Method for the identification of activators of g protein-coupled receptors and nucleic acids encoding those receptors
OwnerFrederick S Hagen, Theo deVos +2inventors
InventorsFrederick S Hagen, Theo deVos, Pieter J Oort, Brett Russell
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Abstract
The present invention provides methods for identifying activators of G protein-coupled receptors (GPCRs) and for identifying nucleic acids encoding the receptor(s) for each identified activator. The present invention is directed at the generation of GPCR-encoding nucleic acid pools for expression in oocytes, the generation of compound pools, and the multiplex screening of both the compound and nucleic acid pools. Through successive subdivision of both the compound and nucleic pools into subpools, both activators and GPCR nucleic acids are identified from complex compound and receptor repertoires.
Claims62
A method for identifying an RNA that encodes a G protein-coupled receptor (GPCR) of unknown function in a library of RNAs and for identifying an activator compound, comprising:
- simultaneously screening and subdividing the library of RNAs in oocytes and a library of compounds to identify the RNA that encodes the GPCR of unknown function and to identify the activator compound that causes a GPCR-mediated response when contacted with the oocytes expressing the RNA that encodes the GPCR of unknown function.
A method for identifying a G protein-coupled receptor (GPCR) of unknown function and an activator compound, comprising:
- (a) introducing a heterogeneous RNA pool into oocytes, an RNA in the RNA pool encoding the GPCR of unknown function;
- (b) contacting the oocytes with a plurality of pools of compounds;
- (c) identifying from the compound pools an activator compound pool that causes a GPCR-mediated electrophysiological response when contacted with the oocytes expressing the pool of heterogeneous RNAs;
- (d) subdividing the activator compound pool into compound subpools;
- (e) subdividing the RNA pool into RNA subpools;
- (f) contacting the compound subpools with oocytes expressing the RNA subpools;
- and
- (g) identifying an activator compound subpool from the compound subpools, and identifying a GPCR RNA subpool, the activator compound subpool causing the GPCR-mediated electrophysiological response when contacted with an oocyte expressing the GPCR RNA subpool.
A method for producing a detectable electrophysiological response in an oocyte that is substantially characteristic of activation through a single, homogeneous type of G protein-coupled receptor (GPCR), comprising:
- expressing a plurality of different GPCRs of unknown function on an oocyte cell surface;
- contacting the oocyte with pools of compounds;
- and identifying the electrophysiological response.
A method for identifying a G protein-coupled receptor (GPCR) of unknown function and an activator compound, comprising:
- (a) providing heterogenous pools of RNAs, at least one of the RNA pools including an RNA encoding the GPCR of unknown function;
- (b) providing pools of compounds;
- (c) introducing the RNA pools into oocytes to express RNAs contained therein;
- (d) contacting the oocytes with the compound pools;
- (e) identifying an activator compound pool and a GPCR RNA pool, the activator compound pool causing a GPCR-mediated electrophysiological response when contacted with the oocytes expressing the GPCR RNA pool;
- (f) subdividing the activator compound pool to form compound subpools, and subdividing the GPCR RNA pool to form RNA subpools;
- and
- (g) contacting the compound subpools with oocytes expressing the RNA subpools to identify the RNA encoding the GPCR and to identify the activator compound that causes the GPCR-mediated electrophysiological response when the activator compound is contacted with an oocyte expressing the RNA.
A method for identifying a G protein-coupled receptor (GPCR) of unknown function and an activator compound, comprising:
- (a) introducing a pool of heterogeneous RNAs from normal cells or tissues into first oocytes;
- (b) introducing a pool of heterogeneous RNAs from cells or tissues having an altered GPCR-phenotype into second oocytes;
- (c) contacting the first and second oocytes with a plurality of pools of compounds;
- (d) comparing GPCR-mediated electrophysiological responses in the first and second oocytes to identify a difference in the electrophysiological response between the first and second oocytes;
- (e) identifying from the compound pools an activator compound pool, and identifying a GPCR RNA pool from the RNA pools from the cells or tissues having an altered GPCR phenotype, the activator compound pool causing the GPCR-mediated electrophysiological response when contacted to the second oocytes expressing the GPCR RNA pool;
- (f) subdividing the activator compound pool into compound subpools;
- (g) subdividing the GPCR RNA pool into RNA subpools;
- (h) contacting the compound subpools with third oocytes expressing the RNA subpools;
- and
- (i) identifying an activator compound subpool from the compound subpools, and identifying a GPCR RNA subpool, the activator compound subpool causing the GPCR-mediated electrophysiological response when contacted with an oocyte expressing the GPCR RNA subpool.
