Rapid de novo discovery of peptidomimetic affinity reagents for human angiotensin converting enzyme 2

Genwei Zhang,Joseph S Brown,Bradley L Pentelute,Amanda E Cowfer,Andrei Loas,Xuyu Tan,Sarah Antilla,Chengxi Li,Anthony J Quartararo,Stephanie Hanna

doi:10.1038/s42004-022-00625-3

Abstract

Rapid discovery and development of serum-stable, selective, and high affinity peptide-based binders to protein targets are challenging. Angiotensin converting enzyme 2 (ACE2) has recently been identified as a cardiovascular disease biomarker and the primary receptor utilized by the severe acute respiratory syndrome coronavirus 2. In this study, we report the discovery of high affinity peptidomimetic binders to ACE2 via affinity selection-mass spectrometry (AS-MS). Multiple high affinity ACE2-binding peptides (ABP) were identified by selection from canonical and noncanonical peptidomimetic libraries containing 200 million members (dissociation constant, KD = 19–123 nM). The most potent noncanonical ACE2 peptide binder, ABP N1 (KD = 19 nM), showed enhanced serum stability in comparison with the most potent canonical binder, ABP C7 (KD = 26 nM). Picomolar to low nanomolar ACE2 concentrations in human serum were detected selectively using ABP N1 in an enzyme-linked immunosorbent assay. The discovery of serum-stable noncanonical peptidomimetics like ABP N1 from a single-pass selection demonstrates the utility of advanced AS-MS for accelerated development of affinity reagents to protein targets.

Highlights

Rapid discovery and development of serum-stable, selective, and high affinity peptide-based binders to protein targets are challenging
Noncanonical amino acids can be extensively used in the preparation of the synthetic libraries utilized in affinity selection-mass spectrometry (AS-MS), provided there is no isobaric monomer mass overlap and sufficient tandem sequencing fidelity
The monomer set utilized in the canonical library (Library 1) is fully proteogenic, except Cys because it could form intra- or intermolecular disulfides and Ile because it is isobaric in mass with Leu, to give 18 amino acids total (Fig. 1b)

Summary

Introduction

Rapid discovery and development of serum-stable, selective, and high affinity peptide-based binders to protein targets are challenging. Discovery via affinity selection using genetically-encoded techniques including phage display[28,29] and mRNA display[30] samples vast libraries up to 1013 members, being amenable for de novo discovery of high affinity reagents These techniques are not well suited to the incorporation of highly noncanonical library members, even in cell-free systems[31,32,33,34]. AS-MS can enable rapid discovery across highly diverse libraries[44,45] With these methods, we set out to perform de novo discovery with synthetic highly noncanonical peptidomimetic libraries against recently identified clinically relevant targets

Methods

Results

Conclusion