Dahring T K, Lu G H, Hamby J M, Batley B L, Kraker A J, Panek R L
Department of Vascular and Cardiac Diseases, Parke-Davis Pharmaceutical Research, Division of Warner-Lambert Company, Ann Arbor, Michigan 48105, USA.
J Pharmacol Exp Ther. 1997 Jun;281(3):1446-56.
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.
PD 089828是一种新型蛋白酪氨酸激酶抑制剂，属于新结构类型的6-芳基吡啶并[2,3-d]嘧啶类。通过用测量蛋白酪氨酸激酶活性的检测方法筛选化合物文库，鉴定出了PD 089828。发现PD 089828能抑制人全长成纤维细胞生长因子（FGF）受体-1（FGFR-1）、血小板衍生生长因子（PDGF）受体β亚基（PDGFR-β）、Src非受体酪氨酸激酶（c-Src）和表皮生长因子（EGF）受体（EGFR）酪氨酸激酶，其半数最大抑制浓度（IC50值）分别为0.15±0.02（n = 4）、0.18±0.04（n = 3）、1.76±0.28（n = 4）和5.47±0.78（n = 6）μM。PD 089828进一步被表征为生长因子受体酪氨酸激酶（FGFR-1、PDGFR-β和EGFR）的ATP竞争性抑制剂，但对c-Src酪氨酸激酶而言是ATP非竞争性抑制剂。此外，PD 089828抑制血管平滑肌细胞（VSMC）中PDGF和EGF刺激的受体自磷酸化以及A121细胞中碱性FGF介导的酪氨酸磷酸化，其IC50值与抑制受体酪氨酸激酶活性时观察到的效力相似。PD 089828对VSMC中PDGF受体自磷酸化的抑制作用迅速，在药物暴露后5分钟内达到最大效应。单次暴露后的抑制作用持久但也可迅速逆转，在药物去除后5分钟内发生。由于PD 089828抑制了PDGF刺激的受体自磷酸化，PDGF诱导的下游信号蛋白，包括磷脂酰肌醇-3-激酶（PI-3K）、生长因子受体结合蛋白-2（GRB2）、SH-2结构域和类胶原（Shc）以及磷脂酶Cγ（PLCγ）与VSMC PDGF受体的结合也被阻断。PD 089828还抑制了PDGF诱导的44 kDa和42 kDa丝裂原活化蛋白激酶同工型的酪氨酸磷酸化。此外，在功能检测中证实了PD 089828的作用，其中PDGF刺激的DNA合成、PDGF引导的迁移以及VSMC的血清刺激生长均受到抑制，抑制程度与PDGF受体自磷酸化相同（IC50分别为0.8、4.5和1.8 μM）。这些结果突出了PD 089828作为一种新型、具有广泛活性的蛋白酪氨酸激酶抑制剂的生物学特性，其具有持久但可逆的细胞效应。这些广泛作用的非选择性抑制剂作为抗增殖和抗迁移剂的潜在治疗用途可能扩展到癌症、动脉粥样硬化和再狭窄等疾病，在这些疾病中已知存在生长信号通路的冗余。
