Inhibition of HIV-1 replication by disrupting the processing of the viral Gag capsid (CA) protein (p24) from the CA-spacer peptide 1 (SP1) protein precursor (p25) is disclosed. Amino acid sequences containing a mutation in the Gag p25 protein, with the mutation resulting in a decrease in the inhibition of processing of p25 to p24 by dimethylsuccinyl betulinic acid or dimethylsuccinyl betulin, polynucleotides encoding such mutated sequences and antibodies that selectively bind such mutated sequences are also included. Methods of inhibiting, inhibitory compounds and methods of discovering inhibitory compounds that target proteolytic processing of the HIV Gag protein are included. In one embodiment, such compounds inhibit the interaction of the HIV protease enzyme with Gag by binding to Gag rather than to the protease enzyme. In another embodiment, viruses or recombinant proteins that contain mutations in the region of the Gag proteolytic cleavage site can be used in screening assays to identify compounds that target proteolytic processing.

Description of the Invention

BRIEF SUMMARY OF THE INVENTION

Generally, the invention provides methods of inhibiting, inhibitory compounds and methods of identifying inhibitory compounds that target proteolytic processing of the HIV-1 Gag protein. In one embodiment, such compounds may directly or indirectly inhibit the interaction of a protease enzyme with HIV-1 Gag protein. In another embodiment, such inhibition of interaction occurs via the binding of a compound to Gag. The inhibition of protease cleavage of the CA-SP1 protein of HIV-1 Gag by 3-O-(3',3'-dimethylsuccinyl)betulinic acid (DSB) is one example, but other proteolytic cleavage sites can be targeted by a similar approach using inhibitory compounds that interact with the substrate in a manner similar to that in which DSB interacts with Gag.

Another aspect of the invention is directed to a method of inhibiting the processing of the viral Gag p25 protein (CA-SP1) to p24 (CA), but having no effect on other Gag processing steps.

A further aspect of the invention is directed to a method for identifying compounds that inhibit processing of the viral Gag p25 protein (CA-SP1) to p24 (CA), but have no effect on other Gag processing steps.

In one aspect, the invention is drawn to a compound or pharmaceutical composition identified by the method for identifying compounds that inhibit HIV-1 replication disclosed herein.

In another aspect, the present invention is directed to a polynucleotide comprising a sequence which encodes an amino acid sequence containing a mutation in the Gag p25 protein, said mutation resulting in a decrease in the inhibition of processing of p25 to p24 by 3-O-(3',3'-dimethylsuccinyl)betulinic acid. This aspect of the invention is also directed to a vector, virus and host cell comprising said polynucleotide, and a method of making said protein.

A further aspect of the present invention is directed to an amino acid sequence containing a mutation in the Gag p25 protein, said mutation resulting in a decrease in the inhibition of processing of p25 to p24 by 3-O-(3',3'-dimethylsuccinyl)betulinic acid.

An additional aspect of the invention is directed to an antibody which selectively binds an amino acid sequence containing a mutation in the Gag p25 protein, said mutation resulting in a decrease in the inhibition of processing of p25 to p24 by 3-O-(3',3'-dimethylsuccinyl)betulinic acid. Also included in this aspect of the invention are a method of making said antibody, a hybridoma producing said antibody and a method of making said hybridoma.

In a further embodiment, the invention is directed to a kit comprising a polynucleotide, polypeptide or antibody disclosed herein.

The invention further relates to a method of inhibiting HIV-1 infection in cells of an animal by contacting said cells with a compound that blocks the maturation of virus particles released from treated infected cells. In one embodiment, the released virus particles exhibit non-condensed cores and a distinctive thin electron-dense layer near the viral membrane and have reduced infectivity. A method is included of contacting animal cells with a compound that both inhibits processing of the viral Gag p25 protein and that disrupts the maturation of virus particles. Also, included is a method of treating HIV-infected cells, wherein the HIV infecting said cells does not respond to other HIV therapies.

This invention further includes a method for identifying compounds that inhibit processing of the viral Gag p25 protein (CA-SP1) to p24 (CA), but have no significant effect on other Gag processing steps. The method involves contacting HIV-1 infected cells with a test compound, and thereafter analyzing virus particles that are released to detect the presence of p25. Methods to detect p25 include western blotting of viral proteins and detecting using an antibody to p25, gel electrophoresis, and imaging of metabolically labeled proteins. Methods to detect p25 also include immunoassays using an antibody to p25 or SP1 (p2) or to an epitope tag inserted into the SP1 sequence.

The invention is further directed to a method for identifying compounds involving contacting HIV-1 infected cells with a compound, and thereafter analyzing virus particles released by the contacted cells, by thin-sectioning and transmission electron microscopy, and determining whether viral particles with non-condensed cores and a distinctive thin electron-dense layer near the viral membrane are present.

The invention is also directed to compounds identified by the aforementioned screening methods. In additional embodiments, the invention is drawn to a method of treating HIV-1 infection in a patient by administering a compound that inhibits processing of the viral Gag p25 protein (CA-SP1) to p24 (CA), but does not significantly affect other Gag processing steps. In related embodiments, such inhibition may be accompanied by a different observable phenotypes. For example, inhibition may not necessarily significantly reduce the quantity of virions released from treated infected cells; and/or said inhibition may have little or no significant effect on the amount of RNA incorporation into the released virions; and/or said inhibition disrupts the maturation of virions released from infected cells treated with said compound. In related embodiments, the virion structure may be affected, and a majority of virions released from treated infected cells exhibit spherical, electron-dense cores that are acentric with respect to the viral particle; and/or possess crescent-shaped electron-dense layers lying just inside the viral membrane; and/or and have reduced or no infectivity.

