Efficacy and Safety of Ensitrelvir for Asymptomatic or Mild COVID-19: An Exploratory Analysis of a Multicenter, Randomized, Phase 2b/3 Clinical Trial.

BACKGROUND: This phase 2b/3, randomized, placebo-controlled trial explored the efficacy and evaluated the safety of ensitrelvir. This trial involved individuals with asymptomatic infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and patients with mild symptoms of coronavirus disease 2019 (COVID-19). METHODS: The trial was conducted at 57 medical institutions in Japan, South Korea, and Vietnam (study period: January 6-August 14, 2022). Eligible participants were randomized (1:1:1) to the ensitrelvir 125-mg, ensitrelvir 250-mg, or placebo group, received the allocated intervention orally, and were followed up until Day 28. Participants self-rated the severity of 14 typical COVID-19 symptoms and recorded the data in an electronic diary. RESULTS: In total, 572 participants (194, 189, and 189 in the ensitrelvir 125-mg, ensitrelvir 250-mg, and placebo groups, respectively) were included in the intention-to-treat population. Ensitrelvir 125-mg group observed a 77% reduction in the risk of developing any of the 14 COVID-19 symptoms or fever and a 29% reduction in the risk of worsening of such symptoms or fever versus placebo (statistically nonsignificant). The viral RNA, viral titer, and time to infectious viral clearance observed a statistically significant decrease versus placebo. Most treatment-related adverse events (TEAEs) were mild to moderate in severity, and the most common TEAE observed across groups was a decrease in high-density lipoprotein. CONCLUSIONS: Our exploratory results suggest a potential reduction in the risk of development or worsening of COVID-19 symptoms with ensitrelvir. Ensitrelvir showed antiviral efficacy and was well tolerated. TRIAL REGISTRATION: Japan Registry of Clinical Trials identifier: jRCT2031210350.

Efficacy and Safety of 5-Day Oral Ensitrelvir for Patients With Mild to Moderate COVID-19: The SCORPIO-SR Randomized Clinical Trial.

Importance: Treatment options for COVID-19 are warranted irrespective of the presence of risk factors for severe disease. Objective: To assess the efficacy and safety of ensitrelvir in patients with mild to moderate COVID-19. Design, Setting, and Participants: This phase 3 part of a phase 2/3, double-blind, placebo-controlled randomized clinical trial was conducted from February 10 to July 10, 2022, with a 28-day follow-up period, at 92 institutions in Japan, Vietnam, and South Korea. Patients (aged 12 to <70 years) with mild to moderate COVID-19 within 120 hours of positive viral test results were studied. Interventions: Patients were randomized (1:1:1) to receive 125 mg of once-daily ensitrelvir (375 mg on day 1), 250 mg of once-daily ensitrelvir (750 mg on day 1), or placebo for 5 days. Main Outcomes and Measures: The primary end point was the time to resolution of the composite of 5 characteristic symptoms of SARS-CoV-2 Omicron infection, assessed using a Peto-Prentice generalized Wilcoxon test stratified by vaccination history. Virologic efficacy and safety were also assessed. Results: A total of 1821 patients were randomized, of whom 1030 (347 in the 125-mg ensitrelvir group, 340 in the 250-mg ensitrelvir group, and 343 in the placebo group) were randomized in less than 72 hours of disease onset (primary analysis population). The mean (SD) age in this population was 35.2 (12.3) years, and 552 (53.6%) were men. A significant difference was observed between the 125-mg ensitrelvir group and the placebo group (P = .04 with a Peto-Prentice generalized Wilcoxon test). The difference in median time was approximately 1 day between the 125-mg ensitrelvir group and the placebo group (167.9 vs 192.2 hours; difference, -24.3 hours; 95% CI, -78.7 to 11.7 hours). Adverse events were observed in 267 of 604 patients (44.2%) in the 125-mg ensitrelvir group, 321 of 599 patients (53.6%) in the 250-mg ensitrelvir group, and 150 of 605 patients (24.8%) in the placebo group, which included a decrease in high-density lipoprotein level (188 [31.1%] in the 125-mg ensitrelvir group, 231 [38.6%] in the 250-mg ensitrelvir group, and 23 [3.8%] in the placebo group). No treatment-related serious adverse events were reported. Conclusions and Relevance: In this randomized clinical trial, 125-mg ensitrelvir treatment reduced the time to resolution of the 5 typical COVID-19 symptoms compared with placebo in patients treated in less than 72 hours of disease onset; the absolute difference in median time to resolution was approximately 1 day. Ensitrelvir demonstrated clinical and antiviral efficacy without new safety concerns. Generalizability to populations outside Asia should be confirmed. Trial Registration: Japan Registry of Clinical Trials Identifier: jRCT2031210350.

