Indolocarbazoles: Potent and Selective Inhibitors of Platelet-Derived Growth Factor Receptor Autophosphorylation

Abstract

A quantitative assay for measuring the autophosphorylation of platelet-derived growth factor (PDGF) receptors in intact vascular smooth muscle cells has been developed and used to screen for novel tyrosine kinase (TK) inhibitors. Several novel inhibitors of PDGF receptor autophosphorylation have been identified from the indolocarbazole series, including the 3,9 dimethoxy derivative, 3744W ( ic 50 = 14.5 ± 2 nM). Tested against a panel of tyrosine and serine/threonine kinases, 3744W is at least 1,000 fold selective for the PDGF receptor tyrosine kinase and was found to inhibit autophosphorylation of both the α and β isoforms of the PDGF receptor in human smooth muscle cells. PDGF-BB-stimulated DNA synthesis in quiescent cultures of human smooth muscle cells was blocked in a concentration-dependent manner by 3744W, ic 50 = 10 nM. Binding studies showed that 3744W did not block the binding of PDGF-BB to cell surface receptors on human airway smooth muscle cells. Furthermore, inhibition of bone marrow stem cell proliferation by 3744W was only observed at concentrations 100–1,000 times greater than those needed to block PDGF-driven DNA synthesis in human smooth muscle cells. 3744W represents a novel, potent and selective inhibitor of PDGF receptor autophosphorylation and a powerful biochemical probe for investigating PDGF-dependent responses in vitro.