Why Certain Repurposed Drugs Are Unlikely to Be Effective Antivirals to Treat SARS-CoV-2 Infections.

Most repurposed drugs have proved ineffective for treating COVID-19. We evaluated median effective and toxic concentrations (EC50, CC50) of 49 drugs, mostly from previous clinical trials, in Vero cells. Ratios of reported unbound peak plasma concentrations, (Cmax)/EC50, were used to predict the potential in vivo efficacy. The 20 drugs with the highest ratios were retested in human Calu-3 and Caco-2 cells, and their CC50 was determined in an expanded panel of cell lines. Many of the 20 drugs with the highest ratios were inactive in human Calu-3 and Caco-2 cells. Antivirals effective in controlled clinical trials had unbound Cmax/EC50 ≥ 6.8 in Calu-3 or Caco-2 cells. EC50 of nucleoside analogs were cell dependent. This approach and earlier availability of more relevant cultures could have reduced the number of unwarranted clinical trials.

Synthesis and biological evaluation of novel peptidomimetic inhibitors of the coronavirus 3C-like protease.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and related variants, are responsible for the devastating coronavirus disease 2019 (COVID-19) pandemic. The SARS-CoV-2 main protease (Mpro) plays a central role in the replication of the virus and represents an attractive drug target. Herein, we report the discovery of novel SARS-CoV-2 Mpro covalent inhibitors, including highly effective compound NIP-22c which displays high potency against several key variants and clinically relevant nirmatrelvir Mpro E166V mutants.

Stably-Inverted Apical-Out Human Upper Airway Organoids for SARS-CoV-2 Infection and Therapeutic Testing.

Apical-out organoids produced through eversion triggered by extra-organoid extracellular matrix (ECM) removal or degradation are generally small, structurally variable, and limited for viral infection and therapeutics testing. This work describes ECM-encapsulating, stably-inverted apical-out human upper airway organoids (AORBs) that are large (~500 µm diameter), consistently spherical, recapitulate in vivo-like cellular heterogeneity, and maintain their inverted morphology for over 60 days. Treatment of AORBs with IL-13 skews differentiation towards goblet cells and the apical-out geometry allows extra-organoid mucus collection. AORB maturation for 14 days induces strong co-expression of ACE2 and TMPRSS2 to allow high-yield infection with five SARS-CoV-2 variants. Dose-response analysis of three well-studied SARS-CoV-2 antiviral compounds [remdesivir, bemnifosbuvir (AT-511), and nirmatrelvir] shows AORB antiviral assays to be comparable to gold-standard air-liquid interface cultures, but with higher throughput (~10-fold) and fewer cells (~100-fold). While this work focuses on SARS-CoV-2 applications, the consistent AORB shape and size, and one-organoid-per-well modularity broadly impacts in vitro human cell model standardization efforts in line with economic imperatives and recently updated FDA regulation on therapeutic testing.

In-vitro and in-silico anti-HSV-1 activity of a marine steroid from the jellyfish Cassiopea andromeda venom.

In this study, we investigated the potential in vitro anti-HSV-1 activities of the Cassiopea andromeda jellyfish tentacle extract (TE) and its fractions, as well as computational work on the thymidine kinase (TK) inhibitory activity of the identified secondary metabolites. The LD50, secondary metabolite identification, preparative and analytical chromatography, and in silico TK assessment were performed using the Spearman-Karber, GC-MS, silica gel column chromatography, RP-HPLC, LC-MS, and docking methods, respectively. The antiviral activity of TE and the two purified compounds Ca2 and Ca7 against HSV-1 in Vero cells was evaluated by MTT and RT-PCR assays. The LD50 (IV, mouse) values of TE, Ca2, and Ca7 were 104.0 ± 4, 5120 ± 14, and 197.0 ± 7 (µg/kg), respectively. They exhibited extremely effective antiviral activity against HSV-1. The CC50 and MNTD of TE, Ca2, and Ca7 were (125, 62.5), (25, 12.5), and (50, 3.125) µg/ml, respectively. GC-MS analysis of the tentacle extract revealed seven structurally distinct chemical compositions. Four of the seven compounds had a steroid structure. According to the docking results, all compounds showed binding affinity to the active sites of both thymidine kinase chains. Among them, the steroid compound Pregn-5-ene-3,11-dione, 17,20:20,21 bis [methylenebis(oxy)]-, cyclic 3-(1,2-ethane diyl acetal) (Ca2) exhibited the highest affinity for both enzyme chains, surpassing that of standard acyclovir. In silico data confirmed the experimental results. We conclude that the oxosteroid Ca2 may act as a potent agent against HSV-1.

