# US 5,700,650 A · Mechanism-based screen for retinoid X receptor agonists and antagonists

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- Number: US 5,700,650 A
- Document ID: US5700650A
- Status: Granted
- Kind: A
- Application number: 08/469,122
- Filed: 1995-06-06
- Granted: 1997-12-23
- Earliest claimed: 1992-12-31
- Assignees: American Cyanamid Company
- Inventors: Paul Mak; Sotirios K. Karathanasis
- Web page: https://patentgrep.com/patent/US5700650A

## 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 14/70567: {Nuclear receptors, e.g. retinoic acid receptor [RAR], RXR, nuclear orphan receptors}
- C07K 14/775: Apolipopeptides
- C12Q 1/6897: involving reporter genes operably linked to promoters
- G01N 2333/395: from Saccharomyces
- G01N 2333/70567: Nuclear receptors, e.g. retinoic acid receptor [RAR], RXR, nuclear orphan receptors
- 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)
- 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

A method is described for screening retinoid X receptor agonists or antagonists using a retinoid X receptor expressed by a yeast expression system to screen a compound having a retinoid X receptor agonist or antagonist activity. Another screen is described for detecting a compound having retinoid X receptor agonist or antagonist activity by: (1) providing a yeast strain which expresses the retinoic acid receptor and activates a reporter plasmid containing apolipoprotein AI gene site A or a mutated variant thereof; (2) incubating the compound in suitable media and a colorless chromogenic substrate; and (3) examining the media for development of color. Mutated variants of apolipoprotein AI gene site A which respond selectively to the retinoid X receptor or respond to receptors other than RXR.alpha. are also described.

## Claims

### Claim 1

A method for screening for a compound having retinoid X receptor agonist or antagonist activity, which comprises the steps of:
(a) providing a yeast strain which expresses the retinoid X receptor and activates a reporter plasmid containing an apolipoprotein AI gene site A mutant comprising a nucleotide sequence selected from the group consisting of SEQ ID NOS: 7-15 and 17;
(b) incubating the compound with the yeast strain of step (a) in suitable media and a colorless chromogenic substrate; and
(c) examining the media for development of color, wherein the presence of color indicates that the compound has retinoid X receptor agonist or antagonist activity.

### Claim 2 (depends on 1)

The method according to claim 1, wherein said retinoid X receptor is fused to a yeast ubiquitin fusion system.

### Claim 3 (depends on 2)

The method according to claim 2, wherein said yeast ubiquitin fusion system contains triosephosphate dehydrogenase as the constitutive yeast promoter.

### Claim 4 (depends on 3)

The method according to claim 3, wherein said retinoid X receptor fused to the yeast ubiquitin fusion system is contained in a yeast strain carrying an expression vector identified as BJ YEpRXR.alpha., having the ATCC designation number 74197.

### Claim 5 (depends on 1)

The method according to claim 1, wherein the chromogenic substrate of step (b) is 5-bromo-4-chloro-3-indolyl-.beta.-D-galactopyranoside.

### Claim 6 (depends on 3)

The method according to claim 3, wherein said retinoid X receptor fused to the yeast ubiquitin fusion system is contained in a yeast strain carrying a reporter plasmid identified as BJ YEpRXR.alpha./YRpA, having the ATCC designation number 74196.