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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.

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Fig. 1: Identification of an ALDH1B1-selective imidazolium.
Fig. 2: Molecular basis of ALDH1B1–imidazolium binding.
Fig. 3: SAR analysis of the imidazolium pharmacophore.
Fig. 4: Imidazolium-based ALDH1B1 inhibitors exhibit off-target mitochondrial toxicity.
Fig. 5: Development of guanidine-based ALDH1B1 antagonists.
Fig. 6: ALDH1B1 inhibitor activities in models of colorectal cancer.

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.

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Acknowledgements

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).

Author information

Authors and Affiliations

Authors

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.

Corresponding author

Correspondence to James K. Chen.

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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.

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Nature Chemical Biology thanks Ronald Buckanovich, John Tanner and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Photoaffinity labeling of imidazolium-binding proteins.

a, Photoaffinity labeling and TAMRA-azide tagging of live NIH-3T3 cells, demonstrating the mitochondrial localization of the imidazolium target. Scale bar: 10 μm. b, Mouse liver mitochondria were photocrosslinked with probe 3 in the absence or presence of competitor 2. The mitochondria were then homogenized, reacted with BODIPY azide (no competitior) or MegaStrokes 673 azide (competitor), and resolved by two-dimensional gel electrophoresis. Protein spots that were specifically labeled by 3 (dashed box, spots 1-5) were then isolated for mass spectrometry-based sequencing.

Extended Data Fig. 2 Inhibition of cellular ALDH1B1 activity by imidazolium derivatives.

a, Immunofluorescence staining of ALDH1A3–/– A375 cells transiently transfected with ALDH1B1, demonstrating the mitochondrial localization of the exogenous protein. Scale bar: 40 µm. b, Flow cytometry-based assays of ALDH1B1 activity and its pharmacological inhibition using ALDH1A3–/– A375 cells. The cells were transiently transfected with ALDH1B1 cDNA or a vector control, incubated with the designated compounds, and then treated with ALDEFLUOR reagent. The cells were gated by ALDEFLUOR signal intensity and side scatter area (SSC-A) to identify those with ALDH1B1 activity, and the percentage of cells outside of the negative control gate is shown for each condition. DMSO and the pan-ALDH inhibitor DEAB were used as negative and positive controls, respectively. c, Chemical structure of 63, which is inactive against ALDH1B1. d, Activity of 63 against selected ALDH isoforms. Fold-change values are relative to a DMSO control and are the average of three biological replicates ± s.d. e, Fluorescence-activated cell sorting (FACS) plots of ALDH1B1-overexpressing ALDH1A3–/– A375 cells that were incubated with DMSO or 63 and then treated with ALDEFLUOR reagent.

Source data

Extended Data Fig. 3 ALDH1B1 promotes SW480 and HCT116 cell growth in spheroid culture.

a,e, Western blot detection of ALDH1B1 protein in individual SW480 and HCT116 cell clones that were transiently transfected with Cas9 cDNA and ALDH1B1 gRNA-1 and gRNA-2. Lysates from the parental lines (P) are also shown, and importin β1 (KPNB1) was used as a loading control. SW480 clone 3 and clone 2 were used as ALDH1B1+/+ and ALDH1B1–/– clones for subsequent studies. b,f, Phase-contrast micrographs of spheroid cultures derived from SW480 and HCT116 cells with differing ALDH1B1 genotypes. c,g, Quantification of spheroid sizes for the micrographs shown in b and f. Each dot represents an individual spheroid with an area that is >500 µm2 in the images. Error bars represent the average spheroid size ± s.e.m. d,h, Viability of the ALDH1B1–/– clone in either adherent or spheroid conditions, as determined by cellular ATP levels and normalized to that of the as ALDH1B1+/+ clone. Data are the average of four (d and h, adherent), eight (d, spheroid) and six (h, spheroid) biological replicates ± s.e.m. Scale bars: 1 mm.