A comprehensive review of highly pathogenic avian influenza (HPAI) H5N1: An imminent threat at doorstep.

Avian influenza viruses (AIVs) are globally challenging due to widespread circulation and high mortality rates. Highly pathogenic avian influenza (HPAI) strains like H5N1 have caused significant outbreaks in birds. Since 2003 to 14 July 2023, the World Health Organization (WHO) has documented 878 cases of HPAI H5N1 infection in humans and 458 (52.16%) fatalities in 23 countries. Recent outbreaks in wild birds, domestic birds, sea lions, minks, and etc., and the occurrence of genetic variations among HPAI H5N1 strains raise concerns about potential transmission and public health risks. This paper aims to provide a comprehensive overview of the current understanding and new insights into HPAI H5N1. It begins with an introduction to the significance of studying this virus and highlighting the need for updated knowledge. The origin and evaluation of HPAI H5N1 are examined, shedding light on its emergence, and spread across different geographic regions. The genome organization and structural biology of the H5N1 virus are explored, providing insights into its molecular composition and key structural features. This manuscript also delves into the phylogeny, evolution, mutational trends, reservoirs, and transmission routes of HPAI H5N1. The immune response against HPAI H5N1 and its implications for vaccine development are analyzed, along with an exploration of the pathogenesis and clinical manifestations of HPAI H5N1 in human cases. Furthermore, diagnostic tools and preventive and therapeutic strategies are discussed, highlighting the current approaches and potential future directions for better management of the potential pandemic.

Botanical inhibitors of SARS-CoV-2 viral entry: a phylogenetic perspective.

Throughout the SARS-CoV-2 pandemic, the use of botanical dietary supplements in the United States has increased, yet their safety and efficacy against COVID-19 remains underexplored. The Quave Natural Product Library is a phylogenetically diverse collection of botanical and fungal natural product extracts including popular supplement ingredients. Evaluation of 1867 extracts and 18 compounds for virus spike protein binding to host cell ACE2 receptors in a SARS-CoV-2 pseudotyped virus system identified 310 extracts derived from 188 species across 76 families (3 fungi, 73 plants) that exhibited ≥ 50% viral entry inhibition activity at 20 µg/mL. Extracts exhibiting mammalian cytotoxicity > 15% and those containing cardiotoxic cardiac glycosides were eliminated. Three extracts were selected for further testing against four pseudotyped variants and infectious SARS-CoV-2 and were then further chemically characterized, revealing the potent (EC50 < 5 µg/mL) antiviral activity of Solidago altissima L. (Asteraceae) flowers and Pteridium aquilinum (L.) Kuhn (Dennstaedtiaceae) rhizomes.

Oncolytic viruses as emerging therapy against cancers including Oncovirus-induced cancers.

There are several human viruses with known potential for causing cancers including, Hepatitis B virus, Hepatitis C virus, Epstein-Barr virus, Kaposi's sarcoma herpesvirus, Human T-cell lymphotropic virus, Human papillomavirus, and Merkel cell polyomavirus. Cancer is the second leading cause of death that affects humans worldwide, especially in developing countries. Surgery, chemotherapy, and radiotherapy can cure about 60% of humans with cancer but recurrent and metastatic diseases remain a major reason for death. In recent years, understanding the molecular characteristics of cancer cells has led to the improvement of therapeutic strategies using novel emerging therapies. Oncolytic viruses with the potential of lysing cancer cells defined the field of oncolytic virology, hence becoming a biotechnology tool rather than just a cause of disease. This study mainly focused on targeting cell proliferation and death pathways in human tumor-inducing viruses by developing innovative therapies for cancer patients based on the natural oncolytic properties of reovirus. To kill tumor cells efficiently and reduce the chance of recurrence both the direct ability of reovirus infection to lyse the tumor cells and the stimulation of a potent host immune response are applied. Hence, bioengineered stem cells can be used as smart carriers to improve the efficacy of oncolytic reovirus and safety profiles.

