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A small-molecule allosteric inhibitor of BAX protects against doxorubicin-induced cardiomyopathy

Abstract

Doxorubicin remains an essential component of many cancer regimens, but its use is limited by lethal cardiomyopathy, which has been difficult to target, owing to pleiotropic mechanisms leading to apoptotic and necrotic cardiac cell death. Here we show that BAX is rate-limiting in doxorubicin-induced cardiomyopathy and identify a small-molecule BAX inhibitor that blocks both apoptosis and necrosis to prevent this syndrome. By allosterically inhibiting BAX conformational activation, this compound blocks BAX translocation to mitochondria, thereby abrogating both forms of cell death. When co-administered with doxorubicin, this BAX inhibitor prevents cardiomyopathy in zebrafish and mice. Notably, cardioprotection does not compromise the efficacy of doxorubicin in reducing leukemia or breast cancer burden in vivo, primarily due to increased priming of mitochondrial death mechanisms and higher BAX levels in cancer cells. This study identifies BAX as an actionable target for doxorubicin-induced cardiomyopathy and provides a prototype small-molecule therapeutic.

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Fig. 1: Deletion of BAX protects against doxorubicin-induced cardiomyopathy.
Fig. 2: BAI1 binds BAX directly to inhibit its mitochondrial insertion and translocation.
Fig. 3: BAI1 inhibits BAX-dependent apoptosis and necrosis.
Fig. 4: BAI1 inhibits apoptosis and necrosis in primary cardiomyocytes.
Fig. 5: BAI1 prevents doxorubicin-induced cardiomyopathy.
Fig. 6: BAI1 does not compromise the reduction of cancer burden by doxorubicin in multiple cancer models, while providing cardiac protection in the same animals.
Fig. 7: Mechanism for BAI1 selectivity in protecting the heart against doxorubicin without compromising killing of cancer cells.

Data availability

Source data for all figures are provided with the paper online. All other data supporting the findings of this study are available from the corresponding authors upon reasonable request.

Code availability

Details and code for the power analyses can be found in the GitHub repository (https://github.com/celldeath/power_analysis).

References

  1. 1.

    Moslehi, J. J. Cardiovascular toxic effects of targeted cancer therapies. N. Engl. J. Med. 375, 1457–1467 (2016).

    CAS  PubMed  Google Scholar 

  2. 2.

    Moslehi, J., Amgalan, D. & Kitsis, R. N. Grounding cardio-oncology in basic and clinical science. Circulation 136, 3–5 (2017).

    PubMed  PubMed Central  Google Scholar 

  3. 3.

    Middleman, E., Luce, J. & Frei, E. 3rd Clinical trials with adriamycin. Cancer 28, 844–850 (1971).

    CAS  PubMed  Google Scholar 

  4. 4.

    Lefrak, E. A., Pitha, J., Rosenheim, S. & Gottlieb, J. A. A clinicopathologic analysis of adriamycin cardiotoxicity. Cancer 32, 302–314 (1973).

    CAS  PubMed  Google Scholar 

  5. 5.

    Tewey, K. M., Rowe, T. C., Yang, L., Halligan, B. D. & Liu, L. F. Adriamycin-induced DNA damage mediated by mammalian DNA topoisomerase II. Science 226, 466–468 (1984).

    CAS  PubMed  Google Scholar 

  6. 6.

    Zhang, S. et al. Identification of the molecular basis of doxorubicin-induced cardiotoxicity. Nat. Med. 18, 1639–1642 (2012).

    PubMed  Google Scholar 

  7. 7.

    Ichikawa, Y. et al. Cardiotoxicity of doxorubicin is mediated through mitochondrial iron accumulation. J. Clin. Invest. 124, 617–630 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. 8.

    Zhou, S., Starkov, A., Froberg, M. K., Leino, R. L. & Wallace, K. B. Cumulative and irreversible cardiac mitochondrial dysfunction induced by doxorubicin. Cancer Res. 61, 771–777 (2001).

    CAS  PubMed  Google Scholar 

  9. 9.

    Lim, C. C. et al. Anthracyclines induce calpain-dependent titin proteolysis and necrosis in cardiomyocytes. J. Biol. Chem. 279, 8290–8299 (2004).

    CAS  PubMed  Google Scholar 

  10. 10.

