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

Abstract

The β-coronavirus SARS-CoV-2 has caused a global pandemic. Affinity reagents targeting the SARS-CoV-2 spike protein 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 K d = 80–970 nM) for RBD and selectivity over human serum proteins. Nanomolar RBD concentrations in a biological matrix could be detected using the biotinylated lead peptide in ELISA format. These peptides do not compete for ACE2 binding, and their site of interaction on the SARS-CoV-2-spike-RBD might be unrelated to the ACE2 binding site, making them potential orthogonal reagents for sandwich immunoassays. These findings serve as a starting point for the development of SARS-CoV-2 diagnostics or conjugates for virus-directed delivery of therapeutics.

Discovery of Cyclic Peptide Ligands to the SARS-CoV-2 Spike Protein Using mRNA Display

“…Interestingly, this molecule is a higher affinity cyclic analogue of a linear RBD ligand recently identified through an affinity selection-mass spectrometry approach. 44 Peptide 5 was also notable in the series with a K D = 76 nM against the RBD. The disulfide-closed peptides showed not only very high apparent RBD affinity but also strong nonspecific binding (data not shown).…”

“…Interestingly, a similar motif is observed in a linear RBD peptide ligand ( 1 TVFG 4 ; Figure 3 D) that was previously identified by affinity selection-mass spectrometry by Pentelute and co-workers. 44 Ala mutagenesis of this 13-residue linear peptide highlighted the importance of the motif, particularly residues Val2 and Gly4, which are also present in the sequences of both the RBD C-terminus and peptide 4 . It is therefore tempting to speculate that a similar mode of binding for the RBD would be observed for the linear peptide that was discovered through an independent affinity selection method.…”

“…Only the RBD is shown for clarity. D) Alignment of peptide 4 , linear RBD-binding peptide ligand, 44 and spike RBD C-terminal β-strand. The most C-terminal amino acid shown from the peptide/RBD is numbered on the right.…”

Evaluation of SARS-CoV-2 Spike S1 Protein Response on PI3K-Mediated IL-8 Release

“…Several studies have shown that the SARS-CoV-2 spike (S) glycoprotein binds with ACE2 with higher affinity and that the S1 subunit containing a receptor-binding domain (RBD) of one protomer in the spike protein trimer tightly interacts with ACE2 extracellular enzymatic domain [38,[44][45][46][47]. To that end, high-affinity peptide sequences and neutralizing antibodies targeting SARS-CoV-2 spike-RBD have been developed with the overall goal of evaluating their efficacy as novel diagnostic or therapeutic modalities [48][49][50]. Thus, it is important to explore the critical signaling pathways involved in regulating the spike protein to gain further mechanistic insights to develop novel therapies for COVID-19 treatment.…”

Accelerating the Discovery of the Beyond Rule of Five Compounds That Have High Affinities Toward SARS-CoV-2 Spike RBD

“…Different approaches have been conducted that are based on interfering with the RBD binding to the hACE2 including neutralizing antibodies, [29][30][31] decoy proteins, 32 miniproteins, 33 peptides, [34][35][36][37][38][39][40] and stapled hACE2 peptides. 41 In addition, many studies have ex-plored drug repurposing or repositioning of vast chemical compounds such as FDA approved drugs, [42][43][44] antivirals, 45,46 phytochemicals, 47 essential free fatty acids, 48 and natural products 49,50 for the sake of disrupting the RBD-hACE2 binding.…”