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Covalent inhibition of pro-apoptotic BAX

Nature Chemical Biology volume 20, pages 1022–1032 (2024)Cite this article

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Abstract

BCL-2-associated X protein (BAX) is a promising therapeutic target for activating or restraining apoptosis in diseases of pathologic cell survival or cell death, respectively. In response to cellular stress, BAX transforms from a quiescent cytosolic monomer into a toxic oligomer that permeabilizes the mitochondria, releasing key apoptogenic factors. The mitochondrial lipid trans-2-hexadecenal (t-2-hex) sensitizes BAX activation by covalent derivatization of cysteine 126 (C126). In this study, we performed a disulfide tethering screen to discover C126-reactive molecules that modulate BAX activity. We identified covalent BAX inhibitor 1 (CBI1) as a compound that selectively derivatizes BAX at C126 and inhibits BAX activation by triggering ligands or point mutagenesis. Biochemical and structural analyses revealed that CBI1 can inhibit BAX by a dual mechanism of action: conformational constraint and competitive blockade of lipidation. These data inform a pharmacologic strategy for suppressing apoptosis in diseases of unwanted cell death by covalent targeting of BAX C126.

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Fig. 1: Disulfide tethering identifies a covalent inhibitor of BAX.
Fig. 2: CBI1 targets BAX at C126.
Fig. 3: NMR analysis of the BAX–CBI1 interaction.
Fig. 4: CBI1 suppresses the membrane-permeabilizing auto-activity of BAX F116A.
Fig. 5: CBI1 reverses the conformational activation of BAX F116A.
Fig. 6: CBI1 inhibits BAX by a dual mechanism of action.

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Data availability

All data generated or analyzed for this study are included in the manuscript and its Supplementary Information. HDX MS data have been deposited to the ProteomeXchange Consortium via the PRIDE74 partner repository with dataset identifier PXD040917 and are also included in the manuscript as Source Data Fig. 5. The NMR structure of full-length BAX, corresponding to PDB ID: 1F16, was used in this study. Source data are provided with this paper.

Code availability

No code was generated for this study.

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Acknowledgements

We thank E. Smith for assistance with figure preparation; J. Lee for performing intact mass spectrometry analysis of tBID at the Dana-Farber Molecular Biology Core; J. Sun for NMR technical support at the Dana-Farber NMR Core and the Harvard Medical School BioNMR Core; and G. Bird and B. Moyer for synthesizing BIM SAHB. This study was funded by National Institutes of Health (NIH) grant R35CA197583 to L.D.W., NIH grant R01AI070292 and the Harry and Dianna Professorship in Pharmaceutical Sciences to J.A.W., NIH grant R01GM67945 to S.P.G., National Science Foundation and Landry Cancer Biology Research pre-doctoral fellowships to M.W.M. and NIH grant 5T32HL007574 to C.M.C.

Author information

Authors and Affiliations

  1. Department of Pediatric Oncology and Linde Program in Cancer Chemical Biology, Dana-Farber Cancer Institute, Boston, MA, USA

    Matthew W. McHenry, Peiwen Shi, Christina M. Camara, Daniel T. Cohen, Utsarga Adhikary, Micah A. Gygi & Loren D. Walensky

  2. Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA

    Matthew W. McHenry

  3. Departments of Pharmaceutical Chemistry and Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA, USA

    T. Justin Rettenmaier & James A. Wells

  4. Department of Cell Biology, Harvard Medical School, Boston, MA, USA

    Ka Yang & Steven P. Gygi

  5. Department of Chemistry and Chemical Biology, Northeastern University, Boston, MA, USA

    Thomas E. Wales & John R. Engen

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  1. Matthew W. McHenry
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Contributions

M.W.M. and L.D.W. designed the study. D.T.C. and T.J.R. conducted the disulfide tethering screen, under the supervision of J.A.W. M.W.M. synthesized the small molecules, produced BAX proteins and performed all biochemical, mitochondrial and structural experiments. P.S. assisted M.W.M. with biochemical and mitochondrial experiments. C.M.C. performed the molecular dynamics simulations and assisted M.W.M. with the HMQC NMR experiments. U.A., M.A.G. and K.Y. conducted the chemoproteomics experiment, under the supervision of S.P.G. M.W.M. and T.E.W. performed the HDX MS analyses, under the supervision of J.R.E. L.D.W. wrote the manuscript, which was reviewed by all co-authors.

