Related Experiment Video
Updated: Dec 21, 2025
Covalent Fragment Screening Using the Quantitative Irreversible Tethering Assay
Published on: February 28, 2025
Identification of a Covalent Molecular Inhibitor of Anti-apoptotic BFL-1 by Disulfide Tethering
Edward P Harvey1, Zachary J Hauseman1, Daniel T Cohen1
1Department of Pediatric Oncology, Dana-Farber Cancer Institute, 450 Brookline Avenue, Boston, MA 02215, USA; Linde Program in Cancer Chemical Biology, Dana-Farber Cancer Institute, 450 Brookline Avenue, Boston, MA 02215, USA.
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.
Insights
Researchers developed small, cysteine-reactive drugs to target the oncogenic BFL-1 protein, overcoming venetoclax resistance and restoring apoptosis. This approach offers a new strategy for cancer therapy.
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- The BCL-2 family regulates apoptosis, with anti-apoptotic members like BFL-1 promoting oncogenesis by inhibiting programmed cell death.
- Overexpression of anti-apoptotic proteins, such as BCL-2, can lead to cancer by sequestering pro-apoptotic "killer domains" and blocking apoptosis.
- Current therapies like venetoclax target BCL-2 but BFL-1 remains undrugged and confers resistance.
Purpose of the Study:
- To investigate the potential of targeting the unique C55 residue in BFL-1's groove for developing novel cancer therapeutics.
- To explore the feasibility of using small, cysteine-reactive molecules to inhibit BFL-1's anti-apoptotic function.
Main Methods:
- Disulfide tethering screen utilizing a unique C55 residue in the BFL-1 groove.
- Synthesis and testing of a disulfide-bearing N-acetyltryptophan analog as a potential BFL-1 inhibitor.
- Structural analyses to elucidate the interaction site and conformational changes induced by the inhibitor.
Main Results:
- A small molecule (304 Da adduct) effectively targeted BFL-1 C55, blocking its BH3-binding functionality.
- The developed drug reversed BFL-1-mediated suppression of mitochondrial apoptosis.
- Structural studies identified the conserved leucine-binding pocket as the interaction site, leading to conformational remodeling.
Conclusions:
- Therapeutic targeting of BFL-1 is achievable through the design of small, cysteine-reactive drugs.
- This strategy offers a promising approach to overcome venetoclax resistance in cancers driven by BFL-1.
- The findings pave the way for developing new small-molecule inhibitors against previously undrugged oncogenic proteins.
