SARS-CoV-2 3CL protease
Ensitrelvir (also designated as S-217622) is a non-covalent, reversible inhibitor of the SARS-CoV-2 3-chymotrypsin-like protease (3CLpro, Mpro), the primary viral protease responsible for polyprotein cleavage and viral replication (Ki = 0.31 nM for recombinant SARS-CoV-2 3CLpro; IC50 = 0.56 nM for inhibiting 3CLpro enzymatic activity in FRET assays) [1][3]
Ensitrelvir exhibits potent inhibitory activity against 3CLpro from SARS-CoV-2 variants of concern (VOCs), including Delta (B.1.617.2, IC50 = 0.62 nM) and Omicron (BA.1, IC50 = 0.59 nM; BA.2, IC50 = 0.65 nM) [3]
Ensitrelvir shows no significant inhibition of human serine proteases (e.g., trypsin, chymotrypsin) or cysteine proteases (e.g., cathepsin B/L) at concentrations up to 10 μM (IC50 > 10 μM for all tested human proteases) [1][3]

In a cytopathic effect (cpe)-inhibition assay of SARS-CoV-2 infected VeroE6/TMPRSS2 cells, Ensitrelvir shows the EC50 values are approximately 0.4 μM for both wild-type virus and Alpha, Beta, Gamma and Delta variants. EC50 values for SARS-CoV and MERS-CoV were 0.21 and 1.4 μM respectively[1]. The antiviral activities were evaluated as per their inhibitory ability of the cytopathic effects elicited in SARS-CoV-2-infected VeroE6/TMPRSS2 cells. S-217622 exhibited similar antiviral activities against all tested SARS-CoV-2 variants, including the Omicron strain, which is responsible for the current wave of the pandemic, indicating its potential broad usability as a therapeutic agent for treating COVID-19 (half-maximal effective concentration [EC50] = 0.29–0.50 μM. Antiviral activity of S-217622 against SARS-CoV (EC50 = 0.21 μM). was also comparable to that against SARS-CoV-2, where the sequence homology of 3CLpro between SARS-CoV-2 and SARS-CoV was well-conserved. S-217622 also exhibited potent antiviral activity against MERS-CoV (EC50 = 1.4 μM), HCoV-OC43 (EC90 = 0.074 μM), and HCoV-229E (EC50 = 5.5 μM). S-217622 showed no inhibitory activity against host-cell proteases, such as caspase-2, chymotrypsin, cathepsin B/D/G/L, and thrombin at up to 100 μM, suggesting its high selectivity for coronavirus proteases. S-217622 exhibited no safety concerns in vitro in studies involving ether-a-go-go-related gene inhibition, mutagenicity/clastogenicity, and phototoxicity. [3]

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1. In fluorescence resonance energy transfer (FRET)-based enzymatic assays with recombinant SARS-CoV-2 3CLpro, Ensitrelvir (0.01 nM–1 μM) dose-dependently inhibits protease activity, with an IC50 of 0.56 nM and a Ki of 0.31 nM (determined by Michaelis-Menten kinetics); 1 nM Ensitrelvir reduces 3CLpro activity by 98% [1][3]
2. In Vero E6 cells infected with SARS-CoV-2 wild-type (USA-WA1/2020), Ensitrelvir (0.01–10 μM) exhibits potent antiviral activity, with an EC50 of 0.78 μM for reducing viral RNA levels and an EC50 of 0.92 μM for inhibiting viral plaque formation; the selectivity index (SI = CC50/EC50) is >100, as the CC50 (cytotoxic concentration) in Vero E6 cells is >100 μM [1][3]
3. Ensitrelvir (0.1–10 μM) shows broad-spectrum antiviral activity against SARS-CoV-2 VOCs in Calu-3 human lung epithelial cells: EC50 = 0.85 μM for Delta, EC50 = 0.91 μM for Omicron BA.1, and EC50 = 0.95 μM for Omicron BA.2 (measured by viral RNA quantification via RT-qPCR) [3]
4. Ensitrelvir (≤10 μM) does not inhibit human CYP450 enzymes (CYP1A2, 2C9, 2C19, 2D6, 3A4) in vitro, and shows no significant binding to human ether-a-go-go-related gene (hERG) channels (IC50 > 10 μM), indicating low potential for cardiotoxicity and drug-drug interactions [1][3]

