GSK923295 is a selective allosteric inhibitor of CENP-E kinesin motor ATPase(Ki=3.2 nM).

CENP-E:3.2 nM(Ki)

In various solid tumor models, including Ewing's sarcoma, sarcoma, and transplanted rhabdomyosarcoma tumors, GSK923295 consistently demonstrates significant antitumor activity. In Colo205 transplanted tumor models, GSK923295 (125 mg/kg) effectively inhibits tumor growth, resulting in a marked increase in mitosis and free apoptotic bodies.

In cancer cell lines, GSK923295 exhibits broad-spectrum antitumor activity, demonstrating effectiveness against SW48 (IC50=17.2 nM), RKO (BRAF mutant) (IC50=55.6 nM), SW620 (KRAS mutant) (IC50=42 nM), and HCT116 (KRAS mutant) (IC50=51.9 nM). Additionally, in human neuroblastoma cell lines, GSK923295 shows an average growth inhibition (IC50) of 41 nM.

Enzymology: Kinesin motor domains are expressed in Escherichia coli BL21(DE3) and purified. CENP-E proteins includes residues 2–340 with a carboxyl-terminal 6-his tag. All studies using MT are conducted in PEM25 buffer [25 mM PipesK+ (pH 6.8), 2 mM MgCl2, 1 mM EGTA] supplemented with 10 μM paclitaxel. The IC50 for steady-state inhibition is determined at 500 μM ATP, 5 μM MT, and 1 nM CENP-E in PEM25 buffer.

Cell-growth inhibition assays are performed by MDS in 384-well plates, and DNA content of fixed cells stained with DAPI using an Incell 1000 is analyzed. DNA content is determined 24 h after seeding (T0) and after exposure to varying concentrations of drug for an additional 72 h (T72). All T72 measurements are normalized to T0. Curves are analyzed using the XLfit curve-fitting tool to determine the concentration of GSK923295 yielding 50% growth inhibition relative to T0 and Ymax values (GI50).(Only for Reference)