WO2004053094A2 - Compositions and methods for inhibiting human immunodeficiency virus infection by down-regulating human cellular genes - Google Patents

Abstract

The invention provides methods for identifiying human cellular genes and their encoded products for use as targets in the design of therapeutic agents for inhibiting or suppressing human immunodeficiency virus (HIV) infection. The invention also provides methods for identifying protective compounds including immunizing agents that inhibit HIV infection. The invention further provides compounds for use in the treatment or prevention of HIV.

Description

COMPOSITIONS AND METHODS FOR INHIBITING HUMAN

IMMUNODEFICIENCY VIRUS INFECTION BY DOWN-REGULATING

HUMAN CELLULAR GENES

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority under 35 U.S.C. 119(e) from

U.S. Provisional Application Serial No. 60/431,522, filed December 6, 2002. The entire disclosure of U.S. Provisional Application Serial No. 60/431,522 is incorporated herein by reference.

BACKGROUND OF THE INVENTION Field of the Invention

The invention relates to methods for identifying human cellular genes and their encoded products for use as targets in the design of therapeutic agents for suppressing human immunodeficiency virus (HIV) infection. In particular, the invention relates to methods for identifying protective compounds that inhibit viral entry of HIV into cells. The invention further relates to compounds for use in the treatment or prevention of HIV. Background of the Invention

The primary cause of acquired immunodeficiency syndrome (AIDS) has been shown to be HIV (Barre-Sinoussi et al, 1983, Science 220:868-70; Gallo et al, 1984, Science 224:500-03). HIV causes immunodeficiency in an individual by infecting important cell types of the immune system, which results in the depletion of these cells. This, in turn, leads to opportunistic infections, neoplastic growth, and death.

HIV is a member of the lentivirus family of retro viruses (Teich et al, 1984, in RNA Tumor Viruses pp. 949-56 (Weiss et al, eds., CSH-Press: New York). Retroviruses are small, enveloped viruses that contain a diploid, single-stranded RNA genome, and replicate via a DNA intermediate produced by a virally encoded reverse transcriptase, an RNA-dependent DNA polymerase (Varmus, 1988, Science 240:1427-39). There are at least two distinct subtypes of HTV: HIV-1 (Barre-Sinoussi et al, 1983, ibid.; Gallo et al, 1984, ibid.) and HIV-2 (Clavel et al, 1986, Science 233:343-46; Guyader et al, 1987, Nature 326:662-69). Genetic heterogeneity exists within each of these HIV subtypes.

CD4⁺ T cells are the major targets of HIV infection because the CD4 cell surface protein acts as a cellular receptor for HIV attachment (Dalgleish et al, 1984, Nature 312:763-67; Klatzmann et al, 1984, Nature 312:767-68; Maddon et al, 1986, Cell 47:333-48). Viral entry into cells is dependent upon the binding of viral protein gpl20 to the cellular CD4 receptor molecule (McDougal et al, 1986, Science 231:382-85; Maddon et al, 1986, Cell 47:333-48).

HIV infection is pandemic and HIV-associated diseases have become a worldwide health problem. Despite considerable efforts in the design of anti-HIV modalities, there is, thus far, no successful prophylactic or therapeutic regimen against

AIDS. However, several stages of the HIV life cycle have been considered as potential targets for therapeutic intervention (Mitsuya et al, 1991, FASEB J. 5:2369- 81).

For example, virally encoded reverse transcriptase has been a major focus of drug development. A number of reverse transcriptase-targeted drugs, including dideoxynucleotide analogs such as AZT, ddl, ddC, and ddT have been shown to be active against HIV (Mitsuya et al, 1990, Science 249:1533-44). While beneficial, these nucleotide analogs are not curative, probably due to the rapid appearance of drug resistant HIV mutants (Lander et al., 1989, Science 243:1731-34). In addition, these drugs often exhibit toxic side effects, such as bone marrow suppression, vomiting, and liver abnormalities.

Another stage of the HIV life cycle that has been targeted is viral entry into cells, the earliest stage of HIV infection. This approach has primarily utilized recombinant soluble CD4 protein to inhibit infection of CD4⁺ T cells by some HIV-1 strains (Smith et al, 1987, Science 238:1704-07). Certain primary HIV-1 isolates, however, are relatively less sensitive to inhibition by recombinant CD4 (Daar et al, 1990, Proc. Natl. Acad. Sci. U.S.A. 87:6574-79). To date, clinical trials of recombinant, soluble CD4 have produced inconclusive results (Schooley et al, 1990, Ann. Int. Med. 112:247-53; Kahn et al, 1990, Ann. Int. Med. 112:254-61; Yarchoan et al, 1989, Proc. Vth Int. Conf. on AIDS 564, MCP 137).

Yet another stage of the HIV life cycle that has been targeted is the integration of the proviral DNA into the host genome. The viral enzyme integrase catalyzes the process of integration, and inhibitors of integrase have been reported (d'Angelo et al, 2001, Pathol Biol. 49:237-46; Farnet and Bushman, 1996, AIDS 10 Supp. A:S3-11). Additionally, the later stages of HIV replication (which involve crucial virus- specific processing of certain viral proteins and enzymes) have been targeted for anti- HIV drug development. Late-stage processing is dependent on the activity of a virally encoded protease, and drugs including saquinavir, ritonavir, and indinavir have been developed to inhibit this protease (Pettit et al, 1993, Persp. Drug Discov. Design 1:69-83). With this class of drugs, the emergence of drug resistant HIV mutants is also a problem; resistance to one inhibitor often confers cross-resistance to other protease inhibitors (Condra et al, 1995, Nature 374:569-71). Also, these drugs often exhibit toxic side effects such as nausea, altered sense of taste, circumoral parethesias, development of fat deposits, diarrhea, and nephrolithiasis.

Antiviral therapy of HIV using different combinations of nucleoside analogs and protease inhibitors have recently been shown to be more effective than the use of a single drug alone (Torres et al, 1997, Infec. Med. 14:142-60). However, despite the ability to achieve significant decreases in viral burden, there is no evidence to date that combinations of available drugs will afford a curative treatment for AIDS.

Other potential approaches for developing treatment for AIDS include the delivery of exogenous genes into infected cells. One such gene therapy approach involves the use of genetically engineered viral vectors to introduce toxic gene products to kill HlV-infected cells. Another form of gene therapy is designed to protect virally infected cells from cytolysis by specifically disrupting viral replication.

Stable expression of RNA-based HIV-1 antiviral agents (e.g., decoys, antisense, or ribozymes) or protein-based HIV-1 antiviral agents (e.g., transdominant mutants) can inhibit certain stages of the viral life cycle. A number of anti-HIV suppressors have been reported, such as decoy RNA of TAR or RRE (Sullenger et al, 1990, Cell 63:601-08; Sullenger et al, 1991, J. Virol. 65:6811-16; Lisziewicz et al, 1993, New

Biol. 3:82-89; Lee et al, 1994, J. Virol. 68:8254-64), ribozymes (Sarver et al, 1990, Science 247:1222-25; Wecrasinghe et al, 1991, J. Virol. 65:5531-34; Dropulic et al, 1992, J. Virol. 66:1432-41; Ojwang et al., 1992, Proc. Natl Acad. Sci. U.S.A. 89:10802-06; Yu et al, 1993, Proc. Natl. Acad. Sci. U.S.A. 90:6340-44; Yu et al, 1995, Proc. Natl. Acad. Sci. U.S.A. 92:699-703; Yamada et al, 1994, Gene Therapy

1:38-45), antisense RNA complementary to the mRNA of viral gag, tat, rev, or env genes (Sezakiel et al, 1991, J. Virol. 65:468-72; Chatterjee et al, 1992, Science 258:1485-88; Rhodes et al, 1990, J. Gen. Virol. 71:1965; Rhodes et al, 1991, AIDS 5:145-51; Sezakiel et al, 1992, J. Virol. 66:5576-81; Joshi et al, 1991, J. Virol. 65:5524-30) and transdominant mutants of Rev (Bevec et al, 1992, Proc. Natl. Acad.

Sci. U.S.A. 89:9870-74), Tat (Pearson et al, 1990, Proc. Natl. Acad. Sci. U.S.A. 87:5079-83; Modesti et al, 1991, New Biol. 3:759-68), Gag (Trono et al, 1989, Cell 59:113-20), Env (Bushschacher et al, 1995, J. Virol. 69:1344-48) and protease (Junker etal, 1996, J. Virol. 70:7765-72). Antisense polynucleotides have been designed to complex with and sequester HIV-1 transcripts (International Pub. Nos. WO 93/11230 and WO 94/10302; European Patent Pub. No. EP 594,881; Chatterjee et al, 1992, Science 258:1485). Furthermore, enzymatically active RNAs (i.e., ribozymes) have been used to cleave viral transcripts. The use of a ribozyme to generate resistance to HIV-1 in a hematopoietic cell line has been reported (Ojwang et al, 1992, Proc. Natl. Acad. Sci. U.S.A. 89:10802-06; Yamada et al, 1994, Gene Therapy 1:38-45; International Pub. Nos. WO 94/26877 and WO 95/13379). In preclinical studies, RevMlO, a transdominant Rev protein, has been transfected ex vivo into CD4 cells of HIV- infected individuals and shown to confer survival advantage over cells transfected with vector alone (Woffendin et al, 1996, Proc. Natl. Acad. Sci. U.S.A. 93:2889-94).

However, despite enormous efforts in the art, reliable, curative anti-HIV therapeutic agents and regimens have not been developed.

In nature, evolution of an intracellular pathogen such as HIV requires the development of interactions of its genes and gene products with multiple cellular components. For instance, the interactions of a virus with a host cell involves the binding of the virus to specific cellular receptors, translocation through the cellular membrane, uncoating, replication of the viral genome, and transcription of the viral genes. Each of these events occurs in a cell and involves interactions with at least one cellular component. Thus, the life cycle of a virus can be completed only if the cell is

"permissive" for viral infection. The availability of amino acids and nucleotides for replication of the viral genome and protein synthesis, the energy status of the cell, and the presence of cellular transcription factors and enzymes all contribute to the propagation of the virus in the cell. Consequently, the cellular components, in part, determine host cell susceptibility to infection and can be used as potential targets for the development of new therapeutic interventions. In the case of HIV, one cellular component that has been used towards this end is the cell surface molecule for HIV attachment, CD4.

Recently, it was reported that HIV entry into a susceptible cell requires the expression of a second type of receptor, the chemokine receptors (CCR2, CCR3,

CCR5, or CXCR4), in addition to CD4 (Moore, 1997, Science 276:51-52). A chemokine receptor normally binds RANTES, MlP-lα, or MlP-lβ as its natural ligand. In the case of HTV infection, it has been proposed that CD4 first binds to the HIV gpl20 protein on the cell surface followed by binding of this complex to a chemokine receptor, resulting in viral entry into the cell (Cohen, 1997, Science 275:1261). Therefore, chemokine receptors can present an additional cellular target for the design of HIV therapeutic agents. Inhibitors of HJN/chemokine receptor interactions are being tested as anti-HIV agents. Thus, there remains a need for the discovery of additional cellular targets for the design of anti-HIV therapeutics, particularly cell surface targets for disrupting viral entry into a cell. There remains a need in the art to isolate and identify human cellular genes that encode products that are necessary for HIV cell entry for use as targets in the design of therapeutic agents for suppressing HIV infection. SUMMARY OF THE INVENTION

The invention relates to methods for identifying human cellular genes that encode products that are necessary for productive HIV entry into a cell for use as targets in the design of therapeutic agents for suppressing HIV infection. The invention further relates to methods for identifying additional human cellular genes that encode products involved in HIV cell entry for use as targets in the design of therapeutic agents for suppressing HIV infection.

The invention also relates to methods for identifying protective compounds that inhibit HIV cell entry. The invention further relates to compounds for use in the treatment or prevention of HIV. One embodiment of the invention is a method of identifying an inhibitor of

HIV cell entry of a human host cell, comprising identifying an inhibitor of the HIV cell entry activity of a cell surface polypeptide selected from the group consisting of CXCR-4, CCR4, CCR7, CD11C, CD47, CD68, CD69, CD74, CSF3R, RARA, GABBR1, P2X1, HELOl, GPRK6 and PTK2B. Preferably, the step of identifying an inhibitor of a comprises the steps of: (a) contacting the human host cell with a putative inhibitor, wherein the human host cell expresses a polypeptide selected from the group consisting of CXCR-4, CCR4, CCR7, CD11C, CD47, CD68, CD69, CD74, CSF3R, RARA, GABBR1, P2X1, HELOl, GPRK6 and PTK2B; and (b) assessing inhibition of said activity of the cell surface polypeptide in an entry assay. In another embodiment, the step of identifying an inhibitor comprises the steps of: (a) contacting the human host cell with a putative inhibitor, wherein the human host cell expresses a polypeptide selected from the group consisting of CXCR-4, CCR4, CCR7, CD11C, CD47, CD68, CD69, CD74, CSF3R, RARA, GABBR1, P2X1, HELOl, GPRK6 and PTK2B; (b) exposing the cell to HIV; and (c) assessing inhibition of HlV-infection in the presence of the putative inhibitor as compared to in the absence of the inhibitor.

Another embodiment of the invention is a method of identifying a cell surface polypeptide in a human host cell, wherein said polypeptide is necessary for entry of HIV into said human host cell, the method comprising the steps of: (a) synthesizing a randomly fragmented cDNA population from total mRNA isolated from a human host cell that is susceptible to HIV infection to yield DNA fragments; (b) transferring the DNA fragments to an expression vector to yield a genetic suppressor element library, wherein each of the DNA fragments is operatively linked to a protein translation initiation codon, and wherein the expression vector expresses the DNA fragments in the human host cell; (c) genetically modifying a population of human host cells by introducing the genetic suppressor element library into the population of human host cells; (d) infecting the population of human host cells with HIV; (e) isolating a genetically modified human host cell containing a genetic suppressor element conferring resistance to HIV infection from the population of human host cells; (f) recovering the genetic suppressor element from the isolated genetically modified human host cell; (g) determining the human cellular gene that corresponds to the genetic suppressor element; (h) determining that the human cellular gene encodes a cell surface polypeptide; and (i) determining that said cell surface polypeptide that is involved in HIV cell entry using the steps comprising: (i) contacting said human host cell with a putative inhibitor, wherein said human host cell expresses a polypeptide selected from the group consisting of CXCR-4, CCR4, CCR7, CD11C, CD47, CD68, CD69, CD74, CSF3R, RARA, GABBR1, P2X1, HELOl, GPRK6 and PTK2B; and (ii) assessing inhibition of said activity of said cell surface polypeptide in an entry assay.