A phase 2/3 study of S-217622 in participants with SARS-CoV-2 infection (Phase 3 part).

BACKGROUND: Limited treatment options exist for patients with mild-to-moderate coronavirus disease 2019 (COVID-19), irrespective of vaccination history or risk status. Ensitrelvir is a novel oral severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 3C-like (3CL) protease inhibitor. While phase 2 studies of ensitrelvir have demonstrated promising results in treating mild-to-moderate COVID-19, evaluation of its clinical efficacy due to shifting vaccination status and emergence of the Omicron variant represents significant challenges. Here, we describe the protocol for a phase 3 study designed to evaluate the efficacy and safety of ensitrelvir in patients with mild-to-moderate COVID-19, regardless of risk status or vaccination history. METHODS: This is a multicenter, randomized, double-blind, placebo-controlled, phase 3 study. Patients with mild-to-moderate COVID-19 within 120 hours from onset will be randomized in a 1:1:1 ratio into 3 treatment arms-ensitrelvir 125 mg (375 mg loading dose on Day 1), ensitrelvir 250 mg (750 mg loading dose on Day 1), and placebo. The study interventions will be administered orally, once-daily, for 5 days. The primary endpoint will be the time to resolution of 5 symptoms of COVID-19 (stuffy or runny nose, sore throat, cough, feeling hot or feverish, and low energy or tiredness), and the key secondary endpoints will include the change from baseline on Day 4 in the amount of SARS-CoV-2 viral ribonucleic acid (RNA) and the time to first negative SARS-CoV-2 viral titer. The primary population for the primary and key secondary endpoints will be patients with <72 hours from COVID-19 onset to randomization and, subsequently, patients in entire patient population (<120 hours) in the ensitrelvir 125 mg group. Closed testing procedure will be used for the primary and key secondary endpoints in both the primary and entire patient populations. All safety assessments and adverse events (AE) will be reported. DISCUSSION: In a post hoc analysis of the phase 2b study, compared with placebo, ensitrelvir demonstrated a reduced time to resolution of 5 symptoms in patients with mild-to-moderate COVID-19. Through this study, we intend to validate and establish the efficacy and safety of ensitrelvir in patients with mild-to-moderate COVID-19.

Efficacy and Safety of Ensitrelvir in Patients With Mild-to-Moderate Coronavirus Disease 2019: The Phase 2b Part of a Randomized, Placebo-Controlled, Phase 2/3 Study.

