# US 7,494,767 B2 · Assay for TSG101 as inhibitors of HIV production

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 7,494,767 B2
- Document ID: US7494767B2
- Status: Granted
- Kind: B2
- Application number: 10/478,753
- Filed: 2002-05-21
- Published: 2005-12-29
- Granted: 2009-02-24
- Earliest claimed: 2001-05-21
- Assignees: Functional Genetics, Inc.
- Inventors: Carol Carter; Arthur Goff; Lorna Ehrlich; Stanley N. Cohen
- Web page: https://patentgrep.com/patent/US7494767B2

## CPC

- G01N 33/502: {for testing non-proliferative effects}
- A61K 38/1709: {from mammals}
- A61P 31/12: Antivirals
- A61P 31/18: for HIV
- C07K 14/005: from viruses
- C07K 14/4702: {Regulators; Modulating activity}
- G01N 33/5008: {for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics}
- G01N 33/56983: {Viruses}
- G01N 33/56988: {HIV or HTLV}
- G01N 33/6878: {in epitope analysis}
- C07K 2319/00: Fusion polypeptide
- C12N 2740/16222: New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
- G01N 2333/16: HIV-1, HIV-2
- G01N 2333/161: gag-pol, e.g. p55, p24/25, p17/18, p.7, p6, p66/68, p51/52, p31/34, p32, p40
- G01N 2500/02: Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)
- G01N 2500/10: involving cells
- G01N 2500/20: cell-free systems
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The present invention provides methods for identifying peptides in a mammalian. Tsg101 protein that binds to the PTAPP (SEQ ID NO: 3) motif or L domain of human immunodeficiency virus type I (HIV-1). Such peptides can be used to inhibit Tsg101-HIV Gag binding, and is therefore effective in reducing HIV particle production. The invention also provides the peptides identified by the method of the invention and to method of using such peptides for treating HIV infection.

## Claims

### Claim 1

A method for identifying a peptide or fragment derived from a mammalian tumor susceptibility gene 101 (Tsg101) protein, wherein the peptide or fragment is effective in reducing HIV particle production, said method comprising:
(a) introducing an expression construct into a mammalian cell or cells, wherein the expression construct comprises a portion of a mammalian tsg101 gene;
(b) introducing one or more expression constructs into the mammalian cell or cells, wherein the one or more expression constructs comprise the HIV gag, pol, and rev gene sequences;
(c) incubating the transfected mammalian cell or cells in a suitable media for a sufficient time and at a temperature of about 37° C. to obtain a mammalian cell culture comprising mammalian cells which express a mammalian Tsg101 peptide or fragment and HIV Gag, Pol and Rev proteins;
(d) measuring the level of particle-associated p24 in the mammalian cell culture; and
(e) correlating a reduced level of particle-associated p24, when compared to a control mammalian cell culture which has not been transfected with the expression construct comprising a portion of a mammalian tsg101 gene, with the identification of a peptide or fragment effective in reducing HIV particle production.

### Claim 2

A method for identifying a peptide or fragment derived from a mammalian Tsg101 protein, wherein the peptide or fragment is effective in reducing HIV particle production, said method comprising:
(a) introducing an expression construct into a mammalian cell or cells, wherein the expression construct comprises a portion of a mammalian tsg101 gene;
(b) introducing HIV into the mammalian cell or cells;
(c) incubating the transfected and infected mammalian cell or cells in a suitable media for a sufficient time and at a temperature of about 37° C. to obtain a mammalian cell culture comprising mammalian cells which express a mammmalian Tsg101 peptide or fragment and which produce HIV particles;
(d) measuring the level of particle-associated p24 in the mammalian cell culture; and
(e) correlating a reduced level of particle-associated p24, when compared to a control mammalian cell culture which has not been transfected with the expression construct comprising a portion of a mammalian tsg101 gene, with the identification of a peptide or fragment effective in reducing HIV particle production.

### Claim 3

A method for identifying a peptide or fragment derived from a mammalian Tsg101 protein, wherein the peptide or fragment is effective in reducing HIV particle production, said method comprising:
(a) introducing an expression construct into a mammalian cell or cells, wherein the expression construct comprises a portion of a mammalian tsg101 gene;
(b) introducing HIV into the mammalian cell or cells;
(c) incubating the transfected and infected mammalian cell or cells in a suitable media for a sufficient time and at a temperature of about 37° C. to obtain a mammalian cell culture comprising mammalian cells which express a mammalian Tsg101 peptide or fragment and which produce HIV particles;
(d) quantifying the HIV particles released from the cells; and
(e) correlating a reduction in HIV particle production, when compared to a control mammalian cell culture which has not been transfected with the expression construct comprising a portion of a mammalian tsg101 gene, with the identification of a peptide or fragment effective in reducing HIV particle production.