Another embodiment of the present invention relates to compositions and methods for inhibiting HIV infection by down-regulating expression of certain human cellular genes and/or inhibiting the activity of products encoded by such genes. In one aspect, it relates to a number of human cell-derived nucleic acid molecules which inhibit the entry of HIV into susceptible cells. The isolated nucleic acid molecules correspond to portions of cellular genes or complements thereof, and are referred to herein as genetic suppressor elements (GSEs). The cellular genes encode cell surface molecules necessary for HIV cell entry. Additionally, small molecule inhibitors of the same cellular genes and their encoded products are preferred embodiments within the scope of the present invention.

Specific preferred embodiments of the invention will become evident from the following more detailed description of certain preferred embodiments and the claims.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The invention includes methods for identifying human cellular genes that encode products that are necessary for productive entry of HIV into a cell for use as targets in the design of therapeutic agents for suppressing HIV infection. The invention further includes methods for identifying additional human cellular genes that encode products involved in HIV cell entry for use as targets in the design of therapeutic agents for suppressing HIV infection.

The invention includes compositions and methods for inhibiting HIV infection by down-regulating the expression of human cell-derived genes encoding proteins that are necessary for the entry of HIV into susceptible cells. These compositions can include nucleic acid molecules comprising genetic suppressor elements that inhibit the expression or activity of the cellular genes, as well as other small molecule and peptide inhibitors of the cellular genes, such as those that can be identified by a method of the invention. The invention also includes methods for identifying protective compounds that inhibit HIV infection. The invention further includes compounds for use in the treatment or prevention of HIV.

The invention is based, in part, on the Applicants' discovery that certain nucleic acid molecules - termed genetic suppressor elements (GSEs) - can be isolated from human cells that prevent activation of latent HIV-1 in a CD4⁺ cell line as well as productive HIV infection in such cells, and that such nucleic acid molecules correspond to fragments of certain human cellular genes. In that regard, any cellular or viral marker associated with HIV infection can be used to select for such nucleic acid molecules. An example of such a marker is CD4, which is conveniently monitored by using a specific antibody. Additional markers include virus-specific gene products, such as gpl20 and p24. The method used to identify the GSEs that inhibit HIV cell entry and cellular gene targets disclosed herein is described in detail in the Examples section. GSEs having the ability to inhibit HIV infection can be isolated that are functional in the sense orientation (and encode a peptide thereby), and also GSEs that are functional in the antisense orientation (and encode antisense RNAs thereby). These GSEs are believed to down-regulate the corresponding cellular gene from which they were derived by different mechanisms. Sense-oriented GSEs exert their effects as transdominant mutants or RNA decoys. Transdominant mutants are expressed proteins or peptides that competitively inhibit the normal function of a wild-type protein in a dominant fashion. RNA decoys are protein-binding sites that titrate out these wild-type proteins. Anti-sense oriented GSEs exert their effects as antisense RNA molecules, i.e., nucleic acid molecules complementary to the mRNA of the target gene. These nucleic acid molecules bind to mRNA and block the translation of the mRNA. In addition, some antisense nucleic acid molecules can act directly at the DNA level to inhibit transcription.

In one embodiment of the invention, the down-regulation of the concentration or activity of a human cellular gene or gene product by a GSE or nucleic acid molecule comprising or consisting of the GSE depletes a cell surface cellular component required for HIV cell entry resulting in an inhibition of HIV infection.

In one embodiment of the invention, the human cellular gene encoding a polypeptide required for HIV cell entry mediates fusion of HIV with the human host cell. In another embodiment, the human cellular gene enhances the binding of HIV to receptors or co-receptors on the surface of the human host cell. Preferred human cellular genes involved in HIV cell entry include those genes listed in Table 1.

Table 1. HIV Cell Surface Targets

While not being bound by theory, the inventors believe that CXCR-4, GPRK6, PTK2B and HELOl are involved in fusion of a virus to a cell, and that CCR4, CCR7 and RARA are involved in binding of a virus to a cell. It will be understood that this invention is not limited to the particular methodology, protocols, cell lines, animal species or genera, constructs, or reagents described herein, as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention that will be limited only by the appended claims. All technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs unless clearly indicated otherwise.

As used herein, the term "HIV infection" refers to the ability of HIV to enter a host cell and/or replicate in the host cell. As used herein, the term "HIV cell entry" refers to the ability of a virus to enter a cell through binding of a virus to a cell and the fusion of the virus to the cell. HTV or virus binding refers to the attachment of a virus to the surface of a cell. HTV or virus fusion refers to the passage of a virus across the cell plasma membrane into the cell.

As used herein, the term "isolated nucleic acid molecule" refers to a nucleic acid molecule that has been removed from its natural milieu (i.e., a molecule that has been subject to human manipulation) and can include DNA, RNA, or derivatives of either DNA or RNA. An isolated nucleic acid molecule can be isolated from its natural source or can be produced using recombinant DNA technology (e.g., polymerase chain reaction amplification) or chemical synthesis. Isolated nucleic acid molecules include natural nucleic acid molecules and homologs thereof, including, but not limited to, natural allelic variants and modified nucleic acid molecules in which nucleotides have been inserted, deleted, substituted, or inverted in such a manner that such modifications do not substantially interfere with the nucleic acid molecule's ability to promote HIV cell entry or inhibit HIV cell entry. It should also be appreciated that reference to an isolated nucleic acid molecule does not necessarily reflect the extent of purity of the nucleic acid molecule. Nucleic acid molecules can be isolated and obtained in substantial purity, generally as other than an intact chromosome. Usually, the nucleic acid molecule will be obtained substantially free of other nucleic acid sequences, generally being at least about 50%, and usually at least about 90% pure. Although the phrase "nucleic acid molecule" primarily refers to the physical nucleic acid molecule and the phrase "nucleic acid sequence" primarily refers to the sequence of nucleotides on the nucleic acid molecule, the two phrases can be used interchangeably.

According to the invention, reference to an "isolated nucleic acid molecule" refers to a nucleic acid molecule that is the size of or smaller than a gene. Thus, an isolated nucleic acid molecule does not encompass isolated genomic DNA or an isolated chromosome. The term isolated nucleic acid molecule does not connote any specific minimum length unless set forth by reference to a minimum number of nucleotides or by a function of the nucleic acid molecule. As used herein, the term "gene" has the meaning that is well known in the art, that is, a nucleic acid sequence that includes the translated sequences that code for a protein ("exons") and the untranslated intervening sequences ("introns"), and any regulatory elements ordinarily necessary to transcribe and/or translate the protein. Included in the invention are nucleic acid molecules that are less than a full-length gene or less than a full-length coding sequence, such as fragments of a gene or coding sequence comprising, consisting essentially of, or consisting of a GSE of the present invention. A coding sequence can include genomic DNA without introns, cDNA or RNA that encodes a protein. An isolated nucleic acid molecule can also include a specified nucleic acid sequence flanked by (i.e., at the 5' and/or the 3' end of the sequence) additional nucleic acids that do not normally flank the specified nucleic acid sequence in nature (i.e., are heterologous sequences).

Preferably, an isolated nucleic acid molecule of the present invention is produced using recombinant DNA technology (e.g., polymerase chain reaction (PCR) amplification, cloning) or chemical synthesis. A nucleic acid molecule homologue can be produced using a number of methods known to those skilled in the art (see, for example, Sambrook et al., ibid.). For example, nucleic acid molecules can be modified using a variety of techniques including, but not limited to, classical mutagenesis techniques and recombinant DNA techniques, such as site-directed mutagenesis, chemical treatment of a nucleic acid molecule to induce mutations, restriction enzyme cleavage of a nucleic acid fragment, ligation of nucleic acid fragments, PCR amplification and/or mutagenesis of selected regions of a nucleic acid sequence, synthesis of oligonucleotide mixtures and ligation of mixture groups to "build" a mixture of nucleic acid molecules and combinations thereof. Nucleic acid molecule homologues can be selected from a mixture of modified nucleic acids by screening for the function of the protein encoded by the nucleic acid and/or by hybridization with a wild-type gene.

The minimum size of a nucleic acid molecule of the present invention is a size sufficient to encode a protein having the desired biological activity, sufficient to have HIV inhibitory activity as described herein (e.g., as in a GSE), or sufficient to form a probe or oligonucleotide primer that is capable of forming a stable hybrid with the complementary sequence of a nucleic acid molecule. As such, the size of a nucleic acid molecule of the present invention can be dependent on nucleic acid composition and percent homology or identity between the nucleic acid molecule and complementary sequence as well as upon hybridization conditions per se (e.g., temperature, salt concentration, and formamide concentration) and the intended use of the nucleic acid molecule. The minimal size of a nucleic acid molecule that is used as an oligonucleotide primer or as a probe is typically at least about 12 to about 15 nucleotides in length if the nucleic acid molecules are GC-rich and at least about 15 to about 18 bases in length if they are AT-rich. There is no limit, other than a practical limit, on the maximal size of a nucleic acid molecule of the present invention, in that the nucleic acid molecule can include a fragment of a gene, a portion of a protein encoding sequence, or a nucleic acid sequence encoding a full-length protein (including a complete gene).

Another embodiment of the present invention includes a recombinant nucleic acid molecule comprising a recombinant vector and a nucleic acid molecule comprising a nucleic acid sequence encoding a cellular gene or fragment thereof as described herein. According to the present invention, a recombinant vector is an engineered (i.e., artificially produced) nucleic acid molecule that is used as a tool for manipulating a nucleic acid sequence of choice and for introducing such a nucleic acid sequence into a host cell. The recombinant vector is therefore suitable for use in cloning, sequencing, and/or otherwise manipulating the nucleic acid sequence of choice, such as by expressing and/or delivering the nucleic acid sequence of choice into a host cell to form a recombinant cell. Such a vector typically contains heterologous nucleic acid sequences, that is nucleic acid sequences that are not naturally found adjacent to nucleic acid sequence to be cloned or delivered, although the vector can also contain regulatory nucleic acid sequences (e.g., promoters, untranslated regions) which are naturally found adjacent to nucleic acid molecules of the present invention or which are useful for expression of the nucleic acid molecules of the present invention (discussed in detail below). The vector can be either RNA or

DNA, either prokaryotic or eukaryotic, and typically is a plasmid. The vector can be maintained as an extrachromosomal element (e.g., a plasmid) or it can be integrated into the chromosome of a recombinant organism (e.g., a microbe or a plant). The entire vector can remain in place within a host cell, or under certain conditions, the plasmid DNA can be deleted, leaving behind the nucleic acid molecule of the present invention. The integrated nucleic acid molecule can be under chromosomal promoter control, under native or plasmid promoter control, or under a combination of several promoter controls. Single or multiple copies of the nucleic acid molecule can be integrated into the chromosome. A recombinant vector of the present invention can contain at least one selectable marker.

In one embodiment, a recombinant vector used in a recombinant nucleic acid molecule of the present invention is an expression vector. As used herein, the phrase "expression vector" is used to refer to a vector that is suitable for production of an encoded product (e.g., a protein of interest). In this embodiment, a nucleic acid sequence encoding the product to be produced is inserted into the recombinant vector to produce a recombinant nucleic acid molecule. The nucleic acid sequence encoding the protein to be produced is inserted into the vector in a manner that operatively links the nucleic acid sequence to regulatory sequences in the vector which enable the transcription and translation of the nucleic acid sequence within the recombinant host cell.

In another embodiment, a recombinant vector used in a recombinant nucleic acid molecule of the present invention is a targeting vector. As used herein, the phrase "targeting vector" is used to refer to a vector that is used to deliver a particular nucleic acid molecule into a recombinant host cell, wherein the nucleic acid molecule is used to delete or inactivate an endogenous gene within the host cell or microorganism (i.e., used for targeted gene disruption or knock-out technology). Such a vector may also be known in the art as a "knock-out" vector. In one aspect of this embodiment, a portion of the vector, but more typically, the nucleic acid molecule inserted into the vector (i.e., the insert), has a nucleic acid sequence that is homologous to a nucleic acid sequence of a target gene in the host cell (i.e., a gene which is targeted to be deleted or inactivated). The nucleic acid sequence of the vector insert is designed to bind to the target gene such that the target gene and the insert undergo homologous recombination, whereby the endogenous target gene is deleted, inactivated or attenuated (i.e., by at least a portion of the endogenous target gene being mutated or deleted).

Typically, a recombinant nucleic acid molecule includes at least one nucleic acid molecule of the present invention operatively linked to one or more expression control sequences, including transcription control sequences and translation control sequences. As used herein, the phrase "recombinant molecule" or "recombinant nucleic acid molecule" primarily refers to a nucleic acid molecule or nucleic acid sequence operatively linked to an expression control sequence, but can be used interchangeably with the phrase "nucleic acid molecule", when such nucleic acid molecule is a recombinant molecule as discussed herein. According to the present invention, the phrase "operatively linked" refers to linking a nucleic acid molecule to an expression control sequence (e.g., a transcription control sequence and/or a translation control sequence) in a manner such that the molecule is able to be expressed when transfected (i.e., transformed, transduced, transfected, conjugated or conduced) into a host cell. Transcription control sequences are sequences which control the initiation, elongation, or termination of transcription. Particularly important transcription control sequences are those which control transcription initiation, such as promoter, enhancer, operator and repressor sequences. Suitable transcription control sequences include any transcription control sequence that can function in a host cell or organism into which the recombinant nucleic acid molecule is to be introduced.