A method for identifying a G protein-coupled receptor (GPCR) of unknown function and an activator compound, comprising:
- (a) introducing a heterogeneous RNA pool into oocytes, an RNA in the RNA pool encoding the GPCR of unknown function;
- (b) contacting the oocytes with a plurality of pools of random or semi-random peptides;
- (c) identifying from the peptide pools an activator peptide pool that causes a GPCR-mediated electrophysiological response when contacted with the oocytes expressing the pool of heterogeneous RNAs;
- (d) subdividing the activator peptide pool into peptide subpools;
- (e) subdividing the RNA pool into RNA subpools;
- (f) contacting the peptide subpools with oocytes expressing the RNA subpools;
- and
- (g) identifying an activator peptide subpool from the peptide subpools, and identifying a GPCR RNA subpool, the activator peptide subpool causing the GPCR-mediated electrophysiological response when contacted with an oocyte expressing the GPCR RNA subpool.
A method for producing a detectable electrophysiological response in an oocyte that is substantially characteristic of activation through a single, homogeneous type of G protein-coupled receptor (GPCR), comprising:
- expressing a plurality of different GPCRs of unknown function on an oocyte cell surface;
- contacting the oocyte with pools of random or semi-random peptides;
- and
- identifying the electrophysiological response.
A method for identifying a G protein-coupled receptor (GPCR) of unknown function and an activator compound, comprising:
- (a) providing heterogenous pools of RNAs, at least one of the RNA pools including an RNA encoding the GPCR of unknown function;
- (b) providing pools of random or semi-random peptides;
- (c) introducing the RNA pools into oocytes to express RNAs contained therein;
- (d) contacting the oocytes with the peptide pools;
- (e) identifying an activator peptide pool and a GPCR RNA pool, the activator peptide pool causing a GPCR-mediated electrophysiological response when contacted with the oocytes expressing the GPCR RNA pool;
- (f) subdividing the activator peptide pool to form peptide subpools, and subdividing the GPCR RNA pool to form RNA subpools;
- and
- (g) contacting the peptide subpools with oocytes expressing the RNA subpools to identify the RNA encoding the GPCR and to identify the activator peptide that causes the GPCR-mediated electrophysiological response when the activator peptide is contacted with an oocyte expressing the RNA.
A method for identifying a G protein-coupled receptor (GPCR) of unknown function and an activator compound, comprising:
- (a) introducing a pool of heterogeneous RNAs from normal cells or tissues into first oocytes;
- (b) introducing a pool of heterogeneous RNAs from cells or tissues having an altered GPCR phenotype into second oocytes;
- (c) contacting the first and second oocytes with a plurality of pools of random or semi-random peptides;
- (d) comparing GPCR-mediated electrophysiological responses in the first and second oocytes to identify a difference in the electrophysiological response between the first and second oocytes;
- (e) identifying from the peptide pools an activator peptide pool, and identifying a GPCR RNA pool from the RNA pools from the cells or tissues having an altered GPCR phenotype, the activator peptide pool causing the GPCR-mediated electrophysiological response when contacted to the second oocytes expressing the GPCR RNA pool;
- (f) subdividing the activator peptide pool into peptide subpools;
- (g) subdividing the GPCR RNA pool into RNA subpools;
- (h) contacting the peptide subpools with third oocytes expressing the RNA subpools;
- and
- (i) identifying an activator peptide subpool from the peptide subpools, and identifying a GPCR RNA subpool, the activator peptide subpool causing the GPCR-mediated electrophysiological response when contacted with an oocyte expressing the GPCR RNA subpool.
Dependencies are inferred from the claim text.