Inhibition of growth factor-mediated tyrosine phosphorylation in vascular smooth muscle by PD 089828, a new synthetic protein tyrosine kinase inhibitor.
PD 089828, a novel protein tyrosine kinase inhibitor of a new structural class, the 6-aryl-pyrido-[2,3-d]pyrimidines, was identified by screening a compound library with assays that measured protein tyrosine kinase activity. PD 089828 was found to inhibit human full-length fibroblast growth factor (FGF) receptor-1 (FGFR-1), platelet-derived growth factor (PDGF) receptor beta subunit (PDGFR-beta), Src nonreceptor tyrosine kinase (c-Src) and epidermal growth factor (EGF) receptor (EGFR) tyrosine kinases with half-maximal inhibitory potencies (IC50 values) of 0.15 +/- 0.02 (n = 4), 0.18 +/- 0.04 (n = 3), 1.76 +/- 0.28 (n = 4) and 5.47 +/- 0.78 (n = 6) microM, respectively. PD 089828 was further characterized as an ATP competitive inhibitor of the growth factor receptor tyrosine kinases (FGFR-1, PDGFR-beta and EGFR) but a noncompetitive inhibitor of c-Src tyrosine kinase with respect to ATP. In addition, PD 089828 inhibited PDGF- and EGF-stimulated receptor autophosphorylation in vascular SMC (VSMC) and basic FGF-mediated tyrosine phosphorylation in A121 cells with IC50 values similar to the potencies observed for inhibition of receptor tyrosine kinase activity. The inhibition of PDGF receptor autophosphorylation in VSMC by PD 089828 occurred rapidly, with maximal effects reached within 5 min of drug exposure. Inhibition after single exposure was long lasting but also rapidly reversible, occurring within 5 min after drug removal. The PDGF-induced association of downstream signaling proteins, including phosphoinositide-3-kinase (PI-3K), growth factor receptor binding protein-2 (GRB2), SH-2 domain and collagen like (Shc) and phospholipase Cgamma (PLCgamma), with VSMC PDGF receptors was also blocked as a result of the inhibition of PDGF-stimulated receptor autophosphorylation by PD 089828. PD 089828 also inhibited the PDGF-induced tyrosine phosphorylation of the 44- and 42-kDa mitogen-activated protein kinase isoforms. Moreover, the effects of PD 089828 were demonstrated in functional assays in which PDGF-stimulated DNA synthesis, PDGF-directed migration and serum-stimulated growth of VSMC were all inhibited to the same extent as PDGF receptor autophosphorylation (IC50 = 0.8, 4.5 and 1.8 microM, respectively). These results highlight the biological characteristics of PD 089828 as a novel, broadly active protein tyrosine kinase inhibitor with long-lasting but reversible cellular effects. The potential therapeutic use of these broadly acting, nonselective inhibitors as antiproliferative and antimigratory agents could extend to such diseases as cancer, atherosclerosis and restenosis in which redundancies in growth-signaling pathways are known to exist.
Dahring
T K
TK
Department of Vascular and Cardiac Diseases, Parke-Davis Pharmaceutical Research, Division of Warner-Lambert Company, Ann Arbor, Michigan 48105, USA.
Lu
G H
GH
Hamby
J M
JM
Batley
B L
BL
Kraker
A J
AJ
Panek
R L
RL
D000818:Animals; D001011:Aorta / Q000187:drug effects*; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006133:Growth Substances / Q000494:pharmacology*; D006801:Humans; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000187:drug effects*; D010766:Phosphorylation / Q000187:drug effects; D011505:Protein-Tyrosine Kinases / Q000187:drug effects*; D011725:Pyridines / Q000494:pharmacology*; D011743:Pyrimidines / Q000494:pharmacology*; D051381:Rats; D017207:Rats, Sprague-Dawley
D004791:Enzyme Inhibitors; D006133:Growth Substances; C106717:PD 089828; D011725:Pyridines; D011743:Pyrimidines; D011505:Protein-Tyrosine Kinases