Dataset Information

Covalent Inhibition of Pro-Apoptotic BAX

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). Here, 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 the physiologic ligand tBID and 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. Hydrogen deuterium exchange mass spectrometry (HDX MS) showed that CBI1 derivatization of BAX C126 reversed the conformational changes caused by the auto-activating mutant F116A These data inform a pharmacologic strategy for blocking apoptosis in diseases of unwanted cell death by covalent targeting of BAX C126.

INSTRUMENT(S): Synapt MS

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: John R. Engen

LAB HEAD: John R. Engen

PROVIDER: PXD040917 | Pride | 2024-01-19

REPOSITORIES: Pride

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Publications

Covalent inhibition of pro-apoptotic BAX.

McHenry Matthew W MW Shi Peiwen P Camara Christina M CM Cohen Daniel T DT Rettenmaier T Justin TJ Adhikary Utsarga U Gygi Micah A MA Yang Ka K Gygi Steven P SP Wales Thomas E TE Engen John R JR Wells James A JA Walensky Loren D LD

Nature chemical biology 20240117

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 ...[more]

PMID: 38233584

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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.

Dataset Information

Covalent Inhibition of Pro-Apoptotic BAX

Publications

Covalent inhibition of pro-apoptotic BAX.

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