While research has identified several inhibitors of the main protease (Mpro) of SARS-CoV-2, a significant portion of these compounds exhibit reduced activity in the presence of reducing agents, raising concerns about their effectiveness in vivo. Furthermore, the conventional biosafety level 3 (BSL-3) for cellular assays using viral particles poses a limitation for the widespread evaluation of Mpro inhibitor efficacy in a cell-based assay. Here, we established a BSL-1 compatible cellular assay to evaluate the in vivo potential of Mpro inhibitors. This assay utilizes mammalian cells expressing a tagged Mpro construct containing N-terminal glutathione S-transferase (GST) and C-terminal hemagglutinin (HA) tags and monitors Mpro autodigestion. Using this method, GC376 and boceprevir effectively inhibited Mpro autodigestion, suggesting their potential in vivo activity. Conversely, carmofur and ebselen did not exhibit significant inhibitory effects in this assay. We further investigated the inhibitory potential of selenoneine on Mpro using this approach. Computational analyses of binding energies suggest that noncovalent interactions play a critical role in facilitating the covalent modification of the C145 residue, leading to Mpro inhibition. Our method is straightforward, cost-effective, and readily applicable in standard laboratories, making it accessible to researchers with varying levels of expertise in infectious diseases.

Feline infectious peritonitis (FIP) is a fatal illness caused by a mutated feline coronavirus (FCoV). This disease is characterized by its complexity, resulting from systemic infection, antibody-dependent enhancement (ADE), and challenges in accessing effective therapeutics. Extract derived from Vigna radiata (L.) R. Wilczek (VRE) exhibits various pharmacological effects, including antiviral activity. This study aimed to investigate the antiviral potential of VRE against FCoV, addressing the urgent need to advance the treatment of FIP. We explored the anti-FCoV activity, antiviral mechanism, and combinational application of VRE by means of in vitro antiviral assays. Our findings reveal that VRE effectively inhibited the cytopathic effect induced by FCoV, reduced viral proliferation, and downregulated spike protein expression. Moreover, VRE blocked FCoV in the early and late infection stages and was effective under in vitro ADE infection. Notably, when combined with VRE, the polymerase inhibitor GS-441524 or protease inhibitor GC376 suppressed FCoV more effectively than monotherapy. In conclusion, this study characterizes the antiviral property of VRE against FCoV in vitro, and VRE possesses therapeutic potential for FCoV treatment.

OBJECTIVE: Feline infectious peritonitis is fatal, and due to lack of approved treatments, unregulated antiviral drugs are used to treat this disease. This study set out to determine the purity of various batches of these drugs from several companies, characterize them, and note any impurities or other unusual characteristics. We also developed a method to qualitatively assess the primary components before administration.
METHODS: We tested 30 vials from 17 brands of GS-441524 and 5 vials from 1 brand of GC376. We compared the GS-441524 to a control standard from Ambeed and the GC376 to a standard from Cayman Chemical.
METHODS: We recorded physical appearance, pH, absorbance, HPLC retention times, and thin-layer chromatography retention factors for all of the samples. Some samples were used for nuclear magnetic resonance and mass spectrometric analysis.
RESULTS: Some of the GS-441524 vials were 10% to 25% more concentrated than advertised, but most of the GS-441524 samples tested were similar in purity and composition, both between batches and between brands. We also tested 5 vials of GC376 and found that 1 of the 5 vials contained GS-441524 rather than GC376 and the other 4 vials contained molnupiravir.
CONCLUSIONS: While all of the GS-441524 vials contained GS-441524, none of the GC376 vials tested contained GC376. GC376 is used in cats that are unresponsive to GS-441524, and use of the wrong antiviral can cause serious side effects. We provide suggested methods for distinguishing one drug from the other in new batches.

Infection with coxsackievirus A10 (CV-A10) can cause hand-foot-mouth disease and is also associated with severe complications, including viral pneumonia, aseptic and viral meningitis. Coxsackievirus infection may also play a role in the pathogenesis of acute myocardial infarction and in the increased risk of type 1 diabetes mellitus in adults. However, there are no approved vaccines or direct antiviral agents available to prevention or treatment of coxsackievirus infection. Here, we reported that GC376 potently inhibited CV-A10 infection in different cell lines without cytotoxicity, significantly suppressed production of viral proteins, and strongly reduced the yields of infectious progeny virions. Further study indicated that GC376, as viral 3C protease inhibitor, had the potential to restrain the cleavage of the viral polyprotein into individually functional proteins, thus suppressed the replication of CV-A10. Furthermore, the drug exhibited antiviral activity against coxsackieviruses of various serotypes including CV-A6, CV-A7 and CV-A16, suggesting that GC376 is a broad-spectrum anti-coxsackievirus inhibitor and the 3C protease is a promising target for developing anti-coxsackievirus agents.

