Ispinesib (SB-715992), a selective, effectvie and reversible inhibitor of kinesin spindle protein (KSP), is derived from quinazolinone, with antineoplastic properties.

KSP (HsEg5):1.7 nM(Ki app)

携带移植瘤的小鼠模型, Ispinesib（4.5-15 mg/kg）能够抑制Colo205, Colo201, HT-29细胞生长.在小鼠实体瘤中,用Ispinesib处理（6 -10 mg/kg ）,Madison 109肺癌, M5076肉瘤,及L1210和P388白血病能够被抑制.携带乳腺癌细胞MCF-7, HCC1954, MDA-MB-468,和KPL4移植瘤的小鼠中,Ispinesib（8-10 mg/kg ）抑制肿瘤生长.

在PC-3前列腺癌细胞中, Ispinesib（5 nM 和 30 nM）通过调节信号的基因表达水平,抑制细胞增殖和诱导细胞凋亡。在乳腺癌细胞系中, Ispinesib（7.4 nM–600 nM）具有广谱抑制活性。在肿瘤细胞系中（Colo205, Colo201, HT-29, M5076, Madison-109,和MX-1）,Ispinesib（IC50=1.2-9.5 nM） 具有很强细胞毒性。

Steady-State Kinetic Analysis of Human KSP ATPase Activity and Inhibition by Ispinesib: Kinesin specificity analysis is carried out using a pyruvate kinase-lactate dehydrogenase detection system that couples the production of ADP to oxidation of NADH. Absorbance changes are monitored at 340 nm. Steady-state studies using nanomolar concentrations of KSP are performed using a sensitive fluorescence-based assay utilizing a pyruvate kinase, pyruvate oxidase, and horseradish peroxidase (HRP) coupled detection system that couples the generation of ADP to oxidation of Amplex Red to fluorescent resorufin. Generation of resorufin is monitored by fluorescence (λexcitation = 520 nm and λemission = 580 nm). Steady-state biochemical experiments are performed in PEM25 buffer [25 mM Pipes-K+ (pH 6.8), 2 mM MgCl2, 1 mM EGTA] supplemented with 10 μM paclitaxel for experiments involving microtubules. The IC50 for steady-state inhibition is determined at 500 μM ATP, 5 μM Microtubules, and 1 nM KSP in PEM25 buffer. Ki app (apparent inhibitor dissociation constant) values of Ispinesib are extracted from the dose-response curves, with explicit correction for enzyme concentration by using the Morrison equation. Inhibitor modality (e.g., competitive, noncompetitive, uncompetitive, or mixed) under steady-state conditions is determined by measuring the effect of inhibitor concentration on initial velocity as a function of substrate concentrations. Data are fit using equations in GraFit to velocity equations for the various modes of inhibition.

Cells are plated in log phase of growth in 96-well plates and treated with Ispinesib for 72 hours. Then, cell growth is measured using CellTiter-Glo, and luminescence is detected using BioTek FLx800. Data are analyzed and the IC50 value, defined as the drug concentration that results in 50% growth inhibition relative to control, is calculated.(Only for Reference)