A radioactive binding assay for inhibitors of trkA kinase.
The high-affinity receptor for nerve growth factor (NGF), trkA, is a receptor-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 differentiation of these cells. On the other hand, the activation of trkA by NGF in other cells has been implicated in several pathologies including inflammation-induced hyperalgesia and several cancers. A radioactive binding assay to evaluate inhibitors of the kinase domain of trkA has been developed and validated. The assay monitors the specific binding of an inhibitor of trkA kinase activity, the indolocarbazole K-252a, to the trkA receptor. [3H]K-252a binds with high affinity to one site on the cytoplasmic kinase domain of the trkA receptor. Binding is saturable and reversible with a dissociation constant (Kd) of 1.5 nM. The binding assay has been used in competition binding experiments to determine the inhibition constants for other indolocarbazole compounds. The IC50 values for compounds obtained in the binding assay correlate very well with the IC50 values obtained in an enzyme-linked immunosorbent assay for trkA tyrosine kinase activity.
Knight
E
E
Cephalon, Inc., West Chester, Pennsylvania 19380, USA.
Connors
T J
TJ
Maroney
A C
AC
Angeles
T S
TS
Hudkins
R L
RL
Dionne
C A
CA
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002227:Carbazoles / Q000032:analysis* / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000032:analysis* / Q000378:metabolism; D005069:Evaluation Studies as Topic; D006801:Humans; D066298:In Vitro Techniques; D026121:Indole Alkaloids; D007700:Kinetics; D011518:Proto-Oncogene Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D011869:Radioligand Assay / Q000379:methods*; D020794:Receptor Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D020917:Receptor, trkA; D017475:Receptors, Nerve Growth Factor / Q000037:antagonists & inhibitors* / Q000378:metabolism
D002227:Carbazoles; D004791:Enzyme Inhibitors; D026121:Indole Alkaloids; D011518:Proto-Oncogene Proteins; D017475:Receptors, Nerve Growth Factor; D000255:Adenosine Triphosphate; C049985:staurosporine aglycone; D020794:Receptor Protein-Tyrosine Kinases; D020917:Receptor, trkA