# US 7,745,196 B1 · Methods and compositions for identifying peptide modulators of cell surface receptors

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- Number: US 7,745,196 B1
- Document ID: US7745196B1
- Status: Granted
- Kind: B1
- Application number: 11/067,065
- Filed: 2005-02-25
- Granted: 2010-06-29
- Earliest claimed: 2004-03-25
- Assignees: Rigel Pharmaceuticals, Inc.
- Inventors: Brian Wong; Donald G. Payan
- Web page: https://patentgrep.com/patent/US7745196B1

## CPC

- G01N 33/5008: {for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics}
- G01N 33/6845: {Methods of identifying protein-protein interactions in protein mixtures}
- 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)

## Abstract

The invention provides methods and compositions for identifying modulators, particularly peptide modulators, of cell surface receptor activity. Specifically, the invention provides a cell containing a recombinant nucleic acid encoding a polypeptide modified for intracellular presentation, which cell may further comprise one or both of a recombinant nucleic acid encoding a binding partner for that polypeptide and recombinant nucleic acid encoding a test polypeptide. The subject cell may be a member of a library of cells, and the library may be used in screening methods for identifying test polypeptides (e.g., conformationally restrained polypeptides) having cell surface receptor modulatory activity. The invention finds use in a variety of drug-discovery applications.

## Claims

### Claim 1

A cell comprising:
a recombinant nucleic acid encoding a first polypeptide modified for intracellular presentation;
and at least one of:
a recombinant nucleic acid encoding a binding partner for said first polypeptide; and
a recombinant nucleic acid encoding a test polypeptide.

### Claim 2 (depends on 1)

The cell of claim 1, wherein said binding partner is a second polypeptide modified for intracellular presentation.

### Claim 3 (depends on 2)

The cell of claim 2, wherein said first and second polypeptides modified for intracellular presentation are receptor subunits modified for intracellular presentation.

### Claim 4 (depends on 1)

The cell of claim 1, wherein said cell comprises:
a recombinant nucleic acid encoding a binding partner for said first polypeptide; and
a recombinant nucleic acid encoding a test polypeptide.

### Claim 5 (depends on 1)

The cell of claim 1, wherein the test polypeptide is a conformationally restricted polypeptide.

### Claim 6 (depends on 5)

The cell of claim 5, wherein said conformationally restricted test polypeptide is a cyclic polypeptide.

### Claim 7 (depends on 5)

The cell of claim 5, wherein said conformationally restricted test polypeptide is a fusion protein.

### Claim 8 (depends on 5)

The cell of claim 5, wherein said conformationally restricted test polypeptide comprises a randomized amino acid sequence.

### Claim 9 (depends on 1)

The cell of claim 1, wherein said first polypeptide and said binding partner, when bound in a polypeptide/binding partner complex, produce a signal.

### Claim 10 (depends on 9)

The cell of claim 9, wherein said signal is a fluorescence resonance energy transfer signal.

### Claim 11 (depends on 9)

The cell of claim 9, wherein said signal is a colorimetric signal.

### Claim 12 (depends on 9)

The cell of claim 9, wherein said signal is a phenotypic signal.

### Claim 13 (depends on 1)

The cell of claim 1, wherein said first polypeptide is a receptor, and said binding partner of said polypeptide is a ligand for said receptor.

### Claim 14 (depends on 1)

The cell of claim 1, wherein said first polypeptide modified for intracellular presentation is associated with an intracellular cell membrane.

### Claim 15 (depends on 1)

The cell of claim 1, wherein said first polypeptide modified for intracellular presentation comprises a transmembrane domain.

### Claim 16 (depends on 1)

The cell of claim 1, wherein said first polypeptide modified for intracellular presentation is cytoplasmically soluble.

### Claim 17

A library of cells, each cell having:
a recombinant nucleic acid encoding a first polypeptide modified for intracellular presentation; and
a recombinant nucleic acid encoding a test polypeptide.

### Claim 18 (depends on 17)

The library of cells of claim 17, wherein each cell further comprises a nucleic acid encoding a binding partner for said first polypeptide.

### Claim 19 (depends on 17)

The library of claim 17, wherein said test polypeptide is a conformationally restricted polypeptide.

### Claim 20 (depends on 17)

The library of cells of claim 17, wherein the test polypeptides comprise randomized amino acid sequences.

### Claim 21

A method of screening, comprising:
assaying a population of cells each cell of said population comprising:
a recombinant nucleic acid encoding a first polypeptide modified for intracellular presentation;
a recombinant nucleic acid encoding a binding partner for said first polypeptide; and,
a recombinant nucleic acid encoding a test polypeptide;
said assaying being for identification of a test polypeptide that modulates binding between said first polypeptide and said binding partner.

### Claim 22 (depends on 21)

The method of claim 21, wherein the test polypeptide is a conformationally restrained polypeptide.

### Claim 23 (depends on 21)

The method of claim 21, wherein said polypeptide modified for intracellular presentation and said binding partner, when complexed, produce a signal, and wherein said assaying comprises assaying said signal.

### Claim 24 (depends on 23)

The method of claim 23, wherein said signal is an optical signal.

### Claim 25 (depends on 21)

The method of claim 21, wherein said assaying involves FACS sorting of said cells.

### Claim 26 (depends on 21)

The method of claim 21, wherein said first polypeptide is a subunit of a receptor, and each cell further comprises a recombinant nucleic acid encoding a ligand for said receptor.

### Claim 27

A method for identifying a cell producing a biologically active polypeptide, comprising: lysing samples of cells from pools of cells to make cell lysates; assaying a plurality of pools of different lysed cells, the lysed cells of each pool producing a plurality of different conformationally restricted test polypeptides, thereby identifying a pool of cells producing a biologically active conformationally restrained test polypeptide; separating the cells in said identified pool; and assaying said isolated cells to identify a cell producing said biologically active conformationally restricted test polypeptide.

### Claim 28 (depends on 27)

The cell of claim 27, wherein said conformationally restricted test polypeptide is a cyclic polypeptide.

### Claim 29 (depends on 27)

The cell of claim 27, wherein said conformationally restricted test polypeptide is a fusion protein.

### Claim 30 (depends on 27)

The method of claim 27, wherein said isolated cells are cultured prior to assaying said isolated cells.

### Claim 31 (depends on 27)

The method of claim 27, wherein said biological activity is modulation of a surface-bound receptor on the surface of a mammalian cell.

### Claim 32 (depends on 27)

The method of claim 27, wherein the conformationally restricted test polypeptides comprise a randomized sequence.

### Claim 33 (depends on 21)

A method of modulating an activity to a cell, comprising: contacting said cell with a polypeptide identified by the method of claim 21.

### Claim 34 (depends on 33)

The method of claim 33, wherein said activity is a receptor-mediated activity.

### Claim 35 (depends on 21)

A method of treating a subject for a receptor-related condition comprising: administering to said subject a polypeptide identified by the method of claim 21, wherein said polypeptide modulates said receptor and thereby treats said receptor-related condition.