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, 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.Abstract Figure

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 3D) 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.…”

“…Several approaches have since been adopted for the discovery of novel RBD binding ligands, including full antibodies, 20−28 as well as antibody fragments such as VH domains, 29,30 nanobodies, 31−35 and monobodies. 36 In addition, engineered ACE2 mimetics/decoy receptors, 37−39 de novo designed miniproteins, 40,41 peptide-based ACE2 mimetics, 42,43 and synthetic peptide libraries 44 have also been reported that are capable of binding the SARS-CoV-2 spike RBD. Macrocyclic peptides are a class of molecules demonstrated to be highly effective at disrupting protein−protein interactions, particularly in cases such as the spike−ACE2 interaction where a defined binding pocket is lacking.…”

Targeting the SARS-CoV-2-spike protein: from antibodies to miniproteins and peptides

“…4 ). 40 Several peptides with a shared residue motif were selected and shown to associate to RBD with nanomolar affinities. While the peptides are not competing against ACE2 binding, their use as diagnostic tools is under investigation (information based on a BioRxiv preprint).…”

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, [24][25][26] miniproteins, 27 peptides, [28][29][30] and small molecules. [31][32][33][34][35] As far as we are aware, no high-throughput virtual screening has been conducted in relatively large molecules in the "beyond rule of 5" chemical space that have high RBD binding affinity.…”