In additional embodiments, the invention is drawn to a method of treating HIV-1 infection in a patient by administering a compound that inhibits the interaction of HIV protease with CA-SP1, which results in the inhibition of the processing of the viral Gag p25 protein (CA-SP1) to p24 (CA), but has no significant effect on other Gag processing steps. Such inhibition may be direct or, alternatively, indirect; and/or may involve said compound binding to the viral Gag protein such that interaction of HIV protease with CA-SP1 is inhibited. The invention is also drawn to a method of treating HIV in a patient with a compound that binds at or near the site of cleavage of the viral Gag p25 protein (CA-SP1) to p24 (CA), thereby inhibiting the interaction of HIV protease with the CA-SP1 cleavage site and resulting in the inhibition of processing of p25 to p24.

In other embodiments, the invention is drawn to a method of treating HJV-1-infection in a patient by administering a compound that inhibits processing of the viral Gag p25 protein (CA-SP1) to p24 (CA), wherein said compound binds to a polypeptide with an amino acid sequence having at least about 40%, 50%, 60%, 70%, 80%, 90% identity, or which is identical to a sequence selected from the group consisting of -- see Original Patent.

In other embodiments, the invention is drawn to a method of treating HIV-1-infection in a patient by administering a compound that inhibits processing of the viral Gag p25 protein (CA-SP1) to p24 (CA), wherein said compound binds to a polypeptide encoded by a polynucleotide sequence having at least about 40%, 50%, 60%, 70%, 80%, 90% identity, or which is identical to a polynucleotide selected from the group consisting of: (a) about nucleotides 1243-1435 of; (b) about nucleotides 1729-1920 of SEQ ID NO: 19; (c) about nucleotides 1344-1435 of SEQ ID NO: 18; (d) about nucleotides 1828-1920 of SEQ ID NO: 19; (e) about nucleotides 1370-1413 of SEQ ID NO: 18; and (f) about nucleotides 1857-1899 of SEQ ID NO: 19.

In another aspect, the invention is drawn to a method of inhibiting processing of the viral Gag p25 protein (CA-SP1) by administration of a compound. In related embodiments, such a compound binds to a polypeptide with an amino acid sequence having at least about 40%, 50%, 60%, 70%, 80%, 90% identity, or which is identical to a sequence selected from the group consisting of -- see Original Patent.

In related embodiments; the invention is drawns to a method of inhibiting processing of the viral Gag p25 protein (CA-SP1) by administration of a compound wherein said compound binds to a polypeptide encoded by a polynucleotide sequence having at least about 40%, 50%, 60%, 70%, 80%, 90% identity, or which is identical to a polynucleotide selected from the group consisting of:

(a) about nucleotides 1243-1435 of SEQ ID NO: 18; (b) about nucleotides 1729-1920 of SEQ ID NO: 19; (c) about nucleotides 1344-1435 of SEQ ID NO: 18; (d) about nucleotides 1828-1920 of SEQ ID NO: 19; (e) about nucleotides 1370-1413 of SEQ ID NO: 18; and (f) about nucleotides 1857-1899 of SEQ ID NO: 19.

The invention may be useful in the treatment of HIV in patients who are not adequately treated by other HIV-1 therapies. Accordingly, the invention is also drawn to a method of treating a patient in need of therapy, wherein the HIV-1 infecting said cells does not respond to other HIV-1 therapies. In another embodiment, methods of the invention are practiced on a subject infected with an HIV that is resistant to a drug used to treat HIV infection. In one application, the HIV is resistant to a protease inhibitor, a polymerase inhibitor, a nucleoside analog, a vaccine, a binding inhibitor, an immunomodulator, or any other inhibitor. In another embodiment, methods of the invention are practiced on a subject infected with an HIV that is resistant to a drug used to treat HIV infection is selected from the group consisting of zidovudine, lamivudine, didanosine, zalcitabine, stavudine, abacavir, nevirapine, delavirdine, emtricitabine, efavirenz, saquinavir, ritonavir, indinavir, nelfinavir, tenofovir, amprenavir, adefovir, atazanavir, fosamprenavir, hydroxyurea, AL-721, ampligen, butylated hydroxytoluene; polymannoacetate, castanospermine; contracan; creme pharmatex, CS-87, penciclovir, famciclovir, acyclovir, cytofovir, ganciclovir, dextran sulfate, D-penicillamine trisodium phosphonoformate, fusidic acid, HPA-23, eflonithine, nonoxynol, pentamidine isethionate, peptide T, phenytoin, isoniazid, ribavirin, rifabutin, ansamycin, trimetrexate, SK-818, suramin, UA001, and combinations thereof.

Compounds of the invention are also useful as part of combination of therapies. Accordingly, in one aspect the invention is drawn to a method of treating HIV in a patient, wherein said patient is administered said compound in combination with at least one anti-viral agent. Anti-viral agents suitable include, but are not limited to: zidovudine, lamivudine, didanosine, zalcitabine, stavudine, abacavir, nevirapine, delavirdine, emtricitabine, efavirenz, saquinavir, ritonavir, indinavir, nelfinavir, amprenavir, tenofovir, adefovir, atazanavir, fosamprenavir, hydroxyurea, AL-721, ampligen, butylated hydroxytoluene; polymannoacetate, castanospermine; contracan; creme pharmatex, CS-87, penciclovir, famciclovir, acyclovir, cytofovir, ganciclovir, dextran sulfate, D-penicillamine trisodium phosphonoformate, fusidic acid, HPA-23, eflornithine, nonoxynol, pentamidine isethionate, peptide T, phenytoin, isoniazid, ribavirin, rifabutin, ansamycin, trimetrexate, SK-818, suramin, UA001, enfuvirtide, gp41-derived peptides, antibodies to CD4, soluble CD4, CD4-containing molecules, CD4-IgG2, and combinations thereof. In another embodiment, the patient is administered said compound in combination with an immunomodulating agent, anticancer agent, antibacterial agent, antifungal agent, or a combination thereof.

