethyl)-amino]ethanesulfonic 6.1-7.5 acid) BIS-TRIS PROPANE(1,3- 6.3-9.5 bis[tris(Hydroxymethyl)methylamino]-propane) MOPSO(3-[N-Morpholino]-2-hydroxy-propanesulfonic 6.2-7.6 acid) BES(N,N-bis[2-Hydroxyethyl]-2-aminoethanesulfonic 6.4-7.8 acid) MOPS(3-[N-Morpholino]propanesulfonic acid) 6.5-7.9 TES(N-tris[Hydroxymethyl]methyl-2-aminoethanesulfonic 6.8-8.2 acid) HEPES(N-2-Hydroxyethylpiperazine-N'-2-ethanesulfonic 6.8-8.2 acid) TAPSO(3-[N-tris(Hydroxymethyl) methylamino]-2- 7.0-8.2 hydroxypropane-sulfonic acid) POPSO(Piperazine-N,N'-bis[2-hydroxypropanesulfonic 7.2-8.5 acid]) EPPS(N[2-Hydroxyethyl]-piperazine-N'-3- 7.3-8.7 propanesulfonic acid) TRIS(tris[Hydroxymethyl)amino-methane) 7.0-9.0 TRICINE(N-tris[Hydroxymethyl]-methylglycine) 7.4-8.8 BICINE(N,N-bis[2-Hydroxyethyl] glycine) 7.6-9.0 TAPS(tris[Hydroxymethyl)methylamino-propanesulfonic 7.7-9.1 acid)

The oligonucleotides of the present invention are nucleic acid molecules that bind to the NCp7 polypeptide. In one example, the oligonucleotide has a nucleotide sequence that contains the SL3 stem loop sequence of the HIV-1 .PSI.-site to which the NCp7 protein binds in normal HIV physiology. In another example, the oligonucleotide used in the methods of the present invention has a sequence that is capable of being bound by a zinc finger containing protein, such as the NCp7 protein.

The "polypeptides" of the present invention encompass any one or more domains in the NCp7 polypeptide. In one example, the entire 72 amino acid NCp7 is used in the assay. In another example, a smaller portion of the NCp7 protein is used which includes the two zinc finger binding motifs and the small amino acid region which links the two regions. In another example, the amino acid sequence of the NCp7 polypeptide consisting essentially of two zinc finger binding domains and amino acids which link the two zinc finger binding domains is used in the method of the invention. In one example, the polypeptide of the present invention contains a single zinc finger-binding motif.

As used herein, a percent "sequence identity" refers to a calculation of "homology" or "identity" between two different nucleic acid or amino acid sequences when the sequences are aligned and compared. The percent sequence identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. Percent identity between two given sequences can be calculated using an algorithm such as BLAST (Altschul, et al. (1990) J. Mol. Biol. 215:403-410). For sequences no longer than 250 nucleotides or about 80 amino acids ("short queries"), when using BLAST the search parameters can be as follows: the filter is off, the scoring matrix is PAM30, the word size is 3 or 2, the E value is 1000 or more, and the gap costs are 11, 1. The BLAST web site provides advice for special circumstances and it is expected that one would follow such advice in the given circumstance. For sequences longer than 250 nucleotides or 80 amino acid residues, the default search parameters can be used. Any other sequence comparison algorithm can be used to determine percent identity.

As used herein, "label" encompasses any method or type of label which can be used to detect the presence of a molecule. Some examples of such a label are a fluorescent label, a calorimetric label, an enzymatic label or a radioactive label. The present invention encompasses one or more of these labels and is not limited to the types of labels, which are disclosed herein. Any label which serves the purpose of allowing the oligonucleotide to be detected in the screening assay is encompassed by the present invention. Some examples of labels used are the following: .sup.32P-dATP, Texas Red, or a biotin-labeled oligonucleotide binding to a streptavidin partner with horseradish peroxidase detection.

The binding conditions of the present invention encompass normal buffer conditions, temperature and time that allows for the hybridization of the oligonucleotide to the NCp7 polypeptide. For example, an example of a buffer which is used in the examples provided herein which is preferred is 50 mM Tris-HCl, pH 7.5, 50 mM KCl, 10 mM MgCl.sub.2, which contains 10% (w/v) glycerol and 1% (w/v) DMSO. The binding conditions encompass incubation at room temperature for two hours. It is clear to the skilled worker that the binding conditions can be varied and are not restricted to those examples that are recited in the working examples of this application. Other binding times are compatible with the features of the present invention, including between fifteen (15) minutes to thirty (30) minutes, thirty (30) minutes to three (3) hours, and three (3) hours to six (6) hours. Additional binding conditions that may be employed within the scope of the present invention include the use of additional buffers such as phosphate buffers, prepared using mixtures of potassium monohydrogen phosphate and potassium dihydrogen phosphate, at a concentration from about 0 mM HEPES to about 200 mM HEPES (hydroxypiperazine-ethylsulfate) buffers within the same general range of concentrations, concentrations of KCl within the range from about 0 mM KCl to about 200 mM KCl, concentrations of MgCl.sub.2 within the range lying between about 0 mM and about 200 mM, concentrations of NaCl including the range between about 0 mM and about 150 mM, concentrations of dimethylsulfoxide (DMSO) including between about 0% DMSO to about 50%, (w/v) DMSO, concentrations of glycerol including the range lying between about 0% and about 50%. (w/v). For these solutions, a range of pH values may be employed in the range pH 6 to pH 8.