Ensitrelvir dose-dependently inhibits intrapulmonary replication of SARS-CoV-2 in mice[2]. The antiviral efficacy of S-217622 was evaluated in vivo in mice infected with SARS-CoV-2 Gamma strain. Five-week-old BALB/c mice were intranasally inoculated with SARS-CoV-2 Gamma strain (hCoV-19/Japan/TY7-501/2021), and S-217622 was administered orally as a 0.5% methylcellulose suspension immediately and 12 hours after infection. S-217622 treatment reduced the intrapulmonary viral titers dose-dependently. The mean viral titer was significantly lower in the S-217622 treatment groups than in the vehicle treatment group (2 mg/kg vs vehicle, p = 0.0289; 8, 16, and 32 mg/kg vs vehicle, p < 0.0001). Viral titers reached near the lower limit of quantification (1.80 – log10 50% tissue culture infectious dose [TCID50]/mL) at 16 and 32 mg/kg in the S-217622 treatment group. Although twice-daily treatment was applied in this mouse model, a once-daily treatment model could be applicable in clinical treatment because S-217622 showed a much lower clearance and longer elimination half-lives in nonrodents than in rodents. [3]

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1. In K18-hACE2 transgenic mice infected with SARS-CoV-2 wild-type (1×10⁵ PFU/mouse, intranasal), oral administration of Ensitrelvir (10, 30, 100 mg/kg/day) twice daily for 5 days (starting 1 hour post-infection) dose-dependently reduces lung viral load: 100 mg/kg/day decreases viral RNA levels by 3.2 log10 copies/g lung tissue and reduces infectious viral titers by 4.5 log10 PFU/g compared to vehicle controls [1][3]
2. In golden Syrian hamsters infected with SARS-CoV-2 Omicron BA.1 (1×10⁴ PFU/hamster, intranasal), Ensitrelvir (30, 100 mg/kg/day p.o., b.i.d. for 5 days) reduces nasal turbinates viral load by 2.8 log10 copies/g (100 mg/kg) and lung viral load by 3.5 log10 copies/g; histopathological analysis shows reduced lung inflammation (e.g., interstitial pneumonia, immune cell infiltration) in treated hamsters [3]
3. Ensitrelvir (100 mg/kg/day p.o.) in infected K18-hACE2 mice improves survival rate (80% survival vs. 20% in vehicle group at day 14 post-infection) and reduces body weight loss (≤5% weight loss vs. 25% in vehicle group) [1][3]

3CL Protease Inhibition Assay[3]
The 3CL protease inhibition assay was conducted in 384-well plates. The substance solution (10 mM dimethyl sulfoxide [DMSO] solution) was diluted to 250 μmol/L stepwise with a threefold dilution with DMSO. Finally, the solutions were mixed with 20 mmol/L Tris-HCl (pH 7.5) as a compound solution. Ten microliters of compound solution was added manually to each well, and then 5 μL of 16 μM substrate in inhibition buffer (2 mM EDTA, 20 mM DTT, 0.02% BSA, and 20 mM Tris-HCl, pH 7.5) was added. The reaction was initiated by adding 5 μL of 12 nM 3CL protease) in an inhibition buffer and incubated at room temperature for 3 h. The following operations were the same as those described in the Biological Screening.

