Studies Of The Middle East Respiratory Syndrome Coronavirus (MERS-CoV) Fusion Inhibitors

In 2012, a novel coronavirus, termed Middle East respiratory syndrome coronavirus (MERS-CoV), emerged in the Middle East, cause SARS-like disease with>40% fatality rate. It was reported that MERS-CoV gained the ability of person-to-person transmission, raising concerns over its pandemic potential. Therefore, it is essential to identify the drug targets in the life cycle of MERS-CoV, and in turn, to develop anti-MERS therapeutics to treat patients with MERS-CoV infection and combat the potential pandemic.Here, we studied the structure and function of the HR1 and HR2 domains in the S protein S2 subunit of MERS-CoV, particularly the 6-HB fusion core formed by the HRl and HR2 domains, in order to elucidate the viral fusion mechanism with the aim of designing novel candidate MERS-CoV fusion inhibitors as therapeutics for treatment of MERS-CoV infection.First, we obtained the full-length amino acid sequence of MERS-CoV (hCoV-EMC) S protein. By multiple sequence alignment with SARS-CoV S protein, we identified the S2 subunit:fusion peptide (aa 943-982), HR1 domain (aa 984-1104), HR2 domain (aa 1246-1295), transmembrane domain (aa 1296-1318), and intracellular domain (aa 1319-1353).To understand the structural basis of the interactions between HR1 and HR2 regions of MERS-CoV, a fusion protein containing the major parts of HR1 (aa 984-1062) and HR2 (aa 1245-1289) with a short linker (SGGRGG) in between was constructed for crystallographic study. The crystal structure of HR1-L6-HR2 shows a canonical 6-HB structure. Taking a rod-like shape with a length of-112 A and a diameter of-27 A, the MERS-CoV S protein fusion core contains a parallel trimeric coiled coil of three HR1 helices around which three HR2 helices are entwined in an oblique antiparallel manner. The helices constituting the 6-HB are formed by aa 987-1062 in the HR1 domain and aa 1263-1279 in the HR2 domain, respectively.Both the N-terminal and C-terminal tails of the HR2 region pack against the hydrophobic grooves of three HR1, mainly through hydrophobic interactions. One HR2 helix with two neighboring HR1 helices consist of 11 hydrogen-bonds, mostly distributed in the regions around the N- and C-terminal ends of the HR2 helices. The relatively concentrated hydrogen-bonds further stabilize its binding with the central hydrophobic grooves.Then, we designed and synthesized two individual peptides, HRIP and HR2P, overlapping the regions involving interaction between the HR1 and HR2 domains in the crystal structure. We used Native-PAGE to investigate whether HR1P could interact with HR2P to form 6-HB. The mixture of HR1P/HR2P showed a new band at the upper position of the HR2P in the gel. We also use the SE-HPLC to analyze the peptides HRIP, HR2P, and HR1P/HR2P complex. The mixture of HR1P/HR2P showed a new peak before the peak of single HR1P and HR2P. We conclude that the HRIP and HR2P could form the oligomeric complex.We also analyzed their complex using 15% SDS polyacrylamide protein gel without heating samples. The result showed that HR1P/HR3P complex corresponding to a molecular mass of ～26 kDa (theoretical mass 25.8 kDa). Circular-dichroism (CD) spectroscopy was then used to investigate the secondary structures and to determine thermal stability of the HR1P/HR2P complex. The mixture of HR1P/HR2P at equimolar concentration showed a helical complex and strong thermal stability in phosphate buffer with a Tm of about 87℃。