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

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, K_D = 19-123 nM). The most potent noncanonical ACE2 peptide binder, ABP N1 (K_D = 19 nM), showed enhanced serum stability in comparison with the most potent canonical binder, ABP C7 (K_D = 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.

Fig. 1. Magnetic bead-based affinity selection-mass spectrometry (AS-MS) enables rapid discovery of both canonical and noncanonical binders in a single experiment.

a Schematic representation of the AS-MS workflow used in this study. In brief, the biotinylated protein (ACE2 represented in orange from PDB: 6M17 or control) was immobilized onto streptavidin (SA)-coated magnetic beads and then sampled in synthetic peptide libraries to enrich peptide binders. Subsequently, unbound peptides were washed away, and bound material was eluted and then sequenced by nLC-MS/MS. Individual hits were synthesized and validated at the final step. b The design for synthetic canonical and noncanonical libraries. c The monomer set used for the synthesis of the noncanonical library (Library 2).

Fig. 2. Nanomolar affinity binders were identified from both canonical and noncanonical libraries.

a–c ACE2-binding traces, measured by bio-layer interferometry, for noncanonical binders ABP N1, ABP N4, and ABP N6, respectively. d–f ACE2-binding traces, measured by bio-layer interferometry, for canonical binders ABP C3, ABP C7, and ABP C8, respectively. g A summary of all individually synthesized peptides. Column headers: ‘ID’, the peptide identifiers; ‘Sequence, X₁₂K’, the peptide sequences with a lysine at the C-terminus; ‘ALC’, the exported average local confidence score from sequence decoding; ‘Error, ppm’, the mass error (in ppm) between the precursor and assigned sequence; ‘K_D, _obs, nM’, the apparent dissociation constant, in nM, measured by bio-layer interferometry. Cyan highlights the canonical (ABP C7) and noncanonical (ABP N1) peptides with the highest binding affinity.

Fig. 3. ABP N1 demonstrates enhanced serum stability relative to the canonical binder ABP C7.

LC-MS chromatograms (total ion current) of ABP N1 (a) and ABP C7 (b) incubated at 37 °C in 1 × PBS (t = 0 h) or 5% normal human serum. Spectra were normalized to the intensity of the peptide at t = 0 h to observe degradation of the original amount of peptide. c Comparison of the serum stability of ABP N1 and ABP C7 by the integral of the extracted ion count (EIC) of the monoisotopic [M + 2H]²⁺ ion of the starting peptide. ABP C7 is quickly degraded and the starting peptide mass disappears over 12 h, with a calculated half-life of approximately 2 h (single phase decay model).

Fig. 4. ABP N1 pulls down ACE2 from human serum selectively.

a Schematic representation of the serum pull-down experiment. ABP N1 was immobilized onto streptavidin-coated magnetic beads and then incubated with ACE2 (represented in orange from PDB: 6M17) in human serum. Bound ACE2 was eluted for subsequent analysis. b The SDS-PAGE image with samples showing from left to right lanes: (1) molecular weight standard; (2) purified ACE2 protein (1.5 µg) as a loading control; (3) normal human serum as a control; (4) normal human serum mixed with ACE2 (1.5 µg); (5) elution of the bound fraction from the magnetic beads. NHS, Normal Human Serum (5%).

Fig. 5. Picomolar ACE2 concentration was detected by ABP N1 by ELISA.

a Schematic representation of the ELISA workflow. b ACE2 in 1 × PBS at different concentrations (100 nM–10 pM) was immobilized on an ELISA plate. c ACE2 in human serum at different concentrations (100 nM–10 pM) was immobilized on an ELISA plate. The plate was incubated with ABP N1 or control peptide, followed by streptavidin-HRP and TMB substrate. Absorbance was measured at 450 nm. The experiment was performed in technical triplicates (n = 3). Each data point, the mean signal as a bar, and error bars from experimental standard deviation, and statistical significance calculated with the unpaired Student’s t-test are shown.