Ensitrelvir is effective against SARS-CoV-2 3CL protease mutants circulating globally.

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been a public health concern worldwide. Ensitrelvir (S-217622) has been evaluated as an antiviral treatment for COVID-19, targeting SARS-CoV-2 3C-like protease (3CLpro). Ensitrelvir has been reported to have comparable antiviral activity against some of the SARS-CoV-2 variants: alpha, beta, gamma, delta, and omicron (BA.1.18). In this paper, we describe that ensitrelvir is effective against newly emerging SARS-CoV-2 variants and globally prevalent 3CLpro mutations. Ensitrelvir exhibited comparable antiviral activity against SARS-CoV-2 variants, including recently emerging ones: omicron (BA1.1, BA.2, BA.2.75, BA.4, BA.5, BQ.1.1, XBB.1, and XE), mu, lambda, and theta. Genetic surveillance of SARS-CoV-2 3CLpro, the target of ensitrelvir, was conducted using a public database and identified 11 major 3CLpro mutations circulating globally (G15S, T21I, T24I, K88R, L89F, K90R, P108S, P132H, A193V, H246Y, and A255V). The 3CLpro mutation from proline to histidine at amino acid position 132 was especially identified in the omicron variant, with prevalence of 99.69%. Enzyme kinetic assay revealed that these 3CLpro mutants have enzymatic activity comparable to that of the wild type (WT). Next, we assessed the inhibitory effect of ensitrelvir against mutated 3CLpro, with it showing inhibitory effects similar to that against the WT. These in vitro data suggest that ensitrelvir will be effective against currently circulating SARS-CoV-2 variants, including omicron variants and those carrying 3CLpro mutations, which emerging novel SARS-CoV-2 variants could carry.

Efficacy comparison of 3CL protease inhibitors ensitrelvir and nirmatrelvir against SARS-CoV-2 in vitro and in vivo.

OBJECTIVES: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become established in the human population, making the need to develop safe and effective treatments critical. We have developed the small-molecule antiviral ensitrelvir, which targets the 3C-like (3CL) protease of SARS-CoV-2. This study evaluated the in vitro and in vivo efficacy of ensitrelvir compared with that of another SARS-CoV-2 3CL PI, nirmatrelvir. METHODS: Cultured cells, BALB/cAJcl mice and Syrian hamsters were infected with various SARS-CoV-2 strains, including the ancestral strain WK-521, mouse-adapted SARS-CoV-2 (MA-P10) strain, Delta strain and Omicron strain. Ensitrelvir efficacy was compared with that of nirmatrelvir. Effective concentrations were determined in vitro based on virus-induced cytopathic effects, viral titres and RNA levels. Lung viral titres, nasal turbinate titres, body-weight changes, and animal survival were also monitored. RESULTS: Ensitrelvir and nirmatrelvir showed comparable antiviral activity in multiple cell lines. Both ensitrelvir and nirmatrelvir reduced virus levels in the lungs of mice and the nasal turbinates and lungs of hamsters. However, ensitrelvir demonstrated comparable or better in vivo efficacy than that of nirmatrelvir when present at similar or slightly lower unbound-drug plasma concentrations. CONCLUSIONS: Direct in vitro and in vivo efficacy comparisons of 3CL PIs revealed that ensitrelvir demonstrated comparable in vitro efficacy to that of nirmatrelvir in cell culture and exhibited equal to or greater in vivo efficacy in terms of unbound-drug plasma concentration in both animal models evaluated. The results suggest that ensitrelvir may become an important resource for treating individuals infected with SARS-CoV-2.

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.