BACKGROUND: This phase 2b part of a randomized phase 2/3 study assessed the efficacy and safety of ensitrelvir for mild-to-moderate coronavirus disease 2019 (COVID-19) during the Omicron epidemic. METHODS: Patients were randomized (1:1:1) to orally receive ensitrelvir fumaric acid 125 mg (375 mg on day 1) or 250 mg (750 mg on day 1) or placebo once daily for 5 days. The co-primary endpoints were the change from baseline in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) titer on day 4 and time-weighted average change from baseline up to 120 hours in the total score of predefined 12 COVID-19 symptoms. Safety was assessed through adverse events. RESULTS: A total of 341 patients (ensitrelvir 125-mg group: 114; ensitrelvir 250-mg group: 116; and placebo group: 111; male: 53.5-64.9%; mean age: 35.3-37.3 years) were included in the efficacy analyses. The change from baseline in SARS-CoV-2 titer on day 4 was significantly greater with both ensitrelvir doses than with placebo (differences from placebo: -0.41 log10 50% tissue-culture infectious dose/mL; P < .0001 for both). The total score of the 12 COVID-19 symptoms did not show a significant difference between the ensitrelvir groups and placebo group. The time-weighted average change from baseline up to 120 hours was significantly greater with ensitrelvir versus placebo in several subtotal scores, including acute symptoms and respiratory symptoms. Most adverse events were mild in severity. CONCLUSIONS: Ensitrelvir treatment demonstrated a favorable antiviral efficacy and potential clinical benefit with an acceptable safety profile. CLINICAL TRIALS REGISTRATION: Japan Registry of Clinical Trials: jRCT2031210350 (https://jrct.niph.go.jp/en-latest-detail/jRCT2031210350).

S-217622, a SARS-CoV-2 main protease inhibitor, decreases viral load and ameliorates COVID-19 severity in hamsters.

In parallel with vaccination, oral antiviral agents are highly anticipated to act as countermeasures for the treatment of the coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Oral antiviral medication demands not only high antiviral activity but also target specificity, favorable oral bioavailability, and high metabolic stability. Although a large number of compounds have been identified as potential inhibitors of SARS-CoV-2 infection in vitro, few have proven to be effective in vivo. Here, we show that oral administration of S-217622 (ensitrelvir), an inhibitor of SARS-CoV-2 main protease (Mpro; also known as 3C-like protease), decreases viral load and ameliorates disease severity in SARS-CoV-2-infected hamsters. S-217622 inhibited viral proliferation at low nanomolar to submicromolar concentrations in cells. Oral administration of S-217622 demonstrated favorable pharmacokinetic properties and accelerated recovery from acute SARS-CoV-2 infection in hamster recipients. Moreover, S-217622 exerted antiviral activity against SARS-CoV-2 variants of concern, including the highly pathogenic Delta variant and the recently emerged Omicron BA.5 and BA.2.75 variants. Overall, our study provides evidence that S-217622, an antiviral agent that is under evaluation in a phase 3 clinical trial (clinical trial registration no. jRCT2031210350), has remarkable antiviral potency and efficacy against SARS-CoV-2 and is a prospective oral therapeutic option for COVID-19.

A Randomized Phase 2/3 Study of Ensitrelvir, a Novel Oral SARS-CoV-2 3C-Like Protease Inhibitor, in Japanese Patients with Mild-to-Moderate COVID-19 or Asymptomatic SARS-CoV-2 Infection: Results of the Phase 2a Part.

This multicenter, double-blind, phase 2a part of a phase 2/3 study assessed the efficacy and safety of ensitrelvir, a novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 3C-like protease inhibitor, in Japanese patients with mild-to-moderate coronavirus disease 2019 (COVID-19) or asymptomatic SARS-CoV-2 infection. Sixty-nine patients were randomized (1:1:1) to orally receive 5-day ensitrelvir fumaric acid (375 mg on day 1 followed by 125 mg daily, or 750 mg on day 1 followed by 250 mg daily) or placebo and followed up until day 28. The primary outcome was the change from baseline in the SARS-CoV-2 viral titer. A total of 16, 14, and 17 patients in the ensitrelvir 125 mg, ensitrelvir 250 mg, and placebo groups, respectively, were included in the intention-to-treat population (mean age: 38.0 to 40.4 years). On day 4, the change from baseline in SARS-CoV-2 viral titer (log10 50% tissue culture infectious dose/mL) in patients with positive viral titer and viral RNA at baseline was greater with ensitrelvir 125 mg (mean [standard deviation], -2.42 [1.42]; P = 0.0712) and 250 mg (-2.81 [1.21]; P = 0.0083) versus placebo (-1.54 [0.74]); ensitrelvir treatment reduced SARS-CoV-2 RNA by -1.4 to -1.5 log10 copies/mL versus placebo. The viral titer and viral RNA were similar across groups on and after day 6. The median time to infectious viral clearance decreased by approximately 50 h with ensitrelvir treatment. All adverse events were mild to moderate. Ensitrelvir treatment demonstrated rapid SARS-CoV-2 clearance and was well tolerated (Japan Registry of Clinical Trials identifier: jRCT2031210350).