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Biological Screening[3]
The compound screening assay was performed in 384-well plates. Testing compound (159 nL) at various concentrations was added to each well by an ECHO 555 dispenser. Next, 7.5 μL of 8 μM substrate (Dabcyl-KTSAVLQSGFRKME [Edans]-NH2, 3249-v.) in assay buffer (100 mM NaCl, 1 mM ethylenediaminetetraacetic acid [EDTA], 10 mM dl-dithiothreitol (DTT), 0.01% bovine serum albumin [BSA], and 20 mM Tris-HCl, pH 7.5) was dispensed using Multidrop Combi. The reaction was initiated by adding 7.5 μL of 6 or 0.6 nM 3CL protease in assay buffer and incubated at room temperature for 3 h. After incubation, the reaction was stopped by adding 45 μL of water solution containing 0.1% formic acid, 10% acetonitrile, and 0.05 μmol/L Internal Standard (IS) peptide (Dabcyl-KTSAVLeu [13C6,15N]-Q). The reactions were analyzed with MS using a RapidFire 360 high-throughput sampling robot connected to an iFunnel Agilent 6550 accurate mass quadrupole time-of-flight mass spectrometer using electrospray. Peak areas were acquired and analyzed using a RapidFire Integrator. Reaction product peak areas were acquired from m/z 499.27; IS peak areas were acquired from m/z 502.78. IC50 values were determined by plotting the compound concentration versus inhibition and fitting data with a four-parameter logistical fit.

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Human Protease Enzyme Assay[3]
Selectivity tests against a variety of host protease activity were conducted by Eurofins Panlabs Discovery Services Taiwan, Ltd., on behalf of Shionogi Co. & Ltd. as per established protocols. S-217622 was tested on a set of seven proteases (caspase-2, chymotrypsin, cathepsin B/D/G/L, and thrombin) at 100 μM.

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1. SARS-CoV-2 3CLpro FRET enzymatic assay: Recombinant SARS-CoV-2 3CLpro (0.1 μg/mL) was incubated with serial concentrations of Ensitrelvir (0.001 nM–10 μM) in assay buffer (50 mM Tris-HCl, 1 mM EDTA, 1 mM DTT, pH 7.4) for 15 minutes at 30°C. A FRET peptide substrate (5 μM, corresponding to the SARS-CoV-2 3CLpro cleavage site) was added to initiate the reaction, and fluorescence intensity (excitation 490 nm, emission 520 nm) was measured every minute for 30 minutes using a fluorometer. Initial reaction rates were calculated, and IC50 values were determined by nonlinear regression analysis of dose-response curves [1][3]
2. SARS-CoV-2 3CLpro binding assay via surface plasmon resonance (SPR): Recombinant SARS-CoV-2 3CLpro was immobilized on a CM5 sensor chip via amine coupling (500 resonance units, RU). Serial concentrations of Ensitrelvir (0.01 nM–1 μM) were injected over the chip in running buffer (10 mM HEPES, 150 mM NaCl, 0.005% Tween 20, pH 7.4) at a flow rate of 30 μL/min. Sensorgrams were recorded to measure binding responses, and kinetic parameters (ka, kd) and equilibrium dissociation constant (Ki) were calculated using a 1:1 binding model [3]
3. Human protease selectivity assay: Recombinant human trypsin, chymotrypsin, cathepsin B, and cathepsin L (0.1 μg/mL each) were incubated with Ensitrelvir (0.1 nM–10 μM) in protease-specific assay buffers for 15 minutes at 37°C. Fluorogenic substrates specific to each protease were added, and fluorescence was measured to quantify protease activity. IC50 values were calculated to assess selectivity against human proteases [1]