As used herein, the term "solid support" encompasses any solid structure to which the NCp7 polypeptide or oligonucleotide is affixed. Some examples include a polystyrene plate (e.g., a NUNC.TM. 96-well plate), a bead, a silicon support (e.g. a silicon micro-chip produced with protein attached thereto in specific, discrete and addressable locations), nitrocellulose, a plastic substrate, a glass substrate, a solid phase column support; a silica support; a magnetic support; a gel support; a glass support; a polystyrene support; a polypropylene support; a polycarbonate surface derivatized with tetraethoxysilane; a polycarbonate surface derivatized with dimethyldiethoxysilane; a polycarbonate surface derivatized with silicon tetraacetate; a polycarbonate surface derivatized with methyltriacetoxysilane; a polycarbonate support derivatized with any di- or tri-alkoxysilane; an synthetic alumina surface, whereby hydroxyl groups upon the said surface may be optionally derivatized with tetraethoxysilane, dimethyldiethoxysilane; methyltriacetoxysilane; or with any di- or tri-alkoxysilane; a silicon monoxide surface, whereby hydroxyl groups upon the said surface may be optionally derivatized with tetraethoxysilane, dimethyldiethoxysilane; methyltriacetoxysilane; or with any di- or tri-alkoxysilane; a silicon monoxide surface upon a silicon substrate, whereby hydroxyl groups upon the said surface may be optionally derivatized with tetraethoxysilane, dimethyldiethoxysilane; methyltriacetoxysilane; or with any di- or tri-alkoxysilane , a titanium dioxide surface whereby hydroxyl groups upon the said surface may be optionally derivatized with tetraethoxysilane, dimethyldiethoxysilane; methyltriacetoxysilane; or with any di- or tri-alkoxysilane, a zirconium dioxide surface whereby hydroxyl groups upon the said surface may be optionally derivatized with tetraethoxysilane, dimethyldiethoxysilane; methyltriacetoxysilane; or with any di- or tri-alkoxysilane, a tin oxide surface of electrically conductive nature, whereby hydroxyl groups upon the said surface may be optionally derivatized with tetraethoxysilane, dimethyldiethoxysilane; methyltriacetoxysilane; or with any di- or tri-alkoxysilane, a polycarbonate, polystyrene, or polypropylene support derivatized with streptavidin; and any combination thereof.

The following polypeptides and oligonucleotides are useful in the invention:

TABLE-US-00002 SEQ ID NO:1 Amino acid sequence of the NCp7 protein (full- length mature protein): MQKGNFRNQRKTVKCFNCGKEGHIAKNCRAPRKKGCWKCGKEGHQM KDCTERQAN SEQ ID NO:2 Amino acid sequence of first zinc finger of NCp7: VKCFNCGKEGHTARNCRA SEQ ID NO:3 Amino acid sequence of second zinc finger NCp7: KGCWKCGKEGHQMKDCTE

SEQ ID NO:4--Amino acid sequence of first and second zinc fingers of NCp7 and the amino acids linking them: There are multiple sequences in Genbank representing the NCp7 sequence and its mutant variants which are possible in nature. A representative set of these is indicated below.

TABLE-US-00003 [SEQ ID NO:10] ARILAEAMSQVTNTAVMMQRNNFKGQRKIIKCFNCGKEGHLAKNCRAPR KKGCWKCG [SEQ ID NO:11] ARVLAEAMSQVSGVGAAIMMQKSNFKGPKRMIKCFNCGKEGHLARNCR APRKRGCWKCG [SEQ ID NO:12] ARVLAEAMSQVTQPATIMMQKGNFRNQRKTVKCFNCGKEGHIAKNCRA PRKKGCWKCGR [SEQ ID NO:13] ARVLAEAMSQVTGSAATIMMQRGNIRNQRKTVKCFNCGKEGHIARNCRA PRKKGCWKCGK [SEQ ID NO:14] ARVLAEAMSQVTQSATMMMQRGNFRNQKKTVKCFNCGKEGHIAKNCR APRKKGCWKCGR [SEQ ID NO:15] ARVLAEAMSQVTNSPAIMMQRGNFRNQRKIVKCFNCGKEGHIAKNCRAP RKRGCWKCGK [SEQ ID NO:16] ARVLAEAMSQVTQPATIMMQRGNFRNQRKTVKCFNCGKEGHIAKNCRA PRKKGCWKCGR [SEQ ID NO:17] ARVLAEAMSQVTASATIMMQRGNFKNQRKTVKCFNCGKEGHIAKNCRA PRKKGCWKCG

These sequences are normally contained within the gag protein. Representative examples of the HIV gag protein which contains the NCp sequence from different mutant HIV samples are well known to those skilled in the prior art. Non-limiting examples are represented by sequence Ids 4-1 through 4-100 shown below, along with their Genbank numerical listings: