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Structural Insights into Chemoresistance Mutants of BCL-2 and their Targeting by Stapled BAD BH3 Helices

ABSTRACT: BCL-2 is a central regulator of apoptosis that inhibits cell death by sequestering pro-apoptotic BH3 alpha-helices within a hydrophobic surface groove. While venetoclax, a BH3-mimetic drug, has transformed the treatment of BCL-2–driven malignancies, its efficacy is increasingly limited by acquired resistance mutations that disrupt binding yet preserve anti-apoptotic function—a remarkable structural adaptation. Here, we employed hydrocarbon-stapled alpha-helices derived from the BAD BH3 motif as conformation-sensitive molecular probes to investigate this therapeutic challenge. The stapled peptides not only retain high-affinity binding to all BCL-2 variants, but also show enhanced potency to select venetoclax-resistant mutants. Structural analyses, including X-ray crystallography and hydrogen-deuterium exchange mass spectrometry (HDX MS), revealed the ability of stapled helices to restore native-like BH3 engagement by reverting the conformational consequences of resistance mutations. Notably, we identified a serendipitous interaction between the α3–α4 hairpin of BCL-2 and the hydrocarbon staple that compensates for altered groove conformation and contributes to mutant binding affinity. Together, these findings offer mechanistic insights into BCL-2 drug resistance and reveal a blueprint for designing next-generation inhibitors that overcome this clinically significant barrier to durable treatment responses.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Thomas Wales

LAB HEAD: Thomas E Wales

PROVIDER: PXD062817 | Pride | 2025-09-30

REPOSITORIES: Pride

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			Action	DRS
	10_flBCL2_A113G_4pt2_10m1.raw.zip	Raw
	10_flBCL2_A113G_4pt2_10m2.raw.zip	Raw
	10_flBCL2_A113G_4pt2_10s1.raw.zip	Raw
	10_flBCL2_A113G_4pt2_10s2.raw.zip	Raw
	10_flBCL2_A113G_4pt2_1m1.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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Structural Insights into Chemoresistance Mutants of BCL-2 and their Targeting by Stapled BAD BH3 Helices

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