# US 9,040,462 B2 · In vivo production of cyclic peptides for inhibiting protein-protein interaction

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- Number: US 9,040,462 B2
- Document ID: US9040462B2
- Status: Granted
- Kind: B2
- Application number: 12/119,283
- Filed: 2008-05-12
- Published: 2008-10-23
- Granted: 2015-05-26
- Earliest claimed: 2000-03-06
- Assignees: Rigel Pharmaceuticals, Inc.
- Applicants: James B. Lorens; Todd R. Pray; Todd M. Kinsella; Mark K. Bennett
- Inventors: James B. Lorens; Todd R. Pray; Todd M. Kinsella; Mark K. Bennett
- Web page: https://patentgrep.com/patent/US9040462B2

## CPC

- C12N 15/10: Processes for the isolation, preparation or purification of DNA or RNA (chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34)
- C12N 15/1055: {Protein x Protein interaction, e.g. two hybrid selection}
- 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 relates to methods and compositions utilizing inteins to generate libraries of cyclic peptides in vivo. The prevent invention also relates to methods for inhibiting protein-protein interaction.

## Claims

### Claim 1

A method comprising:
a) introducing a library of fusion nucleic acids into a plurality of cells, wherein said cells comprise a first interacting protein and a second interacting protein and wherein said library of fusion nucleic acids comprises, from 5′ to 3′:
i) nucleic acid encoding a C-terminal intein motif;
ii) nucleic acid encoding a peptide; and
iii) nucleic acid encoding an N-terminal intein motif;
wherein said introducing provides for production of a cyclic peptide in said cells; and;
b) identifying a cell comprising a cyclic peptide capable of preventing interaction of said interacting proteins.

### Claim 2 (depends on 1)

The method according to claim 1, wherein said first interacting protein is a ubiquitin activating agent and said second interacting protein is a ubiquitin conjugating agent.

### Claim 3 (depends on 1)

The method according to claim 1, wherein said first interacting protein is a ubiquitin conjugating agent and said second interacting protein is a ubiquitin ligating agent.

### Claim 4 (depends on 3)

The method according to claim 3, wherein said ubiquitin conjugating agent is an E2 and said ubiquitin ligating agent is mdm2.

### Claim 5 (depends on 1)

The method according to claim 1, wherein said first interacting protein is a ubiquitin ligating agent and said second interacting protein is a substrate of said ubiquitin ligating agent.

### Claim 6 (depends on 5)

The method according to claim 5, wherein said ubiquitin ligating agent is mdm2 and said substrate of said ubiquitin ligating agent is p53.

### Claim 7 (depends on 1)

The method of claim 1, wherein said first interacting protein is a first subunit of a ubiquitin activating agent and said second interacting protein is a second subunit of a ubiquitin activating protein.

### Claim 8 (depends on 1)

The method of claim 1, wherein said first interacting protein is a first subunit of a ubiquitin ligating agent and said second interacting protein is a substrate of said ubiquitin ligating agent.

### Claim 9 (depends on 1)

The method of claim 1, wherein said first interacting protein is a ubiquitin conjugating agent and said second interacting protein is a substrate of said ubiquitin conjugating agent.

### Claim 10 (depends on 1)

The method according to claim 1, wherein said first and second interacting proteins are signal transduction proteins.

### Claim 11 (depends on 1)

The method according to claim 1, wherein said first interacting protein is an enzyme and said second interacting protein is a substrate for said enzyme.

### Claim 12 (depends on 1)

The method according to claim 1, wherein said cyclic peptide is a competitive inhibitor of binding of said second interacting protein to said first interacting protein.

### Claim 13 (depends on 1)

The method according to claim 1, wherein said cyclic peptide is a non-competitive inhibitor of binding of said second interacting protein to said first interacting protein.

### Claim 14 (depends on 13)

The method according to claim 13, wherein said cyclic peptide binds to an allosteric regulatory site of said second interacting protein.

### Claim 15 (depends on 1)

The method of claim 1, wherein one of said first and second interacting proteins comprises a first FRET label and the other of said interacting proteins comprises a second FRET label or a quencher.

### Claim 16 (depends on 15)

The method of claim 15, wherein said identifying comprises identifying a change in fluorescence as compared with a control cell.

### Claim 17 (depends on 1)

The method of claim 1, wherein said first interacting protein comprises a first subunit of a transcription activation protein pair and said second interacting protein comprises a second subunit of said transcription activation protein pair, wherein binding of said first subunit and said second subunit of said transcription activation protein pair results in transcription of a reporter.

### Claim 18 (depends on 17)

The method of claim 17, wherein said identifying comprises identifying a reduction or absence of expression of said reporter.