### Claim 4

A method for identifying a peptide or fragment derived from a mammalian Tsg101 protein, wherein the peptide or fragment is effective in reducing HIV particle production, said method comprising:
(a) introducing one or more expression constructs into a mammalian cell or cells, wherein the one or more expression constructs comprise the HIV gag, pol, and rev gene sequences;
(b) introducing into the mammalian cell or cells a peptide or fragment derived from a mammalian Tsg101 protein;
(c) incubating the mammalian cell or cells transfected with the one or more expression constructs and permeated with a peptide or fragment derived from a mammalian Tsg101 protein in a suitable media for a sufficient time and at a temperature of about 37° C. to obtain a cell culture;
(d) measuring the level of particle-associated p24 in the mammalian cell culture; and
(e) correlating a reduced level of particle-associated p24, when compared to a control mammalian cell culture which has not been permeated with a peptide or fragment derived from a mammalian Tsg101 protein, with the identification of a peptide or fragment effective in reducing HIV particle production.

### Claim 5

A method for identifying a peptide or fragment derived from a mammalian Tsg101 protein, wherein the peptide or fragment is effective in reducing HIV particle production, said method comprising:
(a) introducing HIV into a mammalian cell or cells;
(b) introducing into the mammalian cell or cells a peptide or fragment derived from a mammalian Tsg101 protein;
(c) incubating the mammalian cell or cells transfected with HIV and permeated with a peptide or fragment derived from a mammalian Tsg101 protein in a suitable media for a sufficient time and at a temperature of about 37° C. to obtain a cell culture;
(d) measuring the level of particle-associated p24 in the cell culture; and
(e) correlating a reduced level of particle-associated p24, when compared to a control mammalian cell culture which has not been permeated with a peptide or fragment derived from a mammalian Tsg101 protein, with the identification of a peptide or fragment effective in reducing HIV particle production.

### Claim 6

A method for identifying a peptide or fragment derived from a mammalian Tsg101 protein, wherein the peptide or fragment is effective in reducing HIV particle production, said method comprising:
(a) introducing HIV into a mammalian cell or cells;
(b) introducing into the mammalian cell or cells a peptide or fragment derived from a mammalian Tsg101 protein;
(c) incubating the mammalian cell or cells transfected with HIV and permeated with a peptide or fragment derived from a mammalian Tsg101 protein in a suitable media and for a sufficient time at a temperature of about 37° C.;
(d) quantifying the HIV particles released from the cells; and
(e) correlating a reduction in HIV particle production, when compared to a control mammalian cell culture which has not been permeated with a peptide or fragment derived from a mammalian Tsg101 protein, which the identification of a peptide or fragment effective in reducing HIV particle production.

### Claim 7 (depends on 1, 2, 3)

The method of any one of claims 1, 2 or 3, wherein the coding sequence for a mammalian tsg101 gene is the coding sequence for the ubiquitin E2 variant (UEV) A domain of Tsg101 protein.

### Claim 8

A method for identifying a peptide or fragment derived from a mammalian Tsg101 protein, wherein said peptide or fragment is effective in reducing HIV particle production, said method comprising:
(a) measuring a level of HIV particles released in a culture of mammalian cells, wherein said mammalian cells comprise an expression construct comprising a portion of a mammalian tsg101 gene such that said mammalian cells express a mammalian Tsg101 peptide or fragment and are infected by HIV virus; and
(b) comparing said level of HIV particles to a level of HIV particles released in a culture of control mammalian cells under similar conditions, wherein said control mammalian cells do not comprise an expression construct comprising a portion of a mammalian tsg101 gene and are infected by HIV virus,
wherein said level of HIV particles measured in step (a) compared to said level of HIV particles of said culture of control mammalian cells below a predetermined thershold level identify said mammalian Tsg101 peptide or fragment as effective in reducing HIV particle production.

### Claim 9

A method for identifying a peptide or fragment derived from a mammalian Tsg101 protein, wherein said peptide or fragment is effective in reducing HIV particle production, said method comprising:
(a) measuring a level of HIV viral particle released in a culture of mammalian cells, wherein said mammalian cells comprise (i) an expression construct comprising a portion of a mammalian tsg101 gene and (ii) an expression construct comprising the HIV gag, pol and rev gene sequence, such that said mammalian cells express a mammalian Tsg101 peptide or fragment and HIV Gag, Pol and Rev proteins; and
(b) comparing said level of HIV particles released to a level of HIV particles released in a culture of control mammalian cells under similar conditions, wherein said control mammalian cells comprise one or more expression constructs comprising the HIV gag, pol and rev gene sequences such that said mammmalian cells express HIV Gag, Pol and Rev protein and wherein said control mammalian cells do not comprise an expression construct comprising a portion of a mammalian tsg101 gene,
wherein said level of HIV particles measured in step (a) compared to said level of HIV particles released in said culture of control mammalian cells below a predetermined thershold level identify said mammalian Tsg101 peptide or fragment as effective in reducing HIV particle production.