The invention is also directed to compounds. Such compounds are useful in a method of treating patients infected with HIV; in a method for inhibiting processing of the viral Gag p25 protein (CA-SP1) to p24 (CA), or in a method for treating human blood and human blood products. Such compounds useful in the present invention include, but are not limited to derivatives of dimethylsuccinyl betulinic acid or dimethylsuccinyl betulin, or is selected from the group consisting of 3-O-(3',3'-dimethylsuccinyl)betulinic acid, 3-O-(3',3'-dimethylsuccinyl) betulin, 3-O-(3',3'-dimethylglutaryl)betulin, 3-O-(3',3'-dimethylsuccinyl)dihydrobetulinic acid, 3-O-(3',3'-dimethylglutaryl)betulinic acid, (3',3'-dimethylglutaryl)dihydrobetulinic acid, 3-O-diglycolyl-betulinic acid, 3-O-diglycolyl-dihydrobetulinic acid and combinations thereof.

Compounds of the invention may be used alone, or administered with additional compounds, including zidovudine, lamivudine, didanosine, zalcitabine, stavudine, abacavir, nevirapine, delavirdine, emtricitabine, efavirenz, saquinavir, ritonavir, indinavir, nelfinavir, amprenavir, tenofovir, adefovir, atazanavir, fosamprenavir, hydroxyurea, AL-721, ampligen, butylated hydroxytoluene; polymannoacetate, castanospermine; contracan; creme pharmatex, CS-87, penciclovir, famciclovir, acyclovir, cytofovir, ganciclovir, dextran sulfate, D-penicillamine trisodium phosphonoformate, fusidic acid, HPA-23, eflornithine, nonoxynol, pentamidine isethionate, peptide T, phenytoin, isoniazid, ribavirin, rifabutin, ansamycin, trimetrexate, SK-818, suramin, UA001, enfuvirtide, gp41-derived peptides, antibodies to CD4, soluble CD4, CD4-containing molecules, CD4-IgG2, and combinations thereof; an antiviral, an immunomodulating agent, anti-cancer agent, antibacterial agent, an anti-fungal agent, or combinations thereof.

In further embodiments, the invention is directed to a method of treating human blood products comprising contacting said blood products with a compound that inhibits processing of the viral Gag p25 protein (CA-SP1) to p24 (CA). In one aspect, said compound does not significantly affect other Gag processing steps. In related embodiments of this method, said inhibition does not significantly reduce the quantity of virions released from treated infected cell; and/or has little or no significant effect on the amount of RNA incorporation into the released virions; and/or inhibits the maturation of virions released from infected cells treated with said compound; and/or affects viral morphology. Such effects on viral morphology include, but are not limited to: the virions released from treated infected cells to exhibit spherical, electron-dense cores that are acentric with respect to the viral particle; and/or possess crescent-shaped electron-dense layers lying just inside the viral membrane; and/or and have reduced or no infectivity. In related embodiments, the method involves the administration of the compound which inhibits the interaction of HIV protease with CA-SP1, which results in the inhibition of the processing of the viral Gag p25 protein (CA-SP1) to p24 (CA) but has no significant effect on other Gag processing steps. This may be via direct, or indirect inhibition of the interaction of HIV protease with CA-SP1; and/or may involve said compound binds to the viral Gag protein such that interaction of HIV protease with CA-SP1 is inhibited; and/or said compound binds at or near the site of cleavage of the viral Gag p25 protein (CA-SP1) to p24 (CA), thereby inhibiting the interaction of HIV protease with the CA-SP1 cleavage site and resulting in the inhibition of processing of p25 to p24.

In a further embodiment, the invention is drawn to a method of treating human blood products comprising contacting said blood products with a compound that inhibits processing of the viral Gag p25 protein (CA-SP1) to p24 (CA), wherein said compound binds to a polypeptide with an amino acid sequence having at least about 40%, 50%, 60%, 70%, 80%, 90% identity, or which is identical to a sequence selected from the group consisting of -- see Original Patent.

In a related embodiment, the invention is drawn to a method of treating human blood products comprising contacting said blood products with a compound that inhibits processing of the viral Gag p25 protein (CA-SP1) to p24 (CA), wherein said compound binds to a polypeptide encoded by a polynucleotide sequence having at least about 40%, 50%, 60%, 70%, 80%, 90% identity, or which is identical a polynucleotide selected from the group consisting of:

(a) about nucleotides 1243-1435 of SEQ ID NO: 18; (b) about nucleotides 1729-1920 of SEQ ID NO: 19; (c) about nucleotides 1344-1435 of SEQ ID NO: 18; (d) about nucleotides 1828-1920 of SEQ ID NO: 19; (e) about nucleotides 1370-1413 of SEQ ID NO: 18; and (f) about nucleotides 1857-1899 of SEQ ID NO: 19.

The invention also embodies methods for identifying compounds that inhibit HIV-1 replication. Accordingly, the invention also includes a method of identifying compounds that inhibit HIV-1 replication in cells of an animal, comprising: contacting a Gag protein comprising a CA-SP1 cleavage site with a test compound; adding a labeled substance that selectively binds near the CA-SP1 cleavage site; and measuring competition between the binding of the test compound and the labeled substance to the CA-SP1 cleavage site. In further embodiments of this method, the compounds inhibits the interaction of HIV-1 protease with a target site by binding to said target site.

