A universal fluorescence polarization high throughput screening assay to target the SAM-binding sites of SARS-CoV-2 and other viral methyltransferases.

SARS-CoV-2 has caused a global pandemic with significant humanity and economic loss since 2020. Currently, only limited options are available to treat SARS-CoV-2 infections for vulnerable populations. In this study, we report a universal fluorescence polarization (FP)-based high throughput screening (HTS) assay for SAM-dependent viral methyltransferases (MTases), using a fluorescent SAM-analogue, FL-NAH. We performed the assay against a reference MTase, NSP14, an essential enzyme for SARS-CoV-2 to methylate the N7 position of viral 5'-RNA guanine cap. The assay is universal and suitable for any SAM-dependent viral MTases such as the SARS-CoV-2 NSP16/NSP10 MTase complex and the NS5 MTase of Zika virus (ZIKV). Pilot screening demonstrated that the HTS assay was very robust and identified two candidate inhibitors, NSC 111552 and 288387. The two compounds inhibited the FL-NAH binding to the NSP14 MTase with low micromolar IC50. We used three functional MTase assays to unambiguously verified the inhibitory potency of these molecules for the NSP14 N7-MTase function. Binding studies indicated that these molecules are bound directly to the NSP14 MTase with similar low micromolar affinity. Moreover, we further demonstrated that these molecules significantly inhibited the SARS-CoV-2 replication in cell-based assays at concentrations not causing cytotoxicity. Furthermore, NSC111552 significantly synergized with known SARS-CoV-2 drugs including nirmatrelvir and remdesivir. Finally, docking suggested that these molecules bind specifically to the SAM-binding site on the NSP14 MTase. Overall, these molecules represent novel and promising candidates to further develop broad-spectrum inhibitors for the management of viral infections.

Therapeutic potential of salicylamide derivatives for combating viral infections.

Since time immemorial human beings have constantly been fighting against viral infections. The ongoing and devastating coronavirus disease 2019 pandemic represents one of the most severe and most significant public health emergencies in human history, highlighting an urgent need to develop broad-spectrum antiviral agents. Salicylamide (2-hydroxybenzamide) derivatives, represented by niclosamide and nitazoxanide, inhibit the replication of a broad range of RNA and DNA viruses such as flavivirus, influenza A virus, and coronavirus. Moreover, nitazoxanide was effective in clinical trials against different viral infections including diarrhea caused by rotavirus and norovirus, uncomplicated influenza A and B, hepatitis B, and hepatitis C. In this review, we summarize the broad antiviral activities of salicylamide derivatives, the clinical progress, and the potential targets or mechanisms against different viral infections and highlight their therapeutic potential in combating the circulating and emerging viral infections in the future.

Editorial of Special Column on Antiviral Drug Discovery and Pharmacology

The COVID-19 pandemic is an overdue reminder of the urgent need for antivirals. Development of antiviral drugs has been historically neglected compared to those for other diseases, including cancer, neurodegeneration diseases, inflammatory diseases and others. Retrospectively, if the 2003 SARS-CoV-1 had raised the alarm of the need for vaccines and antivirals against coronaviruses, we would have been in a much better position fighting against the SARS-CoV-2.

Allosteric inhibitors of the main protease of SARS-CoV-2.

SARS-CoV-2 has raised the alarm to search for effective therapy for this virus. To date several vaccines have been approved but few available drugs reported recently still need approval from FDA. Remdesivir was approved for emergency use only. In this report, the SARS-CoV-2 3CLpro was expressed and purified. By using a FRET-based enzymatic assay, we have screened a library consisting of more than 300 different niclosamide derivatives and identified three molecules JMX0286, JMX0301, and JMX0941 as potent allosteric inhibitors against SARS-CoV-2 3CLpro, with IC50 values similar to that of known covalent inhibitor boceprevir. In a cell-based antiviral assay, these inhibitors can inhibit the virus growth with EC50 in the range of 2-3 µM. The mechanism of action of JMX0286, JMX0301, and JMX0941 were characterized by enzyme kinetics, affinity binding and protein-based substrate digestion. Molecular docking, molecular dynamics (MD) simulations and hydration studies suggested that JMX0286, JMX0301, JMX0941 bind specifically to an allosteric pocket of the SARS-CoV-2 3CL protease. This study provides three potent compounds for further studies.

Inteins as Drug Targets and Therapeutic Tools.

