PF-03814735 is a novel, potent and reversible inhibitor of Aurora A/B with IC50of 0.8 nM/5 nM, is less potent to Flt3, FAK, TrkA, and minimally active to Met and FGFR1. Phase 1.

Aurora A:0.8 nM, Aurora B:5 nM

在完整细胞中，PF-03814735对Aurora1和Aurora2激酶的抑制作用降低了磷酸化Aurora1（Thr 232，Aurora1活性的敏感标志，IC50约为20 nM）、磷酸化组蛋白H3（IC50约为50 nM）和磷酸化Aurora2（IC50约为150 nM）的水平。PF-03814735引发细胞分裂阻塞，导致细胞增殖受抑和多倍体多核细胞的形成。[1] 最近的研究表明，小细胞肺癌（SCLC）和在较小程度上的结肠癌细胞线对PF-03814735特别敏感。Myc基因家族的状态和视网膜母细胞瘤途径成员的表达与PF-03814735的效力显著相关。[1]

每日一次口服PF-03814735，剂量≥20 mg/kg，连续10天给予携带HCT-116异种移植瘤的小鼠治疗，能够与对照组（车辆处理的小鼠）相比，显著并且剂量依赖性地抑制肿瘤生长≥50%。这种抑制与磷酸化组蛋白H3水平的降低相关。在包括A2780卵巢癌、MDA-MB-231乳腺癌、colo-205和SW620结直肠癌、以及HL-60急性早幼粒细胞性白血病等五种额外的异种移植瘤模型中观察到显著的单药抗肿瘤效果。[1] 两种SCLC异种移植模型的体内实验确认了Myc基因驱动模型对PF-03814735的敏感性以及MYC/c-Myc驱动肿瘤可能的时间依赖性。[1]

Recombinant Kinase Assays: Aurora1 and Aurora2 proteins are produced as full-length His-tag recombinant proteins expressed in insect cells. For the Aurora2 kinase assay, phosphorylation of the substrate peptide by recombinant Aurora2 protein is assessed by a Z'-LYTE assay at 3 to 300 μM ATP and various concentrations of PF-03814735 over 60 minutes, at a substrate peptide concentration of 2 μM (biotinylated LRRWSLG, ×4). Phosphorylation is linear over this time for all conditions. For the Aurora1 kinase assay, phosphorylation of the substrate peptide by recombinant Aurora1 protein is assessed by a scintillation proximity assay in a 96-well plate format in which the incorporation of 33P into the peptide substrate (biotinylated LRRWSLG, ×4) is measured by capturing the peptide on a streptavidin scintillation proximity assay bead.

Cell lines are grown in appropriate media and evaluated after 48 h of exposure to either PF-03814735 or vehicle, followed by cell number determination in a Coulter Counter. Proliferation (as measured by an increase in cell number) is expressed as a percent of untreated controls. To evaluate the PF-03814735 exposure time required for antiproliferative activity, HL-60 cell cultures are cultured in RPMI medium supplemented with 15% heat-inactivated fetal bovine serum and exposed to various PF-03814735 concentrations for 4, 8, 12, 24, and 48 hours, followed by a washout step and incubation with growth media without PF-03814735 for the remainder of the 72-h assay period. Continuous exposure to PF-03814735 for 72 hours is also evaluated. Cell counts are determined by a Coulter Counter.(Only for Reference)