### Claim 10

A method for identifying a peptide or fragment derived from a mammalian Tsg101 protein, wherein said peptide or fragment is effective in reducing HIV particle production, said method comprising:
(a) measuring a level of HIV viral particle released in a culture of mammalian cells, wherein said mammalian cells comprise (i) an expression construct comprising a portion of a mammalian tsg101 gene and (ii) an expression construct comprising the HIV gag gene sequence, such that said mammalian cells express a mammalian Tsg101 peptide or fragment and HIV Gag protein; and
(b) comparing said level of HIV viral particle released to a level of HIV viral particle released in a culture of control mammalian cells under similar conditions, wherein said control mammalian cells comprise one or more expression constructs comprising said HIV gag gene sequence such that said mammalian cells express HIV Gag and wherein said control mammalian cells do not comprise an expression construct comprising a portion of a mammalian tsg101 gene,
wherein said level of HIV viral particle measured in step (a) compared to said level of HIV viral particle released in said culture of control mammalian cells below a predetermined threshold level identify said mammalian Tsg101 peptide or fragment as effective in reducing HIV particle production.

### Claim 11 (depends on 8, 9)

The method of claim 8, 9, or 10, wherein said level of HIV particles released in a culture of mammalian cells and said level of HIV particles released in a culture of control mammalian cells are represented by measured levels of particle-associated p24.

### Claim 12 (depends on 8, 9)

The method of claim 8, 9, or 10, wherein said portion of mammalian tsg101 gene is a coding sequence for the UEV A domain of said Tsg101 protein.

### Claim 13 (depends on 12)

The method of claim 12, wherein said predetermined threshold level is a two-fold reduction of said level of HIV particles measured in step (a) compared to said level of HIV particles of said culture of control mammalian cells.

### Claim 14 (depends on 12)

The method of claim 12, wherein said predetermined threshold level is a four-fold reduction of said level of HIV particles measured in step (a) compared to said level of HIV particles of said culture of control mammalian cells.

### Claim 15 (depends on 14)

The method of claim 14, wherein said predetermined threshold level is a 90% reduction of said level of HIV particles measured in step (a) compared to said level of HIV particles of said culture of control mammalian cells.

### Claim 16 (depends on 15)

The method of claim 15, wherein said predetermined threshold level is a 95% reduction of said level of HIV particles measured in step (a) compared to said level of HIV particles of said culture of control mammalian cells.

### Claim 17 (depends on 16)

The method of claim 16, wherein said predetermined threshold level is a 99% reduction of said level of HIV particles measured in step (a) compared to said level of HIV particles of said culture of control mammalian cells.

### Claim 18 (depends on 17)

The method of claim 17, wherein said predetermined threshold level is a 99.5% reduction of said level of HIV particles measured in step (a) compared to said level of HIV particles of said culture of control mammalian cells.

### Claim 19

A method for identifying a peptide or fragment derived from a mammalian Tsg101 protein, wherein said peptide or fragment is effective in reducing retrovirus production, said method comprising identifying a peptide that binds to a peptide comprising the PTAP (SEQ ID NO: 39) motif of a retroviral protein.

### Claim 20 (depends on 19)

The method of claim 19, wherein said retrovirus is a lentivirus.

### Claim 21 (depends on 20)

The method of claim 20, wherein said lentivirus is HIV-1 virus.

### Claim 22 (depends on 20)

The method of claim 20, wherein said lentivirus is HIV-2 virus.

### Claim 23 (depends on 20)

The method of claim 20, wherein said lentivirus is simian immunodeficiency virus.

### Claim 24 (depends on 19)

The method of claim 19, wherein said peptide comprising the PTAP (SEQ ID NO: 39) motif is a HIV Gag protein.

### Claim 25 (depends on 19)

The method of claim 19, wherein said peptide comprising the PTAP (SEQ ID NO: 39) motif is the peptide of SEQ ID NO:4.

### Claim 26 (depends on 8, 9, 10)

The method of any one of claims 8-10, wherein said tsg101 gene is a human tsg101 gene.