Discovery of high affinity binders for SARS-CoV-2 spike protein

An exciting, 'tour-de-force' paper by researchers from the Massachusetts Institute of Technology describes de novo discovery of high-affinity peptide binders for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein, which opens the door for the development of rapid viral identification or conjugates for virus-directed delivery of therapeutics. The findings are currently available on the bioRxiv* preprint server.

A novel beta-coronavirus SARS-CoV-2 has caused a global pandemic of coronavirus disease (COVID-19) with significant health, social and economic implications. Improved diagnostics are a matter of intense research endeavors towards reliable and early detection.

At the moment, the reverse-transcriptase polymerase chain reaction (RT-PCR) represents the gold standard of SARS-CoV2 detection, while serologic detection is used mostly to track viral progression and population immunity.

Direct detection of SARS-CoV-2 has already been proposed in scalable and rapidly deployed formats; however, it was shown that it often suffers from low sensitivity that hampers effectivity in general population testing. Therefore, the discovery of additional reagents which would enable early and swift SARS-CoV-2 detection or neutralization is vital for stopping the pandemic.

The unsung potential of peptide sequences

Fundamental viral research has already shown how soluble human and modified angiotensin-converting enzyme 2 (ACE2) displays a high affinity to the SARS-CoV-2 spike glycoprotein receptor-binding domain (RBD) and exhibits neutralizing activity in live virus infection models.

Consequently, the affinity reagents targeting the SARS-CoV-2 spike glycoprotein – basically the most exposed surface structure of the whole virus – are of substantial interest in the development of novel therapeutics and diagnostics methods.

In this new study, researchers from the Massachusetts Institute of Technology (led by Dr. Sebastian Pomplun) report the discovery of synthetic peptides (harboring 13 residues) with nanomolar affinity for the SARS-CoV-2 spike-RBD.

SARS-CoV-2-spike-RBD binding peptides with nanomolar affinity were identified by affinity selection-mass spectrometry. A) Schematic representation of the AS-MS workflow and enriched sequences. In brief: biotinylated SARS-CoV-2-spike-RBD was immobilized on magnetic streptavidin beads and then incubated with peptide libraries. Unbound members were removed by washing. Peptides bound to SARS-CoV-2-spike-RBD were eluted and analyzed by nanoLC-MS/MS. B) BLI curves for association/dissociation of 1, 2, and 4 to SARS-CoV-2-spike-RBD (in kinetic buffer: 1x PBS, pH = 7.2, 0.1% bovine serum albumin, 0.02% Tween-20). While peptide 4 had somewhat higher affinity, peptide 1, compared to 2 and 4, had the best solubility and was used for all further investigations. Peptides 2 and 4 precipitated within hours at concentrations greater than 10 μM. Kinetic binding results are reported in SI Table 1. C) BLI curves for 1 (blue line) and scrambled analogs of 1 (light and dark grey lines respectively, sc1: GSVKRWLTYVKNFK and sc2: RFYVTKGWSNKVLK). D) Self-competition analysis (BLI association) of 1 to SARS-CoV-2-spike-RBD : 1-biotin immobilized on BLI tips was dipped into solutions containing SARS-CoV- 2-spike-RBD and 1 ([RBD] = 500 nM; [1] = 0 – 16 μM). Increasing the concentration of 1 in solution causes less free RBD available in solution (due to RBD-1 complex formation) and results in a concentration dependent decrease in BLI response.
SARS-CoV-2-spike-RBD binding peptides with nanomolar affinity were identified by affinity selection-mass spectrometry. A) Schematic representation of the AS-MS workflow and enriched sequences. In brief: biotinylated SARS-CoV-2-spike-RBD was immobilized on magnetic streptavidin beads and then incubated with peptide libraries. Unbound members were removed by washing. Peptides bound to SARS-CoV-2-spike-RBD were eluted and analyzed by nanoLC-MS/MS. B) BLI curves for association/dissociation of 1, 2, and 4 to SARS-CoV-2-spike-RBD (in kinetic buffer: 1x PBS, pH = 7.2, 0.1% bovine serum albumin, 0.02% Tween-20). While peptide 4 had somewhat higher affinity, peptide 1, compared to 2 and 4, had the best solubility and was used for all further investigations. Peptides 2 and 4 precipitated within hours at concentrations greater than 10 μM. Kinetic binding results are reported in SI Table 1. C) BLI curves for 1 (blue line) and scrambled analogs of 1 (light and dark grey lines respectively, sc1: GSVKRWLTYVKNFK and sc2: RFYVTKGWSNKVLK). D) Self-competition analysis (BLI association) of 1 to SARS-CoV-2-spike-RBD : 1-biotin immobilized on BLI tips was dipped into solutions containing SARS-CoV- 2-spike-RBD and 1 ([RBD] = 500 nM; [1] = 0 – 16 μM). Increasing the concentration of 1 in solution causes less free RBD available in solution (due to RBD-1 complex formation) and results in a concentration dependent decrease in BLI response.