    Li, D. L. et al. Doxorubicin blocks cardiomyocyte autophagic flux by inhibiting lysosome acidification. Circulation 133, 1668–1687 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. 11.

    Renu, K., V, G. A., P, B. T. & Arunachalam, S. Molecular mechanism of doxorubicin-induced cardiomyopathy - An update. Eur. J. Pharmacol. 818, 241–253 (2018).

    CAS  PubMed  Google Scholar 

  12. 12.

    Zhang, Y. W., Shi, J., Li, Y. J. & Wei, L. Cardiomyocyte death in doxorubicin-induced cardiotoxicity. Arch. Immunol. Ther. Exp. (Warsz) 57, 435–445 (2009).

    CAS  Google Scholar 

  13. 13.

    Walensky, L. D. & Gavathiotis, E. BAX unleashed: the biochemical transformation of an inactive cytosolic monomer into a toxic mitochondrial pore. Trends Biochem. Sci. 36, 642–652 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. 14.

    Whelan, R. S., Kaplinskiy, V. & Kitsis, R. N. Cell death in the pathogenesis of heart disease: mechanisms and significance. Annu. Rev. Physiol. 72, 19–44 (2010).

    CAS  PubMed  Google Scholar 

  15. 15.

    Whelan, R. S. et al. BAX regulates primary necrosis through mitochondrial dynamics. Proc. Natl Acad. Sci. USA 109, 6566–6571 (2012).

    CAS  PubMed  Google Scholar 

  16. 16.

    Karch, J. et al. BAX and BAK function as the outer membrane component of the mitochondrial permeability pore in regulating necrotic cell death in mice. eLife 2, e00772 (2013).

    PubMed  PubMed Central  Google Scholar 

  17. 17.

    Gavathiotis, E. et al. BAX activation is initiated at a novel interaction site. Nature 455, 1076–1081 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. 18.

    Gavathiotis, E., Reyna, D. E., Davis, M. L., Bird, G. H. & Walensky, L. D. BH3-triggered structural reorganization drives the activation of proapoptotic BAX. Mol. Cell 40, 481–492 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. 19.

    Ferreira de Souza, T. et al. Anthracycline therapy is associated with cardiomyocyte atrophy and preclinical manifestations of heart disease. JACC Cardiovasc. Imaging 11, 1045–1055 (2018).

    PubMed  Google Scholar 

  20. 20.

    Neilan, T. G. et al. Left ventricular mass in patients with a cardiomyopathy after treatment with anthracyclines. Am. J. Cardiol. 110, 1679–1686 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. 21.

    Jordan, J. H. et al. Left ventricular mass change after anthracycline chemotherapy. Circ. Heart Fail. 11, e004560 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. 22.

    Zhu, W. et al. Acute doxorubicin cardiotoxicity is associated with p53-induced inhibition of the mammalian target of rapamycin pathway. Circulation 119, 99–106 (2009).

    CAS  PubMed  Google Scholar 

  23. 23.

    Scaffidi, P., Misteli, T. & Bianchi, M. E. Release of chromatin protein HMGB1 by necrotic cells triggers inflammation. Nature 418, 191–195 (2002).

    CAS  PubMed  Google Scholar 

  24. 24.

    Bombrun, A. et al. 3,6-dibromocarbazole piperazine derivatives of 2-propanol as first inhibitors of cytochrome c release via BAX channel modulation. J. Med. Chem. 46, 4365–4368 (2003).

    CAS  PubMed  Google Scholar 

  25. 25.

    Peixoto, P. M., Ryu, S. Y., Bombrun, A., Antonsson, B. & Kinnally, K. W. MAC inhibitors suppress mitochondrial apoptosis. Biochem. J. 423, 381–387 (2009).

    CAS  PubMed  Google Scholar 

  26. 26.

    Garner, T. P. et al. Small-molecule allosteric inhibitors of BAX. Nat. Chem. Biol. 15, 322–330 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. 27.

    Hsu, Y. T. & Youle, R. J. Nonionic detergents induce dimerization among members of the Bcl-2 family. J. Biol. Chem. 272, 13829–13834 (1997).

    CAS  PubMed  Google Scholar 

  28. 28.

    Antonsson, B., Montessuit, S., Sanchez, B. & Martinou, J. C. BAX is present as a high molecular weight oligomer/complex in the mitochondrial membrane of apoptotic cells. J. Biol. Chem. 276, 11615–11623 (2001).

