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

Abstract

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.

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

“…Nonetheless, improving potency, selectivity, and overall drug likeness (i.e., cell permeability) of these agents remain a major challenge. More recently, introduction of covalent warheads on peptide or peptide mimetics to target Cys (de Araujo, Lim, Good, Skerlj, & Fairlie, ; Baggio et al, ; Barile et al, ; Harvey et al, ; Huhn, Guerra, Harvey, Bird, & Walensky, ; Stebbins et al, ), and more recently also Lys and Tyr residues (Baggio et al, ), is emerging strategies to increase affinity and selectivity of these agents. A large number of therapeutically viable PPIs are mediated by an alpha helix (Bullock, Jochim, & Arora, ; Sawyer, Watkins, & Arora, ), suggesting that helical peptides could in principle also be used as starting point for the design of new therapeutic agents.…”

Precision Targeting of BFL-1/A1 and an ATM Co-dependency in Human Cancer

“…We then conducted an i, i+4 and i, i+7 ‘‘staple scan’’ of the 15-amino acid sequence (aa 26–40), whereby all-hydrocarbon staples spanning one or two turns of the α-helix were sequentially substituted along the length of the peptide (Figure 1C; Table S1). For each construct, the N terminus was capped with (R)-1 acryloylpiperidine-3-carboxamide, an acrylamide-bearing D-nipecotic acid derivative (D-NA) that we previously found to be optimally reactive with BFL-1 C55 when incorporated into stapled BIM and NOXA BH3 helices (Harvey et al, 2018; Huhn et al, 2016) (Figure 1C). …”

“…Recombinant anti-apoptotic BFL-1ΔC (aa 1–151) bearing an N-terminal hexahistidine tag and its cysteine to serine mutants generated by PCR-based site directed mutagenesis (Q5 Site-Directed Mutagenesis Kit, NEB) were cloned into pET17b (Novagen), expressed in Escherichia coli LOBSTR BL21(DE3) (Kerafast), and purified by sequential Ni-affinity and size-exclusion chromatography as described (Harvey et al, 2018). Protein expression was induced using 0.5 mM isopropyl β-D-1–thiogalactopyranoside (IPTG) overnight at 16°C.…”

“…We and others recently discovered a selectivity factor for targeting BFL-1 based on the presence of a unique cysteine in its BH3-binding groove (Barile et al, 2017; de Araujo et al, 2016; Harvey et al, 2018; Huhn et al, 2016; Jenson et al, 2017). By incorporating acrylamide moieties into stapled BH3 peptides, we demonstrated efficient and specific covalent targeting of BFL-1 and its inhibitory complexes in lysates and cells, which resulted in selective apoptosis induction of BFL-1-driven melanoma cells (Huhn et al, 2016).…”

A novel computational method to design BH3-mimetic peptide inhibitors that can bind specifically to Mcl-1 or Bcl-XL

“…As part of the strategy to use structure-based design, high-affinity peptide-binders were designed based on "recurring tertiary structural motifs" from known protein structures available in PDB and this was applied to Bcl-X L , Mcl-1 and Bfl-1 proteins 29 . Staples were introduced that enhanced the stability of α-helical structure of peptides that inhibited anti-apoptotic proteins with increased affinity 24,[37][38] . In this paper, we have developed a novel computational method to generate BH3-like sequences that can bind Mcl-1 or Bcl-X L with high affinity and specificity.…”