FIP is a fatal feline disease caused by FIPV. Two drugs (GS441524 and GC376) target FIPV and have good therapeutic effect when administered by subcutaneous injection. However, subcutaneous injection has limitations compared with oral administration. Additionally, the oral efficacy of the two drugs has not been determined. Here, GS441524 and GC376 were shown to efficiently inhibit FIPV-rQS79 (recombination virus with a full-length field type I FIPV and the spike gene replaced with type II FIPV) and FIPV II (commercially available type II FIPV 79-1146) at a noncytotoxic concentration in CRFK cells. Moreover, the effective oral dose was determined via the in vivo pharmacokinetics of GS441524 and GC376. We conducted animal trials in three dosing groups and found that while GS441524 can effectively reducing the mortality of FIP subjects at a range of doses, GC376 only reducing the mortality rate at high doses. Additionally, compared with GC376, oral GS441524 has better absorption, slower clearance and a slower rate of metabolism. Furthermore, there was no significant difference between the oral and subcutaneous pharmacokinetic parameters. Collectively, our study is the first to evaluate the efficacy of oral GS441524 and GC376 using a relevant animal model. We also verified the reliability of oral GS441524 and the potential of oral GC376 as a reference for rational clinical drug use. Furthermore, the pharmacokinetic data provide insights into and potential directions for the optimization of these drugs.

ABSTRACTThe main protease (3-chymotrypsin-like protease, 3CLpro) of SARS-CoV-2 has become a focus of anti-coronavirus research. Despite efforts, drug development targeting 3CLpro has been hampered by limitations in the currently available activity assays. Additionally, the emergence of 3CLpro mutations in circulating SARS-CoV-2 variants has raised concerns about potential resistance. Both emphasize the need for a more reliable, sensitive, and facile 3CLpro assay. Here, we report an orthogonal dual reporter-based gain-of-signal assay for measuring 3CLpro activity in living cells. It builds on the finding that 3CLpro induces cytotoxicity and reporter expression suppression, which can be rescued by its inhibitor or mutation. This assay circumvents most limitations in previously reported assays, especially false positives caused by nonspecific compounds and signal interference from test compounds. It is also convenient and robust for high throughput screening of compounds and comparing the drug susceptibilities of mutants. Using this assay, we screened 1789 compounds, including natural products and protease inhibitors, with 45 compounds that have been reported to inhibit SARS-CoV-2 3CLpro among them. Except for the approved drug PF-07321332, only five of these inhibit 3CLpro in our assays: GC376; PF-00835231; S-217622; Boceprevir; and Z-FA-FMK. The susceptibilities of seven 3CLpro mutants prevalent in circulating variants to PF-07321332, S-217622, and GC376 were also assessed. Three mutants were identified as being less susceptible to PF-07321322 (P132H) and S-217622 (G15S, T21I). This assay should greatly facilitate the development of novel 3CLpro-targeted drugs and the monitoring of the susceptibility of emerging SARS-CoV-2 variants to 3CLpro inhibitors.

With the spread of SARS-CoV-2 throughout the globe causing the COVID-19 pandemic, the threat of zoonotic transmissions of coronaviruses (CoV) has become even more evident. As human infections have been caused by alpha- and beta-CoVs, structural characterization and inhibitor design mostly focused on these two genera. However, viruses from the delta and gamma genera also infect mammals and pose a potential zoonotic transmission threat. Here, we determined the inhibitor-bound crystal structures of the main protease (Mpro) from the delta-CoV porcine HKU15 and gamma-CoV SW1 from the beluga whale. A comparison with the apo structure of SW1 Mpro, which is also presented here, enabled the identification of structural arrangements upon inhibitor binding at the active site. The cocrystal structures reveal binding modes and interactions of two covalent inhibitors, PF-00835231 (active form of lufotrelvir) bound to HKU15, and GC376 bound to SW1 Mpro. These structures may be leveraged to target diverse coronaviruses and toward the structure-based design of pan-CoV inhibitors.