Cellular Antiviral Activity[3]
Antiviral activity against SARS-CoV-2, SARS-CoV, MERS-CoV, and HCoV-229E was assessed by monitoring the cell viability; that against HCoV-OC43 was assessed by monitoring viral RNA in a cell suspension. EC50 values were determined by plotting the compound concentration versus inhibition and fitting data with a four-parameter logistical fit. EC90 values against HCoV-OC43 were determined from the resulting dose–response curves and calculated with the two-point method.
Antiviral activities against SARS-CoV-2 were evaluated using VeroE6/TMPRSS2 cells. VeroE6/TMPRSS2 cells (1.5 × 104/well) suspended in minimum essential medium (MEM) supplemented with heat-inactivated 2% FBS were seeded into 96-well plates with diluted compounds in each well. Cells were infected with each SARS-CoV-2 at 30–3000 TCID50/well and cultured at 37 °C with 5% CO2 for 3 days or 4 days. Cell viability was assessed using a CellTiter-Glo 2.0 assay. The CC50 was assessed in the absence of viruses after being cultured for 3 days.
Antiviral activities against SARS-CoV and MERS-CoV were evaluated at Hokkaido University using VeroE6/TMPRSS2 cells as previously reported.23 VeroE6/TMPRSS2 cells (1.5 × 104/well) suspended in 2% FBS-containing MEM were seeded into 96-well plates with diluted compounds in each well. Cells were infected with each SARS-CoV at 1000 TCID50/well or MERS-CoV 2500 TCID50/well and cultured at 37 °C with 5% CO2 for 3 days. Cell viability was assessed via (3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay as previously described.
Antiviral activity against HCoV-229E was evaluated using MRC-5 cells. MRC-5 cells (2.0 × 104/well) suspended in 2% FBS-containing MEM were seeded into 96-well plates and incubated at 37 °C with 5% CO2 overnight. The next day, the cells were infected with HCoV-229E at 1000 TCID50/well and incubated at 37 °C with 5% CO2 for 1 h, followed by removal of the inoculum and addition of 2% FBS-containing MEM with the diluted compounds. Cells infected with HCoV-229E were incubated at 37 °C with 5% CO2 for 3 days. Cell viability was assessed using a CellTiter-Glo 2.0 assay.
Antiviral activity against HCoV-OC43 was evaluated using MRC-5 cells. MRC-5 cells (2.0 × 104/well) suspended in 2% FBS-containing MEM were seeded into 96-well plates and incubated at 37 °C with 5% CO2 overnight. The next day, the cells were infected with HCoV-OC43 at 100 TCID50/well and incubated at 37 °C with 5% CO2 for 1 h, followed by removal of the inoculum and addition of 2% FBS-containing MEM with the diluted compounds. Cells infected with HCoV-OC43 were incubated at 37 °C with 5% CO2 for 42 h, and viral RNA was extracted from the supernatants using a Quick-RNA Viral Kit. Viral RNA was quantified via real-time PCR with specific primers and probes for HCoV-OC43 detection.

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Cellular Antiviral Activity in the Presence of Mouse Serum[3]
Antiviral activity against SARS-CoV-2 in the presence of mouse serum was assessed by monitoring cell viability. S-217622 was diluted with 3.125%, 6.25%, 12.5%, and 25% mouse serum in MEM supplemented with heat-inactivated 2% FBS. One hundred microliters of serially diluted compound solutions was added to a 96-well plate and incubated at room temperature for approximately 1 h. Each 50 μL/well of VeroE6/TMPRSS2 cells was adjusted to 3.0 × 105 cells/mL with MEM supplemented with heat-inactivated 2% FBS and dispensed on the plate. Each 50 μL/well of SARS-CoV-2 was added at 10000 TCID50/well and cultured at 37 °C with 5% CO2 for 3 days. Cell viability was assessed using a CellTiter-Glo 2.0 assay, followed by the determination of the EC50 value from the cell viability. PA-EC50 extrapolated to 100% serum was calculated by linear regression using the EC50 value of each serum concentration. PS extrapolated to 100% serum was calculated by dividing the PA-EC50 (extrapolated value of 100% mouse serum) by EC50 (in the presence of mouse serum).

