SARS-CoV-2 3CL protease
The target of Ensitrelvir fumarate (development code: S-217622) is the 3-chymotrypsin-like protease (3CLpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It inhibits SARS-CoV-2 3CLpro with an IC50 of 0.31 μM (enzyme assay) and suppresses SARS-CoV-2 (wild-type) replication in Vero/hACE2 cells with an EC50 of 0.46 μM. It also shows potent activity against SARS-CoV-2 variants: Delta (EC50: 0.29 μM) and Omicron (BA.1) (EC50: 0.62 μM) in Vero/hACE2 cells [2]
; No relevant information about Ensitrelvir fumarate was found in reference [1].

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. SARS-CoV-2 3CLpro inhibition: Ensitrelvir fumarate potently inhibited the catalytic activity of recombinant SARS-CoV-2 3CLpro. Using a fluorogenic peptide substrate, enzyme activity was monitored via fluorescence intensity (excitation: 355 nm, emission: 460 nm) at 37°C, and the IC50 was determined to be 0.31 μM with concentration-dependent inhibition [2]
2. SARS-CoV-2 replication inhibition: In Vero/hACE2 cells infected with SARS-CoV-2 (wild-type, MOI=0.01), Ensitrelvir fumarate (0.03–30 μM) was incubated for 48 hours. Viral load (quantified by qRT-PCR targeting the N gene) was significantly reduced, with an EC50 of 0.46 μM. For Delta and Omicron (BA.1) variants, EC50 values were 0.29 μM and 0.62 μM, respectively. The compound’s CC50 (cell cytotoxicity) in Vero/hACE2 cells was >30 μM, resulting in a therapeutic index (TI=CC50/EC50) of >65 for wild-type virus [2]
3. Cross-activity against other coronaviruses: Ensitrelvir fumarate inhibited human coronavirus OC43 (HCoV-OC43) replication in MRC-5 cells (EC50: 1.8 μM) after 72 hours of incubation at 33°C (assessed via cytopathic effect, CPE) [2]

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. Efficacy in SARS-CoV-2 (wild-type)-infected hACE2 mice: Female hACE2 transgenic mice (6–8 weeks old) were intranasally inoculated with 10^4 PFU of SARS-CoV-2. Ensitrelvir fumarate (10, 30, 100 mg/kg, oral) was administered once daily for 5 days starting at 1 hour post-infection (vehicle: 0.5% methylcellulose). On day 6 post-infection, lung viral load (qRT-PCR) was reduced by 1.6 log10, 2.2 log10, and 2.8 log10 copies/g tissue in the 10, 30, and 100 mg/kg groups, respectively (vs. vehicle). Histopathology showed reduced lung inflammation (inflammatory cell infiltration, alveolar damage, interstitial edema) in the 30 and 100 mg/kg groups [2]
2. Efficacy in SARS-CoV-2 Omicron (BA.1)-infected hACE2 mice: Mice were inoculated with 10^5 PFU of Omicron (BA.1). Ensitrelvir fumarate (30, 100 mg/kg, oral) was dosed once daily for 5 days starting at 24 hours post-infection. On day 6, lung viral load was reduced by 1.9 log10 and 2.5 log10 copies/g tissue in the 30 and 100 mg/kg groups, respectively (vs. vehicle) [2]
;

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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SARS-CoV-2 3CLpro inhibitory activity assay: Recombinant SARS-CoV-2 3CLpro (catalytic domain) was used. The reaction mixture contained the enzyme, Ensitrelvir fumarate (0.01–10 μM), and a fluorogenic peptide substrate (mimicking the natural 3CLpro cleavage site) in Tris-HCl buffer (pH 7.5, with DTT and glycerol). The reaction was initiated by adding the substrate, incubated at 37°C, and fluorescence intensity (excitation: 355 nm, emission: 460 nm) was monitored for 30 minutes to measure substrate cleavage. The percentage of enzyme activity (vs. vehicle) was plotted against log drug concentration, and IC50 was calculated via four-parameter logistic regression [2]
;

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.

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1. SARS-CoV-2 replication inhibition assay (Vero/hACE2 cells): Vero/hACE2 cells (80–90% confluent in 96-well plates) were infected with SARS-CoV-2 (wild-type/Delta/Omicron BA.1, MOI=0.01) and adsorbed for 1 hour at 37°C (5% CO2). After washing with PBS, Ensitrelvir fumarate (0.03–30 μM) was added, and cells were incubated for 48 hours. Viral load in supernatants was quantified by qRT-PCR (N gene), and EC50 was calculated via four-parameter logistic regression (percentage of replication inhibition vs. log drug concentration) [2]
2. Cell cytotoxicity assay (CC50): Vero/hACE2 cells in 96-well plates were treated with Ensitrelvir fumarate (0.1–100 μM) and incubated for 48 hours. Cell viability was measured via a tetrazolium salt-based kit (absorbance at 450 nm). CC50 (50% viability reduction) was calculated via four-parameter logistic regression (percentage of viability vs. log drug concentration) [2]
3. HCoV-OC43 replication inhibition assay (MRC-5 cells): Confluent MRC-5 cells were infected with HCoV-OC43 (MOI=0.1) for 1 hour. After washing, Ensitrelvir fumarate (0.1–30 μM) was added, and cells were incubated for 72 hours at 33°C. Viral replication was assessed via CPE (viability kit), and EC50 was calculated as described above [2]
;

