The SARS-CoV-2 pandemic that began in 2019 has posed a significant threat worldwide. In the past two decades, three coronaviruses have emerged and endangered public health, including the severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome CoV (MERS-CoV), and SARS-CoV-2 (Peeri
The viral CoV genome encodes four structural proteins: spike, membrane (M), envelope (E), and nucleocapsid (N). Viral membrane fusion is an essential step of virus replication, which is accomplished by the viral spike and leads to the fusion of the viral and cell membranes (Bosch
From a therapeutic perspective, fusion inhibitors have been successfully used in inhibiting viral infections as they harbor intrinsic ability to prevent the fusion of viral and cellular membranes. In this study, we investigated a current knowledge gap in the discovery of anti-SARS-CoV-2 fusion drugs. First, the inhibitory properties of a truncated 24-mer peptide (peptide #1) and a 36-mer peptide (peptide #2) were compared. The results indicated strong inhibition of S-mediated cell-cell fusion and SARS-CoV-2 pseudovirus activity. In addition, ten peptide #2 analogs were developed. The structural and molecular aspects of the associated inhibitory properties are discussed.
Previous studies of SARS-CoV, MERS-CoV, and other viruses such as HIV and Ebola showed that HR1 analogs are usually weaker inhibitors than HR2-derived peptides (Liu
MDs were used to investigate the stability of the complex between the HR2-modified peptides #2-12 with HR1. The mutations were generated by Schrodinger Maestro package version 12 and saved as PDB files. MDs were performed as previously described with slight modifications (Kandeel
HEK293T is an immortalized cell line derived from a human fetal kidney. A pair of previously described 293FT-based reporter cell lines that constitutively express individual split proteins (DSP1-7 and DSP8-11 proteins) (Wang
The DSP assay using 293FT cells was performed as described previously (Yamamoto
293T cells were transfected with an expression plasmid for SARS-CoV-2-S, VSV-G, or control expression plasmid by calcium-phosphate precipitation. At 16 h posttransfection, the cells were inoculated with a replication-deficient VSV, VSV-ΔG-Luciferase, which lacks the VSV-G gene and encodes firefly luciferase, at an MOI=1 as described previously (Tani
For an infection assay, target Calu-3 cells were seeded in 96-well plates (5×104 cells/100 µL) one day before the assay. Cells were pre-treated with peptides for 1 h before infection. Pseudotyped viral particles were added to cells with the peptides. After 2 h of incubation, the culture supernatant was removed, and cells were washed with EMEM. Cells were further incubated in EMEM containing 10% FBS without peptides and pseudotyped viral particles. At 16 h postinfection, luciferase activity was measured using the Bright-Glo Luciferase Assay System (Promega) and Centro xS960 luminometer (Berthold).
Statistically significant differences between the mean values were determined using a two-tailed Student’s
The peptide design comprised testing the hypothesis of potential inhibition of SARS-CoV fusion by truncated or short peptide, a 24-mer (peptide #1). Furthermore, the 36-mer peptides were designed based on our previous research on MERS-CoV fusion as well as several fusion inhibition reports. All peptides contained the central helix of HR2 (Fig. 1). Peptides #2-12 contained several mutations of the WT 36-mer peptide #2 (Fig. 1). The inserted mutations were calculated to increase the binding free energy by ~0.5 kcal/mol.
We evaluated the effects of the twelve different peptides on SARS-CoV-2 S protein-mediated membrane fusion by DSP assay, which we previously established (Yamamoto
We then examined the effect of peptide #2 on infection of human lung epithelial cells, Calu-3 cells with SARS-CoV-2-S, or VSV-G pseudotyped VSV viral particles. To analyze the effect of peptide #2 on the virus entry step, we treated Calu-3 cells with peptide #2 for 1 h before infection and for an additional 2 h during infection. After the pseudotyped viral particles were removed, cells were incubated for 16 h without the peptide to induce luciferase expression. The luciferase assay showed that the infectious activity of SARS-CoV-2-S pseudotyped viral particles was significantly suppressed by the treatment with peptide #2 (IC50=1.49 µM) (Fig. 3A), while infection with VSV-G pseudotyped viral particles was not affected by peptide #2 (Fig. 3B). These results indicate that peptide #2 potently inhibits infection with SARS-CoV-2-S pseudotyped viral particles by preventing viral-cell fusion, possibly through binding of peptide #2 to the SARS-CoV-2 Spike.
The initial structure of the fusion complex contained a trimer of the 6-helix coiled-coil bundle of the HR1-HR2 fusion core. HR1 comprised the residues 912-989 of SARS-CoV S1, and HR2 contained 38 residues (1164-1202). MDs were used to evaluate the structural stability of the designed peptides and HR1 complexes. The RMSD of peptides-HR1 complexes is provided in Fig. 4A. Table 1 summarizes the average RMSD for every peptide-HR1 complex. The notable feature is the stability of the peptide complexes with low RMSD values (<0.2 nm). Overall, the results indicate tight binding of the peptides complexes. RMSF values revealed that all peptides had a similar profile of low RMSF values, and the peak of high fluctuations corresponds to the loop connecting HR1 and HR2 (residues no. 70-80, Fig. 4B), as previously observed (Kandeel
The sequence and molecular descriptors of the composition of the peptides were analyzed to understand the observed inhibitory activity of the peptides on a molecular basis. In addition, multiple correlation analysis was adopted by investigating the relationship of each molecular descriptor with the corresponding activity (Table 2). Within the examined descriptors, only hydrophilic substitution showed a significant correlation with the obtained S-mediated cell-cell fusion % to DMSO (r=–0.63,
The discovery of new therapeutic agents against emerging viruses is the most effective tool to fight disease and decrease morbidity and mortality. In this context, fusion inhibitors have demonstrated substantial promise for both prophylaxis and treatment of viral infections. In this study, we introduced the first generation of small chemical molecules with anti-MERS-CoV fusion activity (Kandeel
The virus entry inhibitors comprise two important steps of the virus replication cycle: attachment and fusion. The viral receptor-binding domain (RBD) is an important attachment target for developing specific antiviral antibodies or vaccines. However, this target is prone to frequent mutation (Xia
Previous report revealed that CoVs can infect the cells via two entry mechanisms, direct membrane fusion or endocytosis (Qinfen
In comparing the HR1-HR2 complexes, the SARS-CoV-2 complex has stronger binding energy than SARS-CoV, determined by the higher α–helicity of HR1 (Zhu
Both SARS and SARS-CoV-2 proteins demonstrate a high identity rate (Kandeel
The use of peptide as chemotherapeutic agents has been widely applied in treating viral diseases e.g. HIV (Yao
In brief, this study investigated the discovery of new peptide inhibitors against SARS-CoV-2 fusion. Peptides # 2 inhibited S-mediated cell-cell fusion at 1 and 4.4 µM, respectively. This suggests the potent inhibition of SARS-CoV-2, probably by inhibition of the membrane fusion mechanism. Peptide #1 can be regarded as a lead compound for further antiviral optimization.
The authors acknowledge the financial support from the Administration of International Cooperation and Knowledge Exchange (ICKEA), King Faisal University, Project No. IC-01-20. This work was supported in part by grants-in-aid from the Japanese Society for the Promotion of Science (16H06575 to J. I., 20K07610 to M. Y.), a Program of Japan Initiative for Global Research Network on Infectious Diseases (JGRID) from AMED (JP20wm0125002 to Y. K.). The authors acknowledge the computational facilities at the college of veterinary medicine. King Faisal University.
The authors declare no conflict of interest.