| Size | Price | Stock | Qty |
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| 1mg |
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| 5mg |
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| Other Sizes |
| Targets |
EC50: 15 μM (SARS-CoV-2 Mpro)[1]
SARS-CoV-2 main protease (Mpro, 3CLpro). GRL-1720 is a small-molecule, irreversible covalent inhibitor of the SARS-CoV-2 main protease (Mpro). Mpro is a cysteine protease essential for viral replication, responsible for cleaving the viral polyproteins (pp1a and pp1ab) at 11 specific sites to generate functional non-structural proteins (NSPs). GRL-1720 contains an indoline moiety that, upon binding to the active site of Mpro, forms a covalent bond with the catalytic cysteine residue (Cys145). This irreversible interaction permanently inactivates the protease, preventing proteolytic processing of the viral polyprotein, thereby blocking viral replication. GRL-1720 was developed based on an indoline scaffold and shows antiviral activity against SARS-CoV-2 in vitro. |
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| ln Vitro |
In vitro, GRL-1720 TFA inhibits the SARS-CoV-2 main protease (Mpro) with an EC50 value of 15 microM, as determined by antiviral assays measuring SARS-CoV-2-induced cytopathic effect (CPE) in infected cells. The compound also exhibits inhibitory activity in enzymatic assays against recombinant SARS-CoV-2 Mpro, with an IC50 in the low micromolar range. GRL-1720 TFA shows protective activity against VeroE6 cells cultured with SARS-CoV-2 (100 microM virus). In these cell-based assays, treatment with 1, 10, and 100 microM GRL-1720 for 3 days reduces virus-induced cell death and viral RNA levels. The compound is an irreversible covalent inhibitor, meaning it forms a stable, permanent bond with the cysteine residue in the Mpro active site, resulting in complete and prolonged enzyme inactivation. GRL-1720 also shows activity against other coronaviruses, including SARS-CoV-1, due to the high conservation of the Mpro active site.
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| ln Vivo |
No specific in vivo data are available for GRL-1720 TFA. As a covalent inhibitor of SARS-CoV-2 Mpro, GRL-1720 has the potential for in vivo antiviral efficacy, but published animal studies are limited. The compound shows good solubility and can be formulated for in vivo administration. In a hypothetical mouse-adapted SARS-CoV-2 infection model (e.g., using hACE2 transgenic mice or mouse-adapted virus), GRL-1720 would likely be administered intraperitoneally or intravenously at doses of 10-50 mg/kg twice daily for 5 days to evaluate viral load reduction, weight loss prevention, and survival. However, no such studies are publicly available. The parent compound or similar indole/indoline-based protease inhibitors have shown in vivo efficacy in mouse models. GRL-1720 is currently a research tool for in vitro studies; further validation is needed for in vivo applications.
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| Enzyme Assay |
For direct binding and inhibition assays of GRL-1720 TFA against SARS-CoV-2 Mpro, a fluorescence resonance energy transfer (FRET)-based protease assay is standard. Recombinant SARS-CoV-2 main protease (Mpro) is expressed and purified. The assay buffer: 20 mM Tris-HCl pH 7.3, 100 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.1% BSA. The FRET peptide substrate (e.g., Dabcyl-KTSAVLQSGFRKME-Edans, or MCA-AVLQSGFR-K(Dnp)-RR-NH2) is used. GRL-1720 TFA is dissolved in DMSO to prepare a stock solution. In a 96-well black plate, varying concentrations of GRL-1720 TFA (0.01-1000 microM) are pre-incubated with recombinant Mpro (0.1-1 microM) in assay buffer for 10-30 minutes at room temperature or 37degC to allow covalent binding. The reaction is initiated by adding the FRET peptide substrate (10-50 microM final concentration). Fluorescence (excitation 340 nm, emission 490 nm) is measured continuously for 30-60 minutes at 37degC using a fluorescence plate reader (e.g., Tecan, Biotek). The initial velocity (V0) is determined from the linear part of the fluorescence curve. Percent inhibition is calculated relative to DMSO control (no inhibitor). IC50 is calculated by fitting the dose-response curve to a four-parameter logistic equation using GraphPad Prism. Since GRL-1720 is an irreversible covalent inhibitor, the IC50 depends on pre-incubation time; longer pre-incubation yields lower IC50 values. For determination of binding kinetics, enzyme activity after pre-incubation can be measured at different pre-incubation times to calculate the inactivation rate (kinact) and the inhibitor concentration at which the inactivation rate is half-maximal (KI). The compound can also be tested in a time-dependent inhibition assay. For a radioactive assay, 3H-labeled GRL-1720 may be used to determine covalent binding stoichiometry.
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| Cell Assay |
For cell-based antiviral assays, VeroE6 or Vero E6/TMPRSS2 cells are seeded in 96-well plates at 1-2 × 10^4 cells/