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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1. Efficacy study (SARS-CoV-2 wild-type, hACE2 mice): 6–8-week-old female hACE2 mice were anesthetized with isoflurane and intranasally inoculated with 10^4 PFU of SARS-CoV-2 (50 μL). Ensitrelvir fumarate (10/30/100 mg/kg) was formulated in 0.5% methylcellulose and orally administered once daily for 5 days (starting at 1 hour post-infection); the control group received vehicle. On day 6, mice were euthanized, and lungs were collected. Lung homogenates (with protease inhibitors) were used for qRT-PCR (viral load: log10 copies/g tissue). Fixed lung tissues (10% formalin) were paraffin-embedded, sectioned, H&E-stained, and scored for inflammation (infiltration, alveolar damage, edema) [2]
2. Efficacy study (SARS-CoV-2 Omicron BA.1, hACE2 mice): Mice were inoculated with 10^5 PFU of Omicron BA.1. Ensitrelvir fumarate (30/100 mg/kg, oral) was dosed once daily for 5 days starting at 24 hours post-infection. On day 6, lung viral load was measured via qRT-PCR [2]
;

1. Oral bioavailability: Mice (10 mg/kg, orally), Cmax = 2.8 μg/mL, AUC0-∞ = 12.6 μg·h/mL, bioavailability approximately 42%. Rats (10 mg/kg, orally), Cmax = 1.9 μg/mL, AUC0-∞ = 9.8 μg·h/mL, bioavailability approximately 35%. Beagle (5 mg/kg, orally), Cmax=3.1 μg/mL, AUC0-∞=18.5 μg·h/mL, bioavailability approximately 58% [2]
2. Plasma half-life (t1/2): Mice (10 mg/kg, orally): 2.3 hours; Rats (10 mg/kg, orally): 3.1 hours; Beagle (5 mg/kg, orally): 4.5 hours [2]
3. Tissue distribution: In mice (10 mg/kg, orally), the lung concentration 1 hour after administration was 3.2 μg/g (higher than the plasma concentration: 2.8 μg/mL), and the lung-to-plasma ratio was approximately 1.1 [2]
4. Metabolism: Human liver microsomes showed that enciterivir fumarate is mainly metabolized by CYP3A4. At concentrations ≤30 μM, it does not inhibit major CYP isoenzymes (1A2, 2C9, 2C19, 2D6, 3A4) (low likelihood of drug interaction) [2] 5. Excretion: In rats (5 mg/kg, intravenously), 28% of the dose was excreted unchanged in the urine (24 hours) and 52% in the feces (72 hours) [2]

1. Acute toxicity: Mice were given a single oral dose of up to 1000 mg/kg of encitevir fumarate. No death or toxic symptoms (behavioral abnormalities, weight loss, organ damage) were observed within 14 days.[2] 2. Plasma protein binding: The binding rates were 92–94% (human plasma), 90–93% (mouse plasma), 88–91% (rat plasma) and 93–95% (dog plasma), respectively.[2] 3. Subacute toxicity: Rats were given encitevir fumarate (30/100/300 mg/kg/day) orally for 14 consecutive days. No deaths occurred; a dose of 300 mg/kg/day resulted in a slight increase in liver weight in male rats (no histopathological changes were observed). No abnormal clinical chemistry (ALT, AST, creatinine) or hematological parameters were found [2]
4. Genotoxicity: At the maximum test concentration, encitelivir fumarate was negative in both the Ames test (bacterial reverse mutation) and the in vitro chromosomal aberration test (CHO cells) [2]
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[1]. COVID-19, Influenza and RSV: Surveillance-informed prevention and treatment - Meeting report from an isirv-WHO virtual conference. Antiviral Res. 2022;197:105227.

[2]. 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.

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. Mechanism and R&D Status: Encitevir fumarate (S-217622) is an oral, non-covalent SARS-CoV-2 3CLpro inhibitor (a clinical candidate for COVID-19). It binds to the 3CLpro active site and inhibits the cleavage of viral polyproteins (pp1a/pp1ab) into functional non-structural proteins, thereby blocking viral replication [2]
2. Selectivity: Encitevir fumarate does not inhibit human proteases (cathepsin L, chymotrypsin, trypsin, papain) at concentrations ≤100 μM, reducing the risk of off-target effects [2]
3. Stability: It exhibits good chemical stability under physiological conditions (pH 1–7.4) and in simulated gastric/intestinal fluids (supporting oral administration) [2]

C26H21CLF3N9O6

647.949853658676

647.125

C, 48.20; H, 3.27; Cl, 5.47; F, 8.80; N, 19.46; O, 14.81

2757470-18-9

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

162623410

White to off-white solid powder

3

12

8

45

1040

0

CN1C=C2C=C(C(=CC2=N1)Cl)NC3=NC(=O)N(C(=O)N3CC4=CC(=C(C=C4F)F)F)CC5=NN(C=N5)C.C(=C/C(=O)O)\C(=O)O

FBOCUALVLIWPNQ-WLHGVMLRSA-N

InChI=1S/C22H17ClF3N9O2.C4H4O4/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)245-3(6)1-2-4(7)8/h3-7,10H,8-9H2,1-2H3,(H,28,29,36)1-2H,(H,5,6)(H,7,8)/b2-1+

(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 fumaric acid

S 217622; S217622; Xocova;S-217622

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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 (~77.17 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (3.86 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 (3.86 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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 (3.86 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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 (Please use freshly prepared in vivo formulations for optimal results.)