UNASSIGNED: The main objectives of this study were to investigate the efficacy of the nucleotide analog GS-441524 in combination with the 3C-like protease inhibitor GC376 for the treatment of naturally aquired feline infectious peritonitis (FIP) and to test whether their combination shortens the dosing period and improves the cure rate.
UNASSIGNED: In total, 46 FIP-affected cats were enrolled in this experiment, including 36 with wet FIP (29 with abdominal effusion, six with thoracic effusion, and one with thoracic+abdominal effusion), and 10 with dry FIP. The cats were aged from 3 to 96 months. Thoracic+abdominal effusion, lymph-node puncture fluid and perirenal puncture fluid was collected from the affected cats for qPCR testing, and all 46 cats were positive for feline coronavirus (FCoV). The cats divided into different dose groups, all treated for 4 weeks: group 1 (GS-441524, 5 mg/kg.sc.q.24 h; GC376, 20 mg/kg.sc.q.12 h), group 2 (GS-441524, 2.5 mg/kg.sc.q.24 h; GC376, 20 mg/kg.sc.q.12 h), group 3 (GS-441524, 2.5 mg/kg.sc.q.24 h; GC376, 10 mg/kg.sc.q.12 h), and group 4 (GS-441524, 5 mg/kg.sc.q.24 h; GC376, 10 mg/kg.sc.q.12 h).
UNASSIGNED: After the 4-week combination treatment, 45 of the 46 (97.8%) cats survived, and 43 of those became clinically normal. Two cats required longer (7 to 12 weeks) treatment to achieve full recovery. As of writing (10 months after completion of the trial), all 45 cats were alive and no relapse was observed.
UNASSIGNED: GS-441524 combined with GC376 can be safely and effectively used to treat FIP and reduces the treatment period to 4 weeks, with an excellent cure rate.

Introduction: The development of effective vaccines has partially mitigated the trend of the SARS-CoV-2 pandemic; however, the need for orally administered antiviral drugs persists. This study aims to investigate the activity of molnupiravir in combination with nirmatrelvir or GC376 on SARS-CoV-2 to verify the synergistic effect. Methods: The SARS-CoV-2 strains 20A.EU, BA.1 and BA.2 were used to infect Vero E6 in presence of antiviral compounds alone or in combinations using five two-fold serial dilution of compound concentrations ≤EC90. After 48 and 72 h post-infection, viability was performed using MTT reduction assay. Supernatants were collected for plaque-assay titration. All experiments were performed in triplicate, each being repeated at least three times. The synergistic score was calculated using Synergy Finder version 2. Results: All compounds reached micromolar EC90. Molnupiravir and GC376 showed a synergistic activity at 48 h with an HSA score of 19.33 (p < 0.0001) and an additive activity at 72 h with an HSA score of 8.61 (p < 0.0001). Molnupiravir and nirmatrelvir showed a synergistic activity both at 48 h and 72 h with an HSA score of 14.2 (p = 0.01) and 13.08 (p < 0.0001), respectively. Conclusion: Molnupiravir associated with one of the two protease-inhibitors nirmatrelvir and GC376 showed good additive-synergic activity in vitro.

Coronaviruses infect a variety of hosts in the animal kingdom, and while each virus is taxonomically different, they all infect their host via the same mechanism. The coronavirus main protease (Mpro, also called 3CLpro), is an attractive target for drug development due to its essential role in mediating viral replication and transcription. An Mpro inhibitor, GC376, has been shown to treat feline infectious peritonitis (FIP), a fatal infection in cats caused by internal mutations in the feline enteric coronavirus (FECV). Recently, our lab demonstrated that the feline drug, GC373, and prodrug, GC376, are potent inhibitors of SARS-CoV-2 Mpro and solved the structures in complex with the drugs; however, no crystal structures of the FIP virus (FIPV) Mpro with the feline drugs have been published so far. Here, we present crystal structures of FIPV Mpro-GC373/GC376 complexes, revealing the inhibitors covalently bound to Cys144 in the active site, similar to SARS-CoV-2 Mpro. Additionally, GC376 has a higher affinity for FIPV Mpro with lower nanomolar Ki values compared to SARS-CoV and SARS-CoV-2 Mpro. We also show that improved derivatives of GC376 have higher potency for FIPV Mpro. Since GC373 and GC376 represent strong starting points for structure-guided drug design, determining the crystal structures of FIPV Mpro with these inhibitors are important steps in drug optimization and structure-based broad-spectrum antiviral drug discovery.