Autophagy is a lysosomal degradation pathway that eliminates aggregated proteins and damaged organelles to maintain cellular homeostasis. A major route for activating autophagy involves inhibition of the mTORC1 kinase, but current mTORC1-targeting compounds do not allow complete and selective mTORC1 blockade. Here, we have coupled screening of a covalent ligand library with activity-based protein profiling to discover EN6, a small-molecule in vivo activator of autophagy that covalently targets cysteine 277 in the ATP6V1A subunit of the lysosomal v-ATPase, which activates mTORC1 via the Rag guanosine triphosphatases. EN6-mediated ATP6V1A modification decouples the v-ATPase from the Rags, leading to inhibition of mTORC1 signaling, increased lysosomal acidification and activation of autophagy. Consistently, EN6 clears TDP-43 aggregates, a causative agent in frontotemporal dementia, in a lysosome-dependent manner. Our results provide insight into how the v-ATPase regulates mTORC1, and reveal a unique approach for enhancing cellular clearance based on covalent inhibition of lysosomal mTORC1 signaling.
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The data sets generated during and/or analyzed during the current study are available from the corresponding authors on reasonable request.
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We thank the members of the Nomura Research Group, the Zoncu laboratory and Novartis Institutes for BioMedical Research for critical reading of the manuscript. This work was supported by Novartis Institutes for BioMedical Research and the Novartis-Berkeley Center for Proteomics and Chemistry Technologies (NB-CPACT) for C.Y.S.C., C.A.B., B.F., C.C.W. and D.K.N., National Institutes of Health (grant no. NIEHS R01ES028096 for D.K.N. and C.Y.S.C., no. NCI F31CA225173 for C.C.W., no. NCI DP2CA195761 for R.Z. and no. NIGMS R01GM112948 for J.A.O.), the Shurl & Kay Curci Foundation Faculty Scholars grant (R.Z.) and the National Research Foundation funded by the South Korean government for H.R.S. (grant no. 2017R1C1B2007409). This study was also supported by the Mark Foundation for Cancer Research ASPIRE award (D.K.N.). Confocal imaging experiments were conducted at the CRL Molecular Imaging Center, supported by the Helen Wills Neuroscience Institute and Gordon and Betty Moore Foundation (UC Berkeley). We would like to thank H. Aaron and F. Ives for their microscopy training and assistance. We also thank R. Zalpuri at the University of California Berkeley Electron Microscope Laboratory for advice and assistance in electron microscopy sample preparation and data collection.
This study was funded by the Novartis Institutes for BioMedical Research and the Novartis-Berkeley Center for Proteomics and Chemistry Technologies. D.K.N. is the director of the Novartis-Berkeley Center for Proteomics and Chemistry Technologies. D.K.N. is a co-founder, share-holder and adviser for Artris Therapeutics and Frontier Medicines. R.Z. is a co-founder, share-holder and adviser for Frontier Medicines.
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Chung, C.YS., Shin, H.R., Berdan, C.A. et al. Covalent targeting of the vacuolar H+-ATPase activates autophagy via mTORC1 inhibition. Nat Chem Biol 15, 776–785 (2019). https://doi.org/10.1038/s41589-019-0308-4
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