Discovery of a 2'-Fluoro,2'-Bromouridine Phosphoramidate Prodrug Exhibiting Anti-Yellow Fever Virus Activity in Culture and in Mice.

Yellow fever virus (YFV) is a potentially lethal, zoonotic, blood-borne flavivirus transmitted to humans and non-human primates by mosquitoes. Owing to multiple deadly epidemics, the WHO classifies YFV as a "high impact, high threat disease" with resurgent epidemic potential. At present, there are no approved antiviral therapies to combat YFV infection. Herein we report on 2'-halogen-modified nucleoside analogs as potential anti-YFV agents. Of 11 compounds evaluated, three showed great promise with low toxicity, high intracellular metabolism into the active nucleoside triphosphate form, and sub-micromolar anti-YFV activity. Notably, we investigated a 2'-fluoro,2'-bromouridine phosphate prodrug (C9), a known anti-HCV agent with good stability in human blood and favorable metabolism. Predictive modeling revealed that C9 could readily bind the active site of the YFV RdRp, conferring its anti-YFV activity. C9 displayed potent anti-YFV activity in primary human macrophages, 3D hepatocyte spheroids, and in mice. In an A129 murine model, shortly after infection, C9 significantly reduced YFV replication and protected against YFV-induced liver inflammation and pathology with no adverse effects. Collectively, this work identifies a potent new anti-YFV agent with strong therapeutic promise.

Synthesis of Novel N4-Hydrocytidine Analogs as Potential Anti-SARS-CoV-2 Agents.

Coronavirus disease 2019 (COVID-19) is an emerging global pandemic with severe morbidity and mortality caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Molnupiravir, an ester prodrug form of N4-hydroxycytidine (NHC), was recently emergency-use approved for the treatment of early SARS-CoV-2 infections. Herein, we report the synthesis and evaluation of a series of novel NHC analogs.

Baricitinib attenuates the proinflammatory phase of COVID-19 driven by lung-infiltrating monocytes.

SARS-CoV-2-infected subjects are generally asymptomatic during initial viral replication but may suffer severe immunopathology after the virus has receded and monocytes have infiltrated the airways. In bronchoalveolar lavage fluid from severe COVID-19 patients, monocytes express mRNA encoding inflammatory mediators and contain SARS-CoV-2 transcripts. We leverage a human small airway model of infection and inflammation, whereby primary blood monocytes transmigrate across SARS-CoV-2-infected lung epithelium to characterize viral burden, gene expression, and inflammatory mediator secretion by epithelial cells and monocytes. In this model, lung-infiltrating monocytes acquire SARS-CoV-2 from the epithelium and upregulate expression and secretion of inflammatory mediators, mirroring in vivo data. Combined use of baricitinib (Janus kinase inhibitor) and remdesivir (nucleoside analog) enhances antiviral signaling and viral clearance by SARS-CoV-2-positive monocytes while decreasing secretion of proneutrophilic mediators associated with acute respiratory distress syndrome. These findings highlight the role of lung-infiltrating monocytes in COVID-19 pathogenesis and their importance as a therapeutic target.

Inactivation of SARS-CoV-2 and COVID-19 Patient Samples for Contemporary Immunology and Metabolomics Studies.

