Allosteric L5-Directed Inhibitors of Kinesin-5 Can Control Different Biochemical Intermediates

Abstract

Kinesin-5 (Eg5) is an essential mitotic motor that couples ATP hydrolysis to different protein-protein association states with microtubules. Human Eg5 is a cancer chemotherapeutic target, having a unique allosteric site covered by loop-5 (L5). This site is capable of binding small-molecule inhibitors with > 100 different chemotypes. The prevailing biochemical model is that all L5-directed drugs inhibit ADP release, but it does not address the 107-fold difference in potency or the lack of chemical homology between inhibitor families. An alternative hypothesis is that the inhibitors act on different catalytic intermediates, which gives rise to their disparate potencies. Here we present our linear free energy relationship (LFER) study of Eg5, a method to determine whether an inhibitor can block the catalytic transition-state. Steady-state kinetic parameters for wildtype Eg5 and eight different L5 mutants were determined in the background of three different L5-directed inhibitors and a mock control. The predominant effect of the L5 residue substitution is alteration of substrate binding (Km), whereas principal outcome of allosteric drug is change in ATP hydrolysis (kcat). Second, our data showed that despite their use of the same binding pocket, one compound can inhibit the transition state and the other two do not. We conclude that the drugs are not synonymous: it is possible for one allosteric modulator to regulate ADP release and another to control transition-state formation. The significance is that this is the first demonstration of allosteric control of more than one catalytic intermediate for any drug target. Furthermore, these results may give insight into the disparity between increased inhibitor potency and success in clinical trials.This work is funded by the support of the National Institutes of Health (R01 GM097350; S.K.) and the LSU School of Graduate Studies (M.L.).