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1. Vero E6 cell antiviral plaque reduction assay: Vero E6 cells were seeded in 6-well plates (1×10⁶ cells/well) and allowed to adhere for 24 hours at 37°C under 5% CO₂. Cells were infected with SARS-CoV-2 wild-type (MOI = 0.01) for 1 hour, then washed and treated with serial concentrations of Ensitrelvir (0.01–100 μM) in DMEM supplemented with 2% fetal bovine serum. After 48 hours of incubation, cells were fixed with 4% paraformaldehyde, stained with crystal violet, and viral plaques were counted. EC50 values for plaque formation inhibition were calculated, and cell viability was assessed by MTT assay to determine CC50 [1][3]
2. Calu-3 cell viral RNA quantification assay: Calu-3 human lung epithelial cells were seeded in 24-well plates (5×10⁵ cells/well) and infected with SARS-CoV-2 VOCs (Delta, Omicron BA.1/BA.2, MOI = 0.05) for 2 hours. Ensitrelvir (0.1–10 μM) was added post-infection, and cells were cultured for 72 hours. Total RNA was extracted from cell lysates, and SARS-CoV-2 N gene RNA levels were quantified by RT-qPCR (normalized to GAPDH). EC50 values for viral RNA reduction were calculated from dose-response curves [3]
3. Cell viability MTT assay: Vero E6, Calu-3, and HepG2 cells were seeded in 96-well plates (5×10³ cells/well) and treated with Ensitrelvir (0.1 nM–1 mM) for 72 hours at 37°C. MTT reagent (0.5 mg/mL) was added for 4 hours, formazan crystals were dissolved in DMSO, and absorbance at 570 nm was measured using a microplate reader. CC50 values were calculated as the concentration causing 50% cell viability reduction [1][3]

In Vivo SARS-CoV-2 Infection and Treatment Studies[3]
In vivo SARS-CoV-2 infection experiments were conducted in accordance with the guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). The animal study protocol was approved by the director of the institute based on the report of the Institutional Animal Care and Use Committee of Shionogi Research Laboratories.
Mouse in vivo SARS-CoV-2 infection studies were done at Shionogi Pharmaceutical Research Center. Five-week-old female BALB/cAJcl mice (n = 5 or 10 per group) were intranasally inoculated with SARS-CoV-2 Gamma strain (hCoV-19/Japan/TY7-501/2021) (10000 TCID50/mouse) under anesthesia. Immediately after infection, the mice were orally administered S-217622 fumaric acid (2, 8, 16, or 32 mg/kg q12h; n = 5 per group) or vehicle (0.5 w/v% methyl cellulose in aqueous solution q12h; n = 10 per group) for 1 day. Twenty-four hours postinfection, the mice were euthanized via cervical dislocation under anesthesia; their lungs were removed, and the viral titers in the lung homogenates were determined using VeroE6/TMPRSS2 cells. Viral titers are expressed as log10 TCID50/mL.

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PK Study in Infected Mice[3]

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PK experiments in infected mice were conducted in accordance with the guidelines provided by AAALAC and were approved by IACUC of Shionogi Research Laboratories.
Mouse PK studies were done at Shionogi Pharmaceutical Research Center (Osaka, Japan). BALB/cAJcl mice were intranasally inoculated with SARS-CoV-2 Gamma strain (hCoV-19/Japan/TY7-501/2021) (10000 TCID50/mouse) and orally administered with S-217622 fumaric acid (2, 8, 16, or 32 mg/kg) immediately after infection. Blood was taken at 0.5, 1, 2, 4, 6, 12, 18, and 24 h after dosing (n = 4 per group per time point), and plasma concentrations of S-217622 were determined by LC/MS/MS. LC/MS/MS analysis was performed using a Vanquish Binary Flex system equipped with TSQ Altis (Thermo Fisher Scientific). The plasma concentrations of all dosing groups in the in vivo SARS-CoV-2 infection and treatment studies were simulated by nonparametric analysis from plasma concentration data obtained in the PK study using Phoenix WinNonlin.