These methods also include embodiments wherein the CA-SP1 cleavage site region is contained within a polypeptide fragment or recombinant peptide; and/or wherein the labeled substance is a labeled antibody specific for CA-SP1, and measuring the change in the amount of labeled antibody bound to the protein in the presence of test compound compared with a control. Labels include, but are not limited to, an enzyme, fluorescent substance, chemiluminescent substance, horseradish peroxidase, alkaline phosphatase, biotin, avidin, electron dense substance, radioisotope and a combination thereof.

The method of identifying compounds that inhibit HIV-1 replication in cells of an animal also comprises, in one embodiment, measuring the change in the amount of labeled 3-O-(3',3'-dimethylsuccinyl)betulinic acid bound to the protein in the presence of test compound, compared with a control, and wherein the labeled substance is 3-O-(3',3'-dimethylsuccinyl)betulinic acid.

In an alternative embodiment, the invention comprises a method for identifying compounds that inhibit HIV-1 replication in the cells of an animal which comprises: contacting a polypeptide comprising a CA-SP1 cleavage site, with a protease in the presence of a test compound. Preferably the protease is related to HIV-1 protease, or is HIV protease. In one embodiment, the method comprises ; contacting a polypeptide comprising a wild type CA-SP1 cleavage site, with a protease in the presence of a test compound and also contacting a polypeptide comprising a mutant CA-SP1 cleavage site or a protein comprising an alternative protease cleavage site with HIV-1 protease in the presence of the test compound, detecting the cleavage, and comparing the amount of cleavage of the native wild-type polypeptide to the amount of cleavage of the mutant polypeptide or to amount of cleavage of the protein comprising an alternative protease cleavage site. In a related aspect of this method, the wild-type CA-SP1 or mutant CA-SP1 or alternative protease cleavage site region is contained within a polypeptide fragment or recombinant peptide. In a further related aspect, the polypeptide is labeled with a fluorescent moiety and a fluorescence quenching moiety, each bound to opposite sides of the CA-SP1 cleavage site, and wherein said detecting comprises measuring the signal from the fluorescent moiety. In another related embodiment, the polypeptide is labeled with two fluorescent moieties, each bound to opposite sides of the CA-SP1 cleavage site, and wherein said detecting comprises measuring the transfer of fluorescent energy from one moiety to the other in the presence of the test compound. In a further embodiment, the effect of the test compound on cleavage of the polypeptide is detected by measuring the amount of a labeled antibody that is bound to SP1 or p24 (CA). In a related aspect, the labeled antibody that binds CA, or the antibody that binds SP1 is labeled with a molecule selected from the group consisting of enzyme, fluorescent substance, chemiluminescent substance, horseradish peroxidase, alkaline phosphatase, biotin, avidin, electron dense substance, radioisotope, and combinations thereof.

The invention is also directed to a method for identifying compounds that inhibit HIV-1 replication in cells of an animal. In one embodiment, the method comprises: contacting a test compound with cells infected with wild-type virus isolates and with cells infected with virus isolates having significantly reduced sensitivity to 3-O-(3',3'-dimethylsuccinyl)betulinic acid; and selecting test compounds that are more active against the wild-type virus isolate compared with virus isolates that have reduced sensitivity to 3-O-(3',3'-dimethylsuccinyl)betulinic acid. In another embodiment, the method comprises contacting HIV-1 infected cells with a test compound; lysing the infected cells or the released viral particles to form a lysate, and analyzing the lysate to determine whether cleavage of the CA-SP1 protein has occurred. In this latter embodiment, said analyzing may comprise measuring the presence or absence of p25; and or performing a western blot of viral proteins and detecting p25 using an antibody to p25; and/or performing a gel electrophoresis of viral proteins and imaging of metabolically labeled proteins; and/or performing an immunoassay. Such an immunoassay may be performed by any methods known in the art, including, but not limited to:

(a) capturing p25 and p24 on a substrate using an antibody that selectively binds p24; and

(b) detecting the presence or absence of p25 on the substrate by using an antibody that selectively binds p25. The invention also includes such modifications of the above assay as would be obvious to one of ordinary skill in the art.

In a further embodiment, the method of identifying a compound according to the invention comprises the use of an epitope tag sequence inserted into SP1 and the selective detection of p25 is performed using an antibody to the epitope tag.

The invention is also directed to a method for identifying compounds that inhibit HIV-1 replication in the cells of an animal comprising: contacting HIV-1 infected cells with a test compound and thereafter analyzing the virus particles using transmission electron microscopy. Such analysis includes for example, looking for the presence of spherical cores that are acentric with respect to the viral particle; and/or having crescent-shaped, electron-dense layers lying just inside the viral membrane.

