Selection for constrained peptides that bind to a single target protein

Andrew M King,Emerson Glassey,Daniel A Anderson,Zhengan Zhang,Amanda C Embree,Katelin Pratt,Thomas H Segall-Shapiro,Christopher A Voigt,D Benjamin Gordon,Thomas L Williams,David L Niquille

doi:10.1038/s41467-021-26350-4

Abstract

Peptide secondary metabolites are common in nature and have diverse pharmacologically-relevant functions, from antibiotics to cross-kingdom signaling. Here, we present a method to design large libraries of modified peptides in Escherichia coli and screen them in vivo to identify those that bind to a single target-of-interest. Constrained peptide scaffolds were produced using modified enzymes gleaned from microbial RiPP (ribosomally synthesized and post-translationally modified peptide) pathways and diversified to build large libraries. The binding of a RiPP to a protein target leads to the intein-catalyzed release of an RNA polymerase σ factor, which drives the expression of selectable markers. As a proof-of-concept, a selection was performed for binding to the SARS-CoV-2 Spike receptor binding domain. A 1625 Da constrained peptide (AMK-1057) was found that binds with similar affinity (990 ± 5 nM) as an ACE2-derived peptide. This demonstrates a generalizable method to identify constrained peptides that adhere to a single protein target, as a step towards “molecular glues” for therapeutics and diagnostics.

Highlights

Peptide secondary metabolites are common in nature and have diverse pharmacologicallyrelevant functions, from antibiotics to cross-kingdom signaling
The circuit converts a binding event into a transcriptional response. It is based on two fusion proteins containing the RiPP and the bait that, upon binding, bring together two halves of a split intein that catalyze the release of a σ factor that recruits RNA polymerase (RNAP) to a promoter (Fig. 1a)
The first fusion protein consists of the N-terminal σ factor fragment placed upstream of the N-terminal Nostoc punctiforme PCC73102 (Npu) fragment (NpuN) and linker followed by the bait protein

Summary

Introduction

Peptide secondary metabolites are common in nature and have diverse pharmacologicallyrelevant functions, from antibiotics to cross-kingdom signaling. Linear peptides are generally not good drugs due to their conformational flexibility, inability to cross the cell membrane, and sensitivity to proteases[1] Bacteria overcome these issues by chemically modifying peptides to introduce functional groups and constrain their structures to improve affinity and stability. The modified peptides are secreted and act on eukaryotic cells by binding to surface proteins or crossing their membrane to interact with intracellular targets[2,3,4]. The peptides can inhibit cellular processes by occluding the active site of an enzyme or blocking a protein–protein interaction by binding to the interface They can act as a “molecular glue” to bring two proteins together, the most wellknown example being rapamycin, which binds to two human proteins involved in signaling in order to suppress the host immune response[5]. It is more difficult to combine peptide modifications with these techniques, but innovative approaches have been applied to cyclize displayed peptides[23,24,25,26,27,28,29,30,31]

Methods

Results

Conclusion