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Discovery of an Enantioselective Covalent Inhibitor of BAX for Cytoprotection in Vivo

ABSTRACT: The pro-apoptotic BCL-2 protein BAX is a central executioner of mitochondrial apoptosis and a driver of acute and chronic cell loss in diverse human diseases. The C126 residue of BAX lies within a key regulatory region and its derivatization by physiologic and pharmacologic ligands can alternatively activate or inhibit BAX function. Here, we report the discovery of an enantioselective covalent BAX inhibitor that confers cytoprotection across multiple cell types and in vivo. Chemoproteomic screening identified tryptoline acrylamide stereoprobes that derivatized BAX at C126 with high selectivity, constraining its conformation, and suppressing apoptosis in a strictly BAX-dependent manner, as confirmed by genetic knockout models. Medicinal chemistry optimization yielded covalent BAX inhibitor 3 (CBI-3), a more stable analog with retained enantioselectivity, improved pharmacokinetics, and dose-responsive cytoprotective activity against ABT-737 and cisplatin-induced apoptosis without intrinsic toxicity. In a murine model of Fas-induced fulminant hepatic failure, CBI-3 significantly reduced hepatocyte apoptosis and preserved liver histology and animal survival, nearly phenocopying Bax deletion. These findings establish enantioselective covalent inhibition of BAX as a viable strategy for cytoprotection with demonstrable in vivo efficacy, opening the door to next-generation therapeutics that directly block pathologic cell death.

INSTRUMENT(S):

ORGANISM(S): Mus Musculus (mouse)

SUBMITTER: Thomas Wales

LAB HEAD: Thomas E. Wales

PROVIDER: PXD069506 | Pride | 2026-06-28

REPOSITORIES: Pride

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			Action	DRS
	1_BAX_WT_DMSO_10min1.raw.zip	Raw
	1_BAX_WT_DMSO_10min2.raw.zip	Raw
	1_BAX_WT_DMSO_10s1.raw.zip	Raw
	1_BAX_WT_DMSO_10s2.raw.zip	Raw
	1_BAX_WT_DMSO_1min1.raw.zip	Raw

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Project description:Src family kinases are often overexpressed and constitutively active in cancer cells, where they represent promising targets for therapeutic intervention. In myeloid hematopoietic cells, Hck, Lyn, and Fgr are associated with both chronic and acute forms of myeloid leukemia. All three of these kinases are expressed in acute myeloid leukemia (AML), where high-level expression correlates with poor survival. The pyrrolopyrimidine Src-family kinase inhibitor A-419259 (also known as RK-20449) has been shown to significantly inhibit patient-derived AML bone marrow cell growth in immunocompromised mice. Recent work has identified an N-phenylbenzamide kinase inhibitor, known as TL02-59, which potently inhibited Fgr kinase activity in vitro and reversed orthotopic engraftment of AML cells in the bone marrow and spleen of immunocompromised mice following oral administration. The inhibitory mechanism of this compound with Fgr or other Src-family members has not been reported. Both wild-type Fgr and a tail mutant (i.e., replacement of Tyr527 with Phe) were shown to produce equal numbers of transformed colonies when expressed in rodent fibroblasts, whereas wild-type Hck transforming activity was completely repressed. In both Hck and Fgr, however, the tail tyrosine residue was shown to be phosphorylated, and hydrogen deuterium exchange mass spectrometry (HDX MS) of near-full-length Fgr was consistent with the closed conformation in solution, with the SH3 domain bound to the SH2-kinase linker and the tail bound to SH2. Here we present the first X-ray crystal structures of near-full-length Fgr bound to the ATP site inhibitors A-419259 and TL02-59. Structural changes induced by the inhibitors in the crystalline state were validated in solution using HDX MS, with A-419259 stabilizing the closed conformation and TL02-59 enhancing exposure of the SH3 domain to solvent. These new structures help to explain the constitutive activity of wild-type Fgr in cells as well as its unique sensitivity to TL02-59, which is currently in development for AML and other maladies.

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Discovery of an Enantioselective Covalent Inhibitor of BAX for Cytoprotection in Vivo

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