According to the present invention, the term "transfection" is used to refer to any method by which an exogenous nucleic acid molecule (i.e., a recombinant nucleic acid molecule) can be inserted into a cell. The term "transformation" can be used interchangeably with the term "transfection" when such term is used to refer to the introduction of nucleic acid molecules into microbial cells or plants. In microbial systems, the term "transformation" is used to describe an inherited change due to the acquisition of exogenous nucleic acids by the microorganism and is essentially synonymous with the term "transfection." However, in animal cells, transformation has acquired a second meaning which can refer to changes in the growth properties of cells in culture (described above) after they become cancerous, for example. Therefore, to avoid confusion, the term "transfection" is preferably used with regard to the introduction of exogenous nucleic acids into animal cells, including human cells, and is used herein to generally encompass transfection of animal cells and transformation of plant cells and microbial cells, to the extent that the terms pertain to the introduction of exogenous nucleic acids into a cell. Therefore, transfection techniques include, but are not limited to, transformation, chemical treatment of cells, particle bombardment, electroporation, microinjection, lipofection, adsorption, infection and protoplast fusion. A recombinant cell is preferably produced by transforming a host cell with one or more recombinant molecules, each comprising one or more nucleic acid molecules operatively linked to an expression vector containing one or more expression control sequences.

"Hybridization" has the meaning that is well known in the art, that is, the formation of a duplex structure by two single-stranded nucleic acids due to complementary base pairing. Hybridization can occur between exactly complementary nucleic acid strands or between nucleic acid strands that contain some regions of mismatch. As used herein, reference to hybridization conditions refers to standard hybridization conditions under which nucleic acid molecules are used to identify similar nucleic acid molecules. Such standard conditions are disclosed, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Labs Press, 1989. Sambrook et al., ibid., is incorporated by reference herein in its entirety (see specifically, pages 9.31-9.62). In addition, formulae to calculate the appropriate hybridization and wash conditions to achieve hybridization permitting varying degrees of mismatch of nucleotides are disclosed, for example, in Meinkoth et al., 1984, Anal. Biochem. 138, 267-284; Meinkoth et al., ibid., is incorporated by reference herein in its entirety. "Stringent hybridization" has a meaning well- established in the art, that is, hybridization performed at a salt concentration of no more than IM and a temperature of at least 25 degrees Celsius. For example, conditions of 5X SSPE (750 mM NaCl, 50 mM Sodium Phosphate, 5 mM EDTA, pH

7.4) and a temperature of 55 degrees to 60 degrees Celsius are suitable. For example, in one embodiment, "moderately stringent conditions" can be defined as hybridizations carried out as described above, followed by washing in 0.2X SSC and 0.1% SDS at 42 degrees Celsius (Ausubel et al, 1989, Current Protocols for Molecular Biology, ibid.).

More particularly, moderate stringency hybridization and washing conditions, as referred to herein, refer to conditions which permit isolation of nucleic acid molecules having at least about 70% nucleic acid sequence identity with the nucleic acid molecule being used to probe in the hybridization reaction (i.e., conditions permitting about 30% or less mismatch of nucleotides). High stringency hybridization and washing conditions, as referred to herein, refer to conditions which permit isolation of nucleic acid molecules having at least about 80% nucleic acid sequence identity with the nucleic acid molecule being used to probe in the hybridization reaction (i.e., conditions permitting about 20%o or less mismatch of nucleotides). Very high stringency hybridization and washing conditions, as referred to herein, refer to conditions which permit isolation of nucleic acid molecules having at least about 90% nucleic acid sequence identity with the nucleic acid molecule being used to probe in the hybridization reaction (i.e., conditions permitting about 10% or less mismatch of nucleotides). As discussed above, one of skill in the art can use the formulae in Meinkoth et al., ibid, to calculate the appropriate hybridization and wash conditions to achieve these particular levels of nucleotide mismatch. Such conditions will vary, depending on whether DNA:RNA or DNA:DNA hybrids are being formed. Calculated melting temperatures for DNA:DNA hybrids are 10 °C less than for

DNA:RNA hybrids. In particular embodiments, stringent hybridization conditions for DNA:DNA hybrids include hybridization at an ionic strength of 6X SSC (0.9 M Na⁺) at a temperature of between about 20 °C and about 35 °C (low stringency), more preferably, between about 28°C and about 42°C (more stringent), and even more preferably, between about 35 °C and about 45 °C (even more stringent), with appropriate wash conditions. In particular embodiments, stringent hybridization conditions for DNA:RNA hybrids include hybridization at an ionic strength of 6X SSC (0.9 M Na⁺) at a temperature of between about 30 °C and about 45 °C, more preferably, between about 38 °C and about 50 °C, and even more preferably, between about 45°C and about 55°C, with similarly stringent wash conditions. These values are based on calculations of a melting temperature for molecules larger than about 100 nucleotides, 0% formamide and a G + C content of about 40%. Alternatively, T_m can be calculated empirically as set forth in Sambrook et al., supra, pages 9.31 to 9.62. In general, the wash conditions should be as stringent as possible, and should be appropriate for the chosen hybridization conditions. For example, hybridization conditions can include a combination of salt and temperature conditions that are approximately 20-25 °C below the calculated T_m of a particular hybrid, and wash conditions typically include a combination of salt and temperature conditions that are approximately 12-20°C below the calculated T_m of the particular hybrid. One example of hybridization conditions suitable for use with DNA:DNA hybrids includes a 2-24 hour hybridization in 6X SSC (50% formamide) at about 42°C, followed by washing steps that include one or more washes at room temperature in about 2X SSC, followed by additional washes at higher temperatures and lower ionic strength (e.g., at least one wash as about 37 °C in about 0.1X-0.5X SSC, followed by at least one wash at about 68°C in about 0.1X-0.5X SSC).

In one embodiment of the present invention, any amino acid sequence described herein can be produced with from at least one, and up to about 20, additional heterologous amino acids flanking each of the C- and/or N-terminal ends of the specified amino acid sequence. The resulting protein or polypeptide can be referred to as "consisting essentially of the specified amino acid sequence. According to the present invention, the heterologous amino acids are a sequence of amino acids that are not naturally found (i.e., not found in nature, in vivo) flanking the specified amino acid sequence, or that are not related to the function of the specified amino acid sequence, or that would not be encoded by the nucleotides that flank the naturally occurring nucleic acid sequence encoding the specified amino acid sequence as it occurs in the gene, if such nucleotides in the naturally occurring sequence were translated using standard codon usage for the organism from which the given amino acid sequence is derived. Similarly, the phrase "consisting essentially of, when used with reference to a nucleic acid sequence herein, refers to a nucleic acid sequence encoding a specified amino acid sequence that can be flanked by from at least one, and up to as many as about 60, additional heterologous nucleotides at each of the 5' and/or the 3' end of the nucleic acid sequence encoding the specified amino acid sequence. The heterologous nucleotides are not naturally found (i.e., not found in nature, in vivo) flanking the nucleic acid sequence encoding the specified amino acid sequence as it occurs in the natural gene or do not encode a protein that imparts any additional function to the protein or changes the function of the protein having the specified amino acid sequence.

One embodiment of the invention relates to a method of identifying a cell surface polypeptide in a human host cell, wherein said polypeptide is necessary for entry of HIV into said human host cell. The method includes the steps of: (a) synthesizing a randomly fragmented cDNA population (including, but not limited to, a normalized cDNA population) from total mRNA isolated from a human host cell that is susceptible to HIV infection to yield DNA fragments; (b) transferring the DNA fragments to an expression vector to yield a genetic suppressor element library, wherein each of the DNA fragments is operatively linked to a protein translation initiation codon, and wherein the expression vector expresses the DNA fragments in the human host cell; (c) genetically modifying a population of human host cells by introducing the genetic suppressor element library into the population of human host cells; (d) infecting the population of human host cells with HIV; (e) isolating a genetically modified human host cell containing a genetic suppressor element conferring resistance to HIV infection from the population of human host cells; (f) recovering the genetic suppressor element from the isolated genetically modified human host cell; (g) determining the human cellular gene that corresponds to the genetic suppressor element; (h) determining that the human cellular gene encodes a cell surface polypeptide; and (i) determining that said cell surface polypeptide that is involved in HIV cell entry.

Step (i) can include the following steps: (i) contacting said human host cell with a putative inhibitor, wherein said human host cell expresses a polypeptide selected from the group consisting of CXCR-4, CCR4, CCR7, CD11C, CD47, CD68, CD69, CD74, CSF3R, RARA, GABBR1, P2X1, HELOl, GPRK6 and PTK2B; and

(ii) assessing inhibition of said activity of said cell surface polypeptide in an entry assay. In this embodiment, the genetic suppressor element can include a sense oriented genetic suppressor element encoding a peptide or an antisense-oriented genetic suppressor element encoding an antisense RNA. The cell surface polypeptide mediates fusion of HIV with the human host cell or mediates binding of HIV with the human host cell.

A cell-derived library (e.g., an RFE library) can be constructed from nucleic acid molecules of any mammalian cells, and preferably from cDNA of HIV- susceptible cells. In that regard, Example 1 demonstrates that GSEs can be selected from HL-60 cells that are naturally susceptible to HIV infection, from HeLa cells which are not naturally susceptible to HIV infection due to the lack of CD4 expression, and from peripheral blood mononuclear cells (PBMCs). It has been shown that expression of CD4 on the surface of HeLa cells by means of a retroviral vector renders the cells susceptible to HIV infection. Therefore, cell types not normally susceptible to HIV infection can still be useful as a source of genetic material for the construction of RFE libraries. It is also preferred that a normalized cDNA library is prepared (Gudkov and Roninson, 1996, Methods in Molecular Biology 69:229-231). Briefly, in one embodiment, DNA is first treated with enzymes to produce randomly cleaved fragments. This can be conveniently performed by

DNase I cleavage in the presence of Mn⁺ (Roninson et al, U.S. Patent No. 5,217,889, column 5, lines 5-20). Thereafter, the randomly-cleaved DNA is size fractionated by gel electrophoresis. Fragments of between 100 and 700 bp are the preferred lengths for constructing REE libraries. Single strand breaks of the size- selected fragments are repaired by methods well known in the art.

The fragments are ligated with 5' and 3' adaptors, which are selected to have non-cohesive restriction sites so that each fragment can be inserted into an expression vector in an oriented fashion. Further, the 5' adaptor contains a start (ATG) codon to allow the translation of the fragments which contain an open reading frame in the correct phase. The fragments are then inserted into appropriate expression vectors.

Any expression vector that results in efficient expression of the fragments in host cells can be used. In a preferred embodiment viral-based vectors such as the retroviral vectors LNCX (Miller and Rosman, 1989, BioTechniques 7:980) and LNGFRM are exemplified. Alternatively, adenovirus, adeno-associated virus and herpes virus vectors can also be used for this purpose.

When viral-based vectors are used, the ligated vectors are first transfected into a packaging cell line to produce viral particles. For retroviral vectors, any amphotropic packaging line such as PA317 (Miller and Buttimore, 1986, Mol. Cell. Biol. 6:2895-2902; ATCC CRL #9078) can be used to efficiently produce virus. In a preferred embodiment of the invention, the viral vector also contains a selectable gene, such as the neo ^r gene or a truncated nerve growth factor receptor (NGFR) gene, which allows isolation of the cells that contain the vector. The number of independent clones present in each RFE expression library can vary. In a preferred embodiment, libraries of cell-derived cDNA of about 10 to 10 independent clones can be used.

In a specific embodiment illustrated by way of example in Example 2, OM10.1 cells are used to select for GSEs, and are maintained in conventional tissue culture as described in Butera, U.S. Patent No. 5,256,534. The purpose of using OM10.1 cells for the selection of GSEs is that they contain a latent HIV-1 provirus which is inducible by TNF-α. Other cell lines can be similarly engineered with an inducible HIV provirus. Examples of cell lines that are infected with latent HIV include, but are not limited to, UI, U33, 8E5, ACH-2, LL58, THP/HIV and UHC4

(Bednarik and Folks, 1992, AIDS 6:3-16). A variety of agents have been shown to be capable of inducing latent HIV-infected cells, and these include TNF-α, TNF-β, interleukins-1, -2, -3, -4 and -6, granulocyte-macrophage colony stimulating factors, macrophage-colony stimulating factors, interferon-α, transforming growth factor-β, PMA, retinoic acid and vitamin D3 (Poli and Fauci, 1992, AIDS Res. Human

Retroviruses 9:191-197). Alternatively, GSEs can be selected on the basis of their ability to directly protect HIV-susceptible cells from HIV infection or inhibit the entry of HIV into cells using methods described herein.

The cell-derived RFE library can be introduced into latently HIV-infected cells or HIV-susceptible cells by any technique well known in the art that is appropriate to the vector system employed. In one embodiment of the invention, the viral vector also contains a selectable marker in addition to a random fragment of cellular DNA. A suitable marker, by way of example only, is the neo ^r gene, which permits selection of cells containing RFE library members using the drug G-418. In a preferred embodiment, the viral vector contains a truncated low affinity nerve growth factor receptor (NGFR) that permits selection of the cells using an anti-NGFR monoclonal antibody. In alternative embodiments,, the multiplicity of infection of the virions of the library is adjusted so that pre-selection for cells that are transduced by the vector is not needed. In the case of OM10.1 cells, the transduced cell population is treated with

TNF-α for a period of between about 24-72 hours (preferably about 24 hours) according to the method of Butera. The activation of the latent HIV-1 provirus in OM10.1 can be detected by the suppression of the cell surface CD4. (Without being bound by theory, it is believed that viral protein gpl20 binds to CD4 in the cytoplasm, which prevents subsequent expression of CD4 on the cell surface.) Clones that are resistant to HIV replication continue to express cell surface CD4. Such clones can be selected, for example, by cell sorting using any antibody staining technique for CD4 and a fluorescence activated cell sorter (FACS). The fraction of CD4⁺ cells that have been transduced with the RFE library can be compared with cells transduced with an expression library consisting of the vector only. An increased relative difference between the cell-derived RFE library and the control library can be found with each additional round of TNF-α induction. Thus, in the preferred embodiment of the invention there are at least two cycles of induction, selection and recloning before the GSEs are recovered from the cells for further characterization.

After selection, specific nucleic acid molecules corresponding to the GSEs can be recovered from cells that continue to express CD4 following induction of the latent HIV provirus by TNF-α. The specific GSEs are recovered from genomic DNA isolated from CD4⁺ cells sorted by FACS after TNF-α induction. The GSEs in this population are preferably recovered by PCR amplification using primers designed from the sequences of the vector.

