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

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the currently ongoing coronavirus disease 2019 (COVID-19) pandemic, has posed a serious threat to global public health. Recently, several SARS-CoV-2 variants of concern (VOCs) have emerged and caused numerous cases of reinfection in convalescent COVID-19 patients, as well as breakthrough infections in vaccinated individuals. This calls for the development of broad-spectrum antiviral drugs to combat SARS-CoV-2 and its VOCs. Pan-coronavirus fusion inhibitors, targeting the conserved heptad repeat 1 (HR1) in spike protein S2 subunit, can broadly and potently inhibit infection of SARS-CoV-2 and its variants, as well as other human coronaviruses. In this review, we summarized the most recent development of pan-coronavirus fusion inhibitors, such as EK1, EK1C4, and EKL1C, and highlighted their potential application in combating current COVID-19 infection and reinfection, as well as future emerging coronavirus infectious diseases.

A Positron Emission Tomography Tracer Targeting the S2 Subunit of SARS-CoV-2 in Extrapulmonary Infections.

Tracking the pathogen of coronavirus disease 2019 (COVID-19) in live subjects may help estimate the spatiotemporal distribution of SARS-CoV-2 infection in vivo. This study developed a positron emission tomography (PET) tracer of the S2 subunit of spike (S) protein for imaging SARS-CoV-2. A pan-coronavirus inhibitor, EK1 peptide, was synthesized and radiolabeled with copper-64 after being conjugated with 1,4,7-triazacyclononane-1,4,7-triyl-triacetic acid (NOTA). The in vitro stability tests indicated that [64Cu]Cu-NOTA-EK1 was stable up to 24 h both in saline and in human serum. The binding assay showed that [64Cu]Cu-NOTA-EK1 has a nanomolar affinity (Ki = 3.94 ± 0.51 nM) with the S-protein of SARS-CoV-2. The cell uptake evaluation used HEK293T/S+ and HEK293T/S- cell lines that showed that the tracer has a high affinity with the S-protein on the cellular level. For the in vivo study, we tested [64Cu]Cu-NOTA-EK1 in HEK293T/S+ cell xenograft-bearing mice (n = 3) and pseudovirus of SARS-CoV-2-infected HEK293T/ACE2 cell bearing mice (n = 3). The best radioactive xenograft-to-muscle ratio (X/Nxenograft 8.04 ± 0.99, X/Npseudovirus 6.47 ± 0.71) was most evident 4 h postinjection. Finally, PET imaging in the surrogate mouse model of beta-coronavirus, mouse hepatic virus-A59 infection in C57BL/6 J mice showed significantly enhanced accumulation in the liver than in the uninfected mice (1.626 ± 0.136 vs 0.871 ± 0.086 %ID/g, n = 3, P < 0.05) at 4 h postinjection. In conclusion, our experimental results demonstrate that [64Cu]Cu-NOTA-EK1 is a potential molecular imaging probe for tracking SARS-CoV-2 in extrapulmonary infections in living subjects.

A bivalent protein targeting glycans and HR1 domain in spike protein potently inhibited infection of SARS-CoV-2 and other human coronaviruses.

BACKGROUND: Our previous studies have shown that combining the antiviral lectin GRFT and the pan-CoV fusion inhibitory peptide EK1 results in highly potent inhibitory activity against SARS-CoV-2 infection. In this study, we aimed to design and construct a bivalent protein consisting of GRFT and EK1 components and evaluate its inhibitory activity and mechanism of action against infection by SARS-CoV-2 and its mutants, as well as other human coronaviruses (HCoVs). METHODS: The bivalent proteins were expressed in E. coli and purified with Ni-NTA column. HIV backbone-based pseudovirus (PsV) infection and HCoV S-mediated cell-cell fusion assays were performed to test their inhibitory activity. ELISA and Native-PAGE were conducted to illustrate the mechanism of action of these bivalent proteins. Five-day-old newborn mice were intranasally administrated with a selected bivalent protein before or after HCoV-OC43 challenge, and its protective effect was monitored for 14 days. RESULTS: Among the three bivalent proteins purified, GL25E exhibited the most potent inhibitory activity against infection of SARS-CoV-2 PsVs expressing wild-type and mutated S protein. GL25E was significantly more effective than GRFT and EK1 alone in inhibiting HCoV S-mediated cell-cell fusion, as well as infection by SARS-CoV-2 and other HCoVs, including SARS-CoV, MERS-CoV, HCoV-229E, HCoV-NL63 and HCoV-OC43. GL25E could inhibit authentic SASR-CoV-2, HCoV-OC43 and HCoV-229E infection in vitro and prevent newborn mice from authentic HCoV-OC43 infection in vivo. GL25E could bind to glycans in the S1 subunit and HR1 in the S2 subunit in S protein, showing a mechanism of action similar to that of GRFT and EK1 alone. CONCLUSIONS: Since GL25E showed highly potent and broad-spectrum inhibitory activity against infection of SARS-CoV-2 and its mutants, as well as other HCoVs, it is a promising candidate for further development as a broad-spectrum anti-HCoV therapeutic and prophylactic to treat and prevent COVID-19 and other emerging HCoV diseases.