# US 7,465,549 B2 · Methods for identifying agents that bind a levetiracetam binding site (LBS) or compete with LEV binding to a LBS of a synaptic vesicle protein 2 (SV2)

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- Number: US 7,465,549 B2
- Document ID: US7465549B2
- Status: Granted
- Kind: B2
- Application number: 10/725,189
- Filed: 2003-12-02
- Published: 2004-10-14
- Granted: 2008-12-16
- Earliest claimed: 2002-12-03
- Assignees: Ucb, S.A.
- Inventors: Berkley Lynch; Karl Nocka; Bruno Fuks
- Web page: https://patentgrep.com/patent/US7465549B2

## CPC

- A61K 31/4015: having oxo groups directly attached to the heterocyclic ring, e.g. piracetam, ethosuximide
- A61P 5/00: Drugs for disorders of the endocrine system
- A61P 25/00: Drugs for disorders of the nervous system
- A61P 25/04: Centrally acting analgesics, e.g. opioids
- A61P 25/06: Antimigraine agents
- A61P 25/08: Antiepileptics; Anticonvulsants
- A61P 25/16: Anti-Parkinson drugs
- A61P 25/28: for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- G01N 33/6896: {Neurological disorders, e.g. Alzheimer's disease}
- G01N 33/9473: {Anticonvulsants, e.g. phenobarbitol, phenytoin}
- G01N 2333/705: Assays involving receptors, cell surface antigens or cell surface determinants
- G01N 2500/00: Screening for compounds of potential therapeutic value
- G01N 2800/28: Neurological disorders
- Y02A 90/10: Information and communication technologies [ICT] supporting adaptation to climate change, e.g. for weather forecasting or climate simulation
- 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
- 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)
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

The present invention is drawn to methods of characterization of the properties and functions of SV2 proteins. The invention further includes methods of identifying compounds or agents which modulate the activity of SV2 proteins. Included in these methods is the identification of compounds or agents which modulate the binding of levetiracetam to SV2 proteins, including SV2A. Additionally, the present invention provides biotinylated ligands as a tool to screen chemical libraries and characterize the SV2 proteins. Further, the present invention provides a method of solubilizing and purifying functionally active membrane associated proteins, such as SV2.

## Claims

### Claim 1

A method of identifying a compound or an agent that binds a levetiracetam binding site (LBS) of a synaptic vesicle protein 2 (SV2) protein comprising;
a) obtaining a cell-free or membrane-free SV2 protein comprising a LBS wherein the SV2 protein is SV2A or SV2C;
b) incubating the SV2 protein with a compound or an agent and levetiracetam (LEV) or an analog or derivative thereof that binds a LBS on an SV2 protein; and
c) determining if the compound or agent competes with LEV or an analog or derivative thereof for binding to the LEV binding site, thereby identifying a compound or an agent that binds the LBS of the SV2 protein.

### Claim 2 (depends on 1)

The method of claim 1, wherein the compound or agent is an analog or derivative of levetiracetam.

### Claim 3 (depends on 2)

The method of claim 2, wherein the analog or derivative of levetiracetam is selected from the group consisting of N-alkylated 2-oxo -pyrrolidine derivatives, N-alkylated 2-oxo-piperidinyl derivatives, and N-alkylated 2-oxo-azepanyl derivatives.

### Claim 4 (depends on 1)

The method of claim 1, wherein the compound or agent is an anti-SV2 antibody or fragment thereof.

### Claim 5 (depends on 4)

The method of claim 4, wherein the anti-SV2 antibody or fragment thereof binds to the levetiracetam binding site of SV2 protein.

### Claim 6 (depends on 4)

The method of claim 4, wherein the anti-SV2 antibody or fragment thereof is selected from the group consisting of a polyclonal antibody and a monoclonal antibody.

### Claim 7 (depends on 6)

The method of claim 6, wherein the antibody fragment is selected from the group consisting of an Fab fragment, Fab′ fragment, F(ab′)2 fragment and an scFv fragment.

### Claim 8 (depends on 6)

The method of claim 6, wherein the monoclonal antibody is selected from the group consisting of a chimeric antibody, a humanized antibody, and a human antibody.

### Claim 9

A method of identifying a compound or an agent that binds a levetiracetam binding site (LBS) of an SV2 protein, comprising:
a) obtaining a cell-free or membrane-free SV2 protein comprising a LBS wherein the SV2 protein is SV2A or SV2C;
b) incubating the SV2 protein with a compound or an agent and levetiracetam or an analog or derivative thereof that binds a LBS on an SV2 protein; and
c) determining if the binding of levetiracetam or an analog or derivative thereof to the protein is inhibited by the compound or agent, thereby identifying a compound or agent that binds the LBS of an SV2.

