Cell-based assay for screening cox-2 inhibitors
View Patent ↗A cell-based assay is provided for screening substances for the capacity to inhibit COX-2 activity. The assay utilizes as a screening reagent a cell line which has been engineered to achieve constitutive or inducible expression of COX-2. In one embodiment, the cells express a GTPase-deficient, constitutively-activated mutant form of the α-subunit of protein G12 which induces the production of arachidonic acid and COX-2. Decreased cell proliferation in the presence of test substance indicates that the substance has COX-2 inhibitory activity.
1. A method for screening a test substance for COX-2 inhibitory activity comprising:
(a) contacting the test substance with indicator cells which express a GTPase-deficient mutant form of the α-subunit of protein G12, wherein the G12 protein α-subunit mutant comprises the Q229L mutation, which mutant α-subunit has the capacity to induce the production of arachidonic acid in the indicator cells, and induces production of COX-2 in the indicator cells; and
(b) determining the level of one or more prostaglandins produced by the indicator cells in the presence and absence of the test substance, a decrease in production of said prostaglandin by the indicator cells in the presence of the test substance indicating that the test substance has COX-2 inhibitory activity.
2. A method according to claim 1 wherein the prostaglandin level is assayed in the media surrounding the indicator cells, in the indicator cells or in a component of the indicator cells.
3. A method according to claim 1 wherein the prostaglandin level is assayed by a prostaglandin immunoassay.
4. A method according to claim 1 wherein the prostaglandin immunoassay comprises a competitive immunoassay.