    CAS  PubMed  Google Scholar 

  29. 29.

    Scorrano, L. et al. BAX and BAK regulation of endoplasmic reticulum Ca2 +: a control point for apoptosis. Science 300, 135–139 (2003).

    CAS  PubMed  Google Scholar 

  30. 30.

    Oakes, S. A. et al. Proapoptotic BAX and BAK regulate the type 1 inositol trisphosphate receptor and calcium leak from the endoplasmic reticulum. Proc. Natl Acad. Sci. USA 102, 105–110 (2005).

    CAS  PubMed  Google Scholar 

  31. 31.

    Hetz, C. et al. Proapoptotic BAX and BAK modulate the unfolded protein response by a direct interaction with IRE1α. Science 312, 572–576 (2006).

    CAS  PubMed  Google Scholar 

  32. 32.

    Liu, Y. et al. Visnagin protects against doxorubicin-induced cardiomyopathy through modulation of mitochondrial malate dehydrogenase. Sci. Transl. Med. 6, 266ra170 (2014).

    PubMed  PubMed Central  Google Scholar 

  33. 33.

    Minn, A. J. et al. Genes that mediate breast cancer metastasis to lung. Nature 436, 518–524 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. 34.

    Patsialou, A. et al. Selective gene-expression profiling of migratory tumor cells in vivo predicts clinical outcome in breast cancer patients. Breast Cancer Res. 14, R139 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. 35.

    Karagiannis, G. S. et al. Neoadjuvant chemotherapy induces breast cancer metastasis through a TMEM-mediated mechanism. Sci. Transl. Med. 9, eaan0026 (2017).

    PubMed  PubMed Central  Google Scholar 

  36. 36.

    Krivtsov, A. V. et al. Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9. Nature 442, 818–822 (2006).

    CAS  PubMed  Google Scholar 

  37. 37.

    Somervaille, T. C. & Cleary, M. L. Identification and characterization of leukemia stem cells in murine MLL-AF9 acute myeloid leukemia. Cancer Cell 10, 257–268 (2006).

    CAS  PubMed  Google Scholar 

  38. 38.

    Letai, A. G. Diagnosing and exploiting cancer’s addiction to blocks in apoptosis. Nat. Rev. Cancer 8, 121–132 (2008).

    CAS  PubMed  Google Scholar 

  39. 39.

    Montero, J. et al. Drug-induced death signaling strategy rapidly predicts cancer response to chemotherapy. Cell 160, 977–989 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. 40.

    Sarosiek, K. A. et al. Developmental regulation of mitochondrial apoptosis by c-Myc governs age- and tissue-specific sensitivity to cancer therapeutics. Cancer Cell 31, 142–156 (2017).

    CAS  PubMed  Google Scholar 

  41. 41.

    Polster, B. M., Basanez, G., Young, M., Suzuki, M. & Fiskum, G. Inhibition of BAX-induced cytochrome c release from neural cell and brain mitochondria by dibucaine and propranolol. J. Neurosci. 23, 2735–2743 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. 42.

    Hetz, C. et al. BAX channel inhibitors prevent mitochondrion-mediated apoptosis and protect neurons in a model of global brain ischemia. J. Biol. Chem. 280, 42960–42970 (2005).

    CAS  PubMed  Google Scholar 

  43. 43.

    Niu, X. et al. A small-molecule inhibitor of BAX and BAK oligomerization prevents genotoxic cell death and promotes neuroprotection. Cell Chem. Biol. 24, 493–506 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. 44.

    Ky, B., Vejpongsa, P., Yeh, E. T., Force, T. & Moslehi, J. J. Emerging paradigms in cardiomyopathies associated with cancer therapies. Circ. Res. 113, 754–764 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. 45.

    Garcia-Pavia, P. et al. Genetic variants associated with cancer therapy-induced cardiomyopathy. Circulation 140, 31–41 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. 46.

    Rambal, A. A., Panaguiton, Z. L., Kramer, L., Grant, S. & Harada, H. MEK inhibitors potentiate dexamethasone lethality in acute lymphoblastic leukemia cells through the pro-apoptotic molecule BIM. Leukemia 23, 1744–1754 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. 47.

    Meng, J. et al. Apoptosis induction by MEK inhibition in human lung cancer cells is mediated by BIM. PLoS ONE 5, e13026 (2010).

    PubMed  PubMed Central  Google Scholar 

  48. 48.