Prophylactic effect of ensitrelvir in mice infected with SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological cause of coronavirus disease 2019 (COVID-19) and continues to be a major health concern worldwide. Strategies to protect individuals at high risk of COVID-19 are critical but are currently a largely unmet need. We evaluated the oral antiviral drug ensitrelvir, which specifically targets the SARS-CoV-2 3CL protease, for its efficacy as a pre-exposure prophylactic treatment. Aged BALB/c mice were subcutaneously treated with various doses of ensitrelvir 24 h prior to a lethal SARS-CoV-2 challenge infection. Mouse body weight changes, survival rates, and viral titers in the lungs were evaluated, and plasma concentrations of ensitrelvir were determined. A single subcutaneous administration of ensitrelvir at 64 mg/kg or greater 24 h prior to SARS-CoV-2 challenge infection significantly protected aged mice against lethality and inhibited body weight loss. Pharmacokinetic analysis of ensitrelvir in the aged mice suggested that plasma concentrations ≥2.99 µg/mL resulted in a significant prophylactic effect against SARS-CoV-2 infection. In the aged mouse prophylaxis model, SARS-CoV-2 titers were suppressed in the lungs of mice treated with ensitrelvir 24 h prior to challenge infection, suggesting that the prophylactic administration of ensitrelvir exerted its prophylactic effect by suppressing viral proliferation. These findings suggest that ensitrelvir is a candidate drug for pre-exposure prophylactic treatment of individuals at high risk of COVID-19.

Pharmacokinetic and Pharmacodynamic Analysis of the 3CL Protease Inhibitor Ensitrelvir in a SARS-CoV-2 Infection Mouse Model.

The small-molecule antiviral drug ensitrelvir targets the 3C-like protease of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This study evaluated its inhibitory effect on viral replication in a delayed-treatment mouse model and investigated the relationship between pharmacokinetic (PK) parameters and pharmacodynamic (PD) effects. SARS-CoV-2 gamma-strain-infected BALB/c mice were orally treated with various doses of ensitrelvir starting 24 h post-infection. Effectiveness was determined 48 h after first administration based on lung viral titers. Ensitrelvir PK parameters were estimated from previously reported plasma concentration data and PK/PD analyses were performed. Ensitrelvir doses ≥ 16 mg/kg once daily, ≥8 mg/kg twice daily, or ≥8 mg/kg thrice daily for two days significantly reduced lung viral titers compared to that of the vehicle. PK/PD analyses revealed that mean AUC0-48h post-first administration, plasma concentration 48 h post-first administration (C48h), and total time above the target plasma concentration (TimeHigh) were PK parameters predictive of viral titer reduction. In conclusion, ensitrelvir dose-dependently reduced lung SARS-CoV-2 titers in mice, suggesting it inhibited viral replication. PK parameters C48h and TimeHigh were associated with sustained ensitrelvir plasma concentrations and correlated with the reduced viral titers. The findings suggest that maintaining ensitrelvir plasma concentration is effective for exerting antiviral activity against SARS-CoV-2.

Peptide-to-Small Molecule: Discovery of Non-Covalent, Active-Site Inhibitors of ß-Herpesvirus Proteases.

Viral proteases, the key enzymes that regulate viral replication and assembly, are promising targets for antiviral drug discovery. Herpesvirus proteases are enzymes with no crystallographically confirmed noncovalent active-site binders, owing to their shallow and polar substrate-binding pockets. Here, we applied our previously reported "Peptide-to-Small Molecule" strategy to generate novel inhibitors of ß-herpesvirus proteases. Rapid selection with a display technology was used to identify macrocyclic peptide 1 bound to the active site of human cytomegalovirus protease (HCMVPro) with high affinity, and pharmacophore queries were defined based on the results of subsequent intermolecular interaction analyses. Membrane-permeable small molecule 19, designed de novo according to this hypothesis, exhibited enzyme inhibitory activity (IC50 = 10-6 to 10-7 M) against ß-herpesvirus proteases, and the design concept was proved by X-ray cocrystal analysis.

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.

[Corporate Efforts to Research and Develop Therapeutic Agents for Infectious Diseases That Threaten Human].

