PF 477736 (PF-736,PF-00477736) is a specifc, effective and ATP-competitive Chk1 inhibitor (Ki: 0.49 nM ) and also inhibits FGFR3, Aurora-A, VEGFR2, Flt3, Fms (CSF1R), Ret and Yes.

Chk2:47 nM(Ki), Chk1:0.49 nM(Ki), YES:14 nM(Ki), FMS:10 nM(Ki), VEGFR2:8 nM(Ki)

PF-477736 (128 nM) 以剂量依赖性方式消除了CA46及HeLa细胞中由喜树碱引起的DNA损伤检查点。PF-477736 在HT29细胞中有效地消除了由吉西他滨引起的S期阻滞，并相应增加了凋亡细胞群体。PF-477736 (540 nM) 以时间和剂量依赖的方式增强了HT29细胞中吉西他滨引起的细胞毒性。PF-477736 增强了一系列化疗化合物在MTT检测中对广泛p53缺陷的人类癌症细胞系的生长抑制活性。PF-477736 (360 nM) 加入到吉西他滨停滞的细胞中，引起H2AX磷酸化强度的显著增加，反映了在DNA损伤附近γ-H2AX分子数量的增加。PF-477736 (0.5 nM) 在HL-60细胞中存在姜黄素时选择性阻断p73和P53的磷酸化。PF-477736 (360 nM) 抑制了COLO205细胞中由多西他赛引起的组蛋白H3 (Ser10) 和Cdc25C (Ser216) 的磷酸化，并增强了凋亡。PF-477736 (250 nM) 与MK-1775联合使用在OVCAR-5细胞中显示出显著的协同细胞毒作用，并导致OVCAR-5细胞中含有2N至4N之间DNA含量的细胞积累，以及DNA损伤导致的凋亡细胞死亡。

PF-477736 (4 mg/kg i.v.) 在大鼠体内的半衰期（T1/2）为2.9小时，AUC为5.72 μg×hr/mL，CLp为11.8 mL/min/kg。PF-477736 剂量依赖性地增强了在Colo205异种移植小鼠模型中最大耐受剂量的gemcitabine的抗肿瘤活性。PF-477736 (12 mg/kg) 在Colo205异种移植小鼠模型中诱导了组蛋白H3（Ser10）和磷酸化组蛋白H2AX的磷酸化增加。[1] PF-477736 (15 mg/kg i.p.) 在COLO205和MDA-MB-231异种移植模型中增强了docetaxel诱导的肿瘤生长抑制和肿瘤生长延迟。[3] PF 477736（10 mg/kg 每天一次 i.p.）与MK-1775（30 mg/kg 每天两次口服）联用在携带OVCAR-5异种移植物的小鼠中实现了更大的肿瘤生长抑制。[4]

Binding assay: The assay is performed in a 96-well plate for 20 minutes at 30℃ in 0.1 mL of assay buffer containing 50 mM TRIS pH 7.5, 0.4 M NaCl, 4 mM PEP, 0.15 mM NADH, 28 units of lactate dehydrogenase/mL, 16 units of pyruvate kinase/mL, 3 mM DTT, 0.125 mM Syntide-2, 0.15 mM ATP and 25 mM magnesium chloride. Assays are initiated with 1 nM of CHK1 kinase domain. The inhibition of CHK1 activity is determined by measuring initial velocities in the presence of varying concentrations of PF-477736. The data is analyzed using Enzyme Kinetic and Excel software and fit to a kinetic model for competitive inhibition to obtain a Ki value. The kinase selectivity of PF-477736 is evaluated by screening the compound at 1 μM or 10 μM against a panel 2 of about 100 protein kinases.

The IC50 assay measures the antiproliferative effects of PF-477736 on p53-defective human cancer cell lines. Cells in each line are seeded in complete medium at an exponentially growing density in 96-well assay plate and allowed to attach for 16 hours. Serial dilutions of PF-477736 are then done, and appropriate controls are added to each plate. Cells are incubated with drug for 96 hours. After incubation, MTT working stock diluted in complete medium is added to each well, and cells are incubated for 4 hours. After centrifugation and supernatant removal, DMSO is added to each well and plates are read on SpectraMax plate reader at 540 nm. (Only for Reference)