Due to the severity of COVID-19 disease, the U.S. Centers for Disease Control and Prevention and World Health Organization recommend that manipulation of active viral cultures of SARS-CoV-2 and respiratory secretions from COVID-19 patients be performed in biosafety level (BSL)3 laboratories. Therefore, it is imperative to develop viral inactivation procedures that permit samples to be transferred to lower containment levels (BSL2), while maintaining the fidelity of complex downstream assays to expedite the development of medical countermeasures. In this study, we demonstrate optimal conditions for complete viral inactivation following fixation of infected cells with commonly used reagents for flow cytometry, UVC inactivation in sera and respiratory secretions for protein and Ab detection, heat inactivation following cDNA amplification for droplet-based single-cell mRNA sequencing, and extraction with an organic solvent for metabolomic studies. Thus, we provide a suite of viral inactivation protocols for downstream contemporary assays that facilitate sample transfer to BSL2, providing a conceptual framework for rapid initiation of high-fidelity research as the COVID-19 pandemic continues.

GNS561 Exhibits Potent Antiviral Activity against SARS-CoV-2 through Autophagy Inhibition.

Since December 2019, SARS-CoV-2 has spread quickly worldwide, leading to more than 280 million confirmed cases, including over 5,000,000 deaths. Interestingly, coronaviruses were found to subvert and hijack autophagic process to allow their viral replication. Autophagy-modulating compounds thus rapidly emerged as an attractive strategy to fight SARS-CoV-2 infection, including the well-known chloroquine (CQ). Here, we investigated the antiviral activity and associated mechanism of GNS561/Ezurpimtrostat, a small lysosomotropic molecule inhibitor of late-stage autophagy. Interestingly, GNS561 exhibited antiviral activity of 6-40 nM depending on the viral strain considered, currently positioning it as the most powerful molecule investigated in SARS-CoV-2 infection. We then showed that GNS561 was located in lysosome-associated-membrane-protein-2-positive (LAMP2-positive) lysosomes, together with SARS-CoV-2. Moreover, GNS561 increased LC3-II spot size and caused the accumulation of autophagic vacuoles and the presence of multilamellar bodies, suggesting that GNS561 disrupted the autophagy mechanism. To confirm our findings, we used the K18-hACE2 mouse model and highlighted that GNS561 treatment led to a decline in SARS-CoV-2 virions in the lungs associated with a disruption of the autophagy pathway. Overall, our study highlights GNS561 as a powerful drug in the treatment of SARS-CoV-2 infection and supports the hypothesis that autophagy blockers could be an alternative strategy for COVID-19.

Elimination of Aicardi-Goutières syndrome protein SAMHD1 activates cellular innate immunity and suppresses SARS-CoV-2 replication.

The lack of antiviral innate immune responses during severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections is characterized by limited production of interferons (IFNs). One protein associated with Aicardi-Goutières syndrome, SAMHD1, has been shown to negatively regulate the IFN-1 signaling pathway. However, it is unclear whether elevated IFN signaling associated with genetic loss of SAMHD1 would affect SARS-CoV-2 replication. In this study, we established in vitro tissue culture model systems for SARS-CoV-2 and human coronavirus OC43 infections in which SAMHD1 protein expression was absent as a result of CRISPR-Cas9 gene KO or lentiviral viral protein X-mediated proteosomal degradation. We show that both SARS-CoV-2 and human coronavirus OC43 replications were suppressed in SAMHD1 KO 293T and differentiated THP-1 macrophage cell lines. Similarly, when SAMHD1 was degraded by virus-like particles in primary monocyte-derived macrophages, we observed lower levels of SARS-CoV-2 RNA. The loss of SAMHD1 in 293T and differentiated THP-1 cells resulted in upregulated gene expression of IFNs and innate immunity signaling proteins from several pathways, with STAT1 mRNA being the most prominently elevated ones. Furthermore, SARS-CoV-2 replication was significantly increased in both SAMHD1 WT and KO cells when expression and phosphorylation of STAT1 were downregulated by JAK inhibitor baricitinib, which over-rode the activated antiviral innate immunity in the KO cells. This further validates baricitinib as a treatment of SARS-CoV-2-infected patients primarily at the postviral clearance stage. Overall, our tissue culture model systems demonstrated that the elevated innate immune response and IFN activation upon genetic loss of SAMHD1 effectively suppresses SARS-CoV-2 replication.

Comparison of anti-SARS-CoV-2 activity and intracellular metabolism of remdesivir and its parent nucleoside.