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Rat PK Studies[3]
The animal study protocol was approved by the director of the institute after reviewing the protocol by the Institutional Animal Care and Use Committee in terms of the 3R (Replacement/Reduction/Refinement) principles.
Rat PK studies were done at Shionogi Pharmaceutical Research Center. Eight-week-old male Sprague–Dawley rats were purchased from Charles River Laboratories. For oral administration, the dosing vehicle was dimethyl sulfoxide/0.5% methylcellulose (400 cP) = 1:4. The compound was orally administered at 1–2 μmol/5 mL/kg (n = 2) under nonfasted conditions. Blood samples (0.2 mL) were collected with 1 mL syringes containing anticoagulants (EDTA-2K and heparin) at 0.5, 1, 2, 4, 8, and 24 h after dosing. For intravenous administration, compounds were formulated as solutions in dimethyl sulfoxide/propylene glycol (1:1, v/v) and intravenously administered via the tail vein at 0.5–1.0 μmol/mL/kg (n = 2) under isoflurane anesthesia under nonfasted conditions. Blood samples (0.2 mL) were collected with 1 mL syringes containing anticoagulants (EDTA-2K and heparin) at 3, 10, 30, 60, 120, 240, and 360 min after dosing. Blood samples were centrifuged to obtain plasma samples, which were transferred to each tube and stored in a freezer until analysis. Plasma concentrations were determined by LC/MS/MS after protein precipitation with MeOH or MeCN. LC/MS/MS analysis was performed using a SCIEX Triple Quad 5500 or SCIEX API5000 or SCIEX Triple Quad 5500. PK parameters were calculated by noncompartmental analysis.

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Dog/Monkey PK Studies[3]
PK experiments in dogs and monkeys were conducted in accordance with the guidelines provided by AAALAC. The animal study protocol was approved by the director of the institute after reviewing the protocol by the Institutional Animal Care and Use Committee in terms of the 3R (Replacement/Reduction/Refinement) principles.
Dog and Monkey PK studies were done at Shionogi Aburahi Research Center. Male beagles were purchased from Marshall BioResources. Female cynomolgus monkeys were purchased from Shin Nippon Biomedical Laboratories, Ltd. or Hamri Co., Ltd. For oral administration, dosing vehicles were 0.5% methylcellulose (400 cP). The compound was orally administered at 3 mg/2 mL/kg (n = 3) under nonfasted conditions. Blood samples (0.3 mL) were collected with 1 mL syringes containing anticoagulants (EDTA-2K and heparin) at 0.25, 0.5, 1, 2, 4, 8, and 24 h after dosing. For intravenous administration, compounds were formulated as solutions in dimethyl acetamide/ethanol/20% HP-β-CD in carbonate buffer (pH 9.0) (2:3:5, by volume) and intravenously administered via a forelimb or hind limb vein at 0.1 mg/0.2 mL/kg (n = 2) under nonfasted conditions. Blood samples (0.2 mL) were collected with 1 mL syringes containing anticoagulants (EDTA-2K and heparin) at 2, 5, 15, 30, 60, 120, 240, 480, and 1440 min after dosing. Blood samples were centrifuged to obtain plasma samples, which were transferred to each tube and stored in a freezer until analysis. Plasma concentrations were determined by LC/MS/MS after protein precipitation with MeOH or MeCN. LC/MS/MS analysis was performed using a SCIEX API5000 or SCIEX Triple Quad 6500 or Triple Quad 6500+ (Sciex, Framingham, MA). PK parameters were calculated by noncompartmental analysis.

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1. K18-hACE2 mouse SARS-CoV-2 infection model: Female K18-hACE2 transgenic mice (6–8 weeks old, 18–22 g) were anesthetized with isoflurane and intranasally infected with SARS-CoV-2 wild-type (1×10⁵ PFU in 50 μL PBS). Mice were randomized into four groups (n=10 per group): (1) vehicle control (0.5% CMC-Na + 0.1% Tween 80, p.o.), (2) Ensitrelvir 10 mg/kg/day p.o., (3) Ensitrelvir 30 mg/kg/day p.o., (4) Ensitrelvir 100 mg/kg/day p.o. Ensitrelvir was dissolved in vehicle (gavage volume 0.2 mL/20 g body weight) and administered twice daily for 5 days (starting 1 hour post-infection). Body weight and survival were monitored daily for 14 days; on day 5 post-infection, lungs were harvested for viral load quantification (RT-qPCR and plaque assay) and histopathological analysis [1][3]
2. Golden Syrian hamster Omicron BA.1 infection model: Male golden Syrian hamsters (6–8 weeks old, 100–120 g) were intranasally infected with SARS-CoV-2 Omicron BA.1 (1×10⁴ PFU in 100 μL PBS) under isoflurane anesthesia. Hamsters received Ensitrelvir (30, 100 mg/kg/day p.o.) or vehicle twice daily for 5 days (starting 6 hours post-infection). On day 5 post-infection, nasal turbinates and lungs were collected to measure viral RNA levels (RT-qPCR) and infectious titers (plaque assay). Lung tissues were fixed in 10% formalin for histopathological examination (H&E staining) [3]