The above results confirmed that the peptides derived from HRl and HR2 domains of MERS-CoV S protein could form 6-HB, which mimics the fusion core structure in vitro. Therefore, the HR1P/HR2P complex provides a useful model as a basis for designing candidate MERS-CoV fusion inhibitors.To determine whether the HR1P and HR2P peptides are able to inhibit MERS-CoV fusion with the target cell, we developed an MERS-CoV S protein-mediated cell-cell fusion assay using 293 T cells that can instantaneously express MERS-CoV S protein and EGFP (293T/MERS/EGFP) as the effector cells and Huh-7 cells that express DPP4 as the target cells. The 293T cells expressing EGFP only (293T/EGFP) were included as the control. After 293T/MERS/EGFP cells and Huh-7 cells were cocultured at 37℃ for 4 h,293T/MERS/EGFP cells could fused with Huh-7 cells.We then tested the inhibitory activity of the peptides HR1P, HR2P, T20 (an anti-HIV-1 peptide), and SC-1 (an anti-SARS-CoV peptide), on MERS-CoV S-mediated cell-cell fusion. While HR1P, T20 and SC-1 exhibited no significant inhibitory activity at the concentration up to 40 μM, HR2P showed potent inhibitory effect (IC50 at-0.8μM).Our result also shown HR2P could significantly inhibit MERS-CoV replication in Vero cells in a dose-dependent manner (IC50 of～0.6 μM), while HRIP, T20 and SC-1 peptides exhibited no significant inhibition at the concentration up to 5 μM. At same time, HR2P could inhibit MERS-CoV infection in Calu-3 cells (IC50= 0.6 uM), while the same peptide was much less effective in inhibiting MERS-CoV infection in HFL cells (1C5O= 13.9 μM), suggesting that MERS-CoV may enter these cells through different pathways, thereby having different sensitivity to the fusion inhibitor HR2P peptide. MERS-CoV HR2P could not inhibit SARS-CoV pseudovirus infection in 293T/ACE2 cells, indicating that HR2P peptide is a MERS-CoV-specific fusion inhibitor.Using time-of-addition and time-of-removal assays, we investigated the putative mechanism of action of HR2P. The results suggest that the HR2P inhibits the fusion between the viral envelope and the target cell membranes, thus blocking viral entry by target the S protein of MERS-CoV.We then tested the potential cytotoxicity of the HRIP and HR2P peptides on the 293T, Huh-7, and Vero cells. Neither peptide had significant cytotoxicity to these cells at the concentration up to 1,000 μM.These results suggested that HR2P is an effective MERS-CoV fusion inhibitor with low, or no, in vitro toxic effect.Then, we introducing Glu (E) and Lys (K) or Arg (R) residues to modify HR2P at the i to i+4 or i to i+3 arrangements. We introduced 2 point mutations into HR2P-M1 peptide, including T1263E and L1267R, and 7 point mutations into HR2P-M2 peptide, including T1263E, L1267K, S1268K, Q1270E, Q1271E, A1275K and N1277E. These mutations would form salt-bridge to stable the helix and increase hydrophilicity, thus enhancing the water-solubility.The a-helicity of HR2P-M1 (36.4%) and HR2P-M2 (42.4%) was significantly higher than that of HR2P (18.2%). The a-helicity and Tm values of HR1P/HR2P-M1 and HR1P/HR2P-M2 complexes were also higher than that of HR1P/HR2P complex, confirming that the introduction of salt-bridges could increase the stability of the helical peptide. The solubility of HR2P-M1 and HR2P-M2 in H2O was increased about 68-fold and 1,786-fold, respectively, while their inhibitory activity on MERS-CoV S-mediated cell-cell fusion was increased about 9% and 69%, respectively.In summary, we solve the crystal structure of the 6-HB formed by MERS-CoV S protein HR1 and HR2 domains. Based on the structure, we design peptides spanning the HR1 and HR2 sequences, respectively. We find HR2P can effectively inhibit MERS-CoV replication and its spike protein-mediated cell-cell fusion. Then we introduce of Glu, Lys or Arg residue into HR2P, resulting in the increase of its stability, solubility and anti-MERS-CoV activity. Therefore, the HR2P analogs have good potential to be further developed into effective viral fusion inhibitor for treating MERS-CoV infection.