    Kirshenbaum, L. A. & Schneider, M. D. Adenovirus E1A represses cardiac gene transcription and reactivates DNA synthesis in ventricular myocytes, via alternative pocket protein- and p300-binding domains. J. Biol. Chem. 270, 7791–7794 (1995).

    CAS  PubMed  Google Scholar 

  49. 49.

    Kirshenbaum, L. A., MacLellan, W. R., Mazur, W., French, B. A. & Schneider, M. D. Highly efficient gene transfer into adult ventricular myocytes by recombinant adenovirus. J. Clin. Invest. 92, 381–387 (1993).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. 50.

    Uchime, O. et al. Synthetic antibodies inhibit Bcl-2-associated X protein (BAX) through blockade of the N-terminal activation site. J. Biol. Chem. 291, 89–102 (2016).

    CAS  PubMed  Google Scholar 

  51. 51.

    Gavathiotis, E., Reyna, D. E., Bellairs, J. A., Leshchiner, E. S. & Walensky, L. D. Direct and selective small-molecule activation of proapoptotic BAX. Nat. Chem. Biol. 8, 639–645 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. 52.

    Youle, R. J. & Strasser, A. The BCL-2 protein family: opposing activities that mediate cell death. Nat. Rev. Mol. Cell Biol. 9, 47–59 (2008).

    CAS  PubMed  Google Scholar 

  53. 53.

    Regula, K. M., Ens, K. & Kirshenbaum, L. A. Inducible expression of BNIP3 provokes mitochondrial defects and hypoxia-mediated cell death of ventricular myocytes. Circ. Res. 91, 226–231 (2002).

    CAS  PubMed  Google Scholar 

  54. 54.

    Santulli, G., Xie, W., Reiken, S. R. & Marks, A. R. Mitochondrial calcium overload is a key determinant in heart failure. Proc. Natl Acad. Sci. USA 112, 11389–11394 (2015).

    CAS  PubMed  Google Scholar 

  55. 55.

    Lombardi, A., Trimarco, B., Iaccarino, G. & Santulli, G. Impaired mitochondrial calcium uptake caused by tacrolimus underlies β-cell failure. Cell Commun. Signal 15, 47 (2017).

    PubMed  PubMed Central  Google Scholar 

  56. 56.

    Rohrbach, P. et al. Quantitative calcium measurements in subcellular compartments of Plasmodium falciparum-infected erythrocytes. J. Biol. Chem. 280, 27960–27969 (2005).

    CAS  PubMed  Google Scholar 

  57. 57.

    Walczysko, P., Wagner, E. & Albrechtova, J. T. Use of co-loaded Fluo-3 and Fura Red fluorescent indicators for studying the cytosolic Ca2+ concentrations distribution in living plant tissue. Cell Calcium 28, 23–32 (2000).

    CAS  PubMed  Google Scholar 

  58. 58.

    Waning, D. L. et al. Excess TGF-β mediates muscle weakness associated with bone metastases in mice. Nat. Med. 21, 1262–1271 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  59. 59.

    Stypmann, J. et al. Echocardiographic assessment of global left ventricular function in mice. Lab Animal 43, 127–137 (2009).

    CAS  Google Scholar 

  60. 60.

    Gao, S., Ho, D., Vatner, D. E. & Vatner, S. F. Echocardiography in mice. Curr. Protoc. Mouse Biol. 1, 71–83 (2011).

    PubMed  PubMed Central  Google Scholar 

  61. 61.

    Medina-Ramirez, C. M. et al. Apoptosis inhibitor ARC promotes breast tumorigenesis, metastasis, and chemoresistance. Cancer Res. 71, 7705–7715 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  62. 62.

    Chen, W. et al. Malignant transformation initiated by Mll-AF9: gene dosage and critical target cells. Cancer Cell 13, 432–440 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  63. 63.

    Ryan, J., Montero, J., Rocco, J. & Letai, A. iBH3: simple, fixable BH3 profiling to determine apoptotic priming in primary tissue by flow cytometry. Biol. Chem. 397, 671–678 (2016).

    CAS  PubMed  Google Scholar 

  64. 64.