Overcoming serious infectious diseases such as malaria, tuberculosis, and other neglected tropical diseases (NTDs) that threaten human life around the world is an important issue in global health. Most of these diseases are concentrated in developing and low-income countries, and in order to reinforce drug discovery activities, pharmaceutical companies are actively promoting industry-academia-government partnerships and utilizing funds to stimulate global health activities. In this presentation, three examples of our drug discovery activities are introduced. The first is participation in the Booster project led by Drugs for Neglected Diseases initiative (DNDi) aimed at creating therapeutic agents for leishmaniasis and Chagas disease, an effort supported by the Global Health Innovative Technology (GHIT) Fund. As domestic and overseas pharmaceutical companies participate in the project and provide their own compounds, it is possible to obtain structure-activity relationship information in a short period of time and improve compound potency. We collaborated with DNDi to create a lead compound from one hit compound, and contributed to further enhancement of its activity. The remaining two are collaborations with academia for the creation of new therapeutic agents or vaccines: a joint research project with Hokkaido University Research Center for Zoonosis Control for emerging viral diseases, and a collaboration with Nagasaki University in malaria. In each case, our researchers were based at the university, establishing close working collaborations with the university researchers. Novel solutions for serious infectious diseases are expected by the combination of the high-level basic research capabilities of academia and the drug discovery know-how and original compound libraries possessed by pharmaceutical companies.

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.

Design and pharmacological evaluation of PF-4840154, a non-electrophilic reference agonist of the TrpA1 channel.

TrpA1 is an ion channel involved in nociceptive and inflammatory pain. It is implicated in the detection of chemical irritants through covalent binding to a cysteine-rich intracellular region of the protein. While performing an HTS of the Pfizer chemical collection, a class of pyrimidines emerged as a non-reactive, non-covalently binding family of agonists of the rat and human TrpA1 channel. Given the issues identified with the reference agonist Mustard Oil (MO) in screening, a new, non-covalently binding agonist was optimized and proved to be a superior agent to MO for screening purposes. Compound 16a (PF-4840154) is a potent, selective agonist of the rat and human TrpA1 channel and elicited TrpA1-mediated nocifensive behaviour in mouse.

N-(2-hydroxyethyl)-N,2-dimethyl-8-{[(4R)-5-methyl-3,4-dihydro-2H-chromen-4-yl]amino}imidazo[1,2-a]pyridine-6-carboxamide (PF-03716556), a novel, potent, and selective acid pump antagonist for the treatment of gastroesophageal reflux disease.

Inhibition of H(+),K(+)-ATPase is accepted as the most effective way of controlling gastric acid secretion. However, current acid suppressant therapy for gastroesophageal reflux disease, using histamine H(2) receptor antagonists and proton pump inhibitors, does not fully meet the needs of all patients because of their mechanism of action. This study sought to characterize the in vitro and in vivo pharmacology of a novel acid pump antagonist, N-(2-Hydroxyethyl)-N,2-dimethyl-8-{[(4R)-5-methyl-3,4-dihydro-2H-chromen-4-yl]amino}imidazo[1,2-a]pyridine-6-carboxamide (PF-03716556), and to compare it with other acid suppressants. Porcine, canine, and human recombinant gastric H(+),K(+)-ATPase activities were measured by ion-leaky and ion-tight assay. The affinities for a range of receptors, ion channels, and enzymes were determined to analyze selectivity profile. Acid secretion in Ghosh-Schild rats and Heidenhain pouch dogs were measured by titrating perfusate and gastric juice samples. PF-03716556 demonstrated 3-fold greater inhibitory activity than 5,6-dimethyl-2-(4-fluorophenylamino)-4-(1-methyl-1,2,3,4-tetrahydroisoquinoline-2-yl)pyrimidine (revaprazan), the only acid pump antagonist that has been available on the market, in ion-tight assay. The compound did not display any species differences, exhibiting highly selective profile including the canine kidney Na(+),K(+)-ATPase. Kinetics experiments revealed that PF-03716556 has a competitive and reversible mode of action. More rapid onset of action than 5-methoxy-2-{[(4-methoxy-3,5-dimethyl-2-pyridyl)methyl]-sulfinyl}-benzimidazole (omeprazole) and 3-fold greater potency than revaprazan were observed in Ghosh-Schild rats and Heidenhain pouch dogs. PF-03716556, a novel acid pump antagonist, could improve upon or even replace current pharmacological treatment for gastroesophageal reflux disease.