Source data

Extended Data Fig. 4 Exogenous ALDH1B1 rescues the spheroid growth of ALDH1B1 knockout colon cancer cells.

a, Western blot analysis of SW480 clones with the indicated ALDH1B1 genotypes and lentivirally transduced with either EGFP or exogenous ALDH1B1. Importin β1 (KNPB1) was used as gel-loading control. b, Spheroid cultures of the SW480 clones described in a. c, Quantification of spheroid sizes for the micrographs shown in b. Each dot represents an individual spheroid with an area that is >500 µm2 in the image. Error bars represent the average spheroid size ± s.e.m. d, Viability of the ALDH1B1–/– SW480 clone transduced with EGFP or ALDH1B1 and cultured in either adherent or spheroid conditions, as determined by cellular ATP levels and normalized to that of the ALDH1B1+/+ clone transduced with EGFP. Data are the average of four biological replicates ± s.e.m.

Source data

Extended Data Fig. 5 Molecular basis of ALDH1B1-IGUANA binding.

a,b, Lineweaver Burk plots demonstrating that IGUANA-4 exhibits non-competitive inhibition with respect to acetaldehyde (a) and uncompetitive inhibition with respect to NAD+ (b). Both enzyme kinetics assays used the same series of inhibitor concentrations, and the data are the average of three biological replicates ± s.e.m. c, Wall-eyed stereoview of the ALDH1B1–NAD+/2 (light green cartoon and green stick model) and ALDH1B1–NAD+/–IGUANA-4 (light blue cartoon and blue stick model) complexes shown as superimposed structures. Residues 143–154 and 490–500 and the NAD+ cofactor were omitted for clarity. d, Wall-eyed stereoview of the electron density map for IGUANA-4 (blue mesh) bound to ALDH1B1. The polder omit map was calculated with coefficients mFo-DFc and is contoured at 4σ. Residues in the inhibitor-binding site are shown, and the red dashed line indicates a potential n-to-π* interaction between the Asn457 backbone carbonyl and the guanidine (3.2 Å).

Source data

Extended Data Fig. 6 IGUANAs engage ALDH1B1 in live cells.

a, Flow cytometry-based ALDEFLUOR assays using ALDH1A3–/– A375 cells, demonstrating the ability of IGUANA-3 to inhibit cellular ALDH1B1 activity. The cells were transiently transfected with ALDH1B1 cDNA or a vector control, incubated with IGUANA-3 or DMSO vehicle alone, and then treated with ALDEFLUOR reagent. The cells were gated by ALDEFLUOR signal intensity and side scatter area (SSC-A) to identify those with ALDH1B1 activity, and the percentage of cells outside of the negative control gate is shown for each condition. b, Cellular thermal shift assay demonstrating that IGUANA-1 stabilizes endogenous ALDH1B1 in live SW480 cells. Western blot signals for ALDH1B1 and total protein staining of the soluble fraction are shown for each condition. c, Corresponding melting curves of endogenous ALDH1B1 in the presence and absence of IGUANA-1. Data are the average of two biological replicates ± s.d., normalized to the DMSO condition at 45 °C.

Source data

Extended Data Fig. 7 IGUANAs suppress colon cancer spheroid growth.

a, Brightfield micrographs of SW480 spheroid cultures treated with IGUANA-3 and then stained with crystal violet. b, Quantification of spheroid sizes for the micrographs shown in b. Each dot represents an individual spheroid with an area that is >500 µm2 in the micrograph. Error bars represent the average spheroid size ± s.e.m. c Brightfield, fluorescent, and merged micrographs of SW480 spheroids treated with IGUANA-1 or DMSO vehicle alone for 3 days and then stained overnight with the viability dye SYTOX Green. Scale bars: a, 1 mm; c, 100 µm.

Source data

Supplementary information

Supplementary Information

Supplementary Tables 1 and 2, Figs. 1–21, Note (synthetic procedures and compound characterization) and full-length blots for Supplementary figures.

Reporting Summary

Supplementary Data 1

Chemical structures of imidazolium and guanidine derivatives.

Supplementary Data 2

Mass spectrometry data for photoaffinity-labeled protein spots.

Supplementary Data 3

TPP data.

Supplementary Data 4

RNA-seq data for the ALDH1B1-dependent transcriptome.

Supplementary Data 5

Statistical source data for Supplementary figures.

Source data

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Feng, Z., Hom, M.E., Bearrood, T.E. et al. Targeting colorectal cancer with small-molecule inhibitors of ALDH1B1. Nat Chem Biol (2022). https://doi.org/10.1038/s41589-022-01048-w

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