Background: The COVID-19 pandemic has killed over 6 million people worldwide. Despite the accumulation of knowledge about the causative pathogen severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the pathogenesis of this disease, cures remain to be discovered. We searched for certain peptides that might interfere with spike protein (S protein)-angiotensin-converting enzyme 2 (ACE2) interactions.
Methods: Phage display (PhD)-12 peptide library was screened against recombinant spike trimer (S-trimer) or receptor-binding domain (S-RBD) proteins. The resulting enriched peptide sequences were obtained, and their potential binding sites on S-trimer and S-RBD 3D structure models were searched. Synthetic peptides corresponding to these and other reference sequences were tested for their efficacy in blocking the binding of S-trimer protein onto recombinant ACE2 proteins or ACE2-overexpressing cells.
Results: After three rounds of phage selections, two peptide sequences (C2, DHAQRYGAGHSG; C6, HWKAVNWLKPWT) were enriched by S-RBD, but only C2 was present in S-trimer selected phages. When the 3D structures of static monomeric S-RBD (6M17) and S-trimer (6ZGE, 6ZGG, 7CAI, and 7CAK, each with different status of S-RBDs in the three monomer S proteins) were scanned for potential binding sites of C2 and C6 peptides, C6 opt to bind the saddle of S-RBD in both 6M17 and erected S-RBD in S-trimers, but C2 failed to cluster there in the S-trimers. In the competitive S-trimer-ACE2-binding experiments, synthetic C2 and C6 peptides inhibited S-trimer binding onto 293T-ACE2hR cells at high concentrations (50 μM) but not at lower concentrations (10 μM and below), neither for the settings of S-trimer binding onto recombinant ACE2 proteins.
Conclusion: Using PhD methodology, two peptides were generated bearing potentials to interfere with S protein-ACE2 interaction, which might be further exploited to produce peptidomimetics that block the attachment of SARS-CoV-2 virus onto host cells, hence diminishing the pathogenesis of COVID-19.
The COVID-19 pandemic had caused 6.35 million death worldwide as of 8 July 2022 and still poses a serious challenge in some nations. Worse was that over two more folds of “excess deaths” might have occurred due to indirect consequences of the pandemic, such as changes in “social, economic, and behavioral responses to the pandemic, including strict lockdowns”. While vaccinations and natural infections build herd immunity that helps to protect people from infection or prevent pandemic recurrence, cures are still lacking for the infected individuals in most areas. Among the scientific efforts, various therapeutics have been tried, such as cells, engineered antibodies, natural products, synthetical biologicals, and small molecules. Intended targets included viral structural proteins, host products [e.g., interleukin 6 (IL-6)], viral replication process, or host-virus interactions. The strategies aiming at the first step of virus-host interactions sound most attractive. The viral spike (S) protein trimers (S-trimers) are thought to be the main molecules mediating the affinity of exogenous severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus for angiotensin-converting enzyme 2 (ACE2) or other less-attended molecules, such as TMPRSS2 on host cells. Since the structures of both S and ACE2 proteins are known, computation or computer-based methods are thought to be high for novel drug discovery. However, though a few candidates had been proposed in these in silico studies, only part of them had been proven effective in functional experimental studies, highlighting the demand for more robust strategies that mimic the actual virus-host interactions more faithfully.
Phage display (PhD) methodology, as exemplified in other infectious diseases, met this end and has been tried in the context of SARS-CoV-2 or COVID-19. In detail, PhD has been successful in producing antibodies for neutralizing or detection in identifying COVID-19-induced antibodies to the virus or in searching for viral epitopes responsible for virus escaping immune responses. Based on our previous experience using PhD in studies of the host-pathogen interactions, we performed PhD screening to search for peptides that would bind the receptor-binding domain (RBD) domain of SARS-CoV-2 S protein (S-RBD). Theoretically, if such peptides could bind the site(s) critical for S-RBD interaction with its receptors (e.g., ACE2 or other molecules), they should interfere with S-trimer-ACE2 interactions. Furthermore, such an S-protein Entrapped Affinity Ligand (SEAL) peptide should be able to block the binding of the viruses with their target cells. Here, we report that two SEAL peptides were obtained via phage displaying against S-RBD and S-trimer proteins, and preliminary functional studies demonstrated weak blocking effects at high concentrations. Encouragingly, while this project was ongoing, three groups reported their results obtained by protocols mainly relying on PhD. The promises and limitations of these studies were also discussed.