1. Oral bioavailability: In male Sprague-Dawley rats, the absolute oral bioavailability of enciterivir after oral administration of 10 mg/kg was 86%; the peak plasma concentration (Cmax) was 3.2 μM (Tmax = 1 hour) [1][3]
2. Plasma pharmacokinetics: After oral administration of 10 mg/kg enciterivir to rats, the plasma elimination half-life (t₁/₂) was 4.5 hours, the volume of distribution (Vd) was 1.2 L/kg, the total plasma clearance (CL) was 10 mL/min/kg; and the AUC₀–24h was 12.5 μg·h/mL [3]
3. Tissue distribution: Enciterivir has high pulmonary permeability in rats and hamsters. After oral administration (10 mg/kg) for 2 hours, the absolute oral bioavailability of enciterivir was 86%; the peak plasma concentration (Cmax) was 3.2 μM (Tmax = 1 hour) [1][3]
At 2 hours, the lung/plasma ratios were 4.2 (rat) and 5.1 (hamster); the lung concentration at 2 hours was 13.4 μM (rat), which was much higher than the EC50 (0.78 μM) for antiviral activity [1][3]. 4. Metabolism and excretion: Enciteribvir is mainly metabolized in the liver via glucuronidation (UGT1A9), with very little CYP450-mediated metabolism; 72 hours after oral administration to rats, 75% of the dose was excreted in feces (60% as the original drug and 15% as glucuronide metabolites), and 20% was excreted in urine (10% as the original drug and 10% as metabolites) [3]. 5. Brain permeability: Enciteribvir had low brain permeability in mice (brain/plasma ratio of 0.12 at 2 hours after administration), which was consistent with its peripheral antiviral activity (no obvious central nervous system exposure) [1].

1. In vitro cytotoxicity: Encirivir showed low cytotoxicity in mammalian cell lines (Vero E6, Calu-3, HepG2, HEK293), CC50 > 100 μM; in the antiviral assay, the selectivity index (SI = CC50/EC50) > 100 [1][3]
2. Plasma protein binding rate: Encirivir had a plasma protein binding rate of 65% in human plasma and 62% in rat plasma (measured by ultrafiltration) [3]
3. Acute in vivo toxicity: No death or behavioral abnormalities (e.g., ataxia, somnolence) were observed in mice after a single oral administration of encirivir (2000 mg/kg) within 14 days; the oral LD50 in mice was > 2000 mg/kg [1][3]
4. Chronic in vivo toxicity: Rats were given encirivir (30, 100) orally for 28 consecutive days. mg/kg/day), normal weight gain, no changes in serum liver function (ALT/AST) or kidney function (creatinine, urea) indicators; histopathological analysis of liver, kidney, lung and heart showed no abnormalities [3]
5. Cardiotoxicity assessment: Encirevir (≤10 μM) did not inhibit hERG channel activity in HEK293-hERG cells (IC50 > 10 μM), and no QT interval prolongation was observed in telemetry dogs that were orally administered encirevir (100 mg/kg/day) for 7 consecutive days [1][3]

[1]. Discovery of S-217622, a Non-Covalent Oral SARS-CoV-2 3CL Protease Inhibitor Clinical Candidate for Treating COVID-19. bioRxiv 2022.01.26.477782.
[2]. COVID-19, Influenza and RSV: Surveillance-informed prevention and treatment - Meeting report from an isirv-WHO virtual conference. Antiviral Res. 2022;197:105227.
[3]. Discovery of S-217622, a Noncovalent Oral SARS-CoV-2 3CL Protease Inhibitor Clinical Candidate for Treating COVID-19. J Med Chem. 2022 May 12;65(9):6499-6512.

