# US 8,541,192 B2 · Methods to identify USP1 deubiquitinating enzyme complex inhibitors

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- Number: US 8,541,192 B2
- Document ID: US8541192B2
- Status: Granted
- Kind: B2
- Application number: 12/835,559
- Filed: 2010-07-13
- Published: 2010-12-30
- Granted: 2013-09-24
- Earliest claimed: 2006-06-20
- Assignees: Dana-Farber Cancer Institute, Inc.
- Applicants: Alan D. D'Andrea
- Inventors: Alan D. D'Andrea
- Web page: https://patentgrep.com/patent/US8541192B2

## CPC

- G01N 33/57595: {involving intracellular compounds}
- C12Q 1/37: involving peptidase or proteinase
- G01N 33/5011: {for testing antineoplastic activity}
- G01N 2500/00: Screening for compounds of potential therapeutic value
- 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)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

The invention provides methods used to identify new inhibitors of USP1 deubiquitinase. The inhibitors can be identified by contacting isolated USP1 with a test compound in the presence of monoubiquitinated proliferating cell nuclear antigen (PCNA), monoubiquitinated human Fanconi anemia group D2 (FANCD2), or ubiquitin-7-amido-4-methylcoumarin, and detecting the deubiquitination of said PCNA, FANCD2, or ubiquitin-7-amido-4-methylcoumarin using an antibody or fluorescence, wherein a decrease in the deubiquitination of said PCNA, FANCD2, or ubiquitin-7-amido-4-methylcoumarin in the presence of the test compound relative to the absence of the test compound identifies said test compound as an inhibitor of USP1 deubiquitinase.

## Claims

### Claim 1

An in vitro, biochemical method of identifying an inhibitor of ubiquitin specific protease 1(USP1) comprising:
(i) contacting an isolated USP1 with a test compound in the presence of monoubiquitinated proliferating cell nuclear antigen (PCNA) or ubiquitin-7-amido-4-methylcoumarin,
wherein said USP1 is selected from the group consisting of SEQ ID NO: 3 or the polypeptide set forth by amino acids 17-785 of SEQ ID NO: 3, and wherein said PCNA comprises SEQ ID NO: 2; and
(ii) detecting the deubiquitination of said PCNA or ubiquitin-7-amido-4-methylcoumarin using an antibody or fluorescence,
wherein a decrease in the deubiquitination of said PCNA or ubiquitin-7-amido-4-methylcoumarin in the presence of the test compound, relative to the absence of the test compound, identifies said test compound as an inhibitor of USP1.

### Claim 2 (depends on 1)

The method of claim 1, wherein USP1 is present in a heterodimeric complex with human USP1 associated factor 1 (UAF1).

### Claim 3

An in vitro, biochemical method of identifying an inhibitor of USP1 comprising:
(i) contacting a composition comprising isolated USP1 and isolated human UAF1 with a test compound in the presence of ubiquitin-7-amido-4-methylcoumarin,
wherein said USP1 is selected from the group consisting of SEQ ID NO: 3 or the polypeptide set forth by amino acids 17-785 of SEQ ID NO: 3, and
(ii) detecting the deubiquitination of said ubiquitin-7-amido-4- methylcoumarin using fluorescence,
wherein a decrease in the deubiquitination of said ubiquitin-7-amido-4- methylcoumarin in the presence of the test compound, relative to the absence of the test compound, identifies said test compound as an inhibitor of USP1.

### Claim 4 (depends on 3)

The method of claim 3, wherein said method is adapted to a high throughput screen.