    Ryan, J. & Letai, A. BH3 profiling in whole cells by fluorimeter or FACS. Methods 61, 156–164 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

The authors thank K. B. Margulies and K. Bedi of the University of Pennsylvania for generously providing human heart samples; M. Graham for analysis of pharmacokinetic data; B. Bartholdy for RNA-sequencing analysis; V. Hadad for statistical analysis for in vivo studies; M. Zheng, H. Guzik and A. Vohra for technical assistance with mouse studies, imaging and zebrafish studies. This work was supported by grants from the National Institutes of Health (R01HL128071, R01HL130861, R01HL138475 and R01CA170911 to R.N.K.; R01CA178394 to E.G.; R01HL146691 and R00DK107895 to G.S.; R01CA100324 to J.S.C.; P30CA013330 to the Albert Einstein Cancer Center; T32CA200561 to L.R.S.; 1S10OD019961 Shared Instrumentation Grant to the Einstein Analytical Imaging Facility); American Heart Association (15CSA26240000 and 18SRG34280018 to R.N.K. and E.G.; and 15PRE25080032 Predoctoral Fellowship to D.A.); Harrington Scholar-Innovator Award to R.N.K.; Fondation Leducq (RA15CVD04 to R.N.K. and E.G.); Department of Defense (PR151134P1 to R.N.K); New York State Stem Cell Science (NYSTEM C029154 to the Einstein Stem Cell Isolation and Xenotransplantation Facility); Canadian Institutes for Health Research Foundation Grant to L.A.K.; Breast Cancer Research Foundation to R.B.H.; and John S. LaDue Memorial Fellowship at Harvard Medical School to A.A. R.N.K. was supported by the Dr. Gerald and Myra Dorros Chair in Cardiovascular Disease. Overall support was provided by the Wilf Family Cardiovascular Research Institute.

Author information

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Authors

Contributions

R.N.K. and E.G. conceived the research study. D.A. and R.N.K. designed the molecular, cellular and in vivo studies, which were executed by D.A., R.P., X.F.J., M.Y., F.G.L., J.J.C., J.L., Y.C., V.M., M.S., G.S. and L.A.K. E.G. and T.P.G. designed the MST studies, which were executed by T.P.G. E.G., R.N.K., A.L. and D.A. designed the BH3 profiling studies, which were executed by A.L. and D.A. V.P. performed echocardiography. R.N.K., R.B.H. and D.A. designed the LM2 xenograft studies, which were executed by D.A. and H.L. R.N.K, M.H.O., J.S.C. and D.A. designed the breast cancer patient-derived xenograft studies, which were executed by G.S.K., L.R.S. and D.A. R.N.K., U.S., E.G. and D.A. designed the leukemia transplant studies, which were executed by D.A., S.R.N. and K.M. A.A. and R.T.P. designed the zebrafish studies, which were executed by A.A., D.A., E.G. and R.N.K wrote the manuscript with contributions by M.Y. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Evripidis Gavathiotis or Richard N. Kitsis.

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Competing interests

R.N.K., E.G., D.A., T.P.G. and L.A.K. are inventors on a patent application PCT/US2018/021644 submitted by Albert Einstein College of Medicine that covers compounds, compositions and methods for BAX inhibition for the treatment of diseases and disorders.

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Extended data

Extended Data Fig. 1 BAI1 binding to BAX as assessed by microscale thermophoresis (MST).

Relates to Fig. 2b. a, c, Examples of MST which measures the thermophoretic movement induced by infrared laser activation of labeled BAX as reflected by relative fluorescence as a function of time. Cold start region indicated by blue box. Warm region indicated by pink box. Each curve denotes a different concentration of BAI1. BAX was labeled with 1 (C1, red curves) or 2 (C2, blue curves) dye molecules/BAX molecule yielding similar results. Representative of 3 independent experiments. b, d, Binding curves determined using the temperature jump region of the curve (pink box) normalized to the cold start region (blue box).

