A RADIOACTIVE BINDING ASSAY FOR INHIBITORS OF TRKA KINASE

The high-affinity receptor for nerve growth factor (NGF), trkA, is a r eceptor-linked tyrosine kinase. The binding of NGF to trkA, depending on the context of its environment, can cause beneficial or deleterious responses in the target cells. For example, the activation of trkA in sympathetic and sensory neurons causes the subsequent survival and di fferentiation of these cells. On the other hand, the activation of trk A by NGF in other cells has been implicated in several pathologies inc luding inflammation-induced hyperalgesia and several cancers. A radioa ctive binding assay to evaluate inhibitors of the kinase domain of trk A has been developed and validated. The assay monitors the specific bi nding of an inhibitor of trkA kinase activity, the indolocarbazole K-2 52a, to the trkA receptor. [H-3]K-252a binds with high affinity to one site on the cytoplasmic kinase domain of the trKA receptor. Binding i s saturable and reversible with a dissociation constant (K-d) of 1.5 n M. The binding assay has been used in competition binding experiments to determine the inhibition constants for other indolocarbazole compou nds. The IC50 values for compounds obtained in the binding assay corre late very well with the IC50 values obtained in an enzyme-linked immun osorbent assay for trkA tyrosine kinase activity. (C) 1997 Academic Pr ess.