In additional aspects, the invention is drawn to an isolated polynucleotide comprising a sequence which encodes an amino acid sequence containing a mutation in an HIV Gag p25 protein (CA SP1), said mutation resulting in a decrease in inhibition of processing of p25 (CA-SP1) to p24 (CA) by 3-O-(3',3'-dimethylsuccinyl)betulinic acid (DSB). This inhibition of processing of p25 may be due to a decrease in inhibition of the interaction of HIV-1 protease with Gag; and/or a decrease in the binding of 3-O-(3',3'-dimethylsuccinyl)betulinic acid to Gag; and/or a decrease in the binding of DSB at or near the CA-SP1 cleavage site of Gag. Suitable polynucleotides also include those encoding a mutation at or near the CA-SP1 cleavage site or in the SP1 domain of CA-SP1; and/or those encoding a mutation at or near the amino acid sequence G/SHKARV/ILAEAMSQV (SEQ ID NO: 1); and/or those encoding the amino acid sequences GHKARVLVEAMSQV (SEQ ID NO: 2) or SHKARILAEAMSQV (SEQ ID NO: 3); and/or isolated polynucleotide which is selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 9; and/or having at least about 95% identity to a polynucleotide selected from the group consisting of SEQ ID NO: 4, and SEQ ID NO: 6; and/or having at least about 80% identity to a polynucleotide selected from the group consisting of SEQ ID NO: 8 and SEQ ID NO: 9; and/or having at least about 95% identity to a polynucleotide selected from the group consisting of SEQ NO: 5 and SEQ ID NO: 7; and/or having at least about 80% identity to a polynucleotide of SEQ ID NO: 10. In additional embodiments, the polynucleotide having more than about 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% identity or which is identical to the polynucleotide sequences listed above.

The invention is also drawn to vectors comprising such polynucleotides as described above; to a host cell comprising such a vector; and to a method of producing a polypeptide comprising incubating the host cell containing such a vector in a medium and recovering the polypeptide from said medium.

In one embodiment, the invention is directed to an antibody. Such an antibody may bind to a polypeptide with an amino acid sequence having at least about 40%, 50%, 60%, 70%, 80%, 90% identity, or which is identical to a sequence selected from the group consisting of -- see Original Patent.

In a further related embodiment, the invention is drawn to an antibody which binds to a polypeptide encoded by a polynucleotide sequence having at least about 40%, 50%, 60%, 70%, 80%, 90% identity, or which is identical to a polynucleotide with a sequence selected from the group consisting of: (a) about nucleotides 1243-1435 of SEQ ID NO: 18; (b) about nucleotides 1729-1920 of SEQ ID NO: 19; (c) about nucleotides 1344-1435 of SEQ ID NO: 18; (d) about nucleotides 1828-1920 of SEQ ID NO: 19; (e) about nucleotides 1370-1413 of SEQ ID NO: 18; and (f) about nucleotides 1857-1899 of SEQ ID NO: 19.

In one embodiment, the antibody binds to amino acids of the CA-SP1 region of the HIV-1 Gag polypeptide, wherein said amino acids comprise: SHKARILAEAMSQV (SEQ ID NO: 25) or GHKARVLAEAMSQV (SEQ ID NO: 26).

In one embodiment, the invention is drawn to an antibody that inhibits the binding of 3-O-(3',3'-dimethylsuccinyl)betulinic acid to the CA-SP1 region of the Gag polypeptide.

The invention is also drawn to mutant HIV-1 viruses. In one such embodiment, the invention is an isolated mutant recombinant HIV-1 virus, wherein the processing of the viral Gag p25 protein (CA-SP1) to p24 (CA) in said virus is not significantly inhibited by 3-O-(3',3'-dimethylsuccinyl)betulinic acid. In related embodiments, this virus is not inhibited by 3-O-(3',3'-dimethylsuccinyl)betulinic acid. In another embodiment, 3-O-(3',3'-dimethylsuccinyl)betulinic acid does not inhibit the interaction of protease with the Gag polypeptide in this virus. In another, the virus does not bind to 3-O-(3',3'-dimethylsuccinyl)betulinic acid. In further embodiments the invention is drawn to viruses wherein the amino acids of the CA-SP1 region are replaced with alternative amino acids, or amino acids are added to the CA-SP1 region, or where amino acids are deleted. In one embodiment, ; one or more amino acids are deleted from the AEAMSQV (amino acid no. 8-14 of SEQ ID NO:26) amino acid sequence in the CA-SP1 region.

A mutant viruses may be used in the methods of the invention described elsewhere herein. For example, such viruses are useful in a method of identifying a compound which inhibits processing of the viral Gag p25 protein (CA-SP1) to p24 (CA), the method comprising comparing the ability of said compound to inhibit HIV-1 replication compared with the replication of a the mutant virus outlined above. Such inhibition may be examined in a cell, or in an animal, or in vitro.

The invention is also drawn to non-HIV-1 retroviruses that are sensitive to 3-O-(3',3'-dimethylsuccinyl)betulinic acid. In some embodiment, said retrovirus encodes a CA-SP1 polypeptide with an amino acid sequence comprising the sequence AEAMSQV (amino acid no. 8-14 of SEQ ID NO: 26) at or near the CA-SP1 cleavage site. In another embodiment, the retrovirus encodes a CA-SP1 polypeptide with an amino acid sequence comprising the sequence VLAEAMSQV (amino acid no. 6-14 of SEQ ID NO: 26) at or near the CA-SP1 cleavage site. In another embodiment, the retrovirus encodes a CA-SP1 polypeptide with an amino acid sequence comprising the sequence GHKARVLAEAMSQV (SEQ ID NO: 26) at or near the CA-SP1 cleavage site; in another the retrovirus comprises the amino acid sequence having at least 60%, 70%, 80%, 90% identity or which is identical to the sequence enocoded by the polynucleotide of SEQ ID NO:26, SEQ ID NO: 90; SEQ ID NO: 92; SEQ ID NO: 94; SEQ ID NO: 96; or SEQ ID NO: 98; in another embodiment the retrovirus comprises the amino acid sequence having at least 60%, 70%, 80%, 90% identity or which is identical to the sequence of SEQ ID NO: 91; SEQ ID NO: 93; SEQ ID NO: 95; SEQ ID NO: 97; or SEQ ID NO: 99. In another embodiment, the retrovirus comprises the nucleic acid sequence having at least 70%, 80%, 90% or which is identical to the sequence of SEQ ID NO: 90; SEQ ID NO: 92; SEQ ID NO: 94; SEQ ID NO: 96; or SEQ ID NO: 98.