Efficacy of ensitrelvir against SARS-CoV-2 in a delayed-treatment mouse model.

OBJECTIVES: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the aetiological agent of coronavirus disease 2019 (COVID-19) and a devastating worldwide health concern. Development of safe and effective treatments is not only important for interventions during the current pandemic, but also for providing general treatment options moving forward. We have developed ensitrelvir, an antiviral compound that targets the 3C-like protease of SARS-CoV-2. In this study, a delayed-treatment mouse model was used to clarify the potential in vivo efficacy of ensitrelvir. METHODS: Female BALB/cAJcl mice of different ages were infected with the SARS-CoV-2 gamma strain (hCoV-19/Japan/TY7-501/2021) or mouse-adapted SARS-CoV-2 MA-P10 and then 24 h post-infection orally administered various doses of ensitrelvir or vehicle. Viral titres and RNA levels in the lungs were quantified using VeroE6/TMPRSS2 cells and RT-qPCR, respectively. Body weight loss, survival, lung weight, cytokine/chemokine production, nucleocapsid protein expression and lung pathology were evaluated to investigate the in vivo efficacy of ensitrelvir. RESULTS: Based on infectious viral titres and viral RNA levels in the lungs of infected mice, ensitrelvir reduced viral loads in a dose-dependent manner. The antiviral efficacy correlated with increased survival, reduced body weight loss, reduced pulmonary lesions and suppression of inflammatory cytokine/chemokine levels. CONCLUSIONS: This was the first evaluation of the in vivo anti-SARS-CoV-2 efficacy of ensitrelvir in a delayed-treatment mouse model. In this model, ensitrelvir demonstrated high antiviral potential and suppressed lung inflammation and lethality caused by SARS-CoV-2 infection. The findings support the continued clinical development of ensitrelvir as an antiviral agent to treat patients with COVID-19.

Discovery of S-217622, a Noncovalent Oral SARS-CoV-2 3CL Protease Inhibitor Clinical Candidate for Treating COVID-19.

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in millions of deaths and threatens public health and safety. Despite the rapid global spread of COVID-19 vaccines, effective oral antiviral drugs are urgently needed. Here, we describe the discovery of S-217622, the first oral noncovalent, nonpeptidic SARS-CoV-2 3CL protease inhibitor clinical candidate. S-217622 was discovered via virtual screening followed by biological screening of an in-house compound library, and optimization of the hit compound using a structure-based drug design strategy. S-217622 exhibited antiviral activity in vitro against current outbreaking SARS-CoV-2 variants and showed favorable pharmacokinetic profiles in vivo for once-daily oral dosing. Furthermore, S-217622 dose-dependently inhibited intrapulmonary replication of SARS-CoV-2 in mice, indicating that this novel noncovalent inhibitor could be a potential oral agent for treating COVID-19.

Characterization of the In Vitro and In Vivo Efficacy of Baloxavir Marboxil against H5 Highly Pathogenic Avian Influenza Virus Infection.