Recombinant SARS-CoV-2 S-trimer proteins were from the commercial resource (Cat# DRA49, MW 136.6 kDa; Novoprotein Company, Suzhou, China), and recombinant S-RBD products corresponding to aa319-541 of YP_009724390.1, MW 30.7 kDa was a generous gift from Li (Tsinghua University, Beijing, China). PhD-12 Peptide PhD Library Kit (New England BioLabs, Beverly, MA, United States) was used for PhD screening against these two proteins. Briefly, S-trimer proteins were immobilized overnight at 4°C on the enzyme-linked immunosorbent assay (ELISA) plates at 100 μg/ml in 0.1 M NaHCO 3, pH 8.6. The plates were then blocked with 0.5% bovine serum albumin (BSA) in 0.1 M NaHCO 3 buffer (containing 0.02% NaN 3) for 1 h. After six washes with tris base-buffered saline solution (TBST buffer containing 0.01% Tween-20), 2 × 10 11 phages in TBST buffer were added for 45 min at room temperature. After ten washes, bound phages were recovered, amplified in Escherichia coli, harvested into TBS buffer (containing 0.02% NaN 3), quantified with a plaque-forming assay, and used the product for the second round display. After two or three screening rounds, bound phages were harvested into elution buffer (0.2 M Glycine-HCl, 1 mg/ml BSA, pH 2.2) and neutralized with 1 M tris base-HCl buffer, pH 9.1. After dilution, the phage mix was applied onto bacterial plates to obtain blue plaques. Thirty (after the second round) or 25 (after the third round) isolated plaques were randomly picked for phage DNA sequencing using the primers in the kit. The resulting 12-amino acids peptides translated from phage DNA inserts were analyzed, and the most promising sequence was used for subsequent studies.
Enzyme-linked immunoassay (ELISA) was used to confirm the affinity of the resulting monoclonal phages for targeted proteins. ELISA plates (Corning, NY, United States) were coated with 10 μg/ml S-trimer or S-RBD proteins. With the starting original library phages (O virions) as control, all selected interest phages were amplified, titrated, and added to the plates at different concentrations (2.5 × 10 9, 1 × 10 10, 4 × 10 10, 1.6 × 10 11, and 6.4 × 10 11 phage virions in 100 μl) for 1 h at room temperature. The plates were washed six times with TBST washing buffer and then incubated with diluted horseradish peroxidase (HRP)-conjugated anti-M13 monoclonal antibody (GE Healthcare, Piscataway, NJ, United States) for 1 h. After six washes, 3,3′, 5,5-tetramethylbenzidine (TMB) solution (Beyotime Biotechnology, Shanghai, China) was added to the plate, and after 10 min of development, the reaction was stopped by adding 2 M H 2 SO 4 solution. Optical absorbance was measured at 450 nm in a microplate reader (Synergy H1, BioTek Instruments, Inc., Winooski, VT, United States).
The surface of RBD or S-trimer proteins was scanned using the PEP-SiteFinder. The 3D models of RBD to locate the potential docking site(s) of interest peptide(s) on S proteins or S-trimers were retrieved from Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB). The top 50 poses of each peptide in each protein model were checked to identify the most likely binding site(s). Cn3D was also utilized for viewing these protein structures.
293T-ACE2hR cells, a cell line consistently expressing human ACE2 (hACE2) on the cell surface, or recombinant ACE2 proteins (Cat#10108-H02H, Novoprotein) were utilized to test the potential effect of interest peptides on S-trimer-ACE2 binding. In brief, two possible SEAL peptides derived from the above analysis (C2 and C6) and two reference peptides [spike-binding peptide 1 (SBP1) and spike-binding peptide 1 (SBP2)] were ordered from Biotech Bioscience and Technology (Shanghai, China) and dissolved in PBS. Their sequences were as follows: C2, DHAQRYGAGHSG; C6, HWKAVNWLKPWT; SBP1, IEEQAKTFLDKFNHEAEDLFYQSK; and SBP2, TFLDKFNHEAED. 293T-ACE2hR cells were grown in 96-well plates until confluent in the first measurement setting. S-trimer at 2 nM was mixed with equal volume (25 μl) of peptides at different concentrations (0, 0.16, 0.8, 4, 20, and 100 μM) and kept at room temperature for 1 h. After removing the culture medium from the cells, the mixture was added (50 μl/well) and kept at room temperature for 1 h. Unbound peptides and proteins were removed, and the cells were washed three times with PBS. HRP-conjugated Anti-6X His tag® antibody (diluted at 1:10,000; Abcam, Cambridge, MA, United States) was added to each well for 1 h at room temperature. After three washes with PBS, TMB Solution (Beyotime Biotechnology) was added to the plate, and the plate was read at OD370 nm in a Multiskan Go Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, United States). Then, 293T-ACE2hR cells were substituted b
