Methods and compositions for identifying receptor effectors
First Claim
1. A recombinant cell which comprises:
- a heterologous formyl peptide receptor like-1 (FPRL-1) receptor expressed in the cell membrane of said cell such that signal transduction activity via said receptor is modulated by interaction of an extracellular region of the receptor with an extracellular signal; and
a ligand agonist of said FPRL-1 receptor comprising a polypeptide or analog thereof, wherein said ligand agonist is transported to a location allowing interaction with the extracellular region of said FPRL-1 receptor expressed in the cell membrane, and is expressed at a level sufficient for said ligand agonist to bind to and activate said FPRL-1 receptor, thereby causing a detectable signal to be generated.
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Abstract
The present invention makes available rapid, effective assays for screening and identifying pharmaceutically effective compounds that specifically interact with and modulate the activity of a cellular receptor or ion channel. The subject assays enable rapid screening of large numbers of polypeptides in a library to identify those polypeptides which induce or antagonize receptor bioactivity. The subject assays are particularly amenable for identifying agonists and antagonists for orphan receptors. In particular the present invention makes available novel ligand agonists of human formyl peptide receptor like-1 (FPRL-1) receptors. These novel ligand agonists are used in the assays of the invention to identify modulators of FPRL-1 receptor.
13 Citations
181 Claims
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1. A recombinant cell which comprises:
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a heterologous formyl peptide receptor like-1 (FPRL-1) receptor expressed in the cell membrane of said cell such that signal transduction activity via said receptor is modulated by interaction of an extracellular region of the receptor with an extracellular signal; and
a ligand agonist of said FPRL-1 receptor comprising a polypeptide or analog thereof, wherein said ligand agonist is transported to a location allowing interaction with the extracellular region of said FPRL-1 receptor expressed in the cell membrane, and is expressed at a level sufficient for said ligand agonist to bind to and activate said FPRL-1 receptor, thereby causing a detectable signal to be generated. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17)
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18. A recombinant yeast cell which comprises:
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a heterologous formyl peptide receptor like-1 (FPRL-1) receptor expressed in the cell membrane of said yeast cell such that signal transduction activity via said receptor is modulated by interaction of an extracellular region of the receptor with an extracellular signal; and
a ligand agonist of said FPRL-1 receptor, wherein said ligand agonist is transported to a location allowing interaction with the extracellular region of said FPRL-1 receptor expressed in the cell membrane, and is expressed at a level sufficient for said ligand agonist to bind to and activate said FPRL-1 receptor, thereby causing a detectable signal to be generated. - View Dependent Claims (19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40)
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41. A method for identifying a modulator of a heterologous formyl peptide receptor like-1 (FPRL-1) receptor expressed by a cell, comprising:
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providing a recombinant cell comprising;
a heterologous FPRL-1 receptor expressed in the cell membrane of said cell such that signal transduction activity via said receptor is modulated by interaction of an extracellular region of the receptor with an extracellular signal, said heterologous FPRL-1 receptor acting as a surrogate for an endogenous cell receptor in a signal transduction pathway of the cell; and
a ligand agonist of said FPRL-1 receptor comprising a polypeptide or analog thereof, wherein said ligand agonist is transported to a location allowing interaction with the extracellular region of said FPRL-1 receptor expressed in the cell membrane, and is expressed at a level sufficient for said ligand agonist to bind to and activate said FPRL-1 receptor, thereby activating the signal transduction activity of said FPRL-1 receptor and generating a detectable signal;
contacting said yeast cell with a test compound; and
detecting an alteration in said signal generated by said ligand agonist to thereby identify a modulator of said receptor. - View Dependent Claims (42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61)
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62. A method for identifying a modulator of a heterologous formyl peptide receptor like-1 (FPRL-1) receptor expressed by a cell, comprising:
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providing a recombinant cell comprising;
