SARS-CoV-2 and HCoV-OC43 belong to the same β genus of the Coronaviridae family. SARS-CoV-2 was responsible for the recent COVID-19 pandemic, and HCoV-OC43 is the etiological agent of mild upper respiratory tract infections. SARS-COV-2 and HCoV-OC43 co-infections were found in children with respiratory symptoms during the COVID-19 pandemic. The two β-coronaviruses share a high degree of homology between the 3CLpro active sites, so much so that the safer HCoV-OC43 has been suggested as a tool for the identification of new anti-SARS-COV-2 agents. Compounds 5 and 24 inhibited effectively both Wuhan and British SARS-CoV-2 patient isolates in Vero E6 cells and the HCoV-OC43 in MRC-5 cells at low micromolar concentrations. The inhibition was apparently exerted via targeting the 3CLpro active sites of both viruses. Compounds 5 and 24 at 100 μM inhibited the SARS-CoV-2 3CLpro activity of 61.78 and 67.30%, respectively. These findings highlight 5 and 24 as lead compounds of a novel class of antiviral agents with the potential to treat SARS-COV-2 and HCoV-OC43 infections.

The SARS-CoV-2 coronavirus is characterized by high mutation rates and significant infectivity, posing ongoing challenges for therapeutic intervention. To address potential challenges in the future, the continued development of effective drugs targeting SARS-CoV-2 remains an important task for the scientific as well as the pharmaceutical community. The main protease (Mpro) of SARS-CoV-2 is an ideal therapeutic target for COVID-19 drug development, leading to the introduction of various inhibitors, both covalent and non-covalent, each characterized by unique mechanisms of action and possessing inherent strengths and limitations. Natural products, being compounds naturally present in the environment, offer advantages such as low toxicity and diverse activities, presenting a viable source for antiviral drug development. Here, we identified a natural compound, rosmarinic acid, which exhibits significant inhibitory effects on the Mpro of the SARS-CoV-2. Through detailed structural biology analysis, we elucidated the precise crystal structure of the complex formed between rosmarinic acid and SARS-CoV-2 Mpro, revealing the molecular basis of its inhibitory mechanism. These findings not only enhance our understanding of the antiviral action of rosmarinic acid, but also provide valuable structural information and mechanistic insights for the further development of therapeutic strategies against SARS-CoV-2.

The main protease (Mpro) remains an essential therapeutic target for COVID-19 post infection intervention given its critical role in processing the majority of viral proteins encoded by the genome of severe acute respiratory syndrome related coronavirus 2 (SARS-CoV-2). Upon viral entry, the +ssRNA genome is translated into two long polyproteins (pp1a or the frameshift-dependent pp1ab) containing all the nonstructural proteins (nsps) required by the virus for immune modulation, replication, and ultimately, virion assembly. Included among these nsps is the cysteine protease Mpro (nsp5) which self-excises from the polyprotein, dimerizes, then sequentially cleaves 11 of the 15 cut-site junctions found between each nsp within the polyprotein. Many structures of Mpro (often bound to various small molecule inhibitors or peptides) have been detailed recently, including structures of Mpro bound to each of the polyprotein cleavage sequences, showing that Mpro can accommodate a wide range of targets within its active site. However, to date, kinetic characterization of the interaction of Mpro with each of its native cleavage sequences remains incomplete. Here, we present a robust and cost-effective FRET based system that benefits from a more consistent presentation of the substrate that is also closer in organization to the native polyprotein environment compared to previously reported FRET systems that use chemically modified peptides. Using this system, we were able to show that while each site maintains a similar Michaelis constant, the catalytic efficiency of Mpro varies greatly between cut-site sequences, suggesting a clear preference for the order of nsp processing.

