458195 Surface Display-Enabled Directed Evolution of Stabilized Alpha Helix Peptides
Surface display-enabled directed evolution of stabilized alpha helix peptides
Keywords: stapled peptides, surface display, directed evolution, PPI inhibitors
The majority of interfaces between interacting proteins include an alpha helix, the most common protein secondary structure motif. Such interfaces have posed attractive drug targets due to their importance in a wide range of diseases, in which inhibition, by blocking interactions, or activation, by triggering function, can result in therapeutic applications. However, due to the large size of protein-protein interaction surface areas, small molecules have seen little success in drug development. Here, we describe an application of bacteria surface display using directed evolution to screen for potent stabilized peptide binders of intracellular targets.
As done in reported development of stabilized peptide inhibitors, the peptideÕs sequence is generally determined from the bound residues observed in a co-crystal structure of the target and one of its known binders. However, when designing a stabilized peptide, introduction of the staple may abrogate binding, as the modified residues and bulk of the staple may interfere with the peptide-target interaction. This has been illustrated by several recent examples and optimization generally requires painstaking staple scanning. The directed evolution method presented here allows for rapid screening of >108 unique stabilized peptide sequences.
By incorporating azidohomoalanine (AHA) residues spaced 7 residues apart in solid phase peptide synthesis, we and others have demonstrated stabilization of peptides through double-click chemistry using a bifunctional alkyne-containing linker. Such design allows for the inclusion of fluorophores for imaging applications or pharmacologic enhancers like PEG chains. Our lab and others have demonstrated that the resulting stabilized peptides have increased protease resistance, subcutaneous bioavailability, and binding affinities. Taking advantage of the ability of methionine auxotrophic E. coli to efficiently incorporate AHA as a methionine surrogate, we constructed libraries of randomized peptides containing two AHA residues for double-click surface stabilization. We demonstrate efficient display of AHA incorporated peptides using the eCPX scaffold, an artificial protein based on endogenous OmpX, and robust reaction yields and specificity. Following surface display and chemical double-click stabilization, the resulting peptides show increased protease resistance. Here, we demonstrate the development of the technique and its application in screening for novel stabilized alpha helix binders.
Figure 1- the directed evolution process for stabilized peptides involves inducing bacteria with AHA non-natural amino acids 7 residues apart, followed by CuAAC for stabilization and selection. This process can be repeated to enrich for strongly binding clones and affinity maturation can also be done to further improve affinity.
Figure 2- surface display levels of exendin-4 , a model peptide, using eCPX with different vectors. pQE-80L shows optimal display of peptide under both methionine and AHA incorporation conditions.
Figure 3- AHA incorporation is exendin-4 specific. Cells were reacted with SulfoCy5-alkyne after induction and run on a reducing SDS-PAGE gel. A - molecular weight ladder . B - 700 nm scan (SulfoCy5 channel) showing one bright band near the expected molecular weight (20 kDa). C – 800 nm scan (anti-exenatide-4 channel) showing one band for the exenatide-4-eCPX fusion. D – overlay showing co-localization of the two channels indicating specific reaction.
Figure 4 - digest trajectories of surface displayed exendin-4 reacted with either propargylamine (single alkyne, non-stabilizing) or propargyl ether (bisalkyne, stabilizing) showing significant increase in protease resistant upon stabilization on the surface of bacteria.