Three out of 800 million

This group of researchers utilized a combinatorial affinity selection-mass spectrometry platform to rapidly identify sequences with a high affinity toward RBD and significant selectivity over human proteins.

After enrichment and synthesis of the identified peptides, biolayer interferometry was used to validate and observe their selective binding activity, alongside magnetic bead pulldown assay. Furthermore, in an enzyme-linked immunosorbent assay (ELISA), these peptides were able to detect picomolar concentrations of RBD within a complex biological matrix.

From the approximately 800 million screened peptides, the researchers were able to pinpoint three peptide sequences. This was followed by the development of an extracted ion chromatogram for each peptide, revealing selective enrichment of each of them against the SARS-CoV-2 spike-RBD.

Triple S: sensitivity, selectivity and solubility

The aforementioned peptides had dissociation constants between 80 to 970 nanomoles and were able to associate with the SARS-CoV-2 spike-RBD at a site that was not related to ACE2 binding – making them potential orthogonal reagents for sandwich immunoassays.

And albeit peptide 1 did not have the highest affinity to SARS-CoV-2 spike-RBD, it displayed the highest solubility, which was the reason why it was used for all further analyses. More specifically, the observed cross-binding of peptide 1 to the MERS coronavirus spike protein suggested a possible binding site far away from the binding site for the human ACE2 receptor.

Hence, adapting peptide 1 to a chemiluminescence enzyme immunoassay (or a similar technique) could improve its sensitivity for detecting SARS-CoV-2. And due to its selectivity in biological media, it could also be utilized for the direct detection of the virus in bodily fluids.

"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", study authors comment on the importance of their findings in this bioRxiv paper.

Possible diagnostic and therapeutic solution

The peptides reported in this study are possible starting points for establishing affinity-based diagnostic tools, which were already researched for other viruses such as influenza, dengue, rotavirus, Epstein-Barr virus, hepatitis C virus, and human immunodeficiency virus (HIV).

"The rapid identification of SARS-CoV-2 is critical for patient contact tracing, identifying hosts, and epidemiologic studies", say study authors. "The peptides discovered by our platform may provide a useful SARS-CoV-2 detection modality to help achieve these goals", they conclude.

However, high-affinity reagents that lack direct competition activity for native receptors could also be used for virus-directed delivery of antivirals or for the creation of proteasome or lysosome targeting chimeras. In the ongoing quest for an efficacious drug, these new possibilities are rather compelling.

*Important Notice

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Journal reference:
Dr. Tomislav Meštrović

Written by

Dr. Tomislav Meštrović

Dr. Tomislav Meštrović is a medical doctor (MD) with a Ph.D. in biomedical and health sciences, specialist in the field of clinical microbiology, and an Assistant Professor at Croatia's youngest university - University North. In addition to his interest in clinical, research and lecturing activities, his immense passion for medical writing and scientific communication goes back to his student days. He enjoys contributing back to the community. In his spare time, Tomislav is a movie buff and an avid traveler.

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