Remdesivir, a monophosphate prodrug of nucleoside analog GS-441524, is widely used for the treatment of moderate to severe COVID-19. It has been suggested to use GS-441524 instead of remdesivir in the clinic and in new inhalation formulations. Thus, we compared the anti-SARS-CoV-2 activity of remdesivir and GS-441524 in Vero E6, Vero CCL-81, Calu-3, Caco-2 â€‹cells, and anti-HCoV-OC43 activity in Huh-7 â€‹cells. We also compared the cellular pharmacology of these two compounds in Vero E6, Vero CCL-81, Calu-3, Caco-2, Huh-7, 293T, BHK-21, 3T3 and human airway epithelial (HAE) cells. Overall, remdesivir exhibited greater potency and superior intracellular metabolism than GS-441524 except in Vero E6 and Vero CCL-81 â€‹cells.

Baicalein and Baicalin Inhibit SARS-CoV-2 RNA-Dependent-RNA Polymerase.

Coronavirus Disease 2019 (COVID-19) is a deadly emerging infectious disease caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Because SARS-CoV-2 is easily transmitted through the air and has a relatively long incubation time, COVID-19 has rapidly developed into a global pandemic. As there are no antiviral agents for the prevention and treatment of this severe pathogen except for remdesivir, development of antiviral therapies to treat infected individuals remains highly urgent. Here, we showed that baicalein and baicalin exhibited significant antiviral activity against SARS-CoV-2, the causative agent of COVID-19 through in vitro studies. Our data through cell-based and biochemical studies showed that both compounds act as SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) inhibitors directly and inhibit the activity of the SARS-CoV-2 RdRp, but baicalein was more potent. We also showed specific binding of baicalein to the SARS-CoV-2 RdRp, making it a potential candidate for further studies towards therapeutic development for COVID-19 as a selective non-nucleoside polymerase inhibitor.

Arthritogenic alphaviruses: epidemiological and clinical perspective on emerging arboviruses.

Mosquito-borne viruses, or arboviruses, have been part of the infectious disease landscape for centuries, and are often, but not exclusively, endemic to equatorial and subtropical regions of the world. The past two decades saw the re-emergence of arthritogenic alphaviruses, a genus of arboviruses that includes several members that cause severe arthritic disease. Recent outbreaks further highlight the substantial public health burden caused by these viruses. Arthritogenic alphaviruses are often reported in the context of focused outbreaks in specific regions (eg, Caribbean, southeast Asia, and Indian Ocean) and cause debilitating acute disease that can extend to chronic manifestations for years after infection. These viruses are classified among several antigenic complexes, span a range of hosts and mosquito vectors, and can be distributed along specific geographical locations. In this Review, we highlight key features of alphaviruses that are known to cause arthritic disease in humans and outline the present findings pertaining to classification, immunogenicity, pathogenesis, and experimental approaches aimed at limiting disease manifestations. Although the most prominent alphavirus outbreaks in the past 15 years featured chikungunya virus, and a large body of work has been dedicated to understanding chikungunya disease mechanisms, this Review will instead focus on other arthritogenic alphaviruses that have been identified globally and provide a comprehensive appraisal of present and future research directions.

Baricitinib treatment resolves lower-airway macrophage inflammation and neutrophil recruitment in SARS-CoV-2-infected rhesus macaques.

SARS-CoV-2-induced hypercytokinemia and inflammation are critically associated with COVID-19 severity. Baricitinib, a clinically approved JAK1/JAK2 inhibitor, is currently being investigated in COVID-19 clinical trials. Here, we investigated the immunologic and virologic efficacy of baricitinib in a rhesus macaque model of SARS-CoV-2 infection. Viral shedding measured from nasal and throat swabs, bronchoalveolar lavages, and tissues was not reduced with baricitinib. Type I interferon (IFN) antiviral responses and SARS-CoV-2-specific T cell responses remained similar between the two groups. Animals treated with baricitinib showed reduced inflammation, decreased lung infiltration of inflammatory cells, reduced NETosis activity, and more limited lung pathology. Importantly, baricitinib-treated animals had a rapid and remarkably potent suppression of lung macrophage production of cytokines and chemokines responsible for inflammation and neutrophil recruitment. These data support a beneficial role for, and elucidate the immunological mechanisms underlying, the use of baricitinib as a frontline treatment for inflammation induced by SARS-CoV-2 infection.