The recovered GSEs can be introduced into an expression vector as discussed in the Examples section herein. The resultant GSEs expression library is known as a secondary library. The secondary library can utilize the same or a different vector from that used for the construction of the primary library. The secondary library can be transduced into another cell population and the resultant population selected, recloned and processed as described herein.

Additionally, each individually recovered GSE can be inserted into cloning vectors for determining its specific nucleotide sequence and its orientation. The sequence of the GSE is then compared with sequences of known genes to determine the portion of the cellular gene with which it corresponds. Alternatively, the PCR products themselves can be directly sequenced to determine their nucleotide sequences. Concurrently, the isolated GSEs can be analyzed to determine their minimal core sequences. A core sequence is a common sequence found by comparison of GSEs with overlapping sequences. The GSEs are further tested for their ability to protect previously uninfected cells from HIV infection.

Once GSEs have been identified, the core sequence of the GSE is determined. This can be done by comparing overlapping sequences of independently derived GSEs. Alternatively, GSEs can be altered by additions, substitutions or deletions and assayed for retention of HIV-suppressive function. Alterations in the GSEs sequences can be generated using a variety of chemical and enzymatic methods which are well known to those skilled in the art. For example, oligonucleotide-directed mutagenesis can be employed to alter the GSE sequence in a defined way and/or to introduce restriction sites in specific regions within the sequence. Additionally, deletion mutants can be generated using DNA nucleases such as Bal 31 or Exo III and SI nuclease. Progressively larger deletions in the GSE sequences can be generated by incubating the DNA with nucleases for increased periods of time (see Ausubel, et al, ibid. , for a review of mutagenesis techniques) .

The altered sequences can be evaluated for their ability to suppress expression of HIV proteins such as ρ24 in appropriate host cells. It is within the scope of the present invention that any altered or shortened GSE nucleic acid molecules that retain their ability to suppress HIV infection can be incorporated into recombinant expression vectors for further use.

In order to confirm that the selected GSEs can protect uninfected cells from HIV infection, the GSEs can be transferred into latently HIV infected or into HIV- susceptible host cells followed by HIV infection. In this connection, GSEs also can be directly selected from a RFE library for their ability to prevent productive infection by HIV. Protection experiments can be performed in any cell type that takes up the potential GSEs and that is otherwise susceptible to HIV infection. In a preferred embodiment by way of example, the CEM-ss cell line is used (Foley et al. 1965, Cancer 18:522-529). The use of CEM-ss cells as targets for quantitative infectivity of HIV-1 has been described by Nara & Fischinger (1988, Nature 322:469-470). Other cell lines that are susceptible to HIV infection include, but are not limited to, HUT-78,

H9, Jurkat E6-1, A3.01, U-937, AA-2, HeLa CD4⁺ and C8166. In addition, freshly isolated peripheral blood leukocytes can be used.

The test of the potential GSEs can be performed using the same expression vector system as that employed in the RFE library transduction of cells during initial selection steps. In other embodiments, the vector system can be modified to achieve higher levels of expression, e.g., the linkers can be employed to introduce a leader sequence that increases the translational efficiency of the message. One such sequence is disclosed by Kozak, 1994, Biochemie 76:815-821. Another way of testing the effectiveness of a potential GSE against HIV infection is to determine how rapidly HIV-1 variants develop that can negate the effects of that element. Such a test includes infection of a culture of susceptible cells such as CEM-ss cells at a low multiplicity of infection and repeatedly assaying the culture to determine whether and how quickly HIV-1 infection becomes widespread.

The range of useful multiplicities of infection is between about 100 to 1000 tissue culture infectious units (TCID₅₀) per 10⁶ CEM-ss cells. The TCID₅₀ is determined by an endpoint method and is important for determining the input multiplicity of infection (moi). A parameter that correlates with the development in the test culture of HIV-1 strains that are resistant to the effects of the potential GSEs is the fraction of cells that are infected in the culture. This fraction can be determined by immunofluorescent staining with an antibody specific for the HIV-1 p24 antigen of fixed permeabilized cells. Commercially available reagents are suitable for performing such tests (Lee et al, 1994, J. Virol. 68:8254-8264).

Methods to test putative GSEs and similar compounds for the ability to inhibit aspects of HIV infection and replication are also described in detail in U.S. Patent No. 6,613,506, which is incorporated herein by reference in its entirety.

Another embodiment of a cell-derived nucleic acid molecule of the present invention comprises a cellular gene that encodes an intracellular product necessary for productive HIV infection, referred to herein as a "target gene." Human cellular genes that are preferred target genes for the methods described herein include any of the genes listed in Table 1 above, an mRNA nucleotide sequence for which is provided in SEQ ID Nos:7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33 or 35. These genes encode the following cellular proteins, respectively: Chemokine receptor (C-X-C) 4

(CXCR-4; SEQ ID NO:8), Chemokine (C-C motif) receptor 4 (CCR4; SEQ ID NO: 10), Chemokine (C-C motif) receptor 7 (CCR7; SEQ ID NO: 12), Integrin, alpha X (CD11C; SEQ ID NO:14), CD47 antigen (CD47; SEQ ID NO:16), CD68 antigen (CD68; SEQ ID NO: 18), Early T-cell activation antigen (CD69; SEQ ID NO:20), Cell division cycle 25B (CD74; SEQ ID NO:22), Colony stimulating factor 3 receptor

(CSF3R; SEQ ID NO:24), Retinoic acid receptor, alpha (RARA; SEQ ID NO:26), gamma-aminobutyric acid B receptor, 1 (GABBRl; SEQ ID NO:28), P2X1 receptor (P2X1; SEQ ID NO: 30), Homolog of yeast long chain polyunsaturated fatty acid elongation enzyme 2 (HELOl; SEQ ID NO:32), G protein-coupled receptor kinase 6 (GPRK6; SEQ ID NO:34) and Protein tyrosine kinase 2 beta (PTK2B; SEQ ID NO:36). In any of the assays described herein, one can use a full-length gene, including a regulatory region of the gene, or a nucleic acid molecule encoding the gene product (protein encoded by the gene) or fragment thereof, or any fragment of such nucleic acid molecules that is suitable for use in an assay to identify inhibitors of the cellular gene for the purpose of inhibition of HIV infection and particularly, HIY entry into a cell.

The subsections below describe such cellular genes that have been identified herein as important targets for the development of HIV therapeutics. Chemokine receptor (C-X-C) 4 (CXCR-4) is a stromal cell derived protein that has been shown to be involved in many immunological reactions and is expressed in many cancer cells which use this receptor efficiently for metastasis formation. CXCR-4 is described, for example, in: Caruz et al., FEBS Lett. 426 (2), 271_278 (1998); Tachibana et al., Nature 393 (6685), 591_594 (1998); Zou et al., Nature 393 (6685), 595_599 (1998); and Secchiero et al., J. Immunol. 164 (8), 4018_4024 (2000).

Chemokine (C-C motif) receptor 4 (CCR4) is Chemokine (C_C) receptor 4 belongs to the G_protein_coupled receptor family . It is a receptor for the CC chemokine _ MIP_1, RANTES, TARC and MCP_1. Chemokines are a group of small polypeptide, structurally related molecules that regulate cell trafficking of various types of leukocytes. The chemokines also play fundamental roles in the development, homeostasis, and function of the immune system, and they have effects on cells of the central nervous system as well as on endothelial cells involved in angiogenesis or angiostasis. CCR4 is described, for example, in Power et al., J. Biol. Chem. 270 (33), 19495_19500 (1995); Imai et al., J. Biol. Chem. 272 (23), 15036 5042 (1997); and Imai et al., J. Biol. Chem. 273 (3), 1764 768 (1998).

Chemokine (C-C motif) receptor 7 (CCR7) is a protein_coupled receptor family. This receptor was identified as a gene induced by the Epstein_Barr virus (EBV), and is thought to be a mediator of EBV effects on B lymphocytes. This receptor is expressed in various lymphoid tissues and activates B and T lymphocytes. It has been shown to control the migration of memory T cells to inflamed tissues, as well as stimulate dendritic cell maturation. The chemokine (C_C motif) ligand 19 (CCL19/ECL) has been reported to be a specific ligand of this receptor. CCR7 is described, for example, in: Schweickart et al., Genomics 23 (3), 643_650 (1994); Yoshida et al., J. Biol. Chem. 272 (21), 13803 3809 (1997); Kim et al., J. Immunol. 161 (5), 2580_2585 (1998); Yanagihara et al., J. Immunol. 161 (6), 3096_3102

(1998).

Integrin, alpha X (CD11C) is a heterodimeric integral membrane protein composed of an alpha chain and a beta chain. This protein combines with the beta 2 chain (ITGB2) to form a leukocyte_specific integrin referred to as inactivated_C3b

(iC3b) receptor 4 (CR4). The alpha X beta 2 complex seems to overlap the properties of the alpha M beta 2 integrin in the adherence of neutrophils and monocytes to stimulated endothelium cells, and in the phagocytosis of complement coated particles.

CD11C is described, for example, in: Corbi et al., EMBO J. 6 (13), 4023_4028 (1987); Corbi et al., J. Biol. Chem. 265 (5), 2782_2788 (1990); Corbi et al., Leuk.

Lymphoma 25 (5_6), 415_425 (1997).

CD47 antigen (CD47) is a transmembrane glycoprotein that is a signal transducer integrin-associated protein. CD47 is physically and functionally associated with the integrin alpha v beta 3 vitronectin receptor and is involved in the increase in intracellular calcium concentration, which occurs upon cell adhesion to extracellular matrix and is involved in macrophage multinucleation, T cell apoptosis, and Fcgamma and complement receptor mediated phagocytosis, among other functions. CD47 is described, for example, in: Campbell et al., Cancer Res. 52 (19), 5416_5420 (1992); Lindberg et al., J. Cell Biol. 123 (2), 485_496 (1993); Lindberg et al., J. Biol. Chem. 269 (3), 1567_1570 (1994); Mawby et al., Biochem. J. 304 (Pt 2), 525_530

(1994); Yoshida et al., J. Immunol. 168 (7), 3213_3220 (2002).

CD68 antigen (CD68) is CD68 is a 110_kD transmembrane glycoprotein that is highly expressed by human monocytes and tissue macrophages. It is a type I integral membrane protein with a heavily glycosylated extracellular domain. CD68 is described, for example, in: Holness et al., Blood 81 (6), 1607 613 (1993); Wahl et al., J. Leukoc. Biol. 68 (3), 303_310 (2000).

Early T-cell activation antigen (CD69) is a type II integral membrane glycoprotein that can only be detected after stimulation of lymphocytes. CD69 is a signal transduction molecule involved in early activation events of T cells. CD69 is described, for example, in: Cambiaggi et al., J-mmunogenetics 36 (2), 117_120

(1992); Hamann et al, J. Immunol. 150 (11), 4920_4927 (1993); Lopez-Cabrera et al., J. Exp. Med. 178 (2), 537_547 (1993); Santis et al., Eur. J. Immunol. 24 (7), 1692 697 (1994). Cell division cycle 25B (CD74) is CDC25B is a member of the CDC25 family of phosphatases. CDC25B activates the cyclin dependent kinase CDC2 by removing two phosphate groups and it is required for entry into mitosis. CDC25B shuttles between the nucleus and the cytoplasm due to nuclear localization and nuclear export signals. The protein is nuclear in the M and GI phases of the cell cycle and moves to the cytoplasm during S and G2. CDC25B has oncogenic properties, although its role in tumor formation has not been determined. At least four transcript variants for this gene exist. CD74 is described, for example, in: Nagata et al., New Biol. 3 (10), 959_968 (1991); Galaktionov et al., Cell 67 (6), 1181 194 (1991); Galaktionov et al., Science 269 (5230), 1575_1577 (1995); Reynolds et al., J. Mol. Biol. 293 (3),

559_568 (1999); Nilsson et al., Prog Cell Cycle Res 4, 107_114 (2000).

Colony stimulating factor 3 receptor (CSF3R) is the receptor for colony stimulating factor 3, a cytokine that controls the production, differentiation, and function of granulocytes. The encoded protein, which is a member of the family of cytokine receptors, may also function in some cell surface adhesion or recognition processes. Four transcript variants encoding four different isoforms have been found for this gene, with three of the isoforms being membrane_bound and the other being secreted and soluble. Mutations in this gene are a cause of Kostmann syndrome, also known as severe congenital neutropenia. CSF3R is described, for example, in: Fukunaga et al., Proc. Natl. Acad. Sci. U.S.A. 87 (22), 8702_8706 (1990); Larsen et al., J. Exp. Med. 172 (6), 1559_1570 (1990); Fukunaga et al., EMBO J. 10 (10), 2855_2865 (1991); Tweardy et al., Blood 79 (5), 1148_1154 (1992); Yamasaki et al., Nat. Struct. Biol. 4 (6), 498_504 (1997); and Layton et al., J. Biol. Chem. 272 (47), 29735_29741 (1997). Retinoic acid receptor, alpha (RARA) is one of at least two receptors for retenoic acid. Retinoic acid is a vitamin A derivative that exhibits major effects on biological processes such as cell differentiation and embryo pattern formation. Fusion of RARA with PML proteins may block RA target genes, impair RA mediated differentiation and lead to transformation, which can result in promyelocytic leukemia. RARA is described, for example, in: Petkovic, Nature 330 (6147),

444_450 (1987); Giguere et al., Nature 330 (6149), 624_629 (1987); de The et al., Nature 347 (6293), 558_561 (1990); de The et al., Cell 66 (4), 675_684 (1991).

Gamma-aminobutyric acid B receptor, 1 (GABBRl) is the main inhibitory neurotransmitter in the mammalian central nervous system. GABA exerts its effects through ionotropic [GABA(A C)] receptors, to produce fast synaptic inhibition, and metabotropic [GABA(B)] receptors, to produce slow, prolonged inhibitory signals. The GABA(B) receptor consists of a heterodimer of two related 7_transmembrane receptors, GABA(B) receptor 1 and GABA(B) receptor 2. The GABA(B) receptor 1 gene is mapped to chromosome 6p21.3 within the HLA class I region close to the

HLAJF gene. Susceptibility loci for multiple sclerosis, epilepsy, and schizophrenia have also been mapped in this region. Alternative splicing of this gene generates 4 transcript variants. GABBRl is described, for example, in: Fraser et al., Glia 11 (2), 83_93 (1994); Kaupmann et al., Nature 386 (6622), 239_246 (1997); Grifa et al., Biochem. Biophys. Res. Commun. 250 (2), 240_245 (1998); Goei et al., Biol.

