Abstract
VCP (also known as p97 or Cdc48p in yeast) is an AAA+ ATPase regulating endoplasmic reticulum–associated degradation. After high-throughput screening, we developed compounds that inhibit VCP via different mechanisms, including covalent modification of an active site cysteine and a new allosteric mechanism. Using photoaffinity labeling, structural analysis and mutagenesis, we mapped the binding site of allosteric inhibitors to a region spanning the D1 and D2 domains of adjacent protomers encompassing elements important for nucleotide-state sensing and ATP hydrolysis. These compounds induced an increased affinity for nucleotides. Interference with nucleotide turnover in individual subunits and distortion of interprotomer communication cooperated to impair VCP enzymatic activity. Chemical expansion of this allosteric class identified NMS-873, the most potent and specific VCP inhibitor described to date, which activated the unfolded protein response, interfered with autophagy and induced cancer cell death. The consistent pattern of cancer cell killing by covalent and allosteric inhibitors provided critical validation of VCP as a cancer target.
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Acknowledgements
We thank S. Messali, J. Malyszko, S. Thieffine, R. Perego, S. Re Depaolini, C. Albanese and D. Borghi for technical support and E. Pesenti, B. Salom and M. Caruso for helpful discussion.
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P.M. wrote the manuscript and coordinated project activities. A.I. wrote the manuscript, supervised the Nerviano Medical Sciences–Genentech collaboration and coordinated the project. P.K.J. revised the manuscript, coordinated Genentech activities and provided critical support to the project. R.D.A. developed the chemical expansion strategy and coordinated chemistry activities. B.V. and U.C. performed spectrometric analysis and photo-affinity labeling. N.A. and D.A. developed biochemical assays and analyzed compound activity. S.R. and C.P. designed constructs and produced recombinant proteins. F.G. collected cellular image analysis data. L.C. performed cellular treatments with siRNA and inhibitors and immunoblot analysis. C.O. performed molecular modeling studies for identification of the binding region of the allosteric inhibitors; P.P. and G.C. performed synthesis the reported VCP inhibitors. D.B. collected antiproliferative data. A.L. supported realization of HTS. E.C. performed ITC experiments. Y.X. performed siRNA rescue experiments. C.W. and D.J.A. performed combination studies with VCP siRNA and on stabilization of GFP-MCL1. A.G. provided support for and discussed cell biology activities. D.D. provided support for the chemical expansion strategy. T.O.B. provided support in allosteric inhibitor identification and coordination of Genentech activities.
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Magnaghi, P., D'Alessio, R., Valsasina, B. et al. Covalent and allosteric inhibitors of the ATPase VCP/p97 induce cancer cell death. Nat Chem Biol 9, 548–556 (2013). https://doi.org/10.1038/nchembio.1313
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DOI: https://doi.org/10.1038/nchembio.1313
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