BPS2026 – L5 determines sensitivity of C. elegans kinesin BMK-1 to the kinesin Eg5-specific inhibitor

The mitotic kinesin Eg5, essential for bipolar spindle formation, is a promising target for anticancer therapy. Eg5 features an unusually long loop L5, and inhibitors such as S-trityl-L-cysteine (STLC) bind to a hydrophobic pocket formed by L5 and the α2/α3 helices, thereby blocking its function. To investigate L5-mediated inhibition, we focused on the nematode kinesin BMK-1, which possesses a comparably long L5. Caenorhabditis elegans provides a valuable model for evaluating in vivo effects of kinesin inhibitors. We expressed BMK-1, characterized its biochemical properties, and examined its response to STLC. STLC suppressed both ATPase and motile activities of BMK-1, although less effectively than Eg5. An L5-shortened BMK-1 mutant, with loop length reduced to that of conventional kinesins, lost sensitivity to STLC while retaining most microtubule-stimulated ATPase activity. These findings indicate that BMK-1 and Eg5 share a loop L5-dependent inhibition mechanism and suggest that modulating L5 length may broaden the inhibitory spectrum of Eg5-targeted compounds. Because BMK-1 is not essential for mitosis, we extended our analysis to KLP-18, a kinesin critical for spindle assembly in C. elegans . A KLP-18 mutant engineered with an Eg5-like loop L5 provides a suitable model to test whether STLC inhibition applies to mitotically essential kinesins. We are currently assessing the effects of STLC on the ATPase and motor activities of KLP-18 to determine conservation of loop L5-mediated inhibition. These studies aim to elucidate structural determinants of kinesin inhibition and lay the groundwork for optogenetic regulation of mitosis in living C. elegans , with potential implications for therapeutic manipulation of cell division.