Retroviruses of this embodiment of the invention include, but are not limited to HIV-2, HTLV-I, HTLV-II, SIV, avian leukosis virus (ALV), endogenous avian retrovirus (EAV), mouse mammary tumor virus (MMTV), feline immunodeficiency virus (FIV), Bovine immunodeficiency virus (BIV), caprine arthritis encephalitis virus (CAEV), Visna-maedi virus, or feline leukemia virus (FeLV).

In a related embodiment, the invention is drawn to a method of making a recombinant non-HIV-1 lentivirus sensitive to DSB. This method comprises: deleting from the genome of said lentivirus the nucleotides which correspond to nucleotides 1370-1413 from SEQ ID NO: 18, in HIV-1; and inserting nucleotides 1370-1413 from SEQ ID NO: 18 or nucleotides 1857-1899 of SEQ ID NO: 19 into said region of said non-HIV-1 lentivirus.

Examples of chimeric lentiviruses that were, are or may be constructed by this method are described in FIG. 10 (see Original Patent).

Such viruses may be used in the methods of the invention described elsewhere herein. For example, such recombinant non-HIV-1 lentiviruses may be used in a method of identifying a compound which inhibit processing of the viral Gag p25 protein (CA-SP1) to p24 (CA), the method consisting of comparing of the ability of said compound to inhibit replication of a wild-type non-HIV-1 lentivirus with the DSB-sensitive recombinant variant thereof. Such inhibition may occur in a cell; in an animal; or in vitro.

The invention is also drawn to an animal model of lentivirus infection comprising a suitable non-human animal host infected with a lentivirus sensitive to 3-O-(3',3'-dimethylsuccinyl)betulinic acid. In such an embodiment, the lentivirus may include, but is not limited to SIV; FIV; EIAV; BIV; CAEV; and Visna-Maedi virus.

The invention is also drawn to isolated polypeptides. In one embodiment, the invention is drawn to a polypeptide containing a mutation in an HIV CA-SP1 protein, said mutation which results in a decrease in inhibition of processing of p25 by 3-O-(3',3'-dimethylsuccinyl)betulinic acid. In a related embodiment, this polypeptide is encoded by a polynucleotide that contains a mutation located at or near the CA-SP1 cleavage site or in the SP1 domain encoded by SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 10 and/or is encoded by a polynucleotide selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 9; and/or comprises a sequence that is selected from the group consisting of GHKARVLVEAMSQV (SEQ ID NO: 2) or SHKARILAEVMSQV (SEQ ID NO: 3); and/or is encoded by an isolated polynucleotide which hybridizes under stringent conditions to a polynucleotide selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 7, and 10; and/or is part of a chimeric or fusion protein.

The invention is also drawn to antibodies which selectively bind to an amino acid sequence containing a mutation in an HIV CA-SP1 protein which results in a decrease in the inhibition of processing of p25 (CA-SP1) to p24 (CA) by 3-O-(3'3'-dimethylsuccinyl)betulinic acid. In one such embodiment, the antibody selectively binds to a mutation located at or near the CA-SP1 cleavage site or in the SP1 domain of CA-SP1; in another, the antibody selectively binds to a mutation comprising a sequence that is selected from the group consisting of GHKARVLVEAMSQV (SEQ ID NO: 2) or SHKARILAEVMSQV (SEQ ID NO: 3); in another embodiment, the antibody selectively binds an amino acid sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 3.

In another embodiment, the invention is drawn to an antibody that selectively binds SP1 but not CA-SP1; another that selectively binds CA-SP1 but not CA; another that selectively binds CA but not CA-SP1; and a further antibody that selectively binds at or near the CA-SP1 cleavage site.

The invention is also directed to a compound identified by any of the methods elucidated herein. In one embodiment, the compounds is not a compound selected from the group consisting of 3-O-(3',3'-dimethylsuccinyl)betulinic acid, 3-O-(3',3'-dimethylsuccinyl)betulin, 3-O-(3',3'-dimethylglutaryl)betulin, 3-O-(3',3'-dimethylsuccinyl)dihydrobetulinic acid, 3-O-(3',3'-dimethylglutaryl)betulinic acid, (3',3'-dimethylglutaryl)dihydrobetulinic acid, 3-O-diglycolyl-betulinic acid, 3-O-diglycolyl-dihydrobetulinic acid, and combinations thereof.