UNASSIGNED: GST-HG171 is a potent, broad-spectrum, orally bioavailable small-molecule 3C like protease inhibitor that has demonstrated greater potency and efficacy compared to Nirmatrelvir in pre-clinical studies. We aimed to evaluate the efficacy and safety of orally administered GST-HG171 plus Ritonavir in patients with coronavirus disease 2019 (COVID-19) infected with emerging XBB and non-XBB variants.
UNASSIGNED: This randomised, double-blind, placebo-controlled phase 2/3 trial was conducted in 47 sites in China among adult patients with mild-to-moderate COVID-19 with symptoms onset ≤72 h. Eligible patients were randomised 1:1 to receive GST-HG171 (150 mg) plus Ritonavir (100 mg) or corresponding placebo tablets twice daily for 5 days, with stratification factors including the risk level of disease progression and vaccination status. The primary efficacy endpoint was time to sustained recovery of clinical symptoms within 28 days, defined as a score of 0 for 11 COVID-19-related target symptoms for 2 consecutive days, assessed in the modified intention-to-treat (mITT) population. This trial was registered at ClinicalTrials.gov (NCT05656443) and Chinese Clinical Trial Registry (ChiCTR2200067088).
UNASSIGNED: Between Dec 19, 2022, and May 4, 2023, 1525 patients were screened. Among 1246 patients who underwent randomisation, most completed basic (21.2%) or booster (74.9%) COVID-19 immunization, and most had a low risk of disease progression at baseline. 610 of 617 who received GST-HG171 plus Ritonavir and 603 of 610 who received placebo were included in the mITT population. Patients who received GST-HG171 plus Ritonavir showed shortened median time to sustained recovery of clinical symptoms compared to the placebo group (13.0 days [95.45% confidence interval 12.0-15.0] vs. 15.0 days [14.0-15.0], P = 0.031). Consistent results were observed in both SARS-CoV-2 XBB (45.7%, 481/1053 of mITT population) and non-XBB variants (54.3%, 572/1053 of mITT population) subgroups. Incidence of adverse events was similar in the GST-HG171 plus Ritonavir (320/617, 51.9%) and placebo group (298/610, 48.9%). The most common adverse events in both placebo and treatment groups were hypertriglyceridaemia (10.0% vs. 14.7%). No deaths occurred.
UNASSIGNED: Treatment with GST-HG171 plus Ritonavir has demonstrated benefits in symptom recovery and viral clearance among low-risk vaccinated adult patients with COVID-19, without apparent safety concerns. As most patients were treated within 2 days after symptom onset in our study, confirming the potential benefits of symptom recovery for patients with a longer duration between symptom onset and treatment initiation will require real-world studies.
UNASSIGNED: Fujian Akeylink Biotechnology Co., Ltd.

Scientists and researchers have been searching for drugs targeting the main protease (Mpro) of SARS-CoV-2, which is crucial for virus replication. This study employed a virtual screening based on molecular docking to identify benzoylguanidines from an in-house chemical library that can inhibit Mpro on the active site and three allosteric sites. Molecular docking was performed on the LaSMMed Chemical Library using 88 benzoylguanidine compounds. Based on their RMSD values and conserved pose, three potential inhibitors (BZG1, BZG2, and BZG3) were selected. These results indicate that BZG1 and BZG3 may bind to the active site, while BZG2 may bind to allosteric sites. Molecular dynamics data suggest that BZG2 selectively targets allosteric site 3. In vitro tests were performed to measure the proteolytic activity of rMpro. The tests showed that BZG2 has uncompetitive inhibitory activity, with an IC50 value of 77 µM. These findings suggest that benzoylguanidines possess potential as Mpro inhibitors and pave the way towards combating SARS-Cov-2 effectively.

The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has presented an enormous challenge to health care systems and medicine. As a result of global research efforts aimed at preventing and effectively treating SARS-CoV-2 infection, vaccines with fundamentally new mechanisms of action and some small-molecule antiviral drugs targeting key proteins in the viral cycle have been developed. The most effective small-molecule drug approved to date for the treatment of COVID-19 is PaxlovidTM, which is a combination of two protease inhibitors, nirmatrelvir and ritonavir. Nirmatrelvir is a reversible covalent peptidomimetic inhibitor of the main protease (Mpro) of SARS-CoV-2, which enzyme plays a crucial role in viral reproduction. In this combination, ritonavir serves as a pharmacokinetic enhancer, it irreversibly inhibits the cytochrome CYP3A4 enzyme responsible for the rapid metabolism of nirmatrelvir, thereby increasing the half-life and bioavailability of nirmatrelvir. In this tutorial review, we summarize the development and pharmaceutical chemistry aspects of Paxlovid, covering the evolution of protease inhibitors, the warhead design, synthesis and the mechanism of action of nirmatrelvir, as well as the synthesis of ritonavir and its CYP3A4 inhibition mechanism. The efficacy of Paxlovid to novel virus mutants is also overviewed.