Human infections caused by the H5 highly pathogenic avian influenza virus (HPAIV) sporadically threaten public health. The susceptibility of HPAIVs to baloxavir acid (BXA), a new class of inhibitors for the influenza virus cap-dependent endonuclease, has been confirmed in vitro, but it has not yet been fully characterized. Here, the efficacy of BXA against HPAIVs, including recent H5N8 variants, was assessed in vitro. The antiviral efficacy of baloxavir marboxil (BXM) in H5N1 virus-infected mice was also investigated. BXA exhibited similar in vitro activities against H5N1, H5N6, and H5N8 variants tested in comparison with seasonal and other zoonotic strains. Compared with oseltamivir phosphate (OSP), BXM monotherapy in mice infected with the H5N1 HPAIV clinical isolate, the A/Hong Kong/483/1997 strain, also caused a significant reduction in viral titers in the lungs, brains, and kidneys, thereby preventing acute lung inflammation and reducing mortality. Furthermore, compared with BXM or OSP monotherapy, combination treatments with BXM and OSP using a 48-h delayed treatment model showed a more potent effect on viral replication in the organs, accompanied by improved survival. In conclusion, BXM has a potent antiviral efficacy against H5 HPAIV infections.

Acsl1 is essential for skin barrier function through the activation of linoleic acid and biosynthesis of ω-O-acylceramide in mice.

The long-chain acyl-CoA synthase1 (Acsl1) is a major enzyme that converts long-chain fatty acids to acyl-CoAs. The role of Acsl1 in energy metabolism has been elucidated in the adipose tissue, heart, and skeletal muscle. Here, we demonstrate that systemic deficiency of Acsl1 caused severe skin barrier defects, leading to embryonic lethality. Acsl1 mRNA and protein are expressed in the Acsl1+/+ epidermis, which are absent in Acsl1-/- mice. In Acsl1-/- mice, epidermal ceramide [EOS] (Cer[EOS]) containing ω-O-esterified linoleic acid, a lipid essential for the skin barrier, was significantly reduced. Conversely, ω-hydroxy ceramide (Cer[OS]), a precursor of Cer[EOS], was increased. Moreover, the levels of triglyceride (TG) species containing linoleic acids were lower in Acsl1-/- mice, whereas those not containing linoleic acid were comparable to Acsl1+/+ mice. As TG is considered to work as a reservoir of linoleic acid for the biosynthesis of Cer[EOS] from Cer[OS], our results suggest that Acsl1 plays an essential role in ω-O-acylceramide synthesis by providing linoleic acid for ω-O-esterification. Therefore, our findings identified a new biological role of Acsl1 as a regulator of the skin barrier.

5-Hydroxymethyltubercidin exhibits potent antiviral activity against flaviviruses and coronaviruses, including SARS-CoV-2.

Newly emerging or re-emerging viral infections continue to cause significant morbidity and mortality every year worldwide, resulting in serious effects on both health and the global economy. Despite significant drug discovery research against dengue viruses (DENVs) and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), no fully effective and specific drugs directed against these viruses have been discovered. Here, we examined the anti-DENV activity of tubercidin derivatives from a compound library from Hokkaido University and demonstrated that 5-hydroxymethyltubercidin (HMTU, HUP1108) possessed both potent anti-flavivirus and anti-coronavirus activities at submicromolar levels without significant cytotoxicity. Furthermore, HMTU inhibited viral RNA replication and specifically inhibited replication at the late stages of the SARS-CoV-2 infection process. Finally, we demonstrated that HMTU 5'-triphosphate inhibited RNA extension catalyzed by the viral RNA-dependent RNA polymerase. Our findings suggest that HMTU has the potential of serving as a lead compound for the development of a broad spectrum of antiviral agents, including SARS-CoV-2.

Air-liquid interphase culture confers SARS-CoV-2 susceptibility to A549 alveolar epithelial cells.

The human lung cell A549 is susceptible to infection with a number of respiratory viruses. However, A549 cells are resistant to Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) infection in conventional submerged culture, and this would appear to be due to low expression levels of the SARS-CoV-2 entry receptor: angiotensin-converting enzyme-2 (ACE2). Here, we examined SARS-CoV-2 susceptibility to A549 cells after adaptation to air-liquid interface (ALI) culture. A549 cells in ALI culture yielded a layer of mucus on their apical surface, exhibited decreased expression levels of the proliferation marker KI-67 and intriguingly became susceptible to SARS-CoV-2 infection. We found that A549 cells increased the endogenous expression levels of ACE2 and TMPRSS2 following adaptation to ALI culture conditions. Camostat, a TMPRSS2 inhibitor, reduced SARS-CoV-2 infection in ALI-cultured A549 cells. These findings indicate that ALI culture switches the phenotype of A549 cells from resistance to susceptibility to SARS-CoV-2 infection through upregulation of ACE2 and TMPRSS2.