a heterologous FPRL-1 receptor expressed in the cell membrane of said cell such that signal transduction activity via said receptor is modulated by interaction of an extracellular region of the receptor with an extracellular signal, said heterologous FPRL-1 receptor acting as a surrogate for an endogenous receptor in a signal transduction pathway of the cell; and
a ligand agonist of said FPRL-1 receptor comprising a polypeptide or analog thereof, wherein said ligand agonist is transported to a location allowing interaction with the extracellular region of said FPRL-1 receptor expressed in the cell membrane, and is expressed at a level sufficient for said ligand agonist to bind to and activate said FPRL-1 receptor, thereby activating the signal transduction activity of said FPRL-1 receptor and generating a detectable signal;
contacting said cell with each member of a library of test compounds; and
detecting an alteration in said signal generated by said ligand agonist to thereby identify a modulator of said receptor. - View Dependent Claims (63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82)
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83. A method for identifying a modulator of a heterologous formyl peptide receptor like-1 (FPRL-1) receptor expressed by a cell, comprising:
- providing a mixture of recombinant cells, each cell of which has a cell membrane and comprises;
a heterologous formyl peptide receptor like-1 (FPRL-1) receptor expressed in the cell membrane of said cell such that signal transduction activity via said receptor is modulated by interaction of an extracellular region of the receptor with an extracellular signal, said heterologous FPRL-1 receptor acting as a surrogate for an endogenous cell receptor in a signal transduction pathway of the cell;
a ligand agonist of said FPRL-1 receptor comprising a polypeptide or analog thereof, wherein said ligand agonist is transported to a location allowing interaction with the extracellular region of said FPRL-1 receptor expressed in the cell membrane, and is expressed at a level sufficient for said ligand agonist to bind to and activate said FPRL-1 receptor, thereby activating the signal transduction activity of said FPRL-1 receptor and generating a detectable signal; and
a heterologous test polypeptide, wherein the heterologous test polypeptide is transported to a location allowing interaction with the extracellular region of said receptor expressed in the cell membrane;
wherein collectively the mixture of cells expresses a library of said heterologous test polypeptides, said library being expressible at a sufficient level such that modulation of the signal transduction activity of said receptor by a heterologous test polypeptide within the library alters the detectable signal generated by said ligand agonist; and
detecting an alteration in said signal generated by said ligand agonist to thereby identify a modulator of said receptor. - View Dependent Claims (84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102)
- providing a mixture of recombinant cells, each cell of which has a cell membrane and comprises;
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103. A method for identifying a modulator of a heterologous formyl peptide receptor like-1 (FPRL-1) receptor expressed by a cell, comprising:
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providing a first mixture of recombinant cells, each cell of which has a cell membrane and comprises;
a heterologous formyl peptide receptor like-1 (FPRL-1) receptor expressed in the cell membrane of said cell such that signal transduction activity via said receptor is modulated by interaction of an extracellular region of the receptor with an extracellular signal, said heterologous FPRL-1 receptor acting as a surrogate for an endogenous cell receptor in a signal transduction pathway of the cell; and
a ligand agonist of said FPRL-1 receptor comprising a polypeptide or analog thereof, wherein said ligand agonist is transported to a location allowing interaction with the extracellular region of said FPRL-1 receptor expressed in the cell membrane, and is expressed at a level sufficient for said ligand agonist to bind to and activate said FPRL-1 receptor, thereby activating the signal transduction activity of said FPRL-1 receptor and generating a detectable signal;
contacting said first mixture with a second mixture of cells, wherein collectively the second mixture of cells expresses a library of heterologous test polypeptides that are transported to a location allowing interaction with the extracellular region of the FPRL-1 receptor expressed in the cell membrane of the cells of the first mixture; and
detecting an alteration by a heterologous test polypeptide of said signal generated by said ligand agonist to thereby identify a modulator of said receptor. - View Dependent Claims (104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122)
