Mitotic Kinesin Eg5 Overcomes Inhibition to the Phase I/II Clinical Candidate SB743921 by an Allosteric Resistance Mechanism

Abstract

Development of drug resistance during cancer chemotherapy is one of the major causes of chemotherapeutic failure for the majority of clinical agents. The aim of this study was to investigate the underlying molecular mechanism of resistance developed by the mitotic kinesin Eg5 against the potent second-generation ispinesib analogue SB743921 (1), a phase I/II clinical candidate. Biochemical and biophysical data demonstrate that point mutations in the inhibitor-binding pocket decrease the efficacy of 1 by several 1000-fold. Surprisingly, the structures of wild-type and mutant Eg5 in complex with 1 display no apparent structural changes in the binding configuration of the drug candidate. Furthermore, ITC and modeling approaches reveal that resistance to 1 is not through conventional steric effects at the binding site but through reduced flexibility and changes in energy fluctuation pathways through the protein that influence its function. This is a phenomenon we have called "resistance by allostery".

Synergism between inhibitors of Aurora A and KIF11 overcomes KIF15‐dependent drug resistance

“…In this connection, it was reported that Ispinesib‐resistant HCT116 cells contain two point mutations in the KIF11 allosteric binding pocket, D130V and A133D, generating inhibitor‐resistant but otherwise catalytically competent KIF11 (Knight and Parrish, 2008). Interestingly, the mutations do not act through conventional steric effects at the binding site, but through reduction of flexibility and allosteric transmissions after inhibitor binding (Talapatra et al., 2013). Finally, another process specific for antimitotic drug‐resistance is mitotic slippage, by which cells exit mitosis prematurely without sister chromatid separation.…”

Clinical Trials of Mitotic Kinesin Inhibitors

“…However, with the exception of ARRY-520, the clinical experience with Eg5 and CENP-E inhibitors in phase I trials has been disappointing, and in the case of ispinesib and AZD-4877, this lack of effi cacy has been confi rmed by subsequent phase II trials. There are several explanations for these results: First, an obvious explanation is the development of tumor resistance, due to point mutations within L5-a loop in the Eg5 enzymatic domain that constitutes part of the drug-binding site [ 58 ]. The genetic instability that is characteristic of most malignant tumors might allow for selection of Eg5 mutations that confer treatmentresistance.…”

DrugTargetSeqR: a genomics- and CRISPR-Cas9–based method to analyze drug targets

“…D130, A133) and deletions (e.g. residues in ‘loop-5’) within this pocket have been shown to confer resistance to ispinesib-analogs in vitro 19 . Therefore, with these data, we can confirm kinesin-5 to be ispinesib’s direct physiologic target in a human cancer cell line.…”