Corresponding author

Correspondence to Loren D. Walensky.

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Nature Chemical Biology thanks Haiming Dai, Glenn Masson, Tudor Moldoveanu 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 Comparative inhibitory effects of small molecule hits from the disulfide tethering screen on tBID-triggered BAX-mediated liposomal release.

a-d, Liposomal release in response to BAX WT, tBID, the indicated molecule, or the combination of tBID and BAX WT pre-incubated with or without increasing doses of 1C18 (a), 1A18 (b), 1E4 (c), or 3A20 (d). Data are mean ± s.e.m. for experiments performed in technical quadruplicate and repeated using independent preparations of liposomes, proteins, and molecules with similar results.

Source data

Extended Data Fig. 2 Effect of CBI1 on BIM SAHB-triggered BAX-mediated liposomal release.

Dose-responsive inhibition of BIM SAHB-triggered, BAX-mediated liposomal poration (dark gray) upon addition of CBI1 (blue). Data are mean ± s.e.m. for experiments performed in technical quadruplicate and repeated using independent preparations of liposomes, protein, and compounds with similar results.

Source data

Extended Data Fig. 3 No inhibitory effect of CBI1 on Fos-12-induced oligomerization of BAX.

SEC profiles of monomeric BAX (gray) and Fos-12-induced BAX oligomer in the presence (blue) or absence (black) of CBI1. The experiment was performed twice with independent preparations of protein, detergent, and small molecule with similar results.

Source data

Extended Data Fig. 4 NMR analysis of the BAX/CBI1 interaction.

a, Measured chemical shift changes of 15N-BAX (20 μM) upon addition of CBI1 (5:1 of CBI1:BAX), plotted as a function of BAX residue number. Chemical shift changes above the 2 s.d. cutoff (significance threshold of 0.0536 p.p.m.) are colored maroon and those above the 1 s.d. cutoff (significance threshold of 0.0347 p.p.m.) are colored red. Residues whose cross peaks experienced prominent signal attenuation or chemical shift perturbation upon CBI1 incubation are colored beige. b-c, Chemical shift perturbations of BAX cross peaks corresponding to residues F116 (b, dashed box) and S118 (c) upon addition of CBI1. Whereas BAX exhibited one cross peak each for F116 and S118 (gray), the addition of CBI1 at a CBI1:BAX ratio of 5:1 resulted in the appearance of a second cross peak for each residue (red). Upon incubation with CBI1 at a CBI1:BAX ratio of 10:1, the original cross peaks shifted completely to the second locations (blue). In contrast, D142 (b) experienced little to no change in its corresponding cross peak upon addition of CBI1. d, Prominent signal attenuation or chemical shift perturbation of A81 in wild-type BAX upon CBI1 titration, as reflected by disappearance of the cross peak. e, In contrast, in the context of BAX C126A, the A81 cross peak demonstrates progressive migration upon CBI1 titration, consistent with fast exchange between the unbound and bound forms of BAX C126A, as expected for non-covalent interaction.

Source data

Extended Data Fig. 5 Influence of CBI1 on the conformational dynamics of the BAX α1-α2 loop.

a, A difference distance matrix plot derived from molecular dynamics simulations of wild-type BAX in the presence or absence of CBI1 demonstrated the greatest effect of small molecule C126-derivatization on the protein dynamics of the α1-α2 loop. b-c, Representative images from the molecular dynamics (MD) simulations of BAX in the absence (b) and presence (c) of CBI1, demonstrating distinct positioning of the α1-α2 loop (purple).

