# US 7,608,408 B2 · Methods of identifying compounds that inhibit Notch cleavage

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- Number: US 7,608,408 B2
- Document ID: US7608408B2
- Status: Granted
- Kind: B2
- Application number: 11/595,721
- Filed: 2006-11-10
- Published: 2007-05-10
- Granted: 2009-10-27
- Earliest claimed: 2005-11-10
- Assignees: Bristol-Myers Squibb Company
- Inventors: Catherine Burton; Charles F. Albright; Judith A. Wardwell-Swanson
- Web page: https://patentgrep.com/patent/US7608408B2

## CPC

- G01N 33/566: using specific carrier or receptor proteins as ligand binding reagents {where possible specific carrier or receptor proteins are classified with their target compounds}
- C07K 14/705: Receptors; Cell surface antigens; Cell surface determinants {(tumour specific antigens C07K14/4748)}
- C12N 5/0693: {Tumour cells; Cancer cells}
- C12N 9/6421: {from mammals}
- C12Q 1/37: involving peptidase or proteinase
- G01N 33/5008: {for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics}
- C12N 2501/42: Notch; Delta; Jagged; Serrate
- C12N 2503/02: Drug screening
- G01N 2333/4725: Mucins, e.g. human intestinal mucin
- 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)
- 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)
- 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

In vitro cell-based methods for identifying compounds that inhibit Notch cleavage and methods for identifying γ-secretase inhibitors that exhibit reduced induction of goblet cell metaplasia are provided. Also provided are methods of identifying compounds that inhibit cleavage of γ-secretase substrates other than Notch and homogeneous compositions or cultures of Notch-expressing cells that undergo mucin-2 or mucin-5AC induction in response to a compound known to inhibit Notch cleavage and methods of their generation.

## Claims

### Claim 1

A method for identifying a compound that inhibits cleavage of Notch comprising:
(a) contacting Notch-expressing HT29 cells with a compound that inhibits γ-secretase; and
(b) detecting mucin-2 induction or mucin-5AC induction in said cells as compared to HT29 cells not contacted with said compound, wherein said mucin-2 induction or mucin-5AC induction is indicative of an inhibitor of Notch cleavage, and wherein the Notch-expressing cells express γ-secretase.

### Claim 2 (depends on 1)

The method of claim 1 wherein said cells are preselected based on their ability to undergo mucin-2 induction or mucin-5AC induction in response to a compound known to inhibit Notch cleavage.

### Claim 3 (depends on 2)

The method of claim 2 wherein said compound known to inhibit Notch cleavage is Compound Z:

### Claim 4 (depends on 1)

The method of claim 1 wherein said step of detecting comprises ELISA, flow cytometry, or a high content screening assay.

### Claim 5 (depends on 1)

The method of claim 1 further comprising a step of determining whether said test compound inhibits cleavage of one or more γ-secretase substrates other than Notch.

### Claim 6 (depends on 5)

The method of claim 5 wherein said γ-secretase substrate other than Notch comprises an amyloid precursor protein, an E-cadherin, a N-cadherin, LRP, Nectin-1α, CD44, ErbB4, Jagged-2, or Delta-1.

### Claim 7 (depends on 5)

The method of claim 5 wherein said γ-secretase substrate other than Notch comprises an amyloid precursor protein.

### Claim 8 (depends on 5)

The method of claim 5 wherein said determining step is performed prior to, after, or simultaneously with said steps (a) and (b).