title: Novel Allosteric Pathway of Eg5 Regulation Identified through Multivariate Statistical Analysis of Hydrogen-Exchange Mass Spectrometry (HX-MS) Ligand Screening Data.
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.
authors.affiliation: From the ‡Department of Chemistry, University of Calgary, Calgary, Alberta, Canada.
authors.forename: Joey G
authors.initials: JG
authors.lastname: Sheff
authors.affiliation: §Department of Surgery, University of Calgary, Calgary, Alberta, Canada.|¶Department of Oncology, University of Calgary, Calgary, Alberta, Canada.
authors.forename: Farshad
authors.initials: F
authors.lastname: Farshidfar
authors.affiliation: §Department of Surgery, University of Calgary, Calgary, Alberta, Canada.|¶Department of Oncology, University of Calgary, Calgary, Alberta, Canada.
authors.forename: Oliver F
authors.initials: OF
authors.lastname: Bathe
authors.affiliation: ‖Department of Mathematics and Statistics, University of Calgary, Calgary, Alberta, Canada.|**Cancer Epidemiology and Prevention Research, Alberta Health Services, Calgary, AB, Canada.
authors.forename: Karen
authors.initials: K
authors.lastname: Kopciuk
authors.affiliation: ‡‡Department of Physics, University of Alberta, Edmonton, Alberta, Canada.
authors.forename: Francesco
authors.initials: F
authors.lastname: Gentile
authors.affiliation: ‡‡Department of Physics, University of Alberta, Edmonton, Alberta, Canada.|§§Department of Oncology, University of Alberta, Edmonton, Alberta, Canada.
authors.forename: Jack
authors.initials: J
authors.lastname: Tuszynski
authors.affiliation: ¶¶Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Canada.
authors.forename: Khaled
authors.initials: K
authors.lastname: Barakat
authors.affiliation: From the ‡Department of Chemistry, University of Calgary, Calgary, Alberta, Canada; dschriem@ucalgary.ca.|‖‖Department of Biochemistry and Molecular Biology, University of Calgary, Alberta Canada.
authors.forename: David C
authors.initials: DC
authors.lastname: Schriemer
mesh_terms: D000494:Allosteric Regulation / Q000187:drug effects; D001665:Binding Sites / Q000187:drug effects; D041961:Deuterium Exchange Measurement / Q000379:methods*; D004224:Diterpenes / Q000494:pharmacology*; D006801:Humans; D007211:Indoles / Q000494:pharmacology*; D016547:Kinesins / Q000737:chemistry / Q000378:metabolism*; D008024:Ligands; D013058:Mass Spectrometry / Q000379:methods*; D008958:Models, Molecular; D062105:Molecular Docking Simulation; D015999:Multivariate Analysis; D017433:Protein Structure, Secondary
chemical_list: D004224:Diterpenes; D007211:Indoles; C470243:KIF11 protein, human; D008024:Ligands; C477863:terpendole E; D016547:Kinesins