SARS-CoV-2 Bearing a Mutation at the S1/S2 Cleavage Site Exhibits Attenuated Virulence and Confers Protective Immunity.

Severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) possesses a discriminative polybasic cleavage motif in its spike protein that is recognized by the host furin protease. Proteolytic cleavage activates the spike protein, thereby affecting both the cellular entry pathway and cell tropism of SARS-CoV-2. Here, we investigated the impact of the furin cleavage site on viral growth and pathogenesis using a hamster animal model infected with SARS-CoV-2 variants bearing mutations at the furin cleavage site (S gene mutants). In the airway tissues of hamsters, the S gene mutants exhibited low growth properties. In contrast to parental pathogenic SARS-CoV-2, hamsters infected with the S gene mutants showed no body weight loss and only a mild inflammatory response, thereby indicating the attenuated variant nature of S gene mutants. This transient infection was sufficient for inducing protective neutralizing antibodies that cross-react with different SARS-CoV-2 lineages. Consequently, hamsters inoculated with S gene mutants showed resistance to subsequent infection with both the parental strain and the currently emerging SARS-CoV-2 variants belonging to lineages B.1.1.7 and P.1. Taken together, our findings revealed that the loss of the furin cleavage site causes attenuation in the airway tissues of hamsters and highlighted the potential benefits of S gene mutants as potential immunogens. IMPORTANCE SARS-CoV-2 uses its spike protein to enter target cells. The spike protein is cleaved by a host protease, and this event facilitates viral entry and broadens cell tropism. In this study, we employed SARS-CoV-2 mutants lacking the S protein cleavage site and characterized their growth and pathogenicity using hamsters, a laboratory animal model for SARS-CoV-2 infection. These mutants exerted low pathogenicity but induced sufficient levels of neutralizing antibodies in hamsters, which protected hamsters from rechallenge with pathogenic clinical SARS-CoV-2 strains. These virus mutants may be used as protective immunogens against SARS-CoV-2 infection.

MRC5 cells engineered to express ACE2 serve as a model system for the discovery of antivirals targeting SARS-CoV-2.

Although the spread of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) has resulted in a worldwide pandemic, there are currently no virus-specific drugs that are fully effective against SARS-CoV-2. Only a limited number of human-derived cells are capable of supporting SARS-CoV-2 replication and the infectivity of SARS-CoV-2 in these cells remains poor. In contrast, monkey-derived Vero cells are highly susceptibility to infection with SARS-CoV-2, although they are not suitable for the study of antiviral effects by small molecules due to their limited capacity to metabolize drugs compared to human-derived cells. In this study, our goal was to generate a virus-susceptible human cell line that would be useful for the identification and testing of candidate drugs. Towards this end, we stably transfected human lung-derived MRC5 cells with a lentiviral vector encoding angiotensin-converting enzyme 2 (ACE2), the cellular receptor for SARS-CoV-2. Our results revealed that SARS-CoV-2 replicates efficiently in MRC5/ACE2 cells. Furthermore, viral RNA replication and progeny virus production were significantly reduced in response to administration of the replication inhibitor, remdesivir, in MRC5/ACE2 cells compared with Vero cells. We conclude that the MRC5/ACE2 cells will be important in developing specific anti-viral therapeutics and will assist in vaccine development to combat SARS-CoV-2 infections.

