A highly potent and stable pan-coronavirus fusion inhibitor as a candidate prophylactic and therapeutic for COVID-19 and other coronavirus diseases

Abstract

The development of broad-spectrum antivirals against human coronaviruses (HCoVs) is critical to combat the current coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variants, as well as future outbreaks of emerging CoVs. We have previously identified a polyethylene glycol-conjugated (PEGylated) lipopeptide, EK1C4, with potent pan-CoV fusion inhibitory activity. However, PEG linkers in peptide or protein drugs may reduce stability or induce anti-PEG antibodies in vivo . Therefore, we herein report the design and synthesis of a series of dePEGylated lipopeptide-based pan-CoV fusion inhibitors featuring the replacement of the PEG linker with amino acids in the heptad repeat 2 C-terminal fragment (HR2-CF) of HCoV-OC43. Among these lipopeptides, EKL1C showed the most potent inhibitory activity against infection by SARS-CoV-2 and its spike (S) mutants, as well as other HCoVs and some bat SARS-related coronaviruses (SARSr-CoVs) tested. The dePEGylated lipopeptide EKL1C exhibited significantly stronger resistance to proteolytic enzymes, better metabolic stability in mouse serum, higher thermostability than the PEGylated lipopeptide EK1C4, suggesting that EKL1C could be further developed as a candidate prophylactic and therapeutic for COVID-19 and other coronavirus diseases.

A Five-Helix-Based SARS-CoV-2 Fusion Inhibitor Targeting Heptad Repeat 2 Domain against SARS-CoV-2 and Its Variants of Concern

“…We previously identified a series of peptide-based fusion/entry inhibitors targeting the HR1 domain of HIV-1 (e.g., SJ-2176) [ 30 ], SARS-CoV (SC-1) [ 31 ], MERS-CoV (MERS-HR2P) [ 32 ], HCoV-OC43 (OC43-HR2P) [ 5 ] and SARS-CoV-2 (2019-nCoV-HR2P) [ 6 ], as well as to pan-CoV, including EK1 [ 5 ], EK1C4 [ 7 , 33 ] and EKL1C [ 8 ]. Notwithstanding these advances, HR2 should be a better target for the development of broad-spectrum HCoV fusion/entry inhibitors since the amino acid sequence of the HR2 domain is more conserved than that of HR1.…”

“…Authentic SARS-CoV-2 WT strain (nCoV-SH01) and Delta variant (B.1.617.2) were maintained in the BSL-3 Laboratory of the Shanghai Medical College, Fudan University [ 8 ]. The gene sequence of the Delta variant was confirmed by next-generation sequencing.…”

“…Plasmids of pNL4-3.luc.RE (the luciferase reporter-expressing HIV-1 backbone) and pcDNA3.1 encoding SARS-CoV-2-S were co-transfected with 293T cells using VigoFect (Vigorous Biotechnology, Beijing, China) [ 8 ]. The spike gene of SARS-CoV-2 wildtype strain and variants were obtained from the GISAID Platform ( , accessed on 29 January 2021, SARS-CoV-2 (IIUM316), GenBank: MW079429.1; Alpha (CUMC-21081002): OM757358.1; Beta (TUS-Siena7): OM736179.1; Gamma (AR-UFRO-0018): OM146072.1; Delta (CA-CDPH-500041690): OM754056.1; Omicron (CA-LACPHL-AF06801): OM757978.1; Lambda (MA-UMASSMED-P026A04): OM486769.1).…”

“…Inhibitory activity against authentic coronavirus infection was conducted in a Biosafety Level 3 (BSL-3) laboratory of Fudan University. An inhibition assay was performed as described previously [ 8 ]. Briefly, Calu-3 cells were seeded into a 96-well plate.…”

“…Consequently, these domains are important targets for the development of viral fusion/entry inhibitors [ 4 ]. In response, we previously developed a series of HR1-targeting SARS-CoV-2 fusion inhibitory peptides, such as EK1 [ 5 ], HR2P [ 6 ], EK1C4 [ 7 ] and EKL1C [ 8 ]. However, no peptide targeting HR2 of the SARS-CoV-2 S protein has been reported so far, even though HR2 has a more conserved sequence than HR1 ( Figure S1 ) [ 6 ].…”