Affinity selection-mass spectrometry (AS-MS) screening of
kinesin spindle
protein (KSP) followed by
enzyme inhibition studies and temperature-dependent circular dichroism (TdCD) characterization was utilized to identify a series of
benzimidazole compounds. This series also binds in the presence of
Ispinesib, a known anticancer KSP inhibitor in phase I/II clinical trials for
breast cancer. TdCD and AS-MS analyses support simultaneous binding implying existence of a novel non-
Ispinesib binding pocket within KSP. Additional TdCD analyses demonstrate direct binding of these compounds to
Ispinesib-resistant mutants (D130V, A133D, and A133D + D130V double mutant), further strengthening the hypothesis that the compounds bind to a distinct binding pocket. Also importantly, binding to this pocket causes uncompetitive inhibition of KSP
ATPase activity. The uncompetitive inhibition with respect to
ATP is also confirmed by the requirement of
nucleotide for binding of the compounds. After preliminary affinity optimization, the
benzimidazole series exhibited distinctive
antimitotic activity as evidenced by blockade of bipolar spindle formation and appearance of monoasters.
Cancer cell growth inhibition was also demonstrated either as a single agent or in combination with
Ispinesib. The combination was additive as predicted by the binding studies using TdCD and AS-MS analyses. The available data support the existence of a KSP inhibitory site hitherto unknown in the literature. The data also suggest that targeting this novel site could be a productive strategy for eluding
Ispinesib-resistant
tumors. Finally, AS-MS and TdCD techniques are general in scope and may enable screening other targets in the presence of known drugs, clinical candidates, or tool compounds that bind to the
protein of interest in an effort to identify potency-enhancing small molecules that increase efficacy and impede resistance in combination
therapy.