TMPRSS11D and TMPRSS13 Activate the SARS-CoV-2 Spike Protein.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) utilizes host proteases, including a plasma membrane-associated transmembrane protease, serine 2 (TMPRSS2) to cleave and activate the virus spike protein to facilitate cellular entry. Although TMPRSS2 is a well-characterized type II transmembrane serine protease (TTSP), the role of other TTSPs on the replication of SARS-CoV-2 remains to be elucidated. Here, we have screened 12 TTSPs using human angiotensin-converting enzyme 2-expressing HEK293T (293T-ACE2) cells and Vero E6 cells and demonstrated that exogenous expression of TMPRSS11D and TMPRSS13 enhanced cellular uptake and subsequent replication of SARS-CoV-2. In addition, SARS-CoV-1 and SARS-CoV-2 share the same TTSPs in the viral entry process. Our study demonstrates the impact of host TTSPs on infection of SARS-CoV-2, which may have implications for cell and tissue tropism, for pathogenicity, and potentially for vaccine development.

SARS-CoV-2 variants with mutations at the S1/S2 cleavage site are generated in vitro during propagation in TMPRSS2-deficient cells.

The spike (S) protein of Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) binds to a host cell receptor which facilitates viral entry. A polybasic motif detected at the cleavage site of the S protein has been shown to broaden the cell tropism and transmissibility of the virus. Here we examine the properties of SARS-CoV-2 variants with mutations at the S protein cleavage site that undergo inefficient proteolytic cleavage. Virus variants with S gene mutations generated smaller plaques and exhibited a more limited range of cell tropism compared to the wild-type strain. These alterations were shown to result from their inability to utilize the entry pathway involving direct fusion mediated by the host type II transmembrane serine protease, TMPRSS2. Notably, viruses with S gene mutations emerged rapidly and became the dominant SARS-CoV-2 variants in TMPRSS2-deficient cells including Vero cells. Our study demonstrated that the S protein polybasic cleavage motif is a critical factor underlying SARS-CoV-2 entry and cell tropism. As such, researchers should be alert to the possibility of de novo S gene mutations emerging in tissue-culture propagated virus strains.

Identification of quinolone derivatives as effective anti-Dengue virus agents.

Dengue virus (DENV) infection is one of the most important infectious diseases in tropical and subtropical regions around the world. Previously, we performed an initial phenotypic screening of 7000 compounds using DENV type 2 (DENV2)-infected BHK-21 cells to identify small molecules which could inhibit virus replication. In this study, we describe two novel compounds with anti-DENV2 activity, tentatively named Compound-X and Compound-Y. Both compounds possess a quinolone skeleton, and the EC50s of Compound-X and Compound-Y against DENV2 were 3.9 µM and 9.2 µM, respectively. Based on a DENV replicon assay, it was suggested that these compounds have anti-DENV2 activity by inhibition of a step in virus replication. Furthermore, using mutational analysis we obtained compounds-resistant to DENV2 infection and identified a mutation, V130A in the NS5 methyltransferase (MTase) domain. However, these compounds did not inhibit MTase activity. In addition, incorporation of an additional NS1 N246D mutation with the NS5 V130A mutation in DENV2 resulted in recovery of viral replication and a further reduction of the sensitivity to the quinolone compounds by an unknown mechanism. Therefore further investigations are required to clarify the antiviral mechanisms of these quinolone compounds.

Inhibition of dengue virus infection by 1-stearoyl-2-arachidonoyl-phosphatidylinositol in vitro.

