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Identification of a Covalent Molecular Inhibitor of Anti-apoptotic BFL-1 by Disulfide Tethering.

ABSTRACT: The BCL-2 family is composed of anti- and pro-apoptotic members that respectively protect or disrupt mitochondrial integrity. Anti-apoptotic overexpression can promote oncogenesis by trapping the BCL-2 homology 3 (BH3) "killer domains" of pro-apoptotic proteins in a surface groove, blocking apoptosis. Groove inhibitors, such as the relatively large BCL-2 drug venetoclax (868 Da), have emerged as cancer therapies. BFL-1 remains an undrugged oncogenic protein and can cause venetoclax resistance. Having identified a unique C55 residue in the BFL-1 groove, we performed a disulfide tethering screen to determine if C55 reactivity could enable smaller molecules to block BFL-1's BH3-binding functionality. We found that a disulfide-bearing N-acetyltryptophan analog (304 Da adduct) effectively targeted BFL-1 C55 and reversed BFL-1-mediated suppression of mitochondrial apoptosis. Structural analyses implicated the conserved leucine-binding pocket of BFL-1 as the interaction site, resulting in conformational remodeling. Thus, therapeutic targeting of BFL-1 may be achievable through the design of small, cysteine-reactive drugs.

SUBMITTER: Harvey EP

PROVIDER: S-EPMC7405809 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Crystal Structures of Anti-apoptotic BFL-1 and Its Complex with a Covalent Stapled Peptide Inhibitor.

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Selective Covalent Targeting of Anti-Apoptotic BFL-1 by Cysteine-Reactive Stapled Peptide Inhibitors.

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A Redox Switch Regulates the Structure and Function of Anti-Apoptotic BFL-1

Project description:BCL-2 family proteins control cell fate by regulating the mitochondrial apoptotic pathway. Anti-apoptotic members suppress cell death by capturing pro-apoptotic α-helices in a surface groove, a mechanism hijacked by cancer cells to enforce cellular immortality. We previously identified and harnessed a unique cysteine (C55) in the canonical groove of anti-apoptotic BFL-1 to selectively neutralize its oncogenic activity using a covalent stapled-peptide inhibitor. Here, we find that disulfide bonding between a native cysteine pair at the interface between the groove (C55) and C-terminal α9 helix (C175) operates as a redox switch to control the accessibility and functionality of the anti-apoptotic pocket. Hydrogen deuterium exchange mass spectrometry was used to characterize a construct of BFL-1 deleting the C-terminus and thus C175 (BFL-1ΔC C55), as well as full length BFL-1 (BFL-1 C55/C175) in both oxidation states. Reducing the C55-C175 disulfide bond triggers a cascade of structure-function changes that includes release of α9 for membrane translocation, exposure of the groove for α-helical interaction, and resultant blockade of mitochondrial membrane permeabilization by pro-apoptotic BAX. Thus, we identify a unique mechanism of conformational control over anti-apoptotic activity by a redox switch in BFL-1.

2020-06-17 | PXD016059 | Pride

Epistatic mutations in PUMA BH3 drive an alternate binding mode to potently and selectively inhibit anti-apoptotic Bfl-1.

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Project description:Recently, it was reported that tetrapeptides cyclized via lactam bond between the amino terminus and a glutamic residue in position 4 (termed here N-lock) can nucleate helix formation in longer peptides. We applied such strategy to derive N-locked covalent BH3 peptides that were designed to selectively target the anti-apoptotic protein Bfl-1. The resulting agents were soluble in aqueous buffer and displayed a remarkable (low nanomolar) affinity for Bfl-1 and cellular activity. The crystal structure of the complex between such N-locked covalent peptide and Bfl-1 provided insights on the geometry of the N-locking strategy and of the covalent bond between the agent and Bfl-1.

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Project description:Anti-apoptotic Bcl-2 family proteins are overexpressed in a wide spectrum of cancers and have become well validated therapeutic targets. Cancer cells display survival dependence on individual or subsets of anti-apoptotic proteins that could be effectively targeted by multimodal inhibitors. We designed a 2,5-substituted benzoic acid scaffold that displayed equipotent binding to Mcl-1 and Bfl-1. Structure-based design was guided by several solved cocrystal structures with Mcl-1, leading to the development of compound 24, which binds both Mcl-1 and Bfl-1 with Ki values of 100 nM and shows appreciable selectivity over Bcl-2/Bcl-xL. The selective binding profile of 24 was translated to on-target cellular activity in model lymphoma cell lines. These studies lay a foundation for developing more advanced dual Mcl-1/Bfl-1 inhibitors that have potential to provide greater single agent efficacy and broader coverage to combat resistance in several types of cancer than selective Mcl-1 inhibitors alone.

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Tertiary Structural Motif Sequence Statistics Enable Facile Prediction and Design of Peptides that Bind Anti-apoptotic Bfl-1 and Mcl-1.

Project description:Understanding the relationship between protein sequence and structure well enough to design new proteins with desired functions is a longstanding goal in protein science. Here, we show that recurring tertiary structural motifs (TERMs) in the PDB provide rich information for protein-peptide interaction prediction and design. TERM statistics can be used to predict peptide binding energies for Bcl-2 family proteins as accurately as widely used structure-based tools. Furthermore, design using TERM energies (dTERMen) rapidly and reliably generates high-affinity peptide binders of anti-apoptotic proteins Bfl-1 and Mcl-1 with just 15%-38% sequence identity to any known native Bcl-2 family protein ligand. High-resolution structures of four designed peptides bound to their targets provide opportunities to analyze the strengths and limitations of the computational design method. Our results support dTERMen as a powerful approach that can complement existing tools for protein engineering.

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Epistatic mutations in PUMA BH3 drive an alternate binding mode to potently and selectively inhibit anti-apoptotic Bfl-1

Project description:We used computational models to design focused combinatorial libraries to identify BH3 peptides that bind tightly and selectively to Bfl-1. The final library was predicted to be Bfl-1 selective by PSSMSPOT and STATIUM (DeBartolo et. al. J Mol Biol. 2012). As a design control, we designed similar libraries to be selective for Bcl-xL and Mcl-1. The libraries were transformed into yeast for surface display, pooled, and screened for tight and selective binding to Bfl-1. Five rounds of positive, negative, and/or competition FACS screening were used to isolate cells that expressed the tightest and most selective peptides. To analyze enrichment trends and to assess the success of our library design, we deep sequenced samples from the naïve pool and from pools collected after 3, 4, 5, and 6 rounds of sorting. Additional detail is available in our corresponding publication.

2017-06-09 | GSE94864 | GEO

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