Structure-Based Optimization of a Series of Covalent, Cell Active Bfl-1 Inhibitors

Area of Science:

• Biochemistry
• Molecular Biology
• Medicinal Chemistry

Background:

• Bcl-2 family proteins, including Bfl-1, regulate apoptosis and are crucial in cancer cell survival.
• Bfl-1 contributes to resistance against venetoclax therapy, a targeted cancer treatment.
• The unique cysteine residue in Bfl-1's BH3 binding site presents an opportunity for targeted covalent inhibition.

Purpose of the Study:

• To optimize a lead-like hit into a potent covalent cellular tool targeting Bfl-1.
• To develop a structure-based design strategy for creating effective Bfl-1 inhibitors.
• To assess the biochemical potency, cellular activity, and in vivo profile of the optimized compound.

Main Methods:

• Structure-based drug design informed by X-ray fragment screening.
• Optimization of interactions with glutamic acid residue (Glu78) and a cryptic binding pocket.
• Biochemical assays to determine binding kinetics (k_i/K_i).
• Cellular assays to measure caspase activation and target engagement.

Main Results:

• Achieved a 1000-fold improvement in biochemical potency.
• Developed a compound with a k_i/K_i of 4600 M^-1 s^-1.
• Demonstrated <1 μM caspase activation in cellular assays and confirmed cellular target engagement.
• The optimized compound exhibits favorable physicochemical properties and a promising in vivo profile.

Conclusions:

• Structure-based design effectively optimized a covalent inhibitor targeting Bfl-1.
• The developed compound demonstrates high potency and cellular activity, suggesting therapeutic potential.
• This approach offers a promising strategy for overcoming Bfl-1-mediated drug resistance in cancer.