Psychiatry 44 (8), 659_666 (1998); White et al., Nature 396 (6712), 679_682 (1998).

P2X1 receptor (P2X1) is a member of the purinoceptor family consisting of ligand-gated ion channels. P2X receptors are ATP -gated ion channels which mediate rapid (within 10 ms) and selective permeability to cations (Na⁺, K⁺ and Ca²⁺). Some agonists (e.g., alphabeta-methylene ATP) and antagonists [e.g., 2',3'-0-(2,4,6- trinitrophenyl)-ATP] are strongly selective for receptors containing P2X1 subunits. All P2X receptors are permeable to small monovalent cations; some have significant calcium or anion permeability. P2X receptors are abundantly distributed, and functional responses are seen in neurons, glia, epithelia, endothelia, bone, muscle, and hemopoietic tissues. On smooth muscles, P2X receptors respond to ATP released from sympathetic motor nerves (e.g., in ejaculation). On sensory nerves, they are involved in the initiation of afferent signals in several viscera (e.g., bladder, intestine) and play a key role in sensing tissue-damaging and inflammatory stimuli. Paracrine roles for ATP signaling through P2X receptors are likely in neurohypophysis, ducted glands, airway epithelia, kidney, bone, and hemopoietic tissues. P2X1 is described, for example, in: North, Physiol Rev. 2002 Oct;82(4): 1013-67; MacKenzie et al., Ann N Y Acad Sci. 1999 Apr 30;868:716-29; Ralevic et al., Pharmacol Rev. 1998 Sep;50(3):413-92.

Homolog of yeast long chain polyunsaturated fatty acid elongation enzyme 2 (HELOl) is involved in the elongation of long-chain polyunsaturated fatty acids, as determined by the conversion of gamma-linolenic acid (C(18:3, n-6)) into dihomo- gamma-linolenic acid (C(20:3, n-6)), arachidonic acid (C(20:4, n-6)) into adrenic acid (C(22:4, n-6)), stearidonic acid (C(18:4, n-3)) into eicosatetraenoic acid (C(20:4, n- 3)), eicosapentaenoic acid (C(20:5, n-3)) into omega3-docosapentaenoic acid (C(22:5, n-3)) and alpha-linolenic acid (C(18:3, n-3)) into omega3-eicosatrienoic acid (C(20:3, n-3)). HELOl is described, for example, in: Leonard et al, Biochem. J. 350 Pt 3, 765 770 (2000).

G protein-coupled receptor kinase 6 (GPRK6) is one of a family of G protein- coupled receptor kinases. Phosphorylation by receptor_specific and second messenger_activated protein kinases is a mechanism for regulation of G protein_coupled receptors. G protein_coupled receptors are 7_transmembrane dornain_containing proteins and are triggered by a variety of signals. GPRK6 is described, for example, in: Benovic et al., J. Biol. Chem. 268 (26), 19521_19527 (1993); Haribabu et al., Proc. Natl. Acad. Sci. U.S.A. 90 (20), 9398_9402 (1993);

Loudon et al., J. Biol. Chem. 269 (36), 22691_22697 (1994); Willets et al., J. Biol. Chem. 277 (18), 15523 5529 (2002).

Protein tyrosine kinase 2 beta (PTK2B) is a cytoplasmic protein tyrosine kinase which is involved in calciumjnduced regulation of ion channels and activation of the map kinase signaling pathway. The encoded protein may represent an important signaling intermediate between neuropeptide_activated receptors or neurotransmitters that increase calcium flux and the downstream signals that regulate neuronal activity. The encoded protein undergoes rapid tyrosine phosphorylation and activation in response to increases in the intracellular calcium concentration, nicotinic acetylcholine receptor activation, membrane depolarization, or protein kinase C activation. This protein has been shown to bind CRK_associated substrate, nephrocystin, GTPase regulator associated with FAK, and the SH2 domain of GRB2. The encoded protein is a member of the FAK subfamily of protein tyrosine kinases but lacks significant sequence similarity to kinases from other subfamilies. Four transcript variants encoding two different isoforms have been found for this gene.

PTK2B is described, for example, in: Musacchio et al, Nature 376 (6543), 737_745 (1995); Calalb et al, Mol. Cell. Biol. 15 (2), 954_963 (1995); Lev et al., Nature 376 (6543), 737_745 (1995); Sasaki et al., J. Biol. Chem. 270 (36), 21206_21219 (1995); Avraham et al., J. Biol. Chem. 270 (46), 27742_27751 (1995). One embodiment of the present invention relates to an isolated nucleic acid molecule comprising, consisting essentially of, or consisting of: a GSE nucleic acid sequence according to the present invention, and to nucleic acid sequence homologues of the GSE that hybridize under stringent conditions to the complement of a GSE of the invention. Such GSEs are listed in Table 2 of the invention, and include: positions 14-214 of SEQ ID NO:7; positions 1505-1566 of SEQ ID NO:9; positions 3-127 of SEQ ID NO:l l; positions 4019-4095 of SEQ ID NO:13; positions 283-375 of SEQ ID NO: 15; positions 234-410 of SEQ ID NO: 17; positions 23-141 of SEQ ID NO: 19; positions 18-238 of SEQ ID NO.21; positions 55-234 of SEQ ID NO:23; positions 98-351 of SEQ ID NO:25; positions 898-1091 of SEQ ID NO:27; positions 16-46 of

SEQ ID NO:29; positions 4-52 of SEQ ID NO:31; positions 532-765 of SEQ ID NO:33; or positions 313-546 of SEQ ID NO:35. Any of the GSE sequences described herein can be used, in one embodiment, as a template or structure on which various other inhibitors of the present invention (e.g., small molecules) can be designed or selected.

Once a human cellular gene has been identified as a potential target for supporting HIV cell entry, an assay can be used for screening and selecting small molecule inhibitor compounds, peptides, antibodies and other inhibitory compounds having activity as an anti-HIV therapeutic based on the ability to inhibit HIV cell entry by inhibiting the activity of a cell surface polypeptide involved in HIV cell entry. For example, a cell line that naturally expresses the cell surface polypeptide of interest or has been transfected with the gene or other recombinant nucleic acid molecule encoding a cell surface polypeptide of interest as disclosed herein is incubated with various compounds. A reduction of the activity (e.g., biological activity, which can include involvement in the ability of HIV to enter a cell) of the cell surface polypeptide of interest, in the ability of HIV to enter the cell, and/or the ability of HIV to infect and replicate in the cell may be used to identify small molecule inhibitors, antibodies or peptides having activity as anti-HIV therapeutics. Compounds identified in this manner can then be retested in other assays that utilize cells capable of enabling HIV cell entry to confirm their activities against HIV cell entry.

In general, the biological activity or biological action of a protein refers to any function(s) exhibited or performed by the protein that is ascribed to the naturally occurring form of the protein as measured or observed in vivo (i.e., in the natural physiological environment of the protein) or in vitro (i.e., under laboratory conditions). Modifications, activities or interactions which result in a decrease in protein expression or a decrease in the activity of the protein, can be referred to as inactivation (complete or partial), down-regulation, reduced action, or decreased action or activity of a protein. Similarly, modifications, activities or interactions which result in an increase in protein expression or an increase in the activity of the protein, can be referred to as amplification, overproduction, activation, enhancement, up-regulation or increased action of a protein. The biological activity of a protein according to the invention can be measured or evaluated using any assay for the biological activity of the protein as known in the art. Such assays can include, but are not limited to, binding assays, assays to determine intemalization of the protein and/or associated proteins, cell signal transduction assays (e.g., phosphorylation assays), and/or assays for determining downstream cellular events that result from activation or binding of the cell surface protein (e.g., expression of downstream genes, production of various biological mediators, etc.). Preferably, the assay measures the ability of the cell surface protein to allow entry of HIV into the cell. Such assays are described in detail herein.

Compounds to be screened in the methods of the invention include known organic compounds such as antibodies, products of peptide libraries, and products of chemical combinatorial libraries. Compounds may also be identified using rational drug design relying on the structure of the gene product of a human cellular gene. Such methods are known to those of skill in the art and involve the use of three- dimensional imaging software programs. For example, various methods of drug design, useful to design or select mimetics or other therapeutic compounds useful in the present invention are disclosed in Maulik et al., 1997, Molecular Biotechnology:

Therapeutic Applications and Strategies, Wiley-Liss, Inc., which is incorporated herein by reference in its entirety.

A mimetic refers to any peptide or non-peptide compound that is able to mimic the biological action of a naturally occurring peptide, often because the mimetic has a basic structure that mimics the basic structure of the naturally occurring peptide and/or has the salient biological properties of the naturally occurring peptide. Mimetics can include, but are not limited to: peptides that have substantial modifications from the prototype such as no side chain similarity with the naturally occurring peptide (such modifications, for example, may decrease its susceptibility to degradation); anti-idiotypic and/or catalytic antibodies, or fragments thereof; non- proteinaceous portions of an isolated protein (e.g., carbohydrate structures); or synthetic or natural organic molecules, including nucleic acids and drugs identified through combinatorial chemistry, for example. Such mimetics can be designed, selected and/or otherwise identified using a variety of methods known in the art. A mimetic can be obtained, for example, from molecular diversity strategies (a combination of related strategies allowing the rapid construction of large, chemically diverse molecule libraries), libraries of natural or synthetic compounds, in particular from chemical or combinatorial libraries (i.e., libraries of compounds that differ in sequence or size but that have the similar building blocks) or by rational, directed or random drug design. See for example, Maulik et al., supra.

In a molecular diversity strategy, large compound libraries are synthesized, for example, from peptides, oligonucleotides, carbohydrates and/or synthetic organic molecules, using biological, enzymatic and/or chemical approaches. The critical parameters in developing a molecular diversity strategy include subunit diversity, molecular size, and library diversity. The general goal of screening such libraries is to utilize sequential application of combinatorial selection to obtain high-affinity ligands for a desired target, and then to optimize the lead molecules by either random or directed design strategies. Methods of molecular diversity are described in detail in Maulik, et al., ibid.

Maulik et al. also disclose, for example, methods of directed design, in which the user directs the process of creating novel molecules from a fragment library of appropriately selected fragments; random design, in which the user uses a genetic or other algorithm to randomly mutate fragments and their combinations while simultaneously applying a selection criterion to evaluate the fitness of candidate ligands; and a grid-based approach in which the user calculates the interaction energy between three dimensional receptor structures and small fragment probes, followed by liriking together of favorable probe sites.

In one embodiment of the invention, irmibitors of HIV cell entry are identified by exposing a mammalian cell expressing a cell surface polypeptide of the present invention to a test compound and assessing inhibition of the activity of a cell surface polypeptide in an entry assay. Preferred cell surface polypeptides of the present invention include CXCR-4, CCR4, CCR7, CD11C, CD47, CD68, CD69, CD74, CSF3R, RARA, GABBRl, P2X1, HELOl, GPRK6 or PTK2B. As used herein, the term "test compound" or "putative inhibitory compound" refers to compounds having an unknown or previously unappreciated regulatory activity in a particular process. As such, the term "identify" with regard to methods to identify compounds is intended to include all compounds, the usefulness of which as a regulatory compound for the purposes of inhibiting HIV cell entry or infection is determined by a method of the present invention.

Entry assays can include any assay known in the art that determines the ability of a virus to enter a cell. An example of an entry assay is a cell-cell fusion assay in which the ability of a virus to fuse to a cell is determined. By way of illustration, viral cell fusion can be determined by assessing the redistribution of cytoplasmic dyes between env-expressing cells and target cells containing siRNA. The env-expressing cells are labeled with 5 -(and -6)-(((4-chloro-methyl) benzoyl) amino) tetramethylrhodamine (CMTR) dye (red) and target cells are labeled with 5- chloromethylfluorescein diacetate (CMFDA) dye (green). The cells are cocultured for 8 hours, then visualized using a fluorescent microscope. When the colocalization of the red and green dyes occurs, fusion has taken place (I Munoz-Barroso et al., 1999, J Virol 73:6089 and S. Himathongkham et al., 2002, Virology 298:189). Another example of an entry assay is a virus cell binding assay in which the ability of a virus to bind to a cell is determined microscopically.

In one embodiment of the invention, inhibitors of HIV infection are identified by exposing a mammalian cell to a test compound; measuring the activity of a cell surface polypeptide in the mammalian cell; and selecting a compound that inhibits the activity of the cell surface polypeptide. In one embodiment of the invention, inhibitors of HIV infection are identified by exposing a mammalian cell to a test compound and to HIV and selecting a compound that inhibits the entry of HIV into the cell and/or that inhibits the infection of the cell with HIV. A preferred mammalian cell to use in an assay is a mammalian cell that either naturally expresses the cell surface polypeptide or has been transfected with a recombinant form of the human cellular gene or other recombinant nucleic acid molecule encoding the cell surface polypeptide. Preferably, the activity of the cell surface polypeptide or the infectivity of HIV is measured in the presence and absence of the candidate compound, or in the presence of another suitable control compound.

In a preferred embodiment, the activity of a cell surface polypeptide is measured by contacting a cell surface polypeptide on the surface of a cell with a blocking agent such as an antibody or peptide that binds to the cell surface polypeptide and determining whether a virus can bind or fuse to that cell. In another preferred embodiment, the activity of a cell surface polypeptide is measured by disrupting the expression in a cell of the gene encoding the cell surface polypeptide and determining if virus can bind or fuse to the cell. In addition to determining the amount of virus binding or fusion, the activity of a cell surface polypeptide can be measured by measuring the amount of virus replication that occurs in a cell expressing the cell surface polypeptide after the cell has been contacted with virus. In another preferred embodiment, the activity of a cell surface polypeptide can be determined by contacting a cell expressing the cell surface polypeptide with virus under conditions by which virus binding is enhanced and measuring the loss of enhanced binding. Methods to enhance binding are known to those of skill in the art. The compounds can be retested in other assays such as in OM10.1 cells or in productive HIV infection to confirm their activities against HIV infection (see

Examples section). Any of the assays used for the identification of GSEs as described herein can also be used to screen for potential inhibitors of the cellular genes. For example, the use of CEM-ss cell line (Foley et al. 1965, Cancer 18:522-529) as targets for quantitative infectivity of HIV-1 has been described by Nara & Fischinger (1988, Nature 322:469-470). Other cell lines that are susceptible to HIV infection include, but are not limited to, HUT-78, H9, Jurkat E6-1, A3.01, U-937, AA-2, HeLa CD4⁺ and C8166. In addition, freshly isolated peripheral blood leukocytes can be used. Another way of testing the effectiveness of a putative inhibitory compound against HIV cellular entry and HIV infection is to determine how rapidly HIV-1 variants develop that can negate the effects of that compound. Such a test includes infection of a culture of susceptible cells such as CEM-ss cells at a low multiplicity of infection and repeatedly assaying the culture to determine whether and how quickly HIV-1 infection becomes widespread. The range of useful multiplicities of infection is between about 100 to 1000 tissue culture infectious units (TCID₅₀) per 10⁶ CEM-ss cells. The TCID₅₀ is determined by an endpoint method and is important for determining the input multiplicity of infection (moi).

