IN-VITRO PHARMACOLOGICAL CHARACTERIZATION OF PD-166285, A NEW NANOMOLAR POTENT AND BROADLY ACTIVE PROTEIN-TYROSINE KINASE INHIBITOR

PD 166285, a novel protein tyrosine kinase inhibitor of a new structur al class, the 6-aryl-pyrido[2,3-d]pyrimidines, was synthesized as the most potent and soluble analog of a series of small molecules original ly identified by screening a compound library with assays that measure d protein tyrosine kinase activity. PD 166285 was found to inhibit Src nonreceptor tyrosine kinase, fibroblast growth factor receptor-1, epi dermal growth factor receptor and platelet-derived growth factor recep tor beta subunit (PDGFR-beta), tyrosine kinases with half-maximal inhi bitory potencies (IC50 values) of 8.4 +/- 2.3 nM (n = 6), 39.3 +/- 2.8 nM (n = 16), 87.5 +/- 13.7 nM (n = 6) and 98.3 +/- 7.9 nM (n = 16), r espectively. PD 166285 also demonstrated inhibitory activity against m itogen-activated protein kinase (IC50 = 5 mu M) and protein kinase C ( IC50 = 22.7 mu M) PD 166285 was further characterized as an ATP compet itive inhibitor of Src nonreceptor tyrosine kinase, PDGFR-beta, fibrob last growth factor receptor-1 and epidermal growth factor receptor tyr osine kinases. In addition, PD 166285 inhibited PDGF- and EGF-stimulat ed receptor autophosphorylation in vascular smooth muscle cells (VSMCs ) and A431 cells, respectively, and basic fibroblast growth factor-med iated tyrosine phosphorylation in Sf9 cells, with IC50 values of 6.5 n M, 1.6 mu M and 97.3 nM, respectively, further establishing a tyrosine kinase mechanism of inhibition. The inhibition of PDGF receptor autop hosphorylation in VSMCs by PD 166285 was long lasting and persisted fo r 4 days after a single I-hr exposure followed by extensive washing. T he PDGF-induced tyrosine phosphorylation of the 44- and 42-kDa mitogen -activated protein kinase isoforms was also blocked as a result of the inhibition of PDGF-stimulated receptor autophosphorylation by PD 1662 85 in VSMCs. The effects of PD 166285 were also demonstrated in functi onal assays of cell attachment, migration and proliferation, in which vascular cell adhesion to vitronectin, PDGF-directed chemotaxis and se rum-stimulated cell growth were all potently inhibited with IC50 value s of 80 yo 120 nM. Finally, PD 166285 uniquely demonstrated potent inh ibition of phorbol ester-induced production of 92-kDa gelatinase A (MM P-2) in VSMC without affecting 72-kDa gelatinase B (MMP-2) as measured by gelatin zymography. These results highlight the biological charact eristics of PD 166285 as a broadly active protein tyrosine kinase capa ble of potently inhibiting a number of kinase mediated cellular functi ons, including cell attachment, movement and replication. The potentia l therapeutic utility of this broadly acting inhibitor as an antiproli ferative and antimigratory agent could extend to such diseases as canc er, atherosclerosis and restenosis, in which redundancies in protein k inase signaling pathways are known to exist.