The ongoing coronavirus infectious disease (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) still urgently requires effective treatments. The 3C-like (3CL) protease of SARS-CoV-2 is a highly conserved cysteine protease that plays an important role in the viral life cycle and host inflammation, providing an ideal target for developing broad-spectrum antiviral drugs. Herein, we describe the discovery of a large number of herbs mainly produced in Heilongjiang Province, China, that exhibited different inhibitory activities against SARS-CoV-2 3CL protease. We confirmed that Syringa reticulata, which is used for clinical treatment of chronic bronchitis and asthma, is a specific and potent inhibitor of 3CL protease. A 70 % ethanol extract of S. reticulata dose-dependently inhibited the cleavage activity of 3CL protease in a fluorescence resonance energy transfer assay with an IC50 value of 0.0018 mg/mL, but had minimal effect in pseudovirus-based cell entry and luciferase-based RNA-dependent RNA polymerase assays. These results suggest that S. reticulata will be a potential leading candidate for COVID-19 treatment.

[This corrects the article DOI: 10.3389/fbioe.2023.1187761.].

SARS-CoV-2 main protease (Mpro) is a validated antiviral drug target of nirmatrelvir, the active ingredient in Pfizer\'s oral drug Paxlovid. Drug-drug interactions limit the use of Paxlovid. In addition, drug-resistant Mpro mutants against nirmatrelvir have been identified from cell culture viral passage and naturally occurring variants. As such, there is a need for a second generation of Mpro inhibitors. In this study, we explored several reactive warheads in the design of Mpro inhibitors. We identified Jun11119R (vinyl sulfonamide warhead), Jun10221R (propiolamide warhead), Jun1112R (4-chlorobut-2-ynamide warhead), Jun10541R (nitrile warhead), and Jun10963R (dually activated nitrile warhead) as potent Mpro inhibitors. Jun10541R and Jun10963R also had potent antiviral activity against SARS-CoV-2 in Calu-3 cells with EC50 values of 2.92 and 6.47 μM, respectively. X-ray crystal structures of Mpro with Jun10541R and Jun10221 revealed covalent modification of Cys145. These Mpro inhibitors with diverse reactive warheads collectively represent promising candidates for further development.

Despite the long history of use and the knowledge of the genetics and biochemistry of E. coli, problems are still possible in obtaining a soluble form of recombinant proteins in this system. Although, soluble protein can be obtained both in the cytoplasm and in the periplasm of the bacterial cell. The latter is a priority strategy for obtaining soluble proteins. The fusion protein technology followed by detachment of the fusion protein with proteases is used to transfer the target protein into the periplasmic space of E. coli. We have continued for the first time to use the main viral protease 3CL of the SARS-CoV-2 virus for this purpose. We obtained a recombinant 3CL protease and studied its complex catalytic properties. The authenticity of the resulting recombinant enzyme, were confirmed by specific activity analysis and activity suppression by the known low-molecular-weight inhibitors. The catalytic efficiency of 3CL (0.17 ± 0.02 µM-1-s-1) was shown to be one order of magnitude higher than that of the widely used tobacco etch virus protease (0.013 ± 0.003 µM-1-s-1). The application of the 3CL gene in genetically engineered constructs provided efficient specific proteolysis of fusion proteins, which we demonstrated using the receptor-binding domain of SARS-CoV-2 spike protein and GST fusion protein. The solubility and immunochemical properties of RBD were preserved. It is very important that in work we have shown that 3CL protease works effectively directly in E. coli cells when co-expressed with the target fusion protein, as well as when expressed as part of a chimeric protein containing the target protein, fusion partner, and 3CL itself. The results obtained in the work allow expanding the repertoire of specific proteases for researchers and biotechnologists.