Extended Data Fig. 2 BAI1 does not affect baseline or doxorubicin-induced Ca2+ handling or IRE1α activation.

a, Cytosolic Ca2+ concentrations assessed with Fluo-4 at baseline and following 1 hr treatment with doxorubicin (DOX) in the absence or presence of 1 hr pretreatment with BAI1 1 µM. Box plots depict IQR, horizontal lines show the median, and whiskers represent the minimum and maximum values. n=15, 16 (control); n=18, 19 (DOX 1 µM); n=17, 20 (DOX 5 µM); n=24, 23 (DOX 10 µM) cells. Representative of 2 independent experiments. One-way ANOVA, **** P<0.0001. b, Time course of mitochondrial Ca2+ concentrations assessed with Rhod-2 at baseline and following 1 hr treatment with DOX in the absence or presence of pretreatment with BAI1 1 µM. n=28 (DOX 1 µM), n=35 (DOX 5 µM), n=32 (DOX 10 µM) cells. Representative of 2 independent experiments. Data presented as mean ± s.e.m. c, Western blot for total and phosphorylated IRE1α in MEFs treated with DOX 10 μM for 12 hr with or without 1 hr pretreatment with BAI1 1 μM. Thapsigargin (TG) 1 μM served as a positive control. Unprocessed images of blots are provided as source data. Representative of 3 independent experiments. Source data

Extended Data Fig. 3 BAI1 prevents doxorubicin-induced cardiomyopathy in zebrafish.

a, Zebrafish 30 hr post-fertilization treated with DMSO (control), doxorubicin (DOX), or DOX plus BAI1. Hearts indicated by arrows. Representative images of 3 independent experiments. b, BAI1 prevents doxorubicin-induced cardiomyopathy in a dose-dependent manner. Prevention indicates the absence of all 3 of the following manifestations assessed 40 hr post-treatment: decreased cardiac contraction, pericardial edema, and decreased tail blood flow. n=3 independent experiments. One-way ANOVA, * P=0.0456, ** P=0.0011. Data presented as mean ± s.e.m. Source data

Extended Data Fig. 4 Pharmacokinetic analysis of BAI1.

a, Plasma BAI1 concentrations as a function of time following 1 mg/kg intravenous injection into adult male mice. Concentration determined by LC-MS/MS. n=3 males/time point. Data presented as mean ± s.e.m. b, Pharmacokinetic parameters estimated by non-compartmental analysis of the plasma BAI1 concentration-time curve. Kel: elimination rate constant; t1/2: half-life; Cmax: maximum plasma concentration; SE_Cmax: standard error of Cmax; Tmax: time to reach Cmax; C0: concentration at t=0; AUClast: area under the curve from t=0 to the time of the last quantifiable concentration; SE_AUClast: standard error of AUClast; AUCinf: AUC from t=0 to infinity; AUCextrap: extrapolated AUC from last to infinity, expressed as percentage of AUCinf; Vz: volume of distribution following administration; Cl: total body clearance following administration, calculated from dose/AUC; AUMCinf: area under the first moment curve from t=0 to infinity; MRTinf: mean residence time, calculated by dividing the AUMCinf by the AUC; Vss: steady-state volume of distribution, calculated from Cl · MRTinf. Source data

Extended Data Fig. 5 BAI1 prevents cardiomyopathy in long-term doxorubicin model (females).

a, Schematic of low-dose, long-term doxorubicin (DOX)-induced cardiomyopathy mouse model. Mice were 12 weeks old at the start of experiment. b, Echocardiographic analysis of systolic function including fractional shortening (FS), left ventricular end-diastolic dimension (LVEDD), left ventricular end-systolic dimension (LVESD), and LVEDD-LVESD prior to initiation of treatment. Saline, n=10; BAI1, n=10; DOX, n=15; DOX+BAI1, n=15 females. Mean values are shown on the graphs. c, Echocardiographic parameters 4 weeks following the 2-week course of treatment. Saline, n=10; BAI1, n=10; DOX, n=13; DOX+BAI1, n=15 females. Mean values are shown on the graphs. One-way ANOVA, FS: ** P=0.0045, ## P=0.0013; LVESD: * P=0.0368; LVEDD-LVESD: ** P=0.0013, ## P=0.0029. d, TUNEL of cardiac sections and quantification to assess apoptosis. Saline, n=10; BAI1, n=10; DOX, n=12; DOX+BAI1, n=11 females. One-way ANOVA, **** P<0.0001. e, Cleaved caspase-3 immunohistochemistry of cardiac sections and quantification to assess apoptosis. Saline, n=5; BAI1, n=5; DOX, n=9; DOX+BAI1, n=11 females. One-way ANOVA, ** P=0.0078, *** P=0.0003. f, Immunofluorescence for loss of nuclear HMGB1 in cardiac sections and quantification to assess necrosis. Saline, n=4; BAI1, n=5; DOX, n=4; DOX+BAI1, n=5 females. One-way ANOVA, **** P<0.0001. All data presented as mean ± s.e.m. One-way ANOVA, ns P>0.05. The data from the Saline, DOX, and DOX+BAI1 groups for females here are also displayed in Fig. 5b–d along with the corresponding male data in which BAI1 alone was not tested. All four groups are displayed together here so that they can be directly compared within one sex. Source data

