Targeting colorectal cancer with small-molecule inhibitors of ALDH1B1
Abstract
Aldehyde dehydrogenases (ALDHs) are promising cancer drug targets, as certain isoforms are required for the survival of stem-like tumor cells. We have discovered selective inhibitors of ALDH1B1, a mitochondrial enzyme that promotes colorectal and pancreatic cancer. We describe bicyclic imidazoliums and guanidines that target the ALDH1B1 active site with comparable molecular interactions and potencies. Both pharmacophores abrogate ALDH1B1 function in cells; however, the guanidines circumvent an off-target mitochondrial toxicity exhibited by the imidazoliums. Our lead isoform-selective guanidinyl antagonists of ALDHs exhibit proteome-wide target specificity, and they selectively block the growth of colon cancer spheroids and organoids. Finally, we have used genetic and chemical perturbations to elucidate the ALDH1B1-dependent transcriptome, which includes genes that regulate mitochondrial metabolism and ribosomal function. Our findings support an essential role for ALDH1B1 in colorectal cancer, provide molecular probes for studying ALDH1B1 functions and yield leads for developing ALDH1B1-targeting therapies.
Additional Information
© 2022 Nature Publishing Group. Received 21 April 2021; Accepted 26 April 2022; Published 04 July 2022. We thank T. Hurley for the vector for bacterial ALDH1B1 expression, E. Patton for ALDH1A3–/– A375 cells, P. Beachy for Shh-LIGHT2 cells, G. Ponhert for BOPIDY azide and S. Swick for assistance with compound characterization. This work was supported by the National Institutes of Health (R35 GM127030, R01 CA244334 and R01 GM113100 to J.K.C.; U01 CA217851 and U54 CA224081 to C.J.K.; R01 AA11147 to D.M.-R. and F32 CA183527 to A.E.O.), SPARK at Stanford (J.K.C.), Weston Havens Foundation (J.K.C.), the Emerson Collective (J.K.C. and C.J.K.), the Human Cancer Models Initiative (C.J.K.) and Stand Up to Cancer (C.J.K.). Flow cytometry experiments were performed at the Stanford Shared FACS Facility supported by an NIH S10 Shared Instrument grant (S10 OD026831). Use of the Stanford University Mass Spectrometry facility is supported, in part, by the National Institutes of Health (P30 CA124435) utilizing the Stanford Cancer Institute Proteomics/Mass Spectrometry Shared Resources. Use of the SSRL/SLAC National Accelerator Laboratory is supported by the U.S. Department of Energy, Office of Science and Office of Basic Energy Sciences, under contract number DE-AC02-76SF00515. The SSRL Structural Molecular Biology Program is supported by the Department of Energy, Office of Biological and Environmental Research, and by the National Institutes of Health (P41 GM103393). NMR experiments included spectra acquired on a Bruker Avance NEO 500 MHz spectrometer supported by an NIH S10 Shared Instrument Grant (S10 OD028697). Data availability: Any data generated or analyzed during this study, associated protocols and materials are available from the corresponding author on reasonable request. X-ray crystal structures have been validated and deposited with the Protein Data Bank with the following entries: 7MJC (ALDH1B1–NAD+), 7MJD (ALDH1B1–NAD+–2) and 7RAD (ALDH1B1–NAD+–IGUANA-4). Raw and processed proteomics data for the TPP study are publicly available in the MassIVE repository (MassIVE ID MSv000088824), which is a member of the ProteomeXchange Consortium (ProteomeXchange ID PXD031630). Raw and processed RNA-seq data for the identification of ALDH1B1-dependent genes are publicly available in the Gene Expression Omnibus database (GEO accession number GSE165621). Source data are provided with this paper. Contributions: Z.F., M.E.H., T.E.B., Z.C.R., D.F., A.E.O., A.K.M., M.G.T., C.J.K. and J.K.C. designed the experiments and analyzed the data. A.E.O., T.K. and J.K.C. designed compounds and synthetic routes, and A.E.O. prepared compounds. A.E.O. conducted photoaffinity cross-linking, click chemistry, two-dimensional gel electrophoresis and mass spectrometry analysis, and C.-H.C. assisted with the two-dimensional gel electrophoresis. Z.F., M.E.H., T.E.B., Z.C.R. and C.-H.C. expressed and purified ALDH proteins. Z.F., M.E.H., T.E.B., Z.C.R. and C.R.M. performed the enzyme kinetics assays and compound activity profiling. Z.F., Z.C.R. and D.F. conducted the X-ray crystallography studies of ALDH1B1 and ALDH1B1–inhibitor complexes. Z.F. and C.R.M. performed computational modeling of ALDH1–inhibitor complexes. Z.F., T.E.B. and Z.C.R. conducted spheroid and adherent culture growth assays. Z.F., M.E.H. and T.E.B. performed the ALDEFLUOR assays. Z.F. and M.E.H. conducted the Seahorse, HEK-293T viability and Shh-LIGHT2 signaling assays. T.E.B. prepared the ALDH1B1 mutants. Z.F. and Y.G. generated the ALDH1B1–/– and ALDH1B1 rescue clones, and Z.F. conducted related growth assays. Z.F. generated the 5-FU-resistant SW480 cells and conducted related growth assays. A.K.M. and M.G.T. conducted the organoid experiments. Z.F. and Y.G. prepared total RNA samples for RNA-seq analysis, and Z.F. and J.K.C. analyzed the transcriptomic data. Z.F. conducted CETSAs and prepared TPP samples. T.E.B analyzed the TPP data. Z.F. and J.K.C. wrote the manuscript. A.E.O., D.M.-R., C.J.K. and J.K.C. acquired funding for the project. Competing interests: J.K.C., Z.F., M.E.H., T.K., C.R.M. and A.E.O. have filed a PCT application related to the compounds described in this study. Peer review information: Nature Chemical Biology thanks Ronald Buckanovich, John Tanner and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.Attached Files
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Additional details
- Eprint ID
- 115536
- Resolver ID
- CaltechAUTHORS:20220714-16668800
- R35 GM127030
- NIH
- R01 CA244334
- NIH
- R01 GM113100
- NIH
- U01 CA217851
- NIH
- U54 CA224081
- NIH
- R01 AA11147
- NIH
- F32 CA183527
- NIH Postdoctoral Fellowship
- Stanford University
- Weston Havens Foundation
- Emerson Collective
- Human Cancer Models Initiative
- Stand Up to Cancer
- S10 OD026831
- NIH
- P30 CA124435
- NIH
- DE-AC02-76SF00515
- Department of Energy (DOE)
- P41 GM103393
- NIH
- S10 OD028697
- NIH
- Created
-
2022-07-14Created from EPrint's datestamp field
- Updated
-
2022-11-30Created from EPrint's last_modified field