Aryl hydrocarbon receptor and estrogen receptor ligand activity of organic extracts from road dust and diesel exhaust particulates.

A wide variety of contaminants derived from diesel and gasoline engines, tire, asphalt, and natural organic compounds is found in road dust. Polycyclic aromatic compounds (PACs) are the important toxic targets among various contents in road dust and diesel exhaust particulates (DEPs), and endocrine-disrupting activity of PACs was suggested. In the present study, aryl hydrocarbon receptor (AhR) ligand activity was confirmed in the extract of both road dust and DEPs. In the separation of the extracts for both road dust and DEPs with reversed-phase HPLC, it was found that polar fractions contributed to significant AhR ligand activity in both a mouse hepatoma (H1L1) cell system and a yeast system. Furthermore, the contribution of these polar fractions was higher in DEPs than in road dust, probably because of the greater concentration of oxy-PAHs in DEPs than in road dust. The contribution of contaminants associated with the polar region to AhR ligand activity was also evident following the separation of road dust with normal-phase HPLC. Additionally, remarkable estrogen receptor (ER) ligand activity was detected in the highly polar region separated with normal-phase HPLC. It is suggested that many unknown AhR or ER ligand active compounds are contained in the polar region.

High throughput solubility measurement with automated polarized light microscopy analysis.

As a high throughput solubility assay, the solution-precipitation method using a dimethylsulfoxide (DMSO) sample stock solution (DMSO-SP) has been widely used in drug discovery. However, the solid form of the precipitant has not been investigated. In this study, we investigated the experimental conditions of the DMSO-SP, focusing on the solid form of the precipitant. The final concentration of DMSO was 1% (v/v). The precipitant of more than a half of the model compounds was observed as crystalline by polarized light microscopy analysis. When the incubation time was 20 h and the precipitant was crystalline, the DMSO-SP solubility was similar to the solubility from a powder material (PWD). However, when the incubation time was 10 min and/or the precipitant was not crystalline, the DMSO-SP solubility was higher than the PWD solubility. These results suggested that the information regarding the solid form of the precipitant is important in interpreting the solubility data. In addition, we developed an automated birefringence diagnose system for drug discovery usage.

Anti-thyroid hormonal activity of tetrabromobisphenol A, a flame retardant, and related compounds: Affinity to the mammalian thyroid hormone receptor, and effect on tadpole metamorphosis.

The thyroid hormone-disrupting activity of tetrabromobisphenol A (TBBPA), a flame retardant, and related compounds was examined. TBBPA, tetrachlorobisphenol A (TCBPA), tetramethylbisphenol A (TMBPA) and 3,3'-dimethylbisphenol A (DMBPA) markedly inhibited the binding of triiodothyronine (T3; 1 x 10(-10) M) to thyroid hormone receptor in the concentration range of 1 x 10(-7)-1 x 10(-4) M, while bisphenol A and 2,2-diphenylpropane were inactive. TBBPA, TCBPA, TMBPA and DMBPA did not exhibit thyroid hormonal activity in a thyroid hormone-responsive reporter assay using a Chinese hamster ovary cell line (CHO-K1) transfected with thyroid hormone receptor alpha1 or beta1, but TBBPA and TCBPA showed significant anti-thyroid hormone effects on the activity of T3 (1 x 10(-8) M) in the concentration range of 3 x 10(-6) - 5 x 10(-5) M. The thyroid hormone-disrupting activity of TBBPA was also examined in terms of the effect on amphibian metamorphosis stimulated by thyroid hormone. TBBPA in the concentration range of 1 x 10(-8) to 1 x 10(-6) M showed suppressive action on T3 (5 x 10(-8) M)-enhancement of Rana rugosa tadpole tail shortening. These facts suggest that TBBPA, TCBPA, TMBPA and DMBPA can act as thyroid hormone-disrupting agents.