The invention is also drawn to a pharmaceutical composition. In one embodiment, the pharmaceutical composition comprises derivatives of dimethylsuccinyl betulinic acid or dimethylsuccinyl betulin; in another, the pharmaceutical composition comprises a compound selected from the group consisting of 3-O-(3',3'-dimethylsuccinyl)betulinic acid, 3-O-(3',3'-dimethylsuccinyl)betulin, 3-O-(3',3'-dimethylglutaryl)betulin, 3-O-(3',3'-dimethylsuccinyl)dihydrobetulinic acid, 3-O-(3',3'-dimethylglutaryl)betulinic acid, (3',3'-dimethylglutaryl)dihydrobetulinic acid, 3-O-diglycolyl-betulinic acid, 3-O-diglycolyl-dihydrobetulinic acid, and combinations thereof. In another embodiment, the pharmaceutical composition comprises one or more compounds identified according to the methods of the invention which are not otherwise listed; or any pharmaceutically acceptable salt, ester or prodrug thereof, and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical composition further comprising an anti-viral agent which may include any one of zidovudine, lamivudine, didanosine, zalcitabine, stavudine, abacavir, nevirapine, delavirdine, emtricitabine, efavirenz, saquinavir, ritonavir, indinavir, nelfinavir, tenofovir, amprenavir, adefovir, atazanavir, fosamprenavir, hydroxyurea, AL-721, ampligen, butylated hydroxytoluene; polymannoacetate, castanospermine; contracan; creme pharmatex, CS-87, penciclovir, famciclovir, acyclovir, cytofovir, ganciclovir, dextran sulfate, D-penicillamine trisodium phosphonoformate, fusidic acid, HPA-23, eflornithine, nonoxynol, pentamidine isethionate, peptide T, phenytoin, isoniazid, ribavirin, rifabutin, ansamycin, trimetrexate, SK-818, suramin, UA001, combinations thereof, any other antiviral, immunomodulating agent, anti-cancer agent, anti-fungal agent, anti-bacterial agent, or combinations thereof.

The invention is also drawn to a method of determining if an individual is infected with HIV-1 that is susceptible to treatment by a compound that inhibits p25 processing. In one embodiment, the method involves taking blood from the patient, genotyping the viral RNA and determining whether the viral RNA contains mutations in the sequence encoding the region of the CA-SP1 cleavage site.

The invention is also drawn to a method of treating a disease in a patient in need thereof comprising:

identifying a compound which inhibits the processing of viral Gag p25 protein (CA-SP1) to p24 (CA), but has no significant effect on other Gag processing steps;

obtaining regulatory approval for the sale and use of said compound;

packaging the compound for sale and treatment of a disease in a patient in need thereof.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to methods of inhibiting HIV-1 replication in the cells of an animal. More specifically, the invention involves methods of inhibiting HIV-1 replication in the cells of a mammal by contacting infected cells with a compound that inhibits the processing of the viral Gag p25 protein (CA-SP1) to the p24 protein (CA). More specifically, such compounds inhibit the processing of the viral Gag p25 protein (CA-SP1) to the p24 protein (CA) without significantly affecting other Gag processing steps.

"A compound that does not significantly affect other Gag processing steps" means that the compound in question predominantly inhibits processing of p25 to p24, but does not necessarily preclude the possibility of having additional minor effects on other Gag processing steps.

"Significant" or "Significantly," where not otherwise defined herein, means an observable or measurable change compared to the process in the absence of a compound. However, not all observable or measureable changes may necessarily be significant.

A number of viral phenotypes may also be observed in practicing the method of the invention. One result of contacting an infected cell with the compounds of the invention may be the formation of noninfectious viral particles. Alternatively, or in addition, contacting infected cells with a compound that inhibits p25 to p24 processing, results in the formation of non-infectious viral particles, but where there is no significant effect on other Gag processing steps. This may not significantly reduce the quantity of virus released from treated cells and/or has no little or no significant effect on the amount of RNA incorporation into the released virions.

Accordingly, the invention is also drawn to a method of inhibiting HIV infection in cells of an animal comprising contacting said cells with a compound that inhibits p25 processing and also affects other viral phenotypes, discribed above.

Mutant viruses defective in CA-SP1 cleavage have been shown to be non-infectious (Wiegers K. et al., J. Virol. 72:2846-2854 (1998)). 3-O-(3',3'-dimethylsuccinyl)betulinic acid (DSB) is an example of a compound that disrupts p25 to p24 processing and potently inhibits HIV-1 replication. This compound's activity is specific for the p25 to p24 processing step, not other steps in Gag processing. Furthermore, DSB treatment results in the aberrant HIV particle morphology as described in FIG. 3 (see Original Patent).

Identification of HIV-1 Determinants Associated with Sensitivity to 3-O-(3',3'-dimethylsuccinyl)betulinic acid

Generation and Selection of HIV-1 Viruses Resistant to DSB.

Mutant forms of HIV-1 have been generated in which the amino acid sequence in the region of the CA-SP1 cleavage site is modified, decreasing the sensitivity of these strains to compounds that disrupt CA-SP1 processing. Data on these mutant viruses have been used to identify the amino acid residues in wild-type Gag that are implicated in the antiviral activity of these compounds. In one embodiment, compounds that disrupt CA-SP1 processing directly or indirectly inhibit the interaction of HIV-1 protease with the region of the Gag protein containing these amino acid residues. In another embodiment, compounds that disrupt CA-SP1 processing bind to the region containing these amino acid residues. As used herein, the terms "bind," "bound" or "binding" refers to binding or attachment including, e.g., ionic interactions, electrostatic hydrophobic interactions, hydrogen bonds, etc; and also includes associations that may be covalent, e.g., by chemically coupling. Covalent bonds can be, for example, ester, ether, phosphoester, thioester, thioether, urethane, amide, amine, peptide, imide, hydrazone, hydrazide, carbon-sulfur bonds, carbon-phosphorus bonds, and the like. The term "bound" is broader than and includes terms such as "coupled," "conjugated" and "attached."

In another embodiment, compounds that disrupt CA-SP1 processing bind to another region of Gag and thereby inhibit the interaction of HIV-1 protease with the region of the CA-SP1 cleavage site. In another embodiment, viruses or recombinant proteins that contain mutations in the region of the CA-SP1 cleavage site can be used in screening assays to identify compounds that disrupt CA-SP1 processing.

