Targeting an evolutionarily conserved “E-L-L” motif in spike protein to identify a small molecule fusion inhibitor against SARS-CoV-2

Abstract

As newer variants of SARS-CoV-2 continue to pose major threats to global human health and economy, identifying novel druggable antiviral targets is the key towards sustenance. Here, we identify an evolutionary conserved “E-L-L” motif present within the HR2 domain of all human and non-human coronavirus spike (S) proteins that play a crucial role in stabilizing its post-fusion six-helix bundle (6-HB) structure and thus, fusion-mediated viral entry. Mutations within this motif reduces the fusogenicity of the S protein without affecting its stability or membrane localization. We found that posaconazole, an FDA-approved drug, binds to this “E-L-L” motif and impedes the formation of 6-HB, thus effectively inhibiting SARS-CoV-2 infection in cells. While posaconazole exhibits high efficacy in blocking S protein-mediated viral entry, mutations within the “E-L-L” motif rendered the protein completely resistant to the drug, establishing its specificity towards this motif. Our data demonstrate that posaconazole restricts early stages of infection through specific inhibition of membrane fusion and viral genome release into the host cell and is equally effective towards all major variants of concerns of SARS-CoV-2 including beta, kappa, delta, and omicron. Together, we show that this conserved essential “E-L-L” motif is an ideal target for the development of prophylactic and therapeutic interventions against SARS-CoV-2.

Targetable elements in SARS-CoV-2 S2 subunit for the design of pan-coronavirus fusion inhibitors and vaccines

“…It can bind to the conserved “ E1182-L1186L-L1193 ” motif located in HR2 with high affinity, thereby interfering the 6-HB fusion core formation and then blocking membrane fusion. 119 In this way, Posaconazole can serve as a broad-spectrum fusion inhibitor against the ongoing SARS-CoV-2 variants. 119 In addition, itraconazole and estradiol benzoate could interact with the HR1 of several HCoVs, including SARS-CoV-2, SARS-CoV, and MERS-CoV, and exhibit broad neutralizing activity in vitro.…”

“… 119 In this way, Posaconazole can serve as a broad-spectrum fusion inhibitor against the ongoing SARS-CoV-2 variants. 119 In addition, itraconazole and estradiol benzoate could interact with the HR1 of several HCoVs, including SARS-CoV-2, SARS-CoV, and MERS-CoV, and exhibit broad neutralizing activity in vitro. 120 Meanwhile, biflavone-based anti-HIV drugs, such as hinokiflavone and robustaflavone, could prevent SARS-CoV-2 S-mediated virus entry by blocking the HR1-HR2 interaction.…”

SELEX based aptamers with diagnostic and entry inhibitor therapeutic potential for SARS-CoV-2

“…Pseudovirus entry assay was performed as described previously by Das Jana et al (2022) 22 . HEK 293T cells were transduced with lentiviral plasmids to express hACE2 protein.…”

“…Hence, we tested their e cacy for inhibiting SARS-CoV-2 entry by using a pseudovirus-based entry assay. Previous reports indicated that HIV-1-based reporter lentiviruses with the re y luciferase ZsGreen reporter genes in their genomic backbone and SARS-CoV-2 spike protein as the sole surface glycoprotein are suitable for studying viral entry into host cells 21,42 . Therefore, we have generated reporter viruses pseudotyped with SARS-CoV-2 S and treated them with different aptamers before infecting HEK293T cells overexpressing ACE2 receptors (HEK293T-ACE2) (Fig.…”

Pan‐coronavirus fusion inhibitors to combat COVID‐19 and other emerging coronavirus infectious diseases

“…For example, Itraconazole (ITZ) and Estradiol benzoate (EB), the clinically approved drugs for the treatment of patients with fungal infections and prostate cancer, respectively, can interact with the HR1 domain in S proteins of SARS‐CoV, MERS‐CoV, and SARS‐CoV‐2, thus inhibiting the entry and infection of these three HCoVs 44 . Posaconazole, an FDA‐approved antifungal drug, was reported to broadly inhibit fusion, entry, and infection of SARS‐CoV‐2 and its variants by binding the conserved E‐L‐L motif in the HR2 domain of SARS‐CoV‐2 responsible for stabilizing post‐fusion 6‐HB structure 45 . Some naturally occurring biflavone‐based anti‐HIV agents, for example, hinokiflavone and robustaflavone, could interact strongly with the residues in HR1 and HR2 regions of SARS‐CoV‐2 S protein and block the 6‐HB formation, thus inhibiting SARS‐CoV‐2‐target cell membrane fusion 46 .…”