A reverse transcription loop-mediated isothermal amplification for broad coverage detection of Asian and African Zika virus lineages.

BACKGROUND: Early detection of Zika virus (ZIKV) infection during the viremia and viruria facilitates proper patient management and mosquito control measurement to prevent disease spread. Therefore, a cost-effective nucleic acid detection method for the diagnosis of ZIKV infection, especially in resource-deficient settings, is highly required. METHODS: In the present study, a single-tube reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the detection of both the Asian and African-lineage ZIKV. The detection limit, strain coverage and cross-reactivity of the ZIKV RT-LAMP assay was evaluated. The sensitivity and specificity of the RT-LAMP were also evaluated using a total of 24 simulated clinical samples. The ZIKV quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay was used as the reference assay. RESULTS: The detection limit of the RT-LAMP assay was 3.73 ZIKV RNA copies (probit analysis, P ≤ 0.05). The RT-LAMP assay detected the ZIKV genomes of both the Asian and African lineages without cross-reacting with other arthropod-borne viruses. The sensitivity and specificity of the RT-LAMP assay were 90% (95% CI = 59.6-98.2) and 100% (95% CI = 78.5-100.0), respectively. The RT-LAMP assay detected ZIKV genome in 9 of 24 (37.5%) of the simulated clinical samples compared to 10 of 24 (41.7%) by qRT-PCR assay with a high level of concordance (κ = 0.913, P < 0.001). CONCLUSION: The RT-LAMP assay is applicable for the broad coverage detection of both the Asian and African ZIKV strains in resource-deficient settings.

Repurposing Nucleoside Analogs for Human Coronaviruses.

Coronavirus disease 2019 (COVID-19) is a serious illness caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2 or CoV-2). Some reports claimed certain nucleoside analogs to be active against CoV-2 and thus needed confirmation. Here, we evaluated a panel of compounds and identified novel nucleoside analogs with antiviral activity against CoV-2 and HCoV-OC43 while ruling out others. Of significance, sofosbuvir demonstrated no antiviral effect against CoV-2, and its triphosphate did not inhibit CoV-2 RNA polymerase.

GNS561 exhibits potent in vitro antiviral activity against SARS-CoV-2 through autophagy inhibition.

Since December 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2/2019-nCoV) has spread quickly worldwide, with more than 29 million cases and 920,000 deaths. Interestingly, coronaviruses were found to subvert and hijack the autophagic process to allow their viral replication. One of the spotlights had been focused on the autophagy inhibitors as a target mechanism effective in the inhibition of SARS-CoV-2 infection. Consequently, chloroquine (CQ) and hydroxychloroquine (HCQ), a derivative of CQ, was suggested as the first potentially be therapeutic strategies as they are known to be autophagy inhibitors. Then, they were used as therapeutics in SARS-CoV-2 infection along with remdesivir, for which the FDA approved emergency use authorization. Here, we investigated the antiviral activity and associated mechanism of GNS561, a small basic lipophilic molecule inhibitor of late-stage autophagy, against SARS-CoV-2. Our data indicated that GNS561 showed the highest antiviral effect for two SARS-CoV-2 strains compared to CQ and remdesivir. Focusing on the autophagy mechanism, we showed that GNS561, located in LAMP2-positive lysosomes, together with SARS-CoV-2, blocked autophagy by increasing the size of LC3-II spots and the accumulation of autophagic vacuoles in the cytoplasm with the presence of multilamellar bodies characteristic of a complexed autophagy. Finally, our study revealed that the combination of GNS561 and remdesivir was associated with a strong synergistic antiviral effect against SARS-CoV-2. Overall, our study highlights GNS561 as a powerful drug in SARS-CoV-2 infection and supports that the hypothesis that autophagy inhibitors could be an alternative strategy for SARS-CoV-2 infection.