Multidrug-resistant pathogens are of significant concern in recent years. Hence new antifungal and anti-bacterial drug targets are urgently needed before the situation goes beyond control. Inteins are polypeptides that self-splice from exteins without the need for cofactors or external energy, resulting in joining of extein fragments. Inteins are present in many organisms, including human pathogens such as Mycobacterium tuberculosis, Cryptococcus neoformans, C. gattii, and Aspergillus fumigatus. Because intein elements are not present in human genes, they are attractive drug targets to develop antifungals and antibiotics. Thus far, a few inhibitors of intein splicing have been reported. Metal-ions such as Zn2+ and Cu2+, and platinum-containing compound cisplatin inhibit intein splicing in M. tuberculosis and C. neoformans by binding to the active site cysteines. A small-molecule inhibitor 6G-318S and its derivative 6G-319S are found to inhibit intein splicing in C. neoformans and C. gattii with a MIC in nanomolar concentrations. Inteins have also been used in many other applications. Intein can be used in activating a protein inside a cell using small molecules. Moreover, split intein can be used to deliver large genes in experimental gene therapy and to kill selected species in a mixed population of microbes by taking advantage of the toxin-antitoxin system. Furthermore, split inteins are used in synthesizing cyclic peptides and in developing cell culture model to study infectious viruses including SARS-CoV-2 in the biosafety level (BSL) 2 facility. This mini-review discusses the recent research developments of inteins in drug discovery and therapeutic research.

Inteins as Drug Targets and Therapeutic Tools

Multidrug-resistant pathogens are of significant concern in recent years. Hence new antifungal and anti-bacterial drug targets are urgently needed before the situation goes beyond control. Inteins are polypeptides that self-splice from exteins without the need for cofactors or external energy, resulting in joining of extein fragments. Inteins are present in many organisms, including human pathogens such as Mycobacterium tuberculosis, Cryptococcus neoformans, C. gattii, and Aspergillus fumigatus. Because intein elements are not present in human genes, they are attractive drug targets to develop antifungals and antibiotics. Thus far, a few inhibitors of intein splicing have been reported. Metal-ions such as Zn2+ and Cu2+, and platinum-containing compound cisplatin inhibit intein splicing in M. tuberculosis and C. neoformans by binding to the active site cysteines. A small-molecule inhibitor 6G-318S and its derivative 6G-319S are found to inhibit intein splicing in C. neoformans and C. gattii with a MIC in nanomolar concentrations. Inteins have also been used in many other applications. Intein can be used in activating a protein inside a cell using small molecules. Moreover, split intein can be used to deliver large genes in experimental gene therapy and to kill selected species in a mixed population of microbes by taking advantage of the toxin-antitoxin system. Furthermore, split inteins are used in synthesizing cyclic peptides and in developing cell culture model to study infectious viruses including SARS-CoV-2 in the biosafety level (BSL) 2 facility. This mini-review discusses the recent research developments of inteins in drug discovery and therapeutic research.

A cross-sectional study of the epidemic situation on COVID-19 in Gansu Province, China - a big data analysis of the national health information platform.

BACKGROUND: In December 2019, a pneumonia caused by SARS-CoV-2 emerged in Wuhan, China and has rapidly spread around the world since then. This study is to explore the patient characteristics and transmission chains of COVID-19 in the population of Gansu province, and support decision-making. METHODS: We collected data from Gansu Province National Health Information Platform. A cross-sectional study was conducted, including patients with COVID-19 confirmed between January 23 and February 6, 2020, and analyzed the gender and age of the patients. We also described the incubation period, consultation time and sources of infection in the cases, and calculated the secondary cases that occurred within Gansu for each imported case. RESULTS: We found thirty-six (53.7%) of the patients were women and thirty-one (46.3%) men, and the median ages were 40 (IQR 31-53) years. Twenty-eight (41.8%) of the 67 cases had a history of direct exposure in Wuhan. Twenty-five (52.2%) cases came from ten families, and we found no clear reports of modes of transmission other than family clusters. The largest number of secondary cases linked to a single source was nine. CONCLUSION: More women than men were diagnosed with COVID-19 in Gansu Province. Although the age range of confirmed cases of COVID-19 in Gansu Province covered almost all age groups, most patients with confirmed COVID-19 tend to be middle aged persons. The most common suspected mode of transmission was through family cluster. Gansu and other settings worldwide should continue to strengthen the utilization of big data in epidemic control.

