Methods of identifying modulators of prostaglandin receptor EP1
AssigneesMerck Frosst Canada, Inc., Vanderbuilt University
InventorsAnthony Ford-Hutchinson, Colin Funk, Richard Grygorczyk, Kathleen Metters
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Abstract
A novel prostaglandin receptor has been identified and DNA encoding the receptor has been isolated, purified, sequenced and expressed in host cells. This DNA encoding the novel prostaglandin receptor and host cells expressing the receptor are used to identify modulators of the prostaglandin receptor.
Claims26
A method of determining whether a test compound modulates a signal transduction activity of a prostaglandin receptor EP1, comprising:
- a) culturing EP1-receptor-expressing host cells under conditions that would allow expression of a recombinant prostaglandin receptor, said host cells being transfected with a nucleic acid molecule encoding said recombinant prostaglandin EP1-receptor comprising the amino acid sequence as set forth in SEQ ID NO:4:
- b) exposing the EP1 receptor-expressing host cells of step a) to the test compound;
- c) exposing control host cells to the test compound of step b), wherein said control host cells do not express recombinant prostaglandin EP1 receptor protein;
- d) measuring the modulating affect of the test compound which interacts with the recombinant EP1 receptor from the host cells of step a) and control host cells of step c);
- and,
- e) comparing the modulating affect of the test compound on the host cells and control host cells.
A method of determining whether a test compound modulates a signal transduction activity of a prostaglandin receptor EP1, comprising:
- a) culturing EP1-receptor-expressing host cells under conditions that would allow expression of a recombinant prostaglandin receptor, said host cells being transfected with a nucleic acid molecule encoding said recombinant prostaglandin EP1-receptor comprising the amino acid sequence as set forth in SEQ ID NO:4;
- b) exposing a first population of EP1 receptor-expressing cells of step a) to a known prostaglandin EP1 receptor ligand;
- c) exposing a second population of EP1 receptor-expressing cells of step a) to the test compound and the known prostaglandin EP1-receptor ligand of step b);
- d) exposing control host cells to the test compound of step c), wherein said control host cells do not express recombinant prostaglandin EP1 receptor protein;
- e) measuring the modulating affect of the test compound, which interacts with recombinant EP1 protein, in the presence and absence of the known EP1 receptor ligand, from the cells of step b), step c) and step d);
- and,
- f) comparing the modulating affect of the test compound as determined from step b), step c) and step d.
A method of determining whether a test compound competes with a known prostaglandin EP1 receptor ligand for binding to a prostaglandin EP1 receptor protein, comprising:
- a) culturing EP1-receptor-expressing host cells under conditions that would allow expression of a recombinant prostaglandin receptor, said host cells being transfected with a nucleic acid molecule encoding said recombinant prostaglandin EP1-receptor comprising the amino acid sequence as set forth in SEQ ID NO:4;
- b) isolating a substantially purified cell membrane preparation from the EP1 receptor-expressing cells of step a);
- c) exposing a first population of the purified cell membrane preparation of step b) to a known, labeled prostaglandin EP1 receptor ligand;
- d) exposing a second population of the purified cell membrane preparation of step b) to the test compound and the known, labeled prostaglandin EP1 receptor ligand;
- e) determining the binding of the known, labeled prostaglandin EP1 receptor ligand to the membrane preparations of step c) and step d);
- and,
- f) comparing the binding of the known, labeled prostaglandin EP1 receptor ligand protein to the membrane preparations:of step c) and step d).
A method of determining whether a test compound competes with a known prostaglandin EP1 receptor ligand for binding to a prostaglandin EP1 receptor protein, comprising:
- a) culturing EP1-receptor-expressing host cells under conditions that would allow expression of a recombinant prostaglandin receptor, said host cells being transfected with a nucleic acid molecule encoding said recombinant prostaglandin EP1-receptor comprising the amino acid sequence as set forth in SEQ ID NO:4;
- b) lysing the EP1 receptor expressing host cells of step a);
- c) exposing a first population of the lysed cells of step b) to a known, labeled prostaglandin EP1 receptor ligand;
- d) exposing a second population of the lysed cells of step b) to the test compound and the known, labeled prostaglandin EP1 receptor ligand;
- e) determining the binding of the known, labeled prostaglandin EP1 receptor ligand to the EP1 receptor from the lysed cells of step c) and step d);
- and,
- f) comparing the binding of the known, labeled prostaglandin EP1 receptor ligand protein to the EP1 receptor from the lysed cells of step c) and step d).
A method of determining whether a test compound competes with a known prostaglandin EP1 receptor ligand for binding to a prostaglandin EP1 receptor protein, comprising:
- a) culturing EP1-receptor-expressing host cells under conditions that would allow expression of a recombinant prostaglandin receptor, said host cells being transfected with a nucleic acid molecule encoding said recombinant prostaglandin EP1-receptor comprising the amino acid sequence as set forth in SEQ ID NO:4;
- b) exposing a first population of EP1 receptor-expressing cells of step a) to a known, labeled prostaglandin EP1 receptor ligand;
- c) exposing a second population of EP1 receptor-expressing cells of step a) to the compound and the known, labeled prostaglandin EP1-receptor ligand of step b);
- d) determining the binding of the known, labeled prostaglandin EP1 receptor ligand to the membrane preparations of step b) and step c);
- and,
- e) comparing the binding of the known, labeled prostaglandin EP1 receptor ligand protein to the membrane preparations of step b) and step c).
Dependencies are inferred from the claim text.