A Comprehensive Technology Platform for the Rapid Discovery of Peptide Inhibitors against SARS-CoV-2 Pseudovirus Infection

Abstract

We developed and validated a technology platform for designing and testing peptides inhibiting the infectivity of SARS-CoV-2 spike protein-based pseudoviruses. This platform integrates target evaluation, in silico inhibitor design, peptide synthesis, and efficacy screening. We generated a cyclic peptide library derived from the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein and the angiotensin-converting enzyme 2 (ACE2) receptor. The cell-free validation process by ELISA competition assays and Surface Plasmon Resonance (SPR) studies revealed that the cyclic peptide c9_05, but not its linear form, binds well to ACE2. Moreover, it effectively inhibited the transduction in HEK293, stably expressing the human ACE2 receptor of pseudovirus particles displaying the SARS-CoV-2 spike in the Wuhan or UK variants. However, the inhibitory efficacy of c9_05 was negligible against the Omicron variant, and it failed to impede the entry of pseudoviruses carrying the B.1.351 (South African) spike. These variants contain three or more mutations known to increase affinity to ACE2. This suggests further refinement is needed for potential SARS-CoV-2 inhibition. Our study hints at a promising approach to develop inhibitors targeting viral infectivity receptors, including SARS-CoV-2's. This platform also promises swift identification and evaluation of inhibitors for other emergent viruses.

Keywords: COVID-19; ELISA and SPR assays; SARS-CoV-2; drug discovery pipeline; macrocyclic peptides; peptide inhibitors; pseudovirus entry assay.

Figure 1

Flow chart of the drug discovery pipeline for designing and testing peptides inhibiting the SARS-CoV-2 infectivity. The pipeline involves target evaluation, inhibitor identification, and peptide synthesis, followed by cell-free and in vitro compound validation. Inhibitors can be designed using in silico tools or from the literature or external partners. Promising compounds are identified through ELISA and SPR assays before undergoing pseudovirus entry assay validation. This pipeline allows for the rapid identification of potential drug candidates for COVID-19.

Figure 2

Identification of potential small peptide inhibitors using the Rosetta Peptiderive protocol. (A) Crystal structure of the RBD (blue) and ACE2 (brown) complex (PDB: 6M0J) with the identified cyclizable peptide shown in light blue and indicated by the arrow. (B) Surface representation of the cyclizable peptide bound to the ACE2 receptor. (C) List of potential cyclizable peptides, including the wild type and 10 mutants with single or double amino acid changes.

Figure 3

Direct binding and inhibitory effect of cyclic peptide c9_05. Binding signal of (A) cyclic and (B) linear c9_05 to ACE2 as measured by SPR, with a sensorgram for four different c9_05 concentrations (3, 10, 30, and 100 μM) and fitting results displayed in black. (C) Concentration-dependent reduction in binding of RBD when ACE2 was preincubated with cyclic c9_05 at different concentrations (10, 30, 100 µM), as measured by ELISA competition assay. (D) Binding reduction in RBD to ACE2 when 30 μM of cyclic or linear c9_05 are present in the ELISA competition assay. *** p < 0.001.

Figure 4

The treatment with cyclic c9_05 reduced pseudoviral transduction. (A) Representative fluorescence microscopy images and (B) percentage of GFP (green) or RFP (red)-transduced HEK293-ACE2 cells pre-incubated with 25 µM c9_05 cyclic peptide in water and then transduced with the pseudoviral particles exposing different SARS-CoV-2 spike isoforms or with pseudoviral particles without (bald). Cells were pre-incubated in the same experimental conditions, with an equivalent volume of Milli-Q water as a negative control (Vehicle). Cells were also pre-incubated with 25 µM c9_05 cyclic peptide in water but not transduced (c9_05 cyclic). (A) Scale bar, 100 µM. (B) Data are the mean ± SD of GFP-positive HEK293-ACE2 compared to control cells incubated with vehicle only. * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. vehicle according to unpaired t-test.