GSK923295

GSK923295 is the first potent and selective inhibitor of the mitotic kinesin centromere-associated protein-E (CENP-E). ^[1][2] GSK923295 is uncompetitive with both ATP and microtubules (MT), inhibiting CENP-E MT-stimulated ATPase activity with a K_i of 3.2 nM, highly selective over other kinesins. GSK923295 inhibits release of inorganic phosphate and stabilizes CENP-E motor domain interaction with microtubules, reduces the rate of ATP-promoted dissociation of CENP-E from MT (k_{off, MT}) by more than 50-fold. GSK923295 causes failure of metaphase chromosome alignment and induces mitotic arrest. GSK923295 is a potent inhibitor of tumor cell growth, with an average GI50 of 253 nM and a median GI50 of 32 nM for 237 tumor cell lines. ^[1] GSK923295 inhibits tumor cell growth more effectively when mitogen-activated protein kinase (MEK/ERK) signaling is also inhibited. ^[4]

GSK923295 produces clear increases in the abundance of mitotic figures and scattered apoptotic bodies in tumors. GSK923295 causes a dose-dependent increase in the ratio of 4n to 2n nuclei. GSK923295 exhibits robust, dose-dependent antitumor activity against Colo205 xenografts, including partial and complete regressions at the 125 mg/kg dose.^[1] GSK923295 demonstrates significant antitumor activity against solid tumor models, inducing CRs in Ewing sarcoma, rhabdoid, and rhabdomyosarcoma xenografts, may be a valuable therapeutic target in pediatric cancer. ^[3]

• 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 [25mM PipesK⁺ (pH 6.8), 2mM MgCl₂, 1mM EGTA] . The IC50 for steady-state inhibition is determined at 500 μM ATP, 5 μM MT, and 1 nM CENP-E in PEM25 buffer.

• Cell lines

  Tumor cell lines

• Concentrations

  ~10 μM

• Incubation Time

  72 h

• Method

  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 Y_max values (GI50).

• Animal Models

  mice bearing xenografts of the Colo205 colon tumor-cell line

• Dosages

  125 mg/kg, two cycles of three daily injections separated by 1 week

• Administration

  i.p.