Drug repurposing approach to combating coronavirus: Potential drugs and drug targets.

In the past two decades, three highly pathogenic human coronaviruses severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus, and, recently, SARS-CoV-2, have caused pandemics of severe acute respiratory diseases with alarming morbidity and mortality. Due to the lack of specific anti-CoV therapies, the ongoing pandemic of coronavirus disease 2019 (COVID-19) poses a great challenge to clinical management and highlights an urgent need for effective interventions. Drug repurposing is a rapid and feasible strategy to identify effective drugs for combating this deadly infection. In this review, we summarize the therapeutic CoV targets, focus on the existing small molecule drugs that have the potential to be repurposed for existing and emerging CoV infections of the future, and discuss the clinical progress of developing small molecule drugs for COVID-19.

The nucleocapsid protein of zoonotic betacoronaviruses is an attractive target for antiviral drug discovery.

Betacoronaviruses are in one genera of coronaviruses including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome-related coronavirus (MERS-CoV), etc. These viruses threaten public health and cause dramatic economic losses. The nucleocapsid (N) protein is a structural protein of betacoronaviruses with multiple functions such as forming viral capsids with viral RNA, interacting with viral membrane protein to form the virus core with RNA, binding to several cellular kinases for signal transductions, etc. In this review, we highlighted the potential of the N protein as a suitable antiviral target from different perspectives, including structure, functions, and antiviral strategies for combatting betacoronaviruses.

Targeting Crucial Host Factors of SARS-CoV-2.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has spread worldwide since its first incidence in Wuhan, China, in December 2019. Although the case fatality rate of COVID-19 appears to be lower than that of SARS and Middle East respiratory syndrome (MERS), the higher transmissibility of SARS-CoV-2 has caused the total fatality to surpass other viral diseases, reaching more than 1 million globally as of October 6, 2020. The rate at which the disease is spreading calls for a therapy that is useful for treating a large population. Multiple intersecting viral and host factor targets involved in the life cycle of the virus are being explored. Because of the frequent mutations, many coronaviruses gain zoonotic potential, which is dependent on the presence of cell receptors and proteases, and therefore the targeting of the viral proteins has some drawbacks, as strain-specific drug resistance can occur. Moreover, the limited number of proteins in a virus makes the number of available targets small. Although SARS-CoV and SARS-CoV-2 share common mechanisms of entry and replication, there are substantial differences in viral proteins such as the spike (S) protein. In contrast, targeting cellular factors may result in a broader range of therapies, reducing the chances of developing drug resistance. In this Review, we discuss the role of primary host factors such as the cell receptor angiotensin-converting enzyme 2 (ACE2), cellular proteases of S protein priming, post-translational modifiers, kinases, inflammatory cells, and their pharmacological intervention in the infection of SARS-CoV-2 and related viruses.

Prospect of SARS-CoV-2 spike protein: Potential role in vaccine and therapeutic development.

The recent outbreak of the betacoronavirus SARS-CoV-2 has become a significant concern to public health care worldwide. As of August 19, 2020, more than 22,140,472 people are infected, and over 781,135 people have died due to this deadly virus. In the USA alone, over 5,482,602 people are currently infected, and more than 171,823 people have died. SARS-CoV-2 has shown a higher infectivity rate and a more extended incubation period as compared to previous coronaviruses. SARS-CoV-2 binds much more strongly than SARS-CoV to the same host receptor, angiotensin-converting enzyme 2 (ACE2). Previously, several methods to develop a vaccine against SARS-CoV or MERS-CoV have been tried with limited success. Since SARS-CoV-2 uses the spike (S) protein for entry to the host cell, it is one of the most preferred targets for making vaccines or therapeutics against SARS-CoV-2. In this review, we have summarised the characteristics of the S protein, as well as the different approaches being used for the development of vaccines and/or therapeutics based on the S protein.

Broad Spectrum Antiviral Agent Niclosamide and Its Therapeutic Potential.

The recent outbreak of coronavirus disease 2019 (COVID-19) highlights an urgent need for therapeutics. Through a series of drug repurposing screening campaigns, niclosamide, an FDA-approved anthelminthic drug, was found to be effective against various viral infections with nanomolar to micromolar potency such as SARS-CoV, MERS-CoV, ZIKV, HCV, and human adenovirus, indicating its potential as an antiviral agent. In this brief review, we summarize the broad antiviral activity of niclosamide and highlight its potential clinical use in the treatment of COVID-19.