Peptide Ligands for Pro-survival Protein Bfl-1 from Computationally Guided Library Screening

Abstract

Pro-survival members of the Bcl-2 protein family inhibit cell death by binding short helical BH3 motifs in pro-apoptotic proteins. Mammalian pro-survival proteins Bcl-xL, Bcl-2, Bcl-w, Mcl-1 and Bfl-1 bind with varying affinities and specificities to native BH3 motifs, engineered peptides and small molecules. Biophysical studies have determined interaction patterns for these proteins, particularly for the most-studied family members Bcl-xL and Mcl-1. Bfl-1 is a pro-survival protein implicated in preventing apoptosis in leukemia, lymphoma and melanoma. Although Bfl-1 is a promising therapeutic target, relatively little is known about its binding preferences. We explored the binding of Bfl-1 to BH3-like peptides by screening a peptide library that was designed to sample a high degree of relevant sequence diversity. Screening using yeast-surface display led to several novel high-affinity Bfl-1 binders and to thousands of putative binders identified through deep sequencing. Further screening for specificity led to identification of a peptide that bound to Bfl-1 with Kd < 1 nM and very slow dissociation from Bfl-1 compared to other pro-survival Bcl-2 family members. A point mutation in this sequence gave a peptide with ~50 nM affinity for Bfl-1 that was selective for Bfl-1 in equilibrium binding assays. Analysis of engineered Bfl-1 binders deepens our understanding of how the binding profiles of pro-survival proteins differ, and may guide the development of targeted Bfl-1 inhibitors.

Potent and Specific Peptide Inhibitors of Human Pro-Survival Protein Bcl-xL

“…Anisotropy was monitored over 2-48 hours to ensure equilibration. As reported previously 44 , due to the extremely slow off-rates of 23-residue unlabeled Bim-BH3 from Mcl-1 and Bfl-1, we do not report any K i value for these interactions. Binding curves were fit using Igor Pro 6.02 (Wavemetrics) as described previously 24, 38 .…”

“…Yeast populations isolated during screening were analyzed to assess binding to different populations of receptors. 44 …”

“…The plates were incubated at room temperature and anisotropy was measured over a period of 4-24 hours to ensure that the reactions were equilibrated. Competition binding experiments were performed using previously published protocols 44 . Briefly, unlabeled peptides were serially diluted, followed by addition of fluorescently labeled Bim-BH3 (or Hrk-BH3) to a final concentration of 10 nM.…”

The Functional Differences between Pro-survival and Pro-apoptotic B Cell Lymphoma 2 (Bcl-2) Proteins Depend on Structural Differences in Their Bcl-2 Homology 3 (BH3) Domains

“…Subtle differences in some of the conserved residues (i.e. different hydrophobic residues at h1, h3, or h4) as well as in the intervening residues contribute to the significant differences in selectivity in interactions between BH3 domains and pro-survival proteins (15,(25)(26)(27)(28)(29). Indeed, some BH3 sequences are promiscuous and engage all pro-survival proteins with high (low nanomolar) affinity, whereas others are more selective, only engaging subsets of pro-survival proteins avidly (30 -32).…”

Locating Herpesvirus Bcl-2 Homologs in the Specificity Landscape of Anti-Apoptotic Bcl-2 Proteins

“…Significant progress has been made identifying peptide inhibitors of individual human Bcl-2 family members including Mcl-1, Bcl-x L and Bfl-1. 25,39,40 For viral Bcl-2 homologs, a computationally designed protein, BINDI, binds BHRF1 with picomolar affinity and >180-fold specificity over the human Bcl-2 homologs. 41 BINDI is a 14 kDa protein that incorporates a BH3 helix but gains much of its specificity from contacts outside of the BH3 binding groove.…”