GSK461364 (GSK461364A)(Ki=2.2 nM) inhibits purified Plk1 .The specificity of GSK461364 for Plk1 is more than 1000-fold over Plk2/3.

PLK1:2.2 nM(Ki)

GSK461364在抑制多来源癌细胞系增殖方面表现出极低的对非分裂人体细胞的毒性。[1] 通过RNA沉默WT p53，可以增加GSK461364的抗增殖活性。鉴于许多癌症疗法在p53 WT患者中效果更佳，对p53缺失肿瘤高度敏感的GSK461364可能为那些对其他化疗药物有抵抗性的肿瘤提供治疗机会，以及作为这些基因型的早期线疗法。GSK461364是一种噻吩酰胺，能够以2 nM的Ki值抑制体外纯化的Plk1酶，并且对Plk1相比Plk2和Plk3有超过100倍的选择性。GSK461364是一个强效的细胞增殖抑制剂，在大多数测试的细胞系中，导致50%的生长抑制（GI50）低于100 nM，并且对人类非增殖细胞的毒性有限。细胞周期进展的抑制是浓度依赖性的，高浓度的GSK461364初始延迟在G2期，低浓度时在M期停滞。目前，GSK461364正在进行首次人体中的剂量递增试验。携带TP53基因突变的细胞系倾向于对GSK461364更敏感，且通过RNA沉默抑制p53反应可以在一些p53野生型(WT)细胞中增加敏感性。此外，这些更敏感的细胞系还显示出增加的染色体不稳定性，这是与TP53突变相关的特征。[2] 在临床前测试中，GSK461364对超过120种肿瘤细胞系显示出抗增殖活性，并且在超过83%和91%的这些细胞系中，具有低于50和100 nM的IC50值，从而强效抑制增殖。[3]

GSK461364通过细胞周期停滞或细胞毒作用抑制细胞培养生长，但在适当剂量安排下可导致异种移植瘤模型中的肿瘤退缩。GSK461364在人类肿瘤异种移植模型中展示了明确的抗肿瘤活性。[1] GSK461364在小鼠异种移植物中表现出剂量依赖性的有丝分裂阻滞，与其对肿瘤生长的影响相关。[2] GSK461364的腹腔内给药在包括Colo205异种移植物在内的不同异种移植模型中引起肿瘤退缩或肿瘤生长延迟。通过使用GSK461364体内抑制Plk1引起有丝分裂阻滞，异常有丝分裂图像包括单极或崩溃的有丝分裂纺锤体。[3]

Enzyme assays: Kinase reactions are performed in a final assay volume of 10 μL using the Z-Lyte Assay kit (Ser/Thr peptide 16). Briefly, reactions contains 50 mM HEPES (pH 7.5), 10 mM MgCl2, 1 mM EGTA, 1 mM DTT, 0.01% Brij 35, 0.01 mg/mL casein, 200 μM ATP, 200 μM Polo Box peptide (NH2-MAGPMQS[pT]PLNGAKK-OH), and 6 nM recombinant Plk1 (H6-tev-PLK 1-603). Plk1 is preincubated for 60 minutes with 0 to 1 μM GSK461364. Reactions are then initiated by the addition of 2 μM peptide. After 15 minutes at 23 °C, reactions are quenched and processed according the Z′-Lyte protocol and read on an EnVision plate reader. Raw fluorescence values are converted to concentration of product formed using substrate and product standards. Because the potency of inhibition for GSK461364 is observed to vary as a function of the ATP concentration in a manner consistent with an ATP-competitive mode of inhibition, an upper limit for the Ki for GSK461364 is determined.

Cancer cell lines are seeded into 384-well microtiter plates. After seeding, the cells are incubated at 37 °C in 5% CO2 for 24 hours. GSK461364 is added to each cell line at 10 nM with a nontreated control. A zero-time (T = 0) value is read for each cell line. After 72 hours, the medium containing GSK461364 or DMSO control is aspirated from all of the remaining cells and the cell nuclei are stained with 4′,6-diamidino-2-phenylindole and the fluorescent intensity measured using an InCell1000 High Content Analyzer. The percent intensity of the 4′,6-diamidino-2-phenylindole stain at 72 hours, relative to intensity at time zero, is calculated for each concentration of GSK461364 in each of the triplicate wells. (Only for Reference)