Design of selective peptide inhibitors of anti-apoptotic Bfl-1 using experimental screening, structure-based design, and data-driven modeling

Experimental Screening: Hunting for Hidden Gems

High-throughput screens of chemical libraries identified early hits like N-aryl maleimides, which disrupt Bfl-1’s interaction with pro-apoptotic BH3 peptides . However, these small molecules had modest specificity, sparking the shift to peptide-based solutions.

Structure-Based Design: Blueprints from Crystal Structures

In 2017, Harvey et al. solved the crystal structure of Bfl-1 bound to a stapled peptide inhibitor, revealing how the peptide’s acrylamide group forms a covalent bond with C55 . This “molecular staple” locks the peptide into Bfl-1’s groove, outcompeting natural binding partners:

Stapled peptides mimic the α-helical BH3 domain, critical for apoptosis signaling.
Covalent targeting via C55 enhances selectivity, reducing off-target effects .

Table 2: Peptide vs. Small-Molecule Inhibitors

Feature	Peptide Inhibitors	Small Molecules
Specificity	High (e.g., >100-fold for Bfl-1)	Moderate
Delivery	Challenged by cell permeability	Easier
Covalent Targeting	Enabled by C55 reactivity	Limited

Data-Driven Modeling: Machine Learning Meets Biology

Using computational tools like dTERMen, researchers analyze amino acid patterns in protein interfaces to predict optimal peptide sequences. For example, Jenson et al. redesigned PUMA BH3 peptides to bind Bfl-1 in a shifted, rotated orientation, achieving unprecedented selectivity .

Breakthroughs in the Lab: From Theory to Therapy

Covalent Stapled Peptides: A NOXA-based peptide modified with a cysteine-reactive staple showed potent activity in melanoma and lymphoma cells, sparing healthy cells .
PUMA Redesign: Engineered PUMA peptides achieved 150-fold selectivity for Bfl-1 by exploiting epistatic mutations that alter binding geometry .
Combination Therapies: Bfl-1 inhibition synergizes with ATM kinase blockers, a promising approach for acute myeloid leukemia .

Table 3: Recent Advances in Bfl-1 Targeting

Study	Approach	Outcome
Harvey et al. (2016)	Cysteine-reactive stapled peptide	Selective Bfl-1 inhibition in vitro
Jenson et al. (2018)	Computational library design	Peptides with shifted binding mode
Guerra et al. (2018)	Stapled peptide + ATM inhibitor	Synergistic tumor cell death

The Road Ahead: Challenges and Opportunities

While Bfl-1 inhibitors are not yet clinically approved, their progress highlights key lessons:

Delivery Hurdles: Peptides often require modifications (e.g., hydrocarbon stapling) for cell penetration .

Resistance Risks: Tumors may upregulate alternate BCL-2 proteins, necessitating combo therapies .

Beyond Cancer: Bfl-1 is implicated in autoimmune diseases, broadening therapeutic potential .

Conclusion: A New Era of Precision Medicine

The race to inhibit Bfl-1 exemplifies how blending structural biology, chemistry, and AI can transform drug discovery. As covalent peptides enter preclinical testing, they carry the promise of smarter, more resilient cancer therapies. By disarming cancer’s survival machinery at the molecular level, scientists are rewriting the rules of the game—one peptide staple at a time.