Panek R L, Lu G H, Dahring T K, Batley B L, Connolly C, Hamby J M, Brown K J
Department of Vascular and Cardiac Diseases, Parke-Davis Pharmaceutical Research, Division of Warner-Lambert Company, Ann Arbor, Michigan, USA.
J Pharmacol Exp Ther. 1998 Jul;286(1):569-77.
Through direct synthetic efforts, we discovered a small molecule that is a nanomolar inhibitor of the human fibroblast growth factor-1 receptor (FGFR) tyrosine kinase. PD 166866, a member of a new structural class of tyrosine kinase inhibitors, the 6-aryl-pyrido[2,3-d]pyrimidines, was identified by screening a compound library with assays that measure protein tyrosine kinase activity. PD 166866 inhibited human full-length FGFR-1 tyrosine kinase with an IC50 value of 52.4 +/- 0.1 nM and was further characterized as an ATP competitive inhibitor of the FGFR-1. In contrast, PD 166866 had no effect on c-Src, platelet-derived growth factor receptor-beta, epidermal growth factor receptor or insulin receptor tyrosine kinases or on mitogen-activated protein kinase, protein kinase C and CDK4 at concentrations as high as 50 microM. PD 166866 was a potent inhibitor of basic fibroblast growth factor (bFGF)-mediated receptor autophosphorylation in NIH 3T3 cells expressing endogenous FGFR-1 and in L6 cells overexpressing the human FGFR-1 tyrosine kinase, confirming a tyrosine kinase-mediated mechanism. PD 166866 also inhibited bFGF-induced tyrosine phosphorylation of the 44- and 42-kDa (ERK 1/2) mitogen-activated protein kinase isoforms in L6 cells, presumably via inhibition of bFGF-stimulated FGFR-1 tyrosine kinase activation. PD 166866 did not inhibit platelet-derived growth factor, epidermal growth factor or insulin-stimulated receptor autophosphorylation in vascular smooth muscle, A431 or NIHIR cells, respectively, further supporting its specificity for the FGFR-1. In addition, daily exposure of PD 166866 to L6 cells at concentrations from 1 to 100 nM resulted in a concentration-related inhibition of bFGF-stimulated cell growth for 8 consecutive days with an IC50 value of 24 nM. In contrast, PD 166866 had little effect on platelet-derived growth factor-BB-stimulated growth of L6 cells or serum-stimulated vascular smooth muscle cell proliferation. Finally, PD 166866 was found to be a potent inhibitor of microvessel outgrowth (angiogenesis) from cultured artery fragments of human placenta. These results highlight the discovery of PD 166866, a new nanomolar potent and selective small molecule inhibitor of the FGFR-1 tyrosine kinase with potential use as antiproliferative/antiangiogenic agent for such therapeutic targets as tumor growth and neovascularization of atherosclerotic plaques.
通过直接的合成研究，我们发现了一种小分子，它是人类成纤维细胞生长因子-1受体（FGFR）酪氨酸激酶的纳摩尔级抑制剂。PD 166866是一种新型结构类别的酪氨酸激酶抑制剂（6-芳基-吡啶并[2,3-d]嘧啶）的成员，通过使用测量蛋白质酪氨酸激酶活性的检测方法筛选化合物文库而得以鉴定。PD 166866抑制人类全长FGFR-1酪氨酸激酶，IC50值为52.4±0.1 nM，并被进一步表征为FGFR-1的ATP竞争性抑制剂。相比之下，在浓度高达50 μM时，PD 166866对c-Src、血小板衍生生长因子受体-β、表皮生长因子受体或胰岛素受体酪氨酸激酶以及对丝裂原活化蛋白激酶、蛋白激酶C和CDK4均无影响。PD 166866是表达内源性FGFR-1的NIH 3T3细胞以及过表达人类FGFR-1酪氨酸激酶的L6细胞中碱性成纤维细胞生长因子（bFGF）介导的受体自磷酸化的有效抑制剂，证实了酪氨酸激酶介导的机制。PD 166866还抑制L6细胞中bFGF诱导的44 kDa和42 kDa（ERK 1/2）丝裂原活化蛋白激酶同工型的酪氨酸磷酸化，推测是通过抑制bFGF刺激的FGFR-1酪氨酸激酶活化。PD 166866分别不抑制血管平滑肌、A431或NIHIR细胞中血小板衍生生长因子、表皮生长因子或胰岛素刺激的受体自磷酸化，进一步支持了其对FGFR-1的特异性。此外，以1至100 nM的浓度将PD 166866每日暴露于L6细胞，连续8天导致bFGF刺激的细胞生长呈浓度相关的抑制，IC50值为24 nM。相比之下，PD 166866对血小板衍生生长因子-BB刺激的L6细胞生长或血清刺激的血管平滑肌细胞增殖影响很小。最后，发现PD 166866是培养人皮胎盘动脉片段微血管生长（血管生成） 的有效抑制剂。这些结果突出了PD 166866的发现，它是一种新型的纳摩尔级强效且选择性的FGFR-1酪氨酸激酶小分子抑制剂，具有作为抗增殖/抗血管生成剂用于肿瘤生长和动脉粥样硬化斑块新生血管形成等治疗靶点的潜在用途。
