Relocation of Aurora B and survivin from centromeres to the central spindle impaired by a kinesin-specific MKLP-2 inhibitor.

Mitotic kinesin-like protein 2 (MKLP-2), a member of the kinesin-6 family, is essential for cytokinesis. We screened 8900 small molecules for inhibition of the ATPase activity of MKLP-2 and identified the first inhibitor, (Z)-2-(1H-indol-3yl)-3-(pyridin-3-yl)acrylonitrile, which we named paprotrain (PAssenger PROteins TRAnsport INhibitor; Figure 1b). Paprotrain is a reversible inhibitor uncompetitive with ATP (inhibitor constant Ki = (3.4 0.1) mm) and noncompetitive with microtubules (MTs; Ki = (1.6 0.1) mm). It is specific for MKLP-2 as it does not inhibit other members of the kinesin superfamily. Paprotrain is cell permeable and incubation with 10 to 50 mm inhibitor results in binucleated cells, a characteristic phenotype similar to that observed following RNA interference (RNAi)-mediated depletion of MKLP-2. Additional mitotic spindle defects were also observed. Notably, paprotrain perturbs MKLP-2-mediated relocation of the chromosome passenger proteins Aurora B and survivin from the centromeres to the central spindle. Lack of relocation of passenger proteins to the central spindle is associated with failure of cytokinesis and generation of binucleated cells. In contrast, paprotrain does not impair kinesin family member 4 (Kif4)-mediated translocation of protein regulating cytokinesis 1 (PRC1), which emphasizes its specificity for MKLP-2. We conclude that paprotrain is a new lead compound targeting MKLP-2 by a novel mechanism of action. The motor domain of human MKLP-2 (residues 1–519) was cloned, expressed, and purified (Figure 1a). Gel filtration and analytical ultracentrifugation showed that the protein is monomeric (data not shown) with an estimated molecular mass of (77 5) kDa (see Table S1 in the Supporting Information). The basal ATPase activity was (0.034 0.012) s , with a KM,ATP of 59.4 mm. The activity decreased with increasing NaCl concentration and inhibitor screening was therefore performed in the absence of salt. The MT-stimulated ATPase activity was 1.8 s , which corresponded to an estimated 53fold stimulation compared to the basal activity. The K0.5,MT was 1.6 mm (see Figure S1 and Table S2 in the Supporting Information). We measured the inhibition of the basal and MTstimulated ATPase activity in the presence of paprotrain (Figure 2). Calculated IC50 values were (1.35 0.2) mm in basal (Figure 2a) and (0.83 0.1) mm in MT-stimulated ATPase assays (Figure 2b). The measured rates of ATP hydrolysis in the presence of varying concentrations of paprotrain and MTs demonstrate that the inhibitor acts through a mixed noncompetitive mechanism with respect to MTs with an inhibitor constant Ki = (1.6 0.07) mm (Figure 2c). We also analyzed the ATP hydrolysis rate at different ATP and inhibitor concentrations. Paprotrain is an ATP uncompetitive inhibitor with Ki = (3.36 0.09) mm (Figure 2d). The ATPase activity of a panel of 12 other kinesins 3] was not inhibited by paprotrain, thus demonstrating high specificity within the kinesin superfamily (see Figure S2 a in the Supporting Information). Notably, paprotrain does not inhibit Figure 1. a) Bar diagram of human MKLP-2 and the protein construct used for inhibitor screening. MKLP-2 has an N-terminal motor domain (amino acids (aa) 56 to 505), an a-helical region (aa 519 to 821), and a C-terminal tail domain (aa 821 to 890). MKLP-2 contains an approximately 100-residue insertion (aa 189 to 288) in the motor domain that is unique for members of the kinesin-6 family. b) Chemical formula of paprotrain. Trx = thioredoxin.