Extended Data Fig. 6 BAI1 prevents cardiomyopathy in acute doxorubicin model.

a, Schematic of high-dose, acute doxorubicin (DOX)-induced cardiomyopathy mouse model (females). Mice were 8 weeks old at the start of experiment. b, Echocardiographic analysis of systolic function including fractional shortening (FS), left ventricular end-diastolic dimension (LVEDD), left ventricular end-systolic dimension (LVESD), and LVEDD-LVESD prior to initiation of treatment. Saline, n=10; BAI1, n=10; DOX, n=15; DOX+BAI1, n=15 females. Mean values are shown on the graphs. c, Echocardiographic parameters 5 days following the single treatment. Saline, n=10; BAI1, n=10; DOX, n=15; DOX+BAI1, n=14 females. Mean values are shown on the graphs. One-way ANOVA, FS: **** P<0.0001; LVEDD: * P=0.0139, ** P=0.0014; LVESD: ** P=0.0043; LVEDD-LVESD: **** P<0.0001. d, Schematic of high-dose, acute DOX-induced cardiomyopathy mouse model (males). Mice were 8 weeks old at the start of experiment. e, Echocardiographic parameters 5 days following the single treatment. Saline, n=8; DOX, n=9; DOX+BAI1, n=12 males. Mean values are shown on the graphs. One-way ANOVA, FS: ** P=0.0011, ## P=0.0025; LVEDD-LVESD: ** P=0.0015, ## P=0.0039. f, TUNEL of cardiac sections and quantification to assess apoptosis. n=3 males/group. One-way ANOVA, ** P=0.0077, ## P=0.0032. g, Immunofluorescence for loss of nuclear HMGB1 in cardiac sections and quantification to assess necrosis. n=3 males/group. One-way ANOVA, * P=0.0235, ** P=0.0031. All data presented as mean ± s.e.m. One-way ANOVA, ns P>0.05. Source data

Extended Data Fig. 7 BAI1 alone does not affect body or organ weights.

Treatment with BAI1 alone in the long-term model as illustrated in the schematic in Extended Data Fig. 5a. Female mice, 12 weeks old at the start of experiment. n=10 mice/group. All data presented as mean ± s.e.m. P-values from two-tailed Student’s t-test shown on the graphs. Source data

Extended Data Fig. 8 BAI1 alone does not affect histology of multiple tissues.

Treatment with BAI1 alone in the long-term model as illustrated in Extended Data Fig. 5a. Female mice, 12 weeks old at the start of experiment. Hematoxylin and eosin staining of mouse tissues. Representative micrographs from n=5 mice/group. Scale bars: a, 1000 μm; b, 50 μm.

Extended Data Fig. 9 BAI1 alone does not affect hematological parameters.

Treatment with BAI1 alone in the long-term model as illustrated in Extended Data Fig. 5a. Female mice, 12 weeks old at the start of experiment. All values are within the normal range for mice. n=10 mice/group. All data presented as mean ± s.e.m. P-values from two-tailed Student’s t-test shown on the graphs. Source data

Extended Data Fig. 10 BAI1 does not attenuate doxorubicin-induced killing of human cancer cells.

a, b, BAI1 alone does not affect cell viability of various cancer cell lines. Cellular viability measured by maintenance of plasma membrane integrity (CytoTox-Glo). c, d, BAI1 does not interfere with the ability of doxorubicin to kill these cells. For panels c and d, untreated cells (no doxorubicin and no BAI1) assigned a viability of 100% (not shown). All panels, n=3 independent experiments. One-way ANOVA, compared with BAI1 0 µM, ** P=0.0091. All data presented as mean ± s.e.m. For comparison, BAI1 at nanomolar concentrations inhibit cardiomyocyte death (Fig. 4). Source data

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Amgalan, D., Garner, T.P., Pekson, R. et al. A small-molecule allosteric inhibitor of BAX protects against doxorubicin-induced cardiomyopathy. Nat Cancer 1, 315–328 (2020). https://doi.org/10.1038/s43018-020-0039-1

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