Antitumor activity of an allosteric inhibitor of centromere-associated protein-E

Abstract

Centromere-associated protein-E (CENP-E) is a kinetochore-associated mitotic kinesin that is thought to function as the key receptor responsible for mitotic checkpoint signal transduction after interaction with spindle microtubules. We have identified GSK923295, an allosteric inhibitor of CENP-E kinesin motor ATPase activity, and mapped the inhibitor binding site to a region similar to that bound by loop-5 inhibitors of the kinesin KSP/Eg5. Unlike these KSP inhibitors, which block release of ADP and destabilize motor-microtubule interaction, GSK923295 inhibited release of inorganic phosphate and stabilized CENP-E motor domain interaction with microtubules. Inhibition of CENP-E motor activity in cultured cells and tumor xenografts caused failure of metaphase chromosome alignment and induced mitotic arrest, indicating that tight binding of CENP-E to microtubules is insufficient to satisfy the mitotic checkpoint. Consistent with genetic studies in mice suggesting that decreased CENP-E function can have a tumor-suppressive effect, inhibition of CENP-E induced tumor cell apoptosis and tumor regression.

Fig. 1.

GSK923295 inhibits microtubule (MT)-stimulated ATPase of CENP-E and promotes formation of a MT-bound complex. (A) Chemical structure of GSK923295. (B) Concentration-dependent inhibition of CENP-E motor domain (1 nM) ATPase by GSK923295 in the presence (•) and absence (○) of 5 μM MT indicates a preference of GSK923295 for CENP-E-MT complex. GSK923295 inhibited CENP-E MT-stimulated ATPase with a K_i of 3.2 ± 0.2 nM in a manner uncompetitive with either MT or ATP (Table 1, Table S1, and Fig. S1). (C) Equilibrium binding and cosedimentation of CENP-E motor domain (CE; 3 μM) with MT (6 μM) in the presence or absence of the indicated nucleotides (1 mM) or in the presence or absence of 20 μM GSK923925 reveals inhibitor-induced loss of CENP-E motor domain from the supernatant and increase in the MT pellet, irrespective of nucleotide state. (Table S2). (D) Presteady-state rates of release of inorganic phosphate (Pi) from CENP-E motor domain in the presence (red) or absence (black) of GSK923295 as a function of ATP indicates that GSK923295 inhibits the production or release of Pi. (E) Summary model of the biochemical mechanism of action of GSK923295.

Fig. 2.

Proposed binding mode of GSK923295 to ATP-bound CENP-E motor domain GSK923295 binding site lies between helices α2 and α3, adjacent to L5 of CENP-E motor domain. GSK923295 is positioned in the center of the left figure, shown predominantly in blue. Switch I and switch II regions are indicated by roman numerals I and II. Loops L5, L9, and L11 are shown in green. Residues on helix α3 subjected to mutational analysis (I182, T183, K184) and the sites of photo-labeling with inhibitor GSK-1 (Met96 and Met97) are shown in red (Fig. S4). A simplified scheme for this proposed binding mode is shown in Right.

Fig. 3.

Inhibition of CENP-E motor domain ATPase causes failure to achieve metaphase chromosome alignment, mitotic cell-cycle delay, and apoptosis. (A) Flow cytometric analysis of DNA content in HCC1954 cell line (GI₅₀ = 27 nM) at indicated times of exposure to 325 nM GSK-2 (Table S1). (B) Western blots of HCC1954 cells treated as in A are stained for mitotic markers cyclin B and phospho-histone H3 (Ser10) and for cleavage of full-length PARP (top band) to yield a PARP fragment (bottom band), a marker of apoptosis. GAPDH is included as control. (C) SKOV3 cell (GI₅₀ = 26 nM) treated with 50 nM GSK-2 for 24 h is stained for chromatin (blue) and MT (red). Similar mitotic figures were apparent in all cell lines treated with GSK-2 or GSK923295. (D) Quantitative analysis of timelapse images of HCC1954 breast carcinoma cells stably expressing GFP-Histone H2B treated with 325 nM GSK-2. The percentage of mitotic nuclei per image in the presence of GSK-2 (blue) and the percentage of apoptotic nuclei per image in the presence (red) and absence (black) of GSK-2 (Movie S1) is shown.

Fig. 4.

Antitumor activity of GSK923295. (A) GSK923295 is a potent inhibitor of tumor cell growth in vitro (Dataset S1). Variation in GI₅₀ values across 237 cell lines suggests intrinsic determinants of sensitivity. (B) Representative photomicrograph of an H&E-stained section of Colo205 tumor xenografts removed 24 h after a single injection of GSK923295 (125 mg/kg). (Scale bar: 20 μm.) GSK923295 induced appearance of mitotic figures consistent with CENP-E inhibition (arrow) and scattered apoptotic bodies (arrowhead). See Fig. S5 for comparison of vehicle control and GSK923295. (C) Dose-dependent increases in the ratio of 4n to 2n nuclei present in dispersed tumor tissue from Colo205 tumor xenografts 24 h after administration of the indicated doses GSK923925. (D) Dose-dependent antitumor activity of GSK923295 administered as three daily doses on 2 consecutive weeks (arrowheads) to mice bearing xenografts of the colon carcinoma cell line Colo-205. Among five animals treated with 125 mg/kg GSK923295, four experienced partial tumor regressions (PR), and one had a complete regression (CR); 62.5 mg/kg produced tumor-growth delay and no regressions. GSK923295 failed to produce detectable host toxicity at doses as high as 500 mg/kg; the maximal tolerated dose (MTD) in mice is unknown. Paclitaxel dosed at its MTD of 30 mg/kg produced regressions in four of five mice treated (2CR and 2PR). Similar results were obtained in several other tumor xenograft models (Table S4).