Pharmaceutical-Grade Rigosertib Is a Microtubule-Destabilizing Agent
Jost, Marco; Chen, Yuwen; Gilbert, Luke A; Horlbeck, Max A; Krenning, Lenno; Menchon, Grégory; Rai, Ankit; Cho, Min Y; Stern, Jacob J; Prota, Andrea E; Kampmann, Martin; Akhmanova, Anna; Steinmetz, Michel O; Tanenbaum, Marvin E; Weissman, Jonathan S
(2020) Molecular Cell, volume 79, issue 1, pp. 191 - 198.e3
(Article)
Abstract
We recently used CRISPRi/a-based chemical-genetic screens and cell biological, biochemical, and structural assays to determine that rigosertib, an anti-cancer agent in phase III clinical trials, kills cancer cells by destabilizing microtubules. Reddy and co-workers (Baker et al., 2020, this issue of Molecular Cell) suggest that a contaminating degradation product in
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commercial formulations of rigosertib is responsible for the microtubule-destabilizing activity. Here, we demonstrate that cells treated with pharmaceutical-grade rigosertib (>99.9% purity) or commercially obtained rigosertib have qualitatively indistinguishable phenotypes across multiple assays. The two formulations have indistinguishable chemical-genetic interactions with genes that modulate microtubule stability, both destabilize microtubules in cells and in vitro, and expression of a rationally designed tubulin mutant with a mutation in the rigosertib binding site (L240F TUBB) allows cells to proliferate in the presence of either formulation. Importantly, the specificity of the L240F TUBB mutant for microtubule-destabilizing agents has been confirmed independently. Thus, rigosertib kills cancer cells by destabilizing microtubules, in agreement with our original findings.
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Keywords: Antineoplastic Agents/pharmacology, Cell Proliferation, Cells, Cultured, Crystallography, X-Ray, Drug Contamination, Glycine/analogs & derivatives, Humans, Microtubules/drug effects, Mutation, Neoplasms/drug therapy, Pharmaceutical Preparations/chemistry, Protein Conformation, Sulfones/pharmacology, Tubulin/chemistry, microtubules, CRISPRi, chemical genetics, rigosertib, CRISPRa, drug mechanism of action, drug target identification, Molecular Biology, Cell Biology, Research Support, Non-U.S. Gov't, Journal Article, Research Support, N.I.H., Extramural
ISSN: 1097-2765
Publisher: Cell Press
Note: Funding Information: We thank Onconova Therapeutics, specifically Dan Fox and Dr. V.J. Rajadhyaksha, for providing pharmaceutical-grade rigosertib and pure ON01500 for this study. This work was funded by the National Institutes of Health (grants P50 GM102706 , U01 CA168370 , and R01 DA036858 to J.S.W.; post-doctoral fellowship F32 GM116331 to M.J.), the Swiss National Science Foundation ( 31003A_166608 to M.O.S.), the European Research Council (Starting grant ERC-STG 677936-RNAREG to M.E.T. and Synergy grant 609822 MODELCELL to A.A.), a fellowship from the Dutch Cancer Society (KWF) to M.E.T., NWO CW ECHO grant 711.015.005 to A.A., NIH/NCI Pathway to Independence Award K99 CA204602 to L.A.G., and NIH/NCI Pathway to Independence Award K99 CA181494 and a Stand Up to Cancer Innovative Research Grant to M.K. J.S.W. is a Howard Hughes Medical Institute Investigator. Funding Information: We thank Onconova Therapeutics, specifically Dan Fox and Dr. V.J. Rajadhyaksha, for providing pharmaceutical-grade rigosertib and pure ON01500 for this study. This work was funded by the National Institutes of Health (grants P50 GM102706, U01 CA168370, and R01 DA036858 to J.S.W.; post-doctoral fellowship F32 GM116331 to M.J.), the Swiss National Science Foundation (31003A_166608 to M.O.S.), the European Research Council (Starting grant ERC-STG 677936-RNAREG to M.E.T. and Synergy grant 609822 MODELCELL to A.A.), a fellowship from the Dutch Cancer Society (KWF) to M.E.T. NWO CW ECHO grant 711.015.005 to A.A. NIH/NCI Pathway to Independence Award K99 CA204602 to L.A.G. and NIH/NCI Pathway to Independence Award K99 CA181494 and a Stand Up to Cancer Innovative Research Grant to M.K. J.S.W. is a Howard Hughes Medical Institute Investigator. M.J. A.R. A.E.P. A.A. M.O.S. M.E.T. and J.S.W. designed experiments. M.J. A.R. and M.E.T. conducted experiments and analyzed data. A.E.P. and M.O.S. analyzed the crystal structure. M.J. M.E.T. and J.S.W. wrote the manuscript. All authors edited the manuscript. M.A.H. L.A.G. M.K. and J.S.W. have filed a patent application related to CRISPRi and CRISPRa screening (PCT/US15/40449). M.E.T. L.A.G. and J.S.W. have filed a patent application for the SunTag technology (PCT/US2015/040439). J.S.W. consults for and holds equity in KSQ Therapeutics, Maze Therapeutics, and Tenaya Therapeutics. J.S.W. is a venture partner at 5AM Ventures and a member of the Amgen Scientific Advisory Board. M.J. and M.A.H. consult for Maze Therapeutics. Publisher Copyright: © 2020 The Authors Copyright: Copyright 2020 Elsevier B.V., All rights reserved.
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