(660a) 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.