Human angiotensin-converting enzyme 2 (ACE2) is recognized as the cell surface structure that allows for binding and host cell entry of SARS-CoV-2. The identification of small molecules that bind ACE2 is important for the development of both diagnostic and therapeutic tools for the prevention and treatment of disease. These small molecules may include peptidomimetics, small structures that have similar sequences to endogenous peptides, but have been modified for increased activity and/or stability. Affinity-selection mass spectrometry (AS-MS) is a high-throughput screening tool that allows users to identify peptides or peptide-like compounds that bind the target of interest. In a recent study by Zhang et al. in Communications Chemistry, the authors used AS-MS to identify library compounds that bound to ACE2. Importantly, the authors used two synthetic libraries containing over 2 million candidates each for the initial assays, one library generated with all canonical amino acids and one containing noncanonical amino acids, and then tested over a dozen of their hits with follow-up bio-layer interferometry assays to confirm low-nanomolar ACE2-binding affinities. Although two of the hits had similarly high binding affinities, the noncanoncial peptidomimetic showed little-to-no signs of degradation in a serum stability test, and selective binding of ACE2 in a serum pull-down experiment. The use of multiple follow-up and validation experiments in this paper show the power of AS-MS to identify novel and biologically relevant binding partners for the development of highly sensitive and specific diagnostic tools and therapeutics.
How was PEAKS used?
PEAKS Studio 8.5 was used for de novo sequencing of the peptidomimetics identified by AS-MS.
Zhang, G., Brown, J.S., Quartararo, A.J. et al. Rapid de novo discovery of peptidomimetic affinity reagents for human angiotensin converting enzyme 2. Commun Chem 5, 8 (2022), doi:10.1038/s42004-022-00625-3.
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.