### Claim 10 (depends on 9)

The method of claim 9, wherein the analog of levetiracetam is (2S)-2-[4-(3-azidophenyl)-2-oxopyrrolidin-1-yl]butanamide.

### Claim 11 (depends on 9)

The method of claim 9, wherein the SV2 protein is purified.

### Claim 12 (depends on 9)

The method of claim 9, wherein the SV2 protein is immobilized.

### Claim 13 (depends on 9)

The method of claim 9, wherein the SV2 protein is expressed on a transformed host cell.

### Claim 14 (depends on 9)

The method of claim 9, wherein the levetiracetam or an analog or derivative thereof is directly or indirectly labeled.

### Claim 15 (depends on 14)

The method of claim 14, wherein the label is a radiolabel.

### Claim 16 (depends on 15)

The method of claim 15, wherein the radiolabel is 3H.

### Claim 17 (depends on 14)

The method of claim 14, wherein the label is a fluorescent label.

### Claim 18 (depends on 14)

The method of claim 14, wherein the label is an enzyme.

### Claim 19 (depends on 9)

The method of claim 9, wherein the SV2 protein is incubated with the levetiracetam or an analog or derivative prior to addition of the agent.

### Claim 20 (depends on 9)

The method of claim 9, wherein the SV2 protein is incubated with levetiracetam or an analog or derivative after addition of the agent.

### Claim 21 (depends on 9)

The method of claim 9, wherein the SV2 protein is incubated with levetiracetam or an analog or derivative concurrent with the agent.

### Claim 22 (depends on 9)

The method of claim 9, wherein the SV2 protein is incubated with levetiracetam.

### Claim 23

A method of identifying a compound or an agent that binds the levetiracetam binding site (LBS) of an SV2 protein, comprising:
a) obtaining a cell-free or membrane-free SV2 protein comprising a LBS wherein the SV2 protein is SV2A or SV2C;
b) incubating the SV2 protein with a compound or an agent and (2S)-2-[4-(3-azidophenyl)-2-oxopyrrolidin-1-yl]butanamide;
c) separately incubating the SV2 protein with (2S)-2-[4-(3-azidophenyl)-2-oxopyrrolidin-1-yl]butanamide; and
d) comparing the binding of (2S)-2-[4-(3-azidophenyl)-2-oxopyrrolidin-1-yl]butanamide to SV2 in steps (b) and (c) to determine if the binding of (2S)-2-[4-(3-azidophenyl)-2-oxopyrrolidin-1-yl]butanamide to the protein is inhibited by the compound or agent, thereby identifying a compound or an agent that binds the LBS of the SV2 protein.

### Claim 24 (depends on 1)

The method of claim 1, wherein the SV2 protein is a human SV2 protein.

### Claim 25 (depends on 24)

The method of claim 24, wherein the SV2 protein is an SV2A protein comprising the amino acid sequence of SEQ ID NO: 2, or an SV2C protein comprising the amino acid sequence of SEQ ID NO: 6.

### Claim 26 (depends on 1)

The method of claim 1, wherein the SV2 protein comprises conservative amino acid substitutions.

### Claim 27 (depends on 1)

The method of claim 1, wherein the SV2 protein is a recombinantly expressed protein.

### Claim 28 (depends on 27)

The method of claim 27, wherein the SV2 protein is recombinantly expressed from COS cells.

### Claim 29 (depends on 9)

The method of claim 9, wherein the SV2 protein is a human SV2 protein.

### Claim 30 (depends on 29)

The method of claim 29, wherein the SV2 protein is an SV2A protein comprising the amino acid sequence of SEQ ID NO: 2, or an SV2C protein comprising the amino acid sequence of SEQ ID NO: 6.

### Claim 31 (depends on 9)

The method of claim 9, wherein the SV2 protein comprises conservative amino acid substitutions.

### Claim 32 (depends on 9)

The method of claim 9, wherein the SV2 protein is a recombinant protein.

### Claim 33 (depends on 32)

The method of claim 32, wherein the SV2 protein is recombinantly expressed from COS cells.

### Claim 34 (depends on 23)

The method of claim 23, wherein the SV2 protein is a human SV2 protein.

### Claim 35 (depends on 34)

The method of claim 34, wherein the SV2 protein is an SV2A protein comprising the amino acid sequence of SEQ ID NO: 2, or an SV2C protein comprising the amino acid sequence of SEQ ID NO: 6.

### Claim 36 (depends on 23)

The method of claim 23, wherein the SV2 protein comprises conservative amino acid substitutions.

### Claim 37 (depends on 23)

The method of claim 23, wherein the SV2 protein is a recombinant protein.

### Claim 38 (depends on 37)

The method of claim 37, wherein the SV2 protein is recombinantly expressed from COS cells.