De Novo Discovery of High Affinity Peptide Binders for the SARS-CoV-2 Spike Protein

The {beta}-coronavirus SARS-CoV-2 has caused a global pandemic. Affinity reagents targeting the SARS-CoV-2 spike protein, the most exposed surface structure of the virus, are of interest for the development of therapeutics and diagnostics. We used affinity selection-mass spectrometry for the rapid discovery of synthetic high affinity peptide binders for the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. From library screening with 800 million synthetic peptides, we identified three sequences with nanomolar affinities (dissociation constants Kd = 80 to 970 nM) for RBD and selectivity over human serum proteins. Picomolar RBD concentrations in biological matrix could be detected using the biotinylated lead peptide in ELISA format. These peptides might associate with the SARS-CoV-2-spike-RBD at a site unrelated to ACE2 binding, making them potential orthogonal reagents for sandwich immunoassays. We envision our discovery as a robust starting point for the development of SARS-CoV-2 diagnostics or conjugates for virus directed delivery of therapeutics. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=100 SRC="FIGDIR/small/317131v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@803e9borg.highwire.dtl.DTLVardef@18e5d74org.highwire.dtl.DTLVardef@1998310org.highwire.dtl.DTLVardef@16ba5cc_HPS_FORMAT_FIGEXP M_FIG C_FIG

The first-in-class peptide binder to the SARS-CoV-2 spike protein

Coronavirus disease 19 (COVID-19) is an emerging global health crisis. With over 7 million confirmed cases to date, this pandemic continues to expand, spurring research to discover vaccines and therapies. SARS-CoV-2 is the novel coronavirus responsible for this disease. It initiates entry into human cells by binding to angiotensin-converting enzyme 2 (ACE2) via the receptor binding domain (RBD) of its spike protein (S). Disrupting the SARS-CoV-2-RBD binding to ACE2 with designer drugs has the potential to inhibit the virus from entering human cells, presenting a new modality for therapeutic intervention. Peptide-based binders are an attractive solution to inhibit the RBD-ACE2 interaction by adequately covering the extended protein contact interface. Using molecular dynamics simulations based on the recently solved cryo-EM structure of ACE2 in complex with SARS-CoV-2-RBD, we observed that the ACE2 peptidase domain (PD) 1 helix is important for binding SARS-CoV-2-RBD. Using automated fast-flow peptide synthesis, we chemically synthesized a 23-mer peptide fragment of the ACE2 PD 1 helix (SBP1) composed entirely of proteinogenic amino acids. Chemical synthesis of SBP1 was complete in 1.5 hours, and after work up and isolation >20 milligrams of pure material was obtained. Bio-layer interferometry (BLI) revealed that SBP1 associates with micromolar affinity to insect-derived SARS-CoV-2-RBD protein obtained from Sino Biological. Association of SBP1 was not observed to an appreciable extent to HEK cell-expressed SARS-CoV-2-RBD proteins and insect-derived variants acquired from other vendors. Moreover, competitive BLI assays showed SBP1 does not outcompete ACE2 binding to Sino Biological insect-derived SARS-CoV-2-RBD. Further investigations are ongoing to gain insight into the molecular and structural determinants of the variable binding behavior to different SARS-CoV-2-RBD protein variants.