APPROACH TO THE DISCOVERY OF NOVEL, SELECTIVE INHIBITORS OF P56(LCK) TYROSINE KINASE - IDENTIFICATION OF NON-HYDROXYLATED CHROMONES AS P56(LCK) INHIBITORS

Abstract

The protein tyrosine kinase p56(lck), which is expressed predominantly in lymphocytes, plays a critical role in optimal T cell activation through the T cell antigen receptor. An approach is presented for the discovery of selective p56(lck) inhibitors, which are potential immunosuppressants. A non-radioactive assay for p56(lck) tyrosine kinase activity has been developed and adapted for high volume screening. This assay does not require purified enzyme. p56(lck) in the plasma membranes of a human T cell line is purified in situ by immobilization onto the wells of a microtiter plate using an antibody specific for p56(lck). Following the kinase reaction in the presence of test compound, autophosphorylated p56(lck) is detected with a biotinylated monoclonal antibody to phosphotyrosine. Using the approach described in this report, three simple chromones have been identified that inhibit p56(lck) autophosphorylation with low micromolar potencies and exhibit some selectivity fdr p56(lck) over the serine/threonine and other tyrosine kinases tested. These compounds constitute a novel group of p56(lck) tyrosine kinase inhibitors. (C) 1995 Wiley-Liss, Inc.