Dengue fever is an acute febrile infectious disease caused by dengue virus (DENV). Despite the significant public health concerns posed by DENV, there are currently no effective anti-DENV therapeutic agents. To develop such drugs, a better understanding of the detailed mechanisms of DENV infection is needed. Both lipid metabolism and lipid synthesis are activated in DENV-infected cells, so we used lipid screening to identify potential antiviral lipid molecules. We identified 1-stearoyl-2-arachidonoyl-phosphatidylinositol (SAPI), which is the most abundant endogenous phosphatidylinositol (PI) molecular species, as an anti-DENV lipid molecule. SAPI suppressed the cytopathic effects induced by DENV2 infection as well as the replication of all DENV serotypes without inhibiting the entry of DENV2 into host cells. However, no other PI molecular species or PI metabolites, including lysophosphatidylinositols and phosphoinositides, displayed anti-DENV2 activity. Furthermore, SAPI suppressed the production of DENV2 infection-induced cytokines and chemokines, including C-C motif chemokine ligand (CCL)5, CCL20, C-X-C chemokine ligand 8, IL-6, and IFN-ß. SAPI also suppressed the TNF-α production induced by LPS stimulation in macrophage cells differentiated from THP-1 cells. Our results demonstrated that SAPI is an endogenous inhibitor of DENV and modulated inflammatory responses in DENV2-infected cells, at least in part via TLR 4.-Sanaki, T., Wakabayashi, M., Yoshioka, T., Yoshida, R., Shishido, T., Hall, W. W., Sawa, H., Sato, A. Inhibition of dengue virus infection by 1-stearoyl-2-arachidonoyl-phosphatidylinositol in vitro.

Contribution of synovial macrophages to rat advanced osteoarthritis pain resistant to cyclooxygenase inhibitors.

Most advanced knee osteoarthritis (OA) patients experience chronic pain resistant to cyclooxygenase (COX) inhibitors. However, the cells and molecules involved in this advanced OA pain remain poorly understood. In this study, we developed a rat model of advanced knee OA by modification of the monoiodoacetate-induced OA pain model and examined involvement of synovial macrophages in advanced OA pain. Cyclooxygenase inhibitors, such as celecoxib and naproxen, and a steroid were ineffective, but an opioid and anti-nerve growth factor (NGF) antibody was effective for pain management in the advanced OA model. Similar to advanced OA patients, histological analysis indicated severe bone marrow damages, synovitis, and cartilage damage and an increase of macrophages with high expression of interleukin-1ß, NGF, nitric oxide synthase (NOS) 1, NOS2, and COX-2 in the knee joint of the advanced OA model. Intravenous injection of clodronate liposomes depleted synovial macrophages, which decreased the level of not only proinflammatory mediator interleukin-1ß but also NGF in the knee joint, leading to pain suppression in the advanced OA model. These data suggest the involvement of synovial macrophages in advanced knee OA pain resistant to COX inhibitors by increasing proinflammatory mediators, and that drugs targeting synovial macrophages might have potent analgesic effects.

Sensitization of transient receptor potential vanilloid 4 and increasing its endogenous ligand 5,6-epoxyeicosatrienoic acid in rats with monoiodoacetate-induced osteoarthritis.

Transient receptor potential vanilloid 4 (TRPV4) receptor modulates pain, and this has been noted in several animal models. However, the involvement of TRPV4 in osteoarthritic (OA) pain remains poorly understood. This study assessed the functional changes in TRPV4 and the expression of its endogenous ligand 5,6-epoxyeicosatrienoic acid (5,6-EET) in a rat monoiodoacetate (MIA)-induced OA pain model (MIA rats). Monoiodoacetate-treated rats showed reduced grip strength as compared to sham-treated rats, and this loss in function could be recovered by the intraarticular administration of a TRPV4 antagonist (HC067047 or GSK2193874). By contrast, the intraarticular administration of the TRPV4 agonist, GSK1016790A, increased the pain-related behaviors in MIA rats but not in sham rats. TRPV4 expression was not increased in knee joints of MIA rats; however, the levels of phosphorylated TRPV4 at Ser824 were increased in dorsal root ganglion neurons. In addition, 5,6-EET was increased in lavage fluids from the knee joints of MIA rats and in meniscectomy-induced OA pain model rats. 5,6-EET and its metabolite were also detected in synovial fluids from patients with OA. In conclusion, TRPV4 was sensitized in the knee joints of MIA rats through phosphorylation in dorsal root ganglion neurons, along with an increase in the levels of its endogenous ligand 5,6-EET. The analgesic effects of the TRPV4 antagonist in the OA pain model rats suggest that TRPV4 may be a potent target for OA pain relief.