COVID-19 Research: MIT Researchers Discover High Affinity Peptide Binders For SARS-CoV-2 Spike Protein
Source: COVID-19 Research Oct 02, 2020 4 years, 1 month, 2 weeks, 5 days, 18 hours, 22 minutes ago
COVID-19 Research: Scientist from the Massachusetts Institute of Technology (MIT) in a new research study have discovered high-affinity peptide binders for the SARS-CoV-2 spike glycoprotein that can inhibit infection. The study also opens the door for the development of rapid viral identification or conjugates for virus-directed delivery of therapeutics.
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The study findings are published on a preprint server and have yet to be peer-reviewed.
https://www.biorxiv.org/content/10.1101/2020.09.29.317131v1
Currently, 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.
The 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.
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Hence the discovery of additional reagents which would enable early and swift SARS-CoV-2 detection or neutralization is vital for stopping the pandemic.
Emerging 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.
As a result, 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.
The study team 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.
The team used a combinatorial affinity selection-mass spectrometry platform to rapidly identify sequences with a high affinity toward RBD and significant selectivity over human proteins.
Upon enrichment and synthesis of the identified peptides, biolayer interferometry was used to validate and observe their selective binding activity, alongside magnetic bead pulldown assay.
Interestingly, in an enzyme-linked immunosorbent assay (ELISA), these peptides were able to detect picomolar concentrations of RBD within a complex biological matrix.
Peptides have been previously investigated as potential SARS-CoV-2 antiviral agents. For example, long α-helical peptides binding to the S2 unit of the coronavirus spike protein have been described as potent fusion inhibitors. Computational and experimental studies supported targeting the spike RBD with linear peptides derived from the ACE2 N-terminus
Investigations in the laboratory, however, indicated that peptides 12 to 23 residues long derived from the ACE2 N-terminus do not associate with high affinity t
o SARS-CoV-2-spikeRBD expressed in human cells. 23 With straightforward handling, preparation, and late-stage modification,24–26 peptides are attractive potential candidates for point-of-care diagnostics.
It was reported that from the approximately 800 million screened peptides, the study team was 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.
These 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.
Although 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.
Simply by 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.
Dr Pomplun told Thailand Medical News, "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.”
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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.
Importantly, these 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).
Dr Pomplun added, "The rapid identification of SARS-CoV-2 is critical for patient contact tracing, identifying hosts, and epidemiologic studies. The peptides discovered by our platform may provide a useful SARS-CoV-2 detection modality to help achieve these goals.”
It should however be noted that 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.
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