IN-VITRO CHARACTERIZATION OF A NOVEL SERIES OF PLATELET-DERIVED GROWTH-FACTOR RECEPTOR TYROSINE KINASE INHIBITORS

In this report, we describe the discovery and characterization of a no vel biarylhydrazone series of platelet-derived growth factor (PDGF) re ceptor tyrosine kinase inhibitors typified by the prototype WIN 41662 (3-phenyl-N-1-[1-(4-pyridyl)pyrimidine]hydrazone. WIN 41662 inhibited PDGF-stimulated autophosphorylation of PDGF receptors from human vascu lar smooth muscle cells (hVSMC) with an IC50, value of 60 nM. The inhi bitor appeared to be competitive with respect to substrate (Mn2+-ATP), having a calculated K-i of 15 +/- 5 nM. WIN 41662 was approximately 5 00-fold more potent in inhibiting the PDGF receptor tyrosine kinase th an the p56(kk) tyrosine kinase. It was inactive against other serine/t hreonine and tyrosine kinases tested. WIN 41662 produced concentration -dependent inhibition of PDGF stimulated receptor autophosphorylation in intact hVSMC with an IC50 < 100 nM. Intracellular Ca2+ mobilization and cell proliferation were events that occurred in hVSMC subsequent to PDGF receptor activation. WIN 41662 inhibited PDGF-stimulated Ca2mobilization and cell proliferation ([H-3]TdR incorporation) with Ic(5 0) values of 430 nM and 2.3 mu M, respectively. These effects appeared to be specifically related to PDGF receptor tyrosine kinase inhibitio n since WIN 41662 was not cytotoxic (in vitro) and since WIN 72039, a close structural analog that does not inhibit PDGF receptor tyrosine k inase, also did not inhibit PDGF-stimulated receptor autophosphorylati on, Ca2+ mobilization, or hVSMC proliferation. Thus, WIN 41662 is repr esentative of a novel class of selective PDGF receptor tyrosine kinase inhibitors that inhibit PDGF-regulated secondary events in intact cel ls.