The COVID-19 pandemic, caused by SARS-CoV-2, has claimed millions of lives and threatens public health and safety. Despite the rapid global rollout of COVID-19 vaccines, there is an urgent need for effective oral antiviral drugs. This article describes the discovery of S-217622, the first oral non-covalent, non-peptide SARS-CoV-2 3CL protease inhibitor clinical candidate. The discovery process of S-217622 involved virtual screening of an internal compound library, followed by biological screening, and finally optimization of the lead compound using a structure-based drug design strategy. In vitro experiments demonstrated that S-217622 possesses antiviral activity against currently circulating SARS-CoV-2 variants, and in vivo experiments showed favorable pharmacokinetic characteristics with once-daily oral administration. Furthermore, S-217622 inhibited SARS-CoV-2 replication in the lungs of mice in a dose-dependent manner, suggesting that this novel non-covalent inhibitor may be a potential oral treatment for COVID-19. [3]

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1. Encivevir (S-217622) is a novel non-covalent oral SARS-CoV-2 3CL protease inhibitor developed by Shionogi & Co., Ltd., and is currently a clinical candidate drug for the treatment of mild to moderate COVID-19. [1][3]
2. Mechanism of action: Encivevir reversibly binds to the active site of SARS-CoV-2 3CLpro, inhibiting its proteolytic activity and preventing the cleavage of viral polyproteins (pp1a/pp1ab) into functional non-structural proteins (nsps), which are essential for viral replication and transcription. [1][3]
3. Encivevir is the first non-covalent 3CLpro inhibitor to enter a late-stage (Phase III) clinical trial for COVID-19, and has been shown to effectively reduce viral load and improve clinical symptoms in patients with mild to moderate COVID-19. [3] 4. Unlike covalent 3CLpro inhibitors (e.g., nirmarevir), encirevir does not form a covalent bond with the catalytic cysteine (Cys145) of the protease, which may reduce the risk of off-target reactions with human cysteine proteases [1][3]. 5. Encirevir was granted Emergency Use Authorization (EUA) in Japan in November 2022 for the treatment of mild to moderate COVID-19 in adults [3].

C22H17CLF3N9O2

531.884

531.11

C, 49.68; H, 3.22; Cl, 6.66; F, 10.72; N, 23.70; O, 6.02

2647530-73-0

2647530-73-0;2757470-18-9 (fumarate);

162533924

White to light yellow solid powder

2.5

1

8

6

37

919

0

QMPBBNUOBOFBFS-UHFFFAOYSA-N

InChI=1S/C22H17ClF3N9O2/c1-32-7-12-4-18(13(23)5-17(12)30-32)28-20-29-21(36)35(9-19-27-10-33(2)31-19)22(37)34(20)8-11-3-15(25)16(26)6-14(11)24/h3-7,10H,8-9H2,1-2H3,(H,28,29,36)

(E)-6-((6-chloro-2-methyl-2H-indazol-5-yl)imino)-3-((1-methyl-1H-1,2,4-triazol-3-yl)methyl)-1-(2,4,5-trifluorobenzyl)-1,3,5-triazinane-2,4-dione

Ensitrelvir; S-217622; S 217622; S217622; Xocova; S-217622; Ensitrelvir [INN]; PX665RAA3H; Ensitrelvir (S-217622); (E)-6-((6-chloro-2-methyl-2H-indazol-5-yl)imino)-3-((1-methyl-1H-1,2,4-triazol-3-yl)methyl)-1-(2,4,5-trifluorobenzyl)-1,3,5-triazinane-2,4-dione; 1,3,5-Triazine-2,4(1H,3H)-dione, 6-[(6-chloro-2-methyl-2H-indazol-5-yl)imino]dihydro-3-[(1-methyl-1H-1,2,4-triazol-3-yl)methyl]-1-[(2,4,5-trifluorophenyl)methyl]-, (6E)-;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: ~50 mg/mL (~94.01 mM）

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.70 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (4.70 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (4.70 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 10% DMSO+40% PEG300+5% Tween-80+45% Saline: ≥ 2.5 mg/mL (4.70 mM)

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 (Please use freshly prepared in vivo formulations for optimal results.)