Browse
Submit Data
Databases
API
Help

Dataset Information

22 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

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

ACCESS DATA

Json Xml

Similar Datasets

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

Project description:BCL-2 family proteins are high-priority cancer targets whose structures provide essential blueprints for drug design. Whereas numerous structures of anti-apoptotic BCL-2 protein complexes with α-helical BH3 peptides have been reported, the corresponding panel of apo structures remains incomplete. Here, we report the crystal structure of apo BFL-1 at 1.69-Å resolution, revealing similarities and key differences among unliganded anti-apoptotic proteins. Unlike all other BCL-2 proteins, apo BFL-1 contains a surface-accessible cysteine within its BH3-binding groove, allowing for selective covalent targeting by a NOXA BH3-based stapled peptide inhibitor. The crystal structure of this complex demonstrated the sulfhydryl bond and fortuitous interactions between the acrylamide-bearing moiety and a newly formed hydrophobic cavity. Comparison of the apo and BH3-liganded structures further revealed an induced conformational change. The two BFL-1 structures expand our understanding of the surface landscapes available for therapeutic targeting so that the apoptotic blockades of BFL-1-dependent cancers can be overcome.

| S-EPMC5947960 | biostudies-literature

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

Project description:Anti-apoptotic BCL-2 family proteins block cell death by trapping the critical ?-helical BH3 domains of pro-apoptotic members in a surface groove. Cancer cells hijack this survival mechanism by overexpressing a spectrum of anti-apoptotic members, mounting formidable apoptotic blockades that resist chemotherapeutic treatment. Drugging the BH3-binding pockets of anti-apoptotic proteins has become a highest-priority goal, fueled by the clinical success of ABT-199, a selective BCL-2 inhibitor, in reactivating apoptosis in BCL-2-dependent cancers. BFL-1 is a BCL-2 homolog implicated in melanoma, lymphoma, and other cancers, and remains undrugged. A natural juxtaposition of two unique cysteines at the binding interface of the NOXA BH3 helix and BFL-1 pocket informed the development of stapled BH3 peptides bearing acrylamide warheads to irreversibly inhibit BFL-1 by covalent targeting. Given the frequent proximity of native cysteines to regulatory binding surfaces, covalent stapled peptide inhibitors provide a new therapeutic strategy for targeting pathologic protein interactions.

| S-EPMC5055752 | biostudies-literature

A redox switch regulates the structure and function of anti-apoptotic BFL-1.

Project description:Apoptosis is regulated by BCL-2 family proteins. Anti-apoptotic members suppress cell death by deploying a surface groove to capture the critical BH3 ?-helix of pro-apoptotic members. Cancer cells hijack this mechanism by overexpressing anti-apoptotic BCL-2 family proteins to enforce cellular immortality. We previously identified and harnessed a unique cysteine (C55) in the 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 groove (C55) and C-terminal ?9 helix (C175) of BFL-1 operates as a redox switch to control the accessibility of the anti-apoptotic pocket. Reducing the C55-C175 disulfide triggers ?9 release, which promotes mitochondrial translocation, groove exposure for BH3 interaction and inhibition of mitochondrial permeabilization by pro-apoptotic BAX. C55-C175 disulfide formation in an oxidative cellular environment abrogates the ability of BFL-1 to bind BH3 domains. Thus, we identify a mechanism of conformational control of BFL-1 by an intramolecular redox switch.

| S-EPMC7544158 | biostudies-literature

Identification of Aloperine as an anti-apoptotic Bcl2 protein inhibitor in glioma cells.

Project description:Objective:Aloperine (ALO), an alkaloid isolated from the leaves of Sophora alopecuroides, has been suggested to exhibit anti-inflammatory and anti-tumor properties and is traditionally used to treat various human diseases, including cancer. However, limited information is available about the mechanisms that determine the anti-tumor activities of ALO. Methods:Herein, through comprehensive bioinformatics methods and in vitro functional analyses, we evaluated the detailed anti-tumor mechanisms of ALO. Results:Using the databases Bioinformatics analysis tool for molecular mechanism of traditional Chinese medicine and PubChem Project, we identified the potential targets of ALO. A protein-protein interaction network was constructed to determine the relationship among these probable targets. Functional enrichment analysis revealed that ALO is potentially involved in the induction of apoptosis. In addition, molecular docking demonstrated that ALO expectedly docks into the active pocket of the Bcl2 protein, suggesting Bcl2 as a direct target of ALO. Moreover, western blot and qPCR analysis showed that ALO downregulated Bcl2 expression in human glioma cell lines, SK-N-AS and U118. Using flow cytometry methods, we further confirmed that ALO significantly promotes apoptosis in SK-N-AS and U118 cell lines, similar to the effect induced by ABT-737, a well-known Bcl2 inhibitor. In addition, Bcl-2 overexpression could rescue ALO-induced Bcl-2 inhibition and suppress pro-apoptotic effects in glioma cells. Conclusion:Taken together, these findings suggest that the natural agent ALO effectively enhances apoptosis by acting as a potential Bcl2 inhibitor in human glioma cells.

| S-EPMC6730530 | biostudies-literature

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.

Project description:Overexpression of anti-apoptotic Bcl-2 family proteins contributes to cancer progression and confers resistance to chemotherapy. Small molecules that target Bcl-2 are used in the clinic to treat leukemia, but tight and selective inhibitors are not available for Bcl-2 paralog Bfl-1. Guided by computational analysis, we designed variants of the native BH3 motif PUMA that are > 150-fold selective for Bfl-1 binding. The designed peptides potently trigger disruption of the mitochondrial outer membrane in cells dependent on Bfl-1, but not in cells dependent on other anti-apoptotic homologs. High-resolution crystal structures show that designed peptide FS2 binds Bfl-1 in a shifted geometry, relative to PUMA and other binding partners, due to a set of epistatic mutations. FS2 modified with an electrophile reacts with a cysteine near the peptide-binding groove to augment specificity. Designed Bfl-1 binders provide reagents for cellular profiling and leads for developing enhanced and cell-permeable peptide or small-molecule inhibitors.

| S-EPMC5464773 | biostudies-literature

N-locking stabilization of covalent helical peptides: Application to Bfl-1 antagonists.

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.

| S-EPMC7568863 | biostudies-literature

Discovery and Characterization of 2,5-Substituted Benzoic Acid Dual Inhibitors of the Anti-apoptotic Mcl-1 and Bfl-1 Proteins.

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.

| S-EPMC8024114 | biostudies-literature

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.

| S-EPMC6447450 | biostudies-literature

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

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data