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123. A method for identifying a modulator of a heterologous formyl peptide receptor like-1 (FPRL-1) receptor expressed by a cell, comprising:
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providing a recombinant cell comprising a heterologous FPRL-1 receptor expressed in the cell membrane of said cell such that signal transduction activity via said receptor is modulated by interaction of an extracellular region of the receptor with an extracellular signal; and
contacting said recombinant cell with a ligand agonist of said FPRL-1 receptor, said ligand agonist comprising a polypeptide, or analog thereof, to permit said ligand agonist to bind to and activate said FPRL-1 receptor, thereby activating the signal transduction activity of said FPRL-1 receptor and generating a detectable signal;
contacting said cell with a test compound; and
detecting an alteration in said signal generated by said ligand agonist to thereby identify a modulator of said receptor. - View Dependent Claims (124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151)
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152. A method for identifying a modulator of a heterologous formyl peptide receptor like-1 (FPRL-1) receptor expressed in the membrane of a cell, said method comprising:
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contacting said cell with a a ligand agonist of said FPRL-1 receptor, said ligand agonist comprising a polypeptide, or analog thereof, in the presence of a test compound under conditions to permit binding of said ligand agonist to said receptor; and
determining the inhibition by said test compound of binding of said ligand agonist to said receptor, by assessing the amount of said ligand agonist bound to said receptor, such that reduction of binding of said ligand agonist to said receptor identifies said test compound as a modulator of said receptor. - View Dependent Claims (153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170)
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171. A method for identifying a modulator of a heterologous formyl peptide receptor like-1 (FPRL-1) receptor expressed by a cell, comprising:
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providing a first mixture of recombinant cells, each cell of which has a cell membrane and comprises;
a heterologous FPRL-1 receptor expressed in the cell membrane of said cell such that signal transduction activity via said receptor is modulated by interaction of an extracellular region of the receptor with an extracellular signal, said heterologous FPRL-1 receptor acting as a surrogate for an endogenous cell receptor in a signal transduction pathway of the cell; and
contacting said first mixture of recombinant cells with a ligand agonist of said FPRL-1 receptor comprising a polypeptide, or analog thereof, to permit said ligand agonist to bind to and activate said FPRL-1 receptor, thereby activating the signal transduction activity of said FPRL-1 receptor and generating a detectable signal;
contacting said first mixture with a second mixture of cells, wherein collectively the second mixture of cells expresses a library of heterologous test polypeptides that are transported to a location allowing interaction with the extracellular region of the FPRL-1 receptor expressed in the cell membrane of the cells of the first mixture; and
detecting an alteration in said signal generated by said ligand agonist to thereby identify a modulator of said receptor. - View Dependent Claims (172, 173, 174)
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175. A ligand agonist of formyl peptide receptor like-1 (FPRL-1) receptor comprising a polypeptide or analog thereof, said polypeptide comprising from 3 to 80 amino acid residues, wherein said ligand agonist binds to and activates said FPRL-1 receptor, and has an EC50 of 2×
- 10−
5 M or less.
- 10−
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176. A ligand agonist of formyl peptide receptor like-1 receptor comprising the amino acid sequence of SEQ ID NO:
- 1 or analog thereof.
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177. A ligand agonist of formyl peptide receptor like-1 receptor comprising the amino acid sequence of SEQ ID NO:
- 2 or analog thereof.
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178. A ligand agonist of formyl peptide receptor like-1 receptor comprising the amino acid sequence of SEQ ID NO:
- 3 or analog thereof.
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179. A ligand agonist of formyl peptide receptor like-1 receptor comprising the amino acid sequence of SEQ ID NO:
- 4 or analog thereof.
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180. A ligand agonist of formyl peptide receptor like-1 receptor comprising the amino acid sequence of SEQ ID NO:
- 5 or analog thereof.
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181. A ligand agonist of formyl peptide receptor like-1 receptor comprising the amino acid sequence of SEQ ID NO:
- 6 or analog thereof.
Specification