Abstract
The mitotic kinesin Eg5 is an important target in cancer chemotherapy. A structurally diverse collection of canonical loop L5 inhibitors engage an allosteric pathway that includes elements of its microtubule binding region. However, recent evidence suggests that Eg5 may permit alternative allosteric mechanisms. Terpendole E, a natural-product Eg5 inhibitor, is active against mutants resistant to canonical loop L5 inhibitors and appears to offer a unique mode of inhibition. To investigate the variety of inhibitor responses, the structure-function properties of eighteen kinesin inhibitors were quantified with hydrogen-exchange mass spectrometry (HX-MS), functional analysis and molecular modeling. A unique strategy for high-density data analysis was implemented, based on a scalable multivariate statistical method, as current HX-MS routines have a limited capacity to guide a characterization of ligands when additional functional data is available. Inhibitor evaluation was achieved using orthogonal partial least squares projection to latent structures discriminant analysis (OPLS-DA). The strategy generated a model that identified functionally-significant conformational elements involved in kinesin inhibition, confirming the canonical allosteric pathway and identifying a novel response pathway. Terpendole E is demonstrated to be an atypical L5 site inhibitor, where binding induces an allosteric effect mediated by a destabilization in the β-sheet core of the molecular motor, an element involved in mechanochemical coupling for structurally-related kinesins. The analysis suggests that a different approach to inhibitor development may be fruitful.
Highlights
From the ‡Department of Chemistry, University of Calgary, Calgary, Alberta, Canada; §Department of Surgery, University of Calgary, Calgary, Alberta, Canada; ¶Department of Oncology, University of Calgary, Calgary, Alberta, Canada; ʈDepartment of Mathematics and Statistics, University of Calgary, Calgary, Alberta, Canada; **Cancer Epidemiology and Prevention Research, Alberta Health Services, Calgary, AB, Canada; ‡‡Department of Physics, University of Alberta, Edmonton, Alberta, Canada; §§Department of Oncology, University of Alberta, Edmonton, Alberta, Canada; ¶¶Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Canada; ʈʈDepartment of Biochemistry and Molecular Biology, University of Calgary, Alberta Canada
Terpendole E is demonstrated to be an atypical L5 site inhibitor, where binding induces an allosteric effect mediated by a destabilization in the -sheet core of the molecular motor, an element involved in mechanochemical coupling for structurally-related kinesins
We show that Terpendole E invokes a noncanonical mechanism of regulation, which appears to derive from altered occupancy of the L5 binding site region
Summary
Eg5 Motor Domain Expression and Purification—The motor domain of Eg5 [1–386] was expressed and purified following a previously described procedure [18], with some modifications. HX-MS-based Ligand Screening Assays—Stock solutions (5–10 mM) of 18 kinesin inhibitors were prepared in DMSO, and frozen at Ϫ80 °C. A single replicate of the control was collected first, followed by each ligand state in the batch This was repeated twice, following the same order of analysis for each replicate. Statistical Analysis—A matrix of shift data from 76 observations (4 replicates from 19 protein states) and 183 variables (unique peptide features) was built with the HX-MS inhibitor screen data, and trimmed of missing data. We included two different mutants, Asp130Ala and Leu214Ala. Protonation states were assigned to the proteins using the Protonate3D tool in Molecular Operating Environment (MOE, Chemical Computing Group Inc, Montreal, Canada); the protonated structures were energetically relaxed in MOE. Trans-24 (Ͼ95%) and trans-HR22C16 (Ͼ95%) were purchased from Enzo Life Sciences, Inc. (Farmingdale, NY)
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