Extended Data Fig. 6 Comparative NMR analyses of the BAX WT/CBI1, BAX WT/N-CBI1, and BAX C126A/CBI1 interactions.

a–c, Measured chemical shift changes of 15N-BAX WT (40 μM) upon addition of CBI1 (5:1 small molecule:protein) (a), 15N-BAX WT (40 μM) upon addition of N-CBI1 (5:1 small molecule:protein) (b), and 15N-BAX C126A (40 μM) upon addition of CBI1 (5:1 small molecule:protein) (c), plotted as a function of BAX residue number. Chemical shift changes above the 2 s.d. cutoff (significance thresholds of 0.0499, 0.0187, 0.01579 p.p.m. for a, b, c, respectively) are colored maroon and those above the 1 s.d. cutoff (significance thresholds of 0.0329, 0.0125, 0.0107 p.p.m. for a, b, c, respectively) are colored red. Residues whose cross peaks experienced prominent signal attenuation or chemical shift perturbation upon small molecule incubation are colored beige.

Source data

Extended Data Fig. 7 Relative impact of covalent vs. non-covalent small molecule interaction on BAX F116A-mediated liposomal permeabilization.

a, b, Comparative inhibitory effects of CBI1 and N-CBI1 on liposomal release by BAX F116A (a) or BAX F116A/C126A (b). Data are mean ± s.e.m. for experiments performed in technical quadruplicate and repeated using independent preparations of liposomes, proteins, and small molecules with similar results.

Source data

Extended Data Fig. 8 CBI1 reverses the conformational activation of BAX F116A.

a, b, Difference distance matrix plots derived from molecular dynamics simulations of BAX F116A compared to BAX WT (a) and BAX F116A in the presence or absence of CBI1 (b) demonstrated striking reversal of the auto-activating conformational changes induced by BAX F116A mutagenesis upon CBI1 covalent derivatization of C126.

Extended Data Fig. 9 CBI1 blocks the mitochondrial translocation of BAX F116A.

a, b, Distribution of BAX F116A (a) or BAX F116A/C126A (b) (2 μM) between supernatant and BAX/BAK-deficient mitochondrial fractions, as detected by BAX western analysis after pre-treating BAX proteins with escalating doses of CBI1 (200 nM–4 μM, lanes 2–7), incubation with mitochondria, isolation of the supernatant and pellet fractions by centrifugation, and SDS PAGE. Isolation of the mitochondrial pellet fraction was verified by VDAC1 western analysis. The experiment was performed three times using independent preparations of mitochondria, proteins, and small molecule.

Source data

Extended Data Fig. 10 CBI1 blocks t-2-hex lipidation and induced homo-oligomerization of BAX F116A.

Incubation of BAX F116A (5 μM) with t-2-hex (2.5 mM) in the presence or absence of increasing amounts (5 μM-100 μM) of CBI1 (lanes 3–7) or N-CBI1 (lanes 9–13) for 2 hours at 37 °C followed by detection of lipidated BAX by addition of Cy5-hydrazide, gel electrophoresis, and fluorescence scan. BAX protein lipidation, t-2-hex-induced homo-oligomerization (as reflected by laddering), and comparative dose-responsive suppression by CBI1 and N-CBI1, was detected by fluorescence scan of the indicated BAX mixtures (top and middle panels). The influence of t-2-hex and co-treatment with CBI1 or N-CBI1 on the level of monomeric BAX F116A was monitored by protein stain of the electrophoresed BAX mixtures (bottom panel). The experiment was performed twice using independent preparations of protein, lipid, and small molecules.

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Supplementary Tables 1–4, Supplementary Figs. 1–6 and Supplementary Note 1.

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Molecular dynamics simulation of the CBI1–BAX interaction.

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Source data for Supplementary Figs. 1–3, 5 and 6.

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McHenry, M.W., Shi, P., Camara, C.M. et al. Covalent inhibition of pro-apoptotic BAX. Nat Chem Biol 20, 1022–1032 (2024). https://doi.org/10.1038/s41589-023-01537-6

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