A Dynamic and Effective Peptide-Based Strategy for Promptly Addressing Emerging SARS-CoV-2 Variants of Concern

Abstract

Genomic surveillance based on sequencing the entire genetic code of SARS-CoV-2 involves monitoring and studying genetic changes and variations in disease-causing organisms such as viruses and bacteria. By tracing the virus, it is possible to prevent epidemic spread in the community, ensuring a 'precision public health' strategy. A peptide-based design was applied to provide an efficacious strategy that is able to counteract any emerging viral variant of concern dynamically and promptly to affect the outcomes of a pandemic at an early stage while waiting for the production of the anti-variant-specific vaccine, which require longer times. The inhibition of the interaction between the receptor-binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and one of the cellular receptors (DPP4) that its receptors routinely bind to infect human cells is an intriguing therapeutic approach to prevent the virus from entering human cells. Among the other modalities developed for this purpose, peptides surely offer unique advantages, including ease of synthesis, serum stability, low immunogenicity and toxicity, and small production and distribution chain costs. Here, we obtained a potent new inhibitor based on the rearrangement of a previously identified peptide that has been rationally designed on a cell dipeptidyl peptidase 4 (DPP4) sequence, a ubiquitous membrane protein known to bind the RBD-SPIKE domain of the virus. This novel peptide (named DPP4-derived), conceived as an endogenous "drug", is capable of targeting the latest tested variants with a high affinity, reducing the VSV* DG-Fluc pseudovirus Omicron's infection capacity by up to 14%, as revealed by in vitro testing in human Calu-3 cells. Surface plasmon resonance (SPR) confirmed the binding affinity of the new DPP4-derived peptide with Omicron variant RBD.

Keywords: RBD variants; SARS-CoV-2; peptides; protein–protein interaction (PPI); pseudovirus; surface plasmon resonance.

Figure 1

Left-hand images show the zoomed-in context of the DPP_4270–295 peptide in complex to (a) B.1.1.529, (c) BA.4/5, and (e) BQ.1.1 RBD. The key contacting elements inside include (b) the B.1.1.529 RBD/DPP4_270–295 conformation, (d) BA.4/5 RBD/DPP4_270–295, and (f) BQ.1.1 RBD/DPP4_270–295. All amino acids involved in the interaction are represented as balls and sticks, while the H-bonds and salt bridges are shown as yellow and purple dashed lines, respectively.

Figure 2

Vero E6 cells were inoculated with pseudotype particles bearing the S proteins of the indicated Vsvpp.SARS-2-S Omicron variants (B.1.1.529, BA.4/5, and BQ.1.1), together with DPP4_270–295. Infection efficiency was quantified by measuring virus-encoded luciferase activity in cell lysates at 48 h post-transduction, expressed as a percentage (Vsvpp.SARS-2/DPP4_270–295; grey bars), and normalized with respect to Omicron variants (Vsvpp.SARS-2-S⁺) alone (black bars). The data presented are the average of three biological replicates. Error bars indicate the standard deviation ± standard error of the mean (SEM). **** p < 0.0001 based on a one-way ANOVA test.

Figure 3

Workflow for the design of novel DPP4-derived peptides against SARS-CoV-2 RBD domain of emerging variants.

Figure 4

Key contacting elements inside pep6 in complex with (a) BA.4/5, (b) BQ.1.1, and (c) XBB1.5 RBD. All amino acids involved in the interaction are represented as balls and sticks, while the H-bonds are shown as yellow dashed lines. BA.4/5, BQ.1.1, and XBB1.5 RBD domains are reported as green carbon, faded salmon, and yellow cartoons, respectively.

Figure 5

(A,B) Sensogram overlay showing the binding of increasing amounts (range from 9.375 to 150 µg/mL) of DPP4_270-295 and pep6 to the immobilized Omicron BA.5 RBD surface. The response, in resonance units (RU), was recorded as a function of time. (C,D) Saturation curve obtained using the values of RU bound at equilibrium from injection of increasing concentrations of free DPP4_270-295 and pep6 onto the immobilized Omicron BA.5 RBD sensorchip.

Figure 6

(A) Effects pep6 on the viability of VERO E6 cells for three days. MTS assay was performed to determine cell viability after pep6 treatment at concentrations of 50, 100, and 200 µg for 72 h compared to untreated cells. P-value: VeroE6 +200 μg vs. VeroE6 at day 2 < 0.05, p-value VeroE6 +200 μg vs. VeroE6 at day 3 < 0.01. (B) Infection efficiency was quantified by measuring virus-encoded luciferase activity in Vero E6 cells infected for 48 h with VSV_Pseudo SARS-CoV-2 S Omicron XBB.1.5 alone and in combination with the synthetic peptide pep6. Data are expressed as percentage of infection and normalized with respect to the XBB.1.5 Omicron variant alone.

Figure 7

(A) Infection efficiency was quantified by measuring virus-encoded luciferase activity in cells infected for 48 h with VSV_Pseudo SARS-CoV-2 S Omicron XBB.1.5 alone and in combination with synthetic peptide DPP4_270–295 and pep6. Data are expressed as percentage of infection and normalized with respect to the XBB.1.5 Omicron variant alone (black bar). The average data from three biological replicates are presented. Error bars indicate the standard error of the mean (±SEM). *** p < 0.001 based on a one-way ANOVA test. (B) RT-qPCR analyses of immunity-related genes in Calu-3 cells after VSV_Pseudo SARS-CoV-2 S Omicron XBB.1.5 infection and treatment with pep6. Data are from three independent experiments and represented as mean ± SD. ** p < 0.01, and *** p < 0.001 based on a one-way ANOVA test.