In one set of experiments, amino acid residues in HIV-1 Gag that are involved in the disruption of CA-SP1 processing by 3-O-(3',3'-dimethylsuccinyl)betulinic acid (DSB) were identified by sequencing the gag-pol gene of virus isolates that had been selected for resistance to DSB. The amino acid sequences from these resistant viruses were compared with the gag-pol gene sequences from DSB-sensitive HIV-1 isolates. Two single amino acid changes were identified in the DSB-resistant viruses, an alanine (Ala) to valine (Val) substitution at residue 364 (SEQ ID NO: 4) and in a second isolate, at residue 366 (SEQ ID NO: 6), in the Gag polyprotein (see FIG. 4 (see Original Patent)). These residues are located immediately downstream of the CA-SP1 cleavage site (at the N-terminus of SP1). Alanine is highly conserved at these positions throughout all HIV-1 subtypes listed in the Los Alamos National Laboratory database. The five amino acid residues upstream and downstream of the CA-SP1 cleavage site are also highly conserved among the various subtypes. However, isoleucine replaces valine at the position two residues upstream of the cleavage site in a number of clades (c.f., FIG. 4, SEQ ID NO. 1). ("HIV Sequence Compendium 2002, " Kuiken et al. eds. Los Alamos National Laboratory, Los Alamos, N. Mex.)

In order to more extensively map the viral genetic determinants for DSB resistance, additional experiments were performed to select for viruses in vitro that are drug resistant. Multiple parallel cultures of Jurkat T cells (5.times.10.sup.5 each) were transfected with the proviral DNA clone pNL4-3 in the presence or absence of 10-50 ng/ml DSB. The cells were passaged every two days, and fresh drug was added at each passage. Virus replication was monitored by measuring reverse transcriptase activity in culture supernatants. Virus was isolated from culture supernatants harvested at selected timepoints, and genomic DNA was amplified by RT-PCR using primers that spanned the coding region between the N-terminus of CA and the N-terminus of RT. The amplified product was then sequenced using the same set of primers.

In one experiment, an A366V mutation was identified in the SP1 region of NL4-3 virus cultured in the presence of DSB (note: numbering is relative to the Gag polyprotein). Upon further passaging, a double mutant was identified that contained a G357S mutation in CA as well as the A366V mutation in SP1. The A366V mutation was identified previously in experiments selecting for resistant variants of the RF isolate. Interestingly, the wild-type RF sequence also contains a serine residue at position 357 in CA (FIG. 4). Since serine is present at this position in isolates (such as RF) that are sensitive to DSB, the CA G357S mutation alone is not sufficient to confer resistance to DSB. To determine the contribution of each of these mutations to drug resistance, the A366V mutation and the A366V/G357S double mutation were re-engineered into the wild-type NL4-3 backbone by site-directed mutagenesis. The resulting constructs were transfected into Jurkat T cells and characterized in a virus replication assay as described above for the selection of resistance. SDS-PAGE analysis of transfected cell lysates and virus released into the media demonstrated that the A366V mutant Gag was processed and released from cells inefficiently (data not shown) and thus replicated very poorly even in the absence of drug (FIG. 11 (see Original Patent)) However, the A366V/G357S double mutant replicated efficiently in the absence or presence of DSB. There data indicate that the resistant mutant, A366V, requires a serine at the 357 position in the CA region of Gag to compensate for a deleterious effect on virus replication (FIG. 11).

In a further experiment, ten different resistant isolates were generated. Sequencing of these isolates identified four additional mutations not previously seen in resistance selection experiments. These were H358Y, L363F and L363M in CA, and A402T in the NC region of Gag. None of these mutations are present in the consensus sequences for HIV-1 clades A-O, reflecting the breadth of activity of DSB against genetically diverse clades of HIV-1. The L363M substitution in CA was found in the consensus sequence for HIV-2, which may, in part, explain the specificity of DSB for HIV-1.

These results demonstrate the presence of specific genetic determinants for DSB activity in HIV-1, and that these determinants are centered around the CA-SP1 cleavage site.

HIV-1 NL4-3 Deletion and SIV Insertion Studies Used to Identify Viral Genetic Determinants of DSB Sensitivity

Results from in vitro resistance selection experiments indicated that the determinants of DSB HIV-1 inhibitory activity map to the region of Gag flanking the CA-SP1 cleavage site. In order to better define the viral genetic determinant for DSB, HIV-1 point-deletion mutagenesis and SIV insertion studies were undertaken to identify the specific amino acid residues associated with compound activity. The study was carried out as follows. Single residue deletions starting with residue E365 and continuing through residue M377 were engineered into the SP1 domain of the infectious HIV-1 molecular clone NL4-3 (FIG. 12 (see Original Patent)). The effect of these point deletions on viral particle production, infectivity, Gag processing and sensitivity to DSB was determined. The results of these experiments were used to identify the Gag residues in the region of the CA-SP1 cleavage site that are associated with DSB activity. The residues associated with activity were inserted into the CA-SP1 cleavage site region of the DSB-resistant virus SIV (Mac 239 isolate) to generate a HIV-1, SIV chimeric virus (SHIV). Point substitution of HIV-1 residues from the N-terminus of the CA protein were made into this chimeric virus until the minimal sequence necessary to rescue DSB activity was identified. This minimal sequence necessary to gain DSB activity is considered a primary viral genetic determinant of DSB activity. It may suggest the molecular determinant of DSB activity.
 

Claim 1 of 40 Claims

1. An isolated polypeptide comprising 10 amino acids downstream from the CA-SP1 cleavage site of HIV-1 Gag, wherein said polypeptide contains a substitution of Ala to Val at a position corresponding to residue 364 of HIV-1 Gag (residue 1 of SP1) as compared to the sequence of either the wild type strain NL4-3 or the wild type strain RF.

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