Rapid Evaluation of Staple Placement in Stabilized α Helices Using Bacterial Surface Display

Abstract

There are a wealth of proteins involved in disease that cannot be targeted by current therapeutics because they are inside cells, inaccessible to most macromolecules, and lack small-molecule binding pockets. Stapled peptides, where two amino acids are covalently linked, form a class of macrocycles that have the potential to penetrate cell membranes and disrupt intracellular protein–protein interactions. However, their discovery relies on solid-phase synthesis, greatly limiting queries into their complex design space involving amino acid sequence, staple location, and staple chemistry. Here, we use stabilized peptide engineering by Escherichia coli display (SPEED), which utilizes noncanonical amino acids and click chemistry for stabilization, to rapidly screen staple location and linker structure to accelerate peptide design. After using SPEED to confirm hotspots in the mdm2–p53 interaction, we evaluated different staple locations and staple chemistry to identify several novel nanomolar and sub-nanomolar antagonists. Next, we evaluated SPEED in the B cell lymphoma 2 (Bcl-2) protein family, which is responsible for regulating apoptosis. We report that novel staple locations modified in the context of BIM, a high affinity but nonspecific naturally occurring peptide, improve its specificity against the highly homologous proteins in the Bcl-2 family. These compounds demonstrate the importance of screening linker location and chemistry in identifying high affinity and specific peptide antagonists. Therefore, SPEED can be used as a versatile platform to evaluate multiple design criteria for stabilized peptide engineering.

Discovery of high affinity and specificity stapled peptide Bcl-xL inhibitors using bacterial surface display

“…Bcl-2 proteins were purified as described previously with modifications. 51 Protein was expressed in E. coli Rosetta2 cells at 37 °C, 250 rpm in terrific broth media supplemented 100 mg/L ampicillin. The purified protein was digested with TEV protease overnight at 4 °C to remove the purification tag in the presence of the following buffer 50mM Tris, pH 7.5, 150mM NaCl, 0.1% β-ME.…”

“…Libraries were displayed using the pqe80L-eCPX2 plasmid as described previously. 51,52 We added an N-terminal HA tag and a peptide linker to mitigate steric clashes between display and binding measurements as described previously, resulting in NH2-eCPX-HA-linker-peptide-COOH. 25…”

“…Bcl-2 peptides were synthesized, stapled, and purified as described previously. 51,52 Peptide structures, mass spectra, and chromatograms are in Figure S2 -Figure S4.…”

“…41 In this work, we use stabilized peptide engineering by E. coli display (SPEED) to rapidly evaluate high affinity and specificity Bcl-xL stapled peptides (Figure 1). 51,52 SPEED enables high-throughput screening of fully stapled peptides in a quantitative format by displaying genetically encoded stapled peptides on the surface of bacteria using non-natural amino acids, enabling evaluation of up to 10 9 peptides. To generate specific Bcl-xL inhibitors, we designed a library of BIM mutants, a naturally occurring peptide that has high affinity for all Bcl-2 proteins but no specificity towards Bcl-xL.…”

“…8 SPEED was used to simultaneously vary both peptide sequence and staple location to determine how staple location governs specificity in the context of a BIM-based library. 51 We sorted the library using a combination of magnetically activated-and fluorescently activated-cell sorting towards highly specific mutations that would target Bcl-xL with both high affinity and specificity. By analyzing the final peptides in our library using next generation sequencing, we identified sequence trends and staple locations that govern specificity.…”