A parameter that correlates with the development in the test culture of HIV-1 strains that are resistant to the effects of the putative inhibitory compound is the fraction of cells that are infected in the culture. This fraction can be determined by immunofluorescent staining with an antibody specific for the HIV-1 p24 antigen of fixed permeabilized cells. Commercially available reagents are suitable for performing such tests (Lee et al, 1994, J. Virol. 68:8254-8264).

In another embodiment of the invention, inhibitors of HIV infection are selected by determining the three-dimensional structure of a human cellular gene product; and determining the three-dimensional structure of an inhibitor of the human cellular gene product. Preferably, the structure of the inhibitor is determined using computer software capable of modeling the interaction of an inhibitor with the human cellular gene product. One of skill in the art can select the appropriate three- dimensional structure, inhibitor, and analytical software based on the identity of the human cellular gene.

For example, suitable candidate chemical compounds can align to a subset of residues described for a target site. Preferably, a candidate chemical compound comprises a conformation that promotes the formation of covalent or noncovalent crosslinking between the target site and the candidate chemical compound.

Preferably, a candidate chemical compound binds to a surface adjacent to a target site to provide an additional site of interaction in a complex. When designing an antagonist, for example, the antagonist should bind with sufficient affinity to the binding site or to substantially prohibit a ligand (i.e., a molecule that specifically binds to the target site) from binding to a target area. It will be appreciated by one of skill in the art that it is not necessary that the complementarity between a candidate chemical compound and a target site extend over all residues specified here in order to inhibit or promote binding of a ligand.

In general, the design of a chemical compound possessing stereochemical complementarity can be accomplished by techniques that optimize, chemically or geometrically, the "fit" between a chemical compound and a target site. Such techniques are disclosed by, for example, Sheridan and Venkataraghavan, Ace. Chem Res., vol. 20, p. 322, 1987: Goodford, J. Med. Chem., vol. 27, p. 557, 1984; Beddell, Chem. Soc. Reviews, vol. 279, 1985; Hoi, Angew. Chem., vol. 25, p. 767, 1986; and Verlinde and Hoi, Structure, vol. 2, p. 577, 1994, each of which are incorporated by this reference herein in their entirety.

As another example, a "geometric approach" is used. In a geometric approach, the number of internal degrees of freedom (and the corresponding local minima in the molecular conformation space) is reduced by considering only the geometric (hard_sphere) interactions of two rigid bodies, where one body (the active site) contains "pockets" or "grooves" that form binding sites for the second body (the complementing molecule, such as a ligand). The geometric approach is described by Kuntz et al., J. Mol. Biol, vol. 161, p. 269, 1982, which is incorporated by this reference herein in its entirety. The algorithm for chemical compound design can be implemented using the software program DOCK Package, Version 1.0 (available from the Regents of the University of California). Pursuant to the Kuntz algorithm, the shape of the cavity or groove on the surface of a structure at a binding site or interface is defined as a series of overlapping spheres of different radii. One or more extant databases of crystallographic data (e.g., the Cambridge Structural Database

System maintained by University Chemical Laboratory, Cambridge University,

Lensfield Road, Cambridge CB2 1EW, U.K.) or the Protein Data Bank maintained by

Brookhaven National Laboratory, is then searched for chemical compounds that approximate the shape thus defined. Chemical compounds identified by the geometric approach can be modified to satisfy criteria associated with chemical complementarity, such as hydrogen bonding, ionic interactions or Van der Waals interactions.

As yet another example, one can determine the interaction of chemical groups ("probes") with an active site at sample positions within and around a binding site or interface, resulting in an array of energy values from which three dimensional contour surfaces at selected energy levels can be generated. This method is referred to herein as a "chemical__probe approach." The chemical_probe approach to the design of a chemical compound useful of the present invention is described by, for example, Goodford, J. Med. Chem., vol. 28, p. 849, 1985, which is incorporated by this reference herein in its entirety, and is implemented using an appropriate software package, including for example, GRID (available from Molecular Discovery Ltd., Oxford OX2 9LL, U.K.). The chemical prerequisites for a site_complementing molecule can be identified at the outset, by probing the active site of a protein with different chemical probes, e.g., water, a methyl group, an amine nitrogen, a carboxyl oxygen and/or a hydroxyl. Preferred sites for interaction between an active site and a probe are determined. Putative complementary chemical compounds can be generated using the resulting three dimensional pattern of such sites.

In still another embodiment of the invention, inhibitors of HIV cell entry are identified by exposing a polypeptide encoded by a human cellular gene to a test compound; measuring the binding of the test compound to the polypeptide; and selecting a compound that binds to the polypeptide at a desired concentration, affinity, or avidity. In a preferred embodiment, the assay is performed under conditions conducive to promoting the interaction or binding of the compound to the polypeptide. One of skill in the art can determine such conditions based on the polypeptide and the compound being used in the assay. In one embodiment, a

BIAcore machine can be used to determine the binding constant of a complex between the target protein (a protein encoded by the target gene) and a natural ligand in the presence and absence of the candidate compound. For example, the target protein or a ligand binding fragment thereof can be immobilized on a substrate. A natural or synthetic ligand is contacted with the substrate to form a complex. The dissociation constant for the complex can be determined by monitoring changes in the refractive index with respect to time as buffer is passed over the chip (O'Shannessy et al. Anal. Biochem. 212:457_468 (1993); Schuster et al, Nature 365:343_347 (1993)). Contacting a test compound at various concentrations with the complex and monitoring the response function (e.g., the change in the refractive index with respect to time) allows the complex dissociation constant to be determined in the presence of the test compound and indicates whether the test compound is either an inhibitor or an agonist of the complex. Alternatively, the test compound can be contacted with the immobilized target protein at the same time as the ligand to see if the test compound inhibits or stabilizes the binding of the ligand to the target protein.

Other suitable assays for measuring the binding of a candidate compound to a target protein or its ligand, and or for measuring the ability of such compound to affect the binding of the target protein to its ligand include, for example, immunoassays such as enzyme linked immunoabsorbent assays (ELISA) and radioimmunoassays (RIA), as well as cell_based assays including, cytokine secretion assays, or intracellular signal transduction assays that determine, for example, protein or lipid phosphorylation, mediator release or intracellular Ca⁺ mobilization upon CR2 binding to a cell signal transduction molecule or coreceptor. In still another embodiment of the invention, inhibitors of HIV cell entry are identified by exposing an enzyme encoded by a human cellular gene to a test compound; measuring the activity of the enzyme encoded by the human cellular gene in the presence and absence of the compound; and selecting a compound that down- regulates the activity of the enzyme encoded by the human cellular gene. Methods to measure enzymatic activity are well known to those skilled in the art and are selected based on the identity of the enzyme being tested. For example, if the enzyme is a kinase, phosphorylation assays can be used.

In addition to methods for identifying and producing small molecule inhibitors, antibodies or peptide inhibitors, methods known in the art that down- regulate expression or function of a human cellular gene as identified by the methods of the invention are also encompassed by this invention. For example, antisense RNA and DNA molecules may be used to directly block translation of mRNA encoded by these cellular genes by binding to targeted mRNA and preventing protein translation. Polydeoxyribonucleotides can form sequence-specific triple helices by hydrogen bonding to specific complementary sequences in duplexed DNA to effect specific down-regulation of target gene expression. Formation of specific triple helices may selectively inhibit the replication or expression of targeted genes by prohibiting the specific binding of functional trans-acting factors. Polynucleotides to be used in triplex helix formation should be single-stranded and composed of deoxynucleotides. The base composition of these polynucleotides must be designed to promote triple helix formation via Hoogsteen base pairing rules, which generally require sizeable stretches of either purines or pyrimidines to be present on one strand of a duplex. Polynucleotide sequences may be pyrimidine- based, which will result in TAT and CGC triplets across the three associated strands of the resulting triple helix. The pyrimidine-rich polynucleotides provide base complementarity to a purine-rich region of a single strand of the duplex in a parallel orientation to that strand. In addition, polynucleotides may be chosen that are purine- rich, for example, containing a stretch of G residues. These polynucleotides will form a triple helix with a DNA duplex that is rich in GC pairs, in which the majority of the purine residues are located on a single strand of the targeted duplex, resulting in GGC triplets across the three strands in the triplex.

Alternatively, sequences that can be targeted for triple helix formation can be increased by creating a so-called "switchback" polynucleotide. Switchback polynucleotides are synthesized in an alternating 5'-3', 3'-5' manner, so that they base pair with first one strand of a duplex and then the other, eliminating the necessity for a sizeable stretch of either purines or pyrimidines to be present on one strand of a duplex.

Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. Ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Within the scope of the invention are ribozyme embodiments including engineered hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of cellular RNA sequences. Antisense RNA molecules showing high-affinity binding to target sequences can also be used as ribozymes by addition of enzymatically active sequences known to those skilled in the art.

Both antisense RNA and DNA molecules, and ribozymes of the invention may be prepared by any method known in the art. These include techniques for chemically synthesizing polynucleotides well known in the art such as solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding the antisense RNA molecule. Such DNA sequences may be incorporated into a wide variety of vectors that incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Alternatively, antisense cDNA constructs that synthesize antisense RNA constitutiveiy or inducibly, depending on the promoter used, can be introduced stably into host cells.

Various modifications to the nucleic acid molecules may be introduced as a means of increasing intracellular stability and half-life. Possible modifications include, but are not limited to, the addition of flanking sequences of ribonucleotides or deoxyribonucleotides to the 5' or 3' ends of the molecule or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the oligodeoxyribonucleotide backbone. Additional linkages include but are not limited to phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate, phosphoroamidate, and the like. See, e.g., LaPlanche et al, 1986, Nucl Acids Res. 14: 9081; Stec et al, 1984, J. Am. Chem. Soc. 106: 6077; Stein et al, 1988, Nucl. Acids Res. 16: 3209; Zon et al, 1991, Anti-Cancer Drug Design 6: 539; Zon et al, 1991, OLIGONUCLEOTIDES AND ANALOGUES: A PRACTICAL APPROACH, (F. Eckstein, ed.), Oxford University Press, Oxford

England, pp. 87-108; Stec et al, U.S. Pat. No. 5,151,510; Uhlmann and Peyman, 1990, Chemical Reviews 90: 543, the disclosures of each of which are hereby incorporated by reference for any purpose.

In one embodiment of the invention, the inhibitors of HIV cell entry are not toxic to a human host cell that is not infected with HTV. In another embodiment, the inhibitors of HIV cell entry prevent fusion of virus to a human host cell.

In one embodiment of the invention, a pharmaceutical composition is prepared from a therapeutically-effective amount of an inhibitor of the invention and a pharmaceutically-acceptable canier. Pharmaceutically-acceptable carriers are well known to those with skill in the art. In another embodiment, resistance to HIV infection is conferred upon an individual by administering a pharmaceutical composition of the invention. The pharmaceutical compositions of the present invention can be manufactured in a manner that is itself known, e.g. , by means of a conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the present invention thus can be formulated in conventional manner using one or more physiologically acceptable caniers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically.

Proper formulation is dependent upon the route of administration chosen.

For injection, the compounds of the invention can be formulated in appropriate aqueous solutions, such as physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer. For transmucosal and transcutaneous administration, penetrants appropriate to the banier to be permeated are used in the formulation. Such penetrants are generally known in the art.

For oral administration, the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such caniers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated. Pharmaceutical preparations for oral use can be obtained with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents can be added, such as the cross-linked polyvinyl pynolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions can be used, which can optionally contain gum arabic, talc, polyvinyl pynolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.

Dyestuffs or pigments can be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds can be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers can be added. All formulations for oral administration should be in dosages suitable for such administration. For buccal administration, the compositions can take the form of tablets or lozenges fonnulated in conventional manner.

For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotefrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g., gelatin for use in an inhaler or insufflator can be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

The compounds can be fonnulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection can be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions can take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical foπnulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds can be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions can contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension can also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. Alternatively, the active ingredient can be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

The compounds can also be fonnulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.

In addition to the foiimilations described previously, the compounds can also be formulated as a depot preparation. Such long acting formulations can be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds can be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

A pharaiaceutical canier for the hydrophobic compounds of the invention is a cosolvent system comprising benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase. The cosolvent system can be the VPD co- solvent system. VPD is a solution of 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant polysorbate 80, and 65% w/v polyethylene glycol 300, made up to volume in absolute ethanol. The VPD co-solvent system (VPD:5W) consists of VPD diluted 1:1 with a 5% dextrose in water solution. This co-solvent system dissolves hydrophobic compounds well, and itself produces low toxicity upon systemic administration. Naturally, the proportions of a co-solvent system can be varied considerably without destroying its solubility and toxicity characteristics.

Furthermore, the identity of the co-solvent components can be varied: for example, other low-toxicity nonpolar surfactants can be used instead of polysorbate 80; the fraction size of polyethylene glycol can be varied; other biocompatible polymers can replace polyetliylene glycol, e.g. polyvinyl pyrrolidone; and other sugars or polysaccharides can substitute for dextrose.

Alternatively, other delivery systems for hydrophobic pharmaceutical compounds can be employed. Liposomes and emulsions are well known examples of delivery vehicles or caniers for hydrophobic drugs. Certain organic solvents such as dimethylsulfoxide also can be employed, although usually at the cost of greater toxicity. Additionally, the compounds can be delivered using a sustained-release system, such as semipenneable matrices of solid hydrophobic polymers containing the therapeutic agent. Various sustained-release materials have been established and are well known by those skilled in the art. Sustained-release capsules can, depending on their chemical nature, release the compounds for a few weeks up to over 100 days.

Depending on the chemical nature and the biological stability of the therapeutic reagent, additional strategies for protein and nucleic acid stabilization can be employed.

The pharmaceutical compositions also can comprise suitable solid or gel phase caniers or excipients. Examples of such caniers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.

The compounds of the invention can be provided as salts with pharmaceutically compatible counterfoils. Phannaceutically compatible salts can be formed with many acids, including but not limited to hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents that are the conesponding free base forms.

Pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve its intended purpose. More specifically, a therapeutically effective amount means an amount effective to prevent development of or to alleviate the existing symptoms of the subject being treated. Detemύnation of the effective amounts is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. For example, a dose can be formulated in animal models to achieve a circulating concentration range that includes the EC50 (effective dose for 50% increase) as determined in cell culture, i.e., the concentration of the test compound which achieves a half-maximal inhibition of HIV replication as assayed by the infected cells to retain CD4 expression, to reduce viral p24 or gpl20, and to prevent syncytia fonnation. Such information can be used to more accurately determine useful doses in humans.

Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio between LD50 and ED50. Compounds which exhibit high therapeutic indices are prefened. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage can vary within this range depending upon the dosage fom employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See, e.g. Fingl et al, 1975, in "The Pharmacological Basis of Therapeutics", Ch.l, p.l).

Dosage amount and interval can be adjusted individually to provide plasma levels of the active moiety which are sufficient to maintain the inhibitory effects. Usual patient dosages for systemic administration range from 100 - 2000 mg/day.

Stated in tenns of patient body surface areas, usual dosages range from 50 - 910 mg/m²/day. Usual average plasma levels should be maintained within 0.1-1000 μM. In cases of local administration or selective uptake, the effective local concentration of the compound can not be related to plasma concentration. The amount of composition administered will, of course, be dependent on the subject being treated, on the subject's body surface area, the severity of the affliction, the manner of administration and the judgment of the prescribing physician.

Suitable routes of administration can, for example, include oral, rectal, transmucosal, transcutaneous, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections. Alternatively, one can administer the compound in a local rather than systemic manner, for example, via injection of the compound directly into a specific tissue, often in a depot or sustained release formulation. Furthermore, one can administer the compound in a targeted drug delivery system, for example, in a liposome and or conjugated with a cell-specific antibody. The liposomes and cell- specific antibody will be targeted to and taken up selectively by HIV-infected cells.

The prefened embodiment of the invention and its advantages over previously investigated detection methods are best understood by refening to Examples 1-4. The Examples, which follow, are illustrative of specific embodiments of the invention, and various uses thereof. They are set forth for explanatory purposes only, and are not to be taken as limiting the invention.

All publications cited herein are incorporated by reference in their entirety.

EXAMPLE 1

Preparation of Random Fragment Libraries for Isolating and Identifying Human Cell-Derived GSEs Exhibiting HIV Suppressive Activity

Three random fragment expression (RFE) libraries were constructed from mRNA isolated from HL-60 and HeLa cells, and from phytohemaglutinin (PHA) stimulated peripheral blood mononuclear cells (PBMCs).

A. HL60 RFE Library

The HL60 RFE library was prepared by isolating mRNA from uninduced HL60 cells (ATCC Ace. No. CCL 240) and then subtracting that mRNA with mRNA isolated from cells induced with TNF-α. This procedure represents a modification of that described by Coche et al. (1994, Nucleic Acids Res. 22:1322-23). Tracer mRNA was isolated from HL-60 cells transduced with the retroviral vector pLNCX (Miller and Rosman, 1989, BioTechniques 7:980-90) at different time points after induction with TNF-α (Boehringer Mannheim; Indianapolis, IN). The pLNCX sequences were used as an internal standard to monitor the enrichment of the sequences present in the tracer after subtraction.

RNA isolated from induced and uninduced cells was annealed separately to oligo-dT magnetic beads (Dynal Biotech; Lake Success, NY) and first strand cDNA was synthesized using reverse transcriptase and an oligo-dT primer. The RNA strand was then hydrolyzed and second strand cDNA synthesized from the induced cell first strand cDNA using a primer containing ATG codons in all three reading frames and an additional ten random nucleotides on the 3' end. Single-stranded cDNA fragments were annealed to an excess of driver cDNA attached to the magnetic beads. This procedure was repeated several times until substantial emichment in the pLNCX sequences were seen. The final population of single-stranded DNA (ssDNA) molecules was amplified using a primer containing TGA codons in all three reading frames and an additional ten random nucleotides on the 3' end. The resulting population of cDNA fragments was then cloned into pLNCX. This step was taken to enrich for cellular sequences encoding products that might be important in supporting certain stages of the HIV life cycle in order to compensate for the low efficiency of retroviral transfer into OM10.1 cells. The HL60 library was found to comprise approximately 1 million transformants. B. HeLa RFE Library

The HeLa RFE library was prepared using the method described by Gudkov et al. (1994, Proc. Natl. Acad. Sci. U.S.A. 91:3744). First, cDNA was prepared from

HeLa cells and then partially digested with DNAse I in the presence of Mn^ (Sambrook et al, 1989, Molecular Cloning A Laboratory Manual, Cold Spring

Harbor Laboratory, New York). Under these conditions, DNAse I is known to produce mostly double-stranded breaks. The resulting fragments were repaired using both the Klenow fragment of DNA polymerase I and T4 polymerase and then the fragments were ligated to synthetic double-stranded adaptors. The 5' adaptor was prepared from the primers 5'-C-T-C-G-G-A-A-T-T-C-A-A-G-C-T-T-A-T-G-G-A-T-

G-G-A-T-G-G-3' (SEQ ID NO: 1) and 5'-C-A-T-C-C-A-T-C-C-A-T-A-A-G-C-T-T- G-A-A-T-T-C-C-3' (SEQ ID NO: 2). The 3' adaptor was prepared from the primers 5 '-T-G-A-G-T-G-A-G-T-G-A-A-T-C-G-A-T-G-G-A-T-C-C-G-T-C-T-3 ' (SEQ ID NO: 3) and 5'-T-C-C-T-A-G-A-C-G-G-A-T-C-C-A-T-C-G-A-T-T-C-A-C-T-C-A-C- T-C-A-3' (SEQ ID NO: 4).

This randomly fragmented cDNA was then subjected to a normalization procedure to provide for the uniforai abundance of different sequences in the population (Gudkov and Roninson, 1997, Methods in Molecular Biology 69:221, Humana Press, New York). This procedure was used to increase the probability of isolating GSEs from rare cDNAs, since total polyA⁺ RNA comprises a mixture of unequally represented sequences.

The randomly fragmented cDNA population was normalized by first denaturing 20 μg of cDNA by boiling for 5 minutes in 25 μl of TE buffer, followed by immediate cooling on ice. Then, 25 μl of 2X hybridization solution was added, and the mixture was divided equally into four aliquots in Eppendorf tubes. One to two drops of mineral oil were added to each sample to avoid evaporation, and the tubes were placed into a 68°C water bath for annealing. One tube was frozen every 12 hours. Following the last time-point, each of the annealing mixtures was diluted with water to a final volume of 500 μl and subjected to hydroxylapatite (HAP) chromatography. HAP suspension equilibrated with 0.01 M phosphate-buffered saline (PBS) was placed into Eppendorf tubes so that the volume of HAP pellet was approximately 100 μl. The tubes with HAP and all the solutions used below were preheated and kept at 65°C. Excess PBS was removed, and diluted annealing solution was added. After mixing by shaking in a 65°C water bath, the tubes were left in the water bath until a HAP pellet was formed (a 15 second centrifugation was used to collect the pellet without exceeding lOOOg in the microcentrifuge to avoid damage of

HAP crystals). The supernatant was carefully replaced with 1 ml of preheated 0.01 M

PBS, and the process was repeated. To elute the ssDNA, the HAP pellet was suspended in 500 μl of PBS at the single-strand elution concentration determined

(e.g., 0.16 M), the supernatant was collected, and the process was repeated. The supernatants were combined and traces of HAP were removed by centrifugation. The ssDNA was concentrated by centrifugation, and washed three times using 1 ml of water on a Centricon-100 column. The isolated ssDNA sequences were amplified by PCR using sense primers from each adapter and a minimal number of cycles to obtain 10 μg of the product. The size of the PCR product that remained within the desired range (200-500 bp) was ascertained. The nonnalization quality was tested by Southern or slot-blot hybridization with ³²P-labeled probes for high, moderate- and low-expressing genes using 0.3-1.0 μg of normalized cDN A/lane, β-actin and β-tubulin cDNAs were used as probes for high-expressing genes, c-myc and topo II cDNAs were used as probes for moderate-expressing genes, and c-fos cDNA was used as a probe for low- expressing genes. The cDNAs isolated after different annealing times were compared with the original unnormalized cDNA. The probes were ensured to have a similar size and specific activity. The best-nomialized ssDNA fraction (i.e., the population which produced the most uniform signal intensity with different probes) was used for large-scale PCR amplification to synthesize at least 20 μg of the product for cloning. More ssDNA template was used to obtain the desired amount by scaling up the number of PCR cycles or the reaction volume. Following nonnalization, the mixture of randomly fragmented cDNA was digested with BamΗl and EcoRI, column purified, and then ligated into either pLNCX or pLNGFRM (pLNGFRM differs from pLNCX in that the neo gene has been replaced with a truncated low affinity NGFR gene). Cells transduced with pLNGFRM express a truncated receptor on tlieir surface that can be easily selected by an anti-NGFR antibody during FACS. The ligation mixture was introduced into E

.coli, and approximately 100,000 transformants were obtained. The size distribution of the cloned fragments was analyzed by PCR using primers derived from vector sequences adjacent to the adapter sequences. C. PBMC RFE Library

The PBMC RFE library was prepared by isolating buffy coats from four healthy donors, from which PBMCs were purified by Ficoll gradient centrifugation followed by stimulation with PHA (1 μg/ml). Cells were removed at 5, 10, and 24 hours following the addition of PHA and total RNA was isolated by Trizol extraction.

The isolated total RNA collected from the four donors was then pooled, yielding three populations conesponding to the time at which the cells were removed following PHA treatment. Poly-A⁺ mRNA was purified from the total RNA using the Gibco Superscript Choice system for cDNA synthesis (Gibco BRL; Bethesda, MD) and a random primer. The cDNA was normalized using the PCR-Select cDNA Subtraction kit (Clontech; Palo Alto, CA), based on the suppression subtractive hybridization methods of Diatchenko et al. (1996, Proc. Natl Acad. Sci. U.S.A. 93:6025-30), and the primers 5'-T-A-G-G-G-C-T-C-G-A-G-C-C-G-C-C-A-C-C-A-T-G-3' (SEQ ID NO: 5) and 5'-A-T-C-C-C-T-G-C-A-G-G-T-C-A-C-T-C-A-C-T-C-A-3' (SEQ ID NO: 6). The normalized random fragments were digested with Xhol and Ssel, purified on quick spin columns (Qiagen; Valencia, CA) and ligated into the Ssel and Xhol sites of a bicistronic retroviral vector, pLXEMCVNgfr. This vector is based on pLXSNgfr. Modifications mcluded the replacement of the SV40 promoter with encephalomyocarditis virus (EMVC) internal ribosomal entry site (IRES) isolated from the plasmid pCITE (Amersham Biosciences; Piscataway, NJ). The ligation mixture was introduced into competent cells, and approximately 50 million transformants were obtained.

EXAMPLE 2 Transduction and Selection of Human Cell-Derived GSEs

HL-60 RFE libraries prepared as described in Example 1 were introduced into a packaging cell line, PA317 (ATCC Ace. No. CRL 9078), and converted into retrovirus for infection of OM10.1 cells (ATCC Ace. No. CRL 10850; U.S. Patent No. 5,256,534). OM10.1 cells transduced with a pLNCX-based HL-60 RFE library were co-cultured and selected with G418. OM10.1 cells transduced with a pLNGFRM-based or pLXEMCVNgfr-based HL-60 RFE library were first subjected to spinoculation (centrifugation of target cells at \200xg for 90 minutes in the presence of filtered retroviral supernatant) and then selected by FACS sorting of the NGFR⁺ population. Following selection, OM10.1 cells harboring the entire RFE library were mduced with 10 U/ml of TNF-α at 37°C for 24 hours, stained with antibody, and then sorted for CD4 expression. Genomic DNA from the CD4⁺ cells was purified and used for PCR amplification of inserts using vector-derived primers.

The amplified mixture was digested with EcoRI and BamΗI and cloned back into the retroviral vector. This selection was repeated for additional rounds.

Normalized RFΕ libraries prepared from HeLa cells or PBMCs as described in

Example 1 were transferred mto CEM-ss cells (Cat. No. 776; NIH AIDS Research and Reference Reagent Program) and neo resistant and NGFR+ populations were isolated. The HeLa and PMBC RFE libraries each comprised 50 x 10⁶ independent recombinant clones. Following introduction of the RFE libraries into CEM-ss cells, the CEM-ss cells were infected with a TCID₅₀ of 3000/10⁶ cells of HIV-l_ιπB (Cat. No. 398; NIH AIDS Research and Reference Reagent Program). Because it has been suggested that syncytia formation can be prevented by blocking the interaction between gρl20 expressed on the surface of an infected cells and CD4 on the surface of an uninfected cells, 3 μg/ml of a purified anti-CD4 monoclonal antibody, L77

(Becton Dickinson), was added at 4 and 7 days following infection. The L77 antibody does not prevent HIV infection of a cell. At 8-10 days after infection, a subpopulation of CD4⁺/p24^" cells conesponding to the uninfected cells was sorted. Genomic DNA from the isolated CD4⁺/p24^" cells was purified and used for PCR amplification of inserts with the vector-derived primers. The amplified mixture was digested with EcoRI and BamΗI and then cloned back into the retroviral vector. This selection was repeated for additional rounds.

EXAMPLE 3 Recovery and Sequencing of Human Cell-Derived GSEs

Genomic DNA was isolated from the selected OM10.1 or CEM-ss cells prepared as described in Example 2 by first centrifuging the selected cells, resuspending the cell pellet in 0.1% Triton X-100, 20 μg/ml proteinase K, and IX PCR buffer, incubating at 55°C for 1 hour, and then boiling for 10 minutes. Genomic DNA was used for PCR amplification using vector-derived primers, cloned into the retroviral vector, and introduced into E. coli using standard transformation techniques. Individual plasmids were purified from E. coli clones using QIAGEN plasmid purification kits. Inserts were sequenced by the dideoxy procedure (using the AutoRead Sequencing Kit, Phamiacia Biotech or the Prism Big Dye Terminator Cycle Sequencing Ready Reaction Kit, ABI) and analyzed on a Pharmacia LKB A.L.F. or ABI 3700 DNA sequencer. Sequences were analyzed using the DNASTAR program or other proprietary data mining procedure.

As described in Example 2, two independent selection strategies were performed on two different cell lines (OM10.1 and CEM-ss) into which three independent RFE libraries were introduced. The GSEs identified using these selection strategies, and the human cellular genes from which these GSEs were derived, are indicated in Table 2.

Table 2 GSEs and Human Cellular Genes Involved in Inhibition of HIV Infection

EXAMPLE 4

Cell Population Sorting Based on p24 Expression Using Immunofluorescence and Flow Cvtometrv

Since intracellular p24 accumulation and surface CD4 down-modulation are associated with HIV-1 replication, successful interference with HIV-1 infection should result in an enrichment in cells displaying a p247CD4⁺ phenotype. Cells possessing this phenotype (i.e., uninfected cells) were identified by immunofluorescence 8-10 days following challenge with HIV. First, CD4+ cells were isolated from the challenged cell population (1 x 10⁷ cells) by washing the cells twice with Assay Buffer (500 ml PBS, 1 ml of 0.5 mM of EDTA, pH 8, 0.5 ml of 10% sodium azide, and 10 ml of fetal bovine serum), and then resuspending the cells in 500 μl PBS containing 50 μl of anti-CD4 antibody (Q4120 PE; Sigma). Following incubation at 4°C for 30 minutes, 5 ml of Assay Buffer was added and the cells were centrifuged at 1200 rpm for 4 minutes. The cells were then washed twice with Assay Buffer and CD4⁺ cells sorted from the population by FACS. The aforementioned procedure was performed under sterile conditions. To identify cells within the CD4⁺ population that do not express p24, the sorted cell population (1 x 10⁶ cells) was washed twice with Assay Buffer, and then suspended in 100 μl of Assay Buffer and 2 ml of Ortho PermeaFix Solution (Ortho Diagnostics). The cells were incubated at room temperature for 40 minutes, centrifuged at 1200 rpm and 4°C for 4 minutes, and then resuspended in 2 ml Wash Buffer (500 ml PBS, 25 ml fetal bovine serum, 1.5% bovine serum albumin and

0.0055% EDTA). Following centrifugation at room temperature for 10 minutes, the cells were resuspended in 50 μl Wash Buffer diluted 1:500 with IgG_2a antibody, and incubated at 4°C for 20 minutes. Following this incubation, 5-10 μl of anti-p24 antibody (KC57-FITC; Coulter) was added and the cells were incubated at 4°C for 30 minutes. The cells were then washed twice with Wash Buffer and analyzed by flow cytometry.

EXAMPLE 5 siRNA Inhibition of Cell Surface Targets

To verify that inhibition of GSE identified genes is detrimental to HIV infection, small interfering RNAs (siRNA; S. M. Elbashir, et al., 2001, Nature

411:494) targeting specified cell surface targets were introduced into infectable cells, infected 24 hours later with HIV-1 strain NL4-3 and shown to have an inhibitory effect on infection at 48 hours post infection. Table 3 summarizes the percent inhibition of 21 -nucleotide RNA duplexes derived from the cDNA of the genes identified using the HIV infection assay generally described above in Example 2 in

CEM-ss cells. In these experiments, the conesponding siRNA were transfected in parallel with a control RNA duplex conesponding to Lamin A/C and percent inhibition was determined 2 days after HIV-1 infection, relative to these control cells. Table 3. siRNA Inhibition of HIV Infection

It should be understood that the foregoing disclosure emphasizes certain specific embodiments of the invention and that all modifications or alternatives equivalent thereto are within the spirit and scope of the invention as set fortli in the appended claims.

Claims

WHAT WE CLAIM IS:

1. A method of identifying an inhibitor of HIV entry into a human host cell, comprising identifying an inhibitor of a cell surface polypeptide selected from the group consisting of CXCR-4 (SEQ ID NO:8), CCR4 (SEQ ID NO.10), CCR7 (SEQ ID NO:12), CD1 IC (SEQ ID NO:14), CD47 (SEQ ID NO: 16), CD68 (SEQ ID

NO:18), CD69 (SEQ ID NO:20), CD74 (SEQ ID TO:22), CSF3R (SEQ ID NO:24), RARA (SEQ ID NO:26), GABBRl (SEQ ID NO:28), P2X1 (SEQ ID NO:30), HELOl (SEQ ID NO:32), GPRK6 (SEQ ID NO:34) and PTK2B (SEQ ID NO:36).

2. The method of Claim 1 , wherein the step of identifying an inhibitor of HIV entry into a human host cell comprises the steps of:

(a) contacting a human host cell with a putative inhibitor, wherein the human host cell expresses a polypeptide selected from the group consisting of CXCR-4, CCR4, CCR7, CD11C, CD47, CD68, CD69, CD74, CSF3R, RARA, GABBRl, P2X1, HELOl, GPRK6 and PTK2B; and (b) assessing inhibition of the activity of the cell surface polypeptide in an entry assay.

3. The method of Claim 2, wherem the entry assay is selected from the group consisting of a virus binding assay and a fusion assay.

4. The method of claim 3, wherein the fusion assay determines the ability of a virus to enter a cell.

5. The method of Claim 3, wherein the binding assay determines the ability of a virus to attach to the surface of a cell.

6. The method of Claim 2, wherein the human host cell is selected from the group consisting of primary T cells, C1866 cells, H9 cells, HUT78 cells, CEM-ss cells and HeLa CD4+ cells.

7. The method of Claim 1, wherein the step of identifying an inhibitor of HIV entry into a human host cell comprises the steps of:

(a) contacting a human host cell that is susceptible to HIV- infection with a putative inhibitor, wherein the human host cell expresses a polypeptide selected from the group consisting of CXCR-4, CCR4, CCR7,

CD11C, CD47, CD68, CD69, CD74, CSF3R, RARA, GABBRl, P2X1, HELOl, GPRK6 and PTK2B;

(b) exposing the cell to HIV; and (c) assessing inhibition of HIV-infection in the presence of the putative inhibitor as compared to in the absence of the inhibitor.

8. The method of Claim 1 , wherem the method identifies an inhibitor of CXCR-4 comprising the steps of: (a) contacting a cell that expresses CXCR-4 polypeptide with a putative inhibitor under conditions suitable for binding of the inhibitor with the polypeptide; and

(b) determining the ability of the putative inhibitor to inhibit HIV cell entry by exposing HIV to the cell and measuring tlie amount of virus fused to the cell.

9. The method of Claim 1, wherein the method identifies an inhibitor of CCR4 comprising the steps of:

(a) contacting a cell that expresses CCR4 polypeptide with a putative inhibitor under conditions suitable for binding of the inhibitor with the polypeptide; and

(b) determining the ability of the inhibitor to inhibit HIV cell entry by exposing HIV to the cell and measuring the amount of virus fused to the cell.

10. The method of Claim 1 , wherein the method identifies an inhibitor of CCR7 comprising the steps of: (a) contacting a cell that expresses CCR7 polypeptide with a putative inhibitor under conditions suitable for binding of the inhibitor with the polypeptide; and

(b) detennining the ability of the inhibitor to inhibit HIV cell entry by exposing HIV to the cell and measuring tlie amount of virus bound to the cell.

11. The method of Claim 1 , wherem the method identifies an inhibitor of

CD11C comprising the steps of:

(a) contacting a cell that expresses CD 11 C polypeptide with a putative inhibitor under conditions suitable for binding of the inhibitor with the polypeptide; and (b) determining tlie ability of the inhibitor to inhibit HIV cell entry by exposing HIV to the cell and measuring tlie amount of virus fused or bound to the cell.

12. The method of Claim 1 , wherem the method identifies an inhibitor of

CD47 comprising the steps of: (a) contacting a cell that expresses a CD47 polypeptide with a putative inhibitor under conditions suitable for binding of the inhibitor with the polypeptide; and

(b) determining the ability of the inhibitor to inhibit HIV cell entry by exposing HIV to the cell and measuring the amount of virus fused or bound to the cell.

13. The method of Claim 1, wherein the method identifies an inhibitor of CD68 comprising the steps of:

(a) contacting a cell that expresses CD68 polypeptide with a putative inhibitor under conditions suitable for binding of the inhibitor with the polypeptide; and

(b) determining the ability of the inhibitor to inhibit HIV cell entry by exposing HIV to the cell and measuring the amount of virus fused or bound to the cell.

14. The method of Claim 1 , wherein the method identifies an inhibitor of

CD69 comprising the steps of:

(a) contacting a cell that expresses CD69 polypeptide with a putative inhibitor under conditions suitable for binding of the inhibitor with the polypeptide; and (b) determining the ability of the inhibitor to inhibit HIV cell entry by exposing HIV to the cell and measuring the amount of virus fused or bound to the cell.

15. The method of Claim 1 , wherein the method identifies an inhibitor of CD74 comprising the steps of: (a) contacting a cell that expresses CD74 polypeptide with a putative inhibitor under conditions suitable for binding of the inhibitor with the polypeptide; and

(b) detennining the ability of the inhibitor to inhibit HIV cell entry by exposing HIV to the cell and measurmg the amount of virus fused or bound to the cell.

16. The method of Claim 1, wherein the method identifies an inhibitor of CSF3R comprising the steps of: (a) contacting a cell that expresses CSF3R polypeptide with a putative inhibitor under conditions suitable for binding of the inhibitor with the polypeptide; and

(b) detennining the ability of the inhibitor to inhibit HIV cell entry by exposing HIV to the cell and measuring the amount of virus fused or bound to the cell.

17. The method of Claim 1, wherein the method identifies an inhibitor of RARA comprising the steps of:

(a) contacting a cell that expresses RARA polypeptide with a putative inhibitor under conditions suitable for binding of the inhibitor with the polypeptide; and

(b) detennining the ability of the inhibitor to inhibit HIV cell entry by exposing HIV to tlie cell and measuring the amount of virus fused to the cell.

18. The method of Claim 1 , wherein the method identifies an inhibitor of GABBRl comprising the steps of:

(a) contacting a cell that expresses GABBRl polypeptide with a putative inhibitor under conditions suitable for binding of the inhibitor with the polypeptide; and

(b) detennining the ability of the inhibitor to inhibit HIV cell entry by exposing HIV to the cell and measuring the amount of virus fused or bound to the cell.

19. The method of Claim 1, wherein the method identifies an inhibitor of P2X1 comprising tlie steps of:

(a) contacting a cell that expresses P2X1 polypeptide with a putative inhibitor under conditions suitable for binding of the inhibitor with the polypeptide; and

(b) determining the ability of the inhibitor to inhibit HIV cell entry by exposing HIV to tlie cell and measuring the amount of virus fused or bound to the cell.

20. The method of Claim 1, wherein tlie method identifies an inhibitor of

HELOl comprising the steps of:

(a) contacting a cell that expresses HELOl polypeptide with a putative inhibitor under conditions suitable for binding of the inhibitor with the polypeptide; and (b) determining tlie ability of tlie inhibitor to inhibit HIV cell entry by exposing HIV to the cell and measuring the amount of virus fused to the cell.

21. The method of Claim 1, wherein the method identifies an inhibitor of

GPRK6 comprising the steps of: (a) contacting a cell that expresses GPRK6 polypeptide with a putative inhibitor under conditions suitable for binding of the inhibitor with the polypeptide; and

(b) detennining the ability of the inhibitor to inhibit HIV cell entry by exposing HIV to the cell and measuring the amount of virus fused to the cell.

22. The method of Claim 1, wherein the method identifies an inhibitor of

PTK2B comprising the steps of:

(a) contacting a cell that expresses PTK2B polypeptide with a putative inhibitor under conditions suitable for binding of the inhibitor with the polypeptide; and (b) detennining the ability of tlie inhibitor to inhibit HIV cell entry by exposing HIV to the cell and measuring the amount of virus fused to the cell.

23. The method of Claim 1, wherein the inhibitor is identified by:

(a) detennining the three-dimensional structure of the cell surface polypeptide; and (d) detennining the three-dimensional structure of an inhibitor by using computer software capable of modeling the interaction of the cell surface polypeptide and putative test compounds.

24. An inhibitor of a cell surface polypeptide in a human host cell preventing HIV entry into the human host cell, the cell surface polypeptide being selected from the group consisting of CXCR-4, CCR4, CCR7, CD 11 C, CD47, CD68,

CD69, CD74, CSF3R, RARA, GABBRl, P2X1, HELOl, GPRK6 and PTK2B.

25. The inhibitor of Claim 24, wherein the inhibitor is identified by a method comprising:

(a) contacting the human host cell with a putative inhibitor, wherein the human host cell expresses a polypeptide selected from the group consisting of CXCR-4, CCR4, CCR7, CDl lC, CD47, CD68, CD69, CD74, CSF3R, RARA, GABBRl, P2X1, HELOl, GPRK6 and PTK2B; and

(b) selecting the inhibitor by the ability to inhibit activity of the cell surface polypeptide in an entry assay.

26. The inhibitor of Claim 24, wherein the inhibitor is not toxic to a human host cell that is not infected with HIV.

27. A pharmaceutical composition comprising a therapeutically-effective amount of the inhibitor of Claim 24 and a pharmaceutically-acceptable carrier.

28. A method of confening resistance to HIV infection in an individual, comprising administering to the mdividual the phaniiaceutical composition of Claim 27.

29. The inhibitor of Claim 24, wherein the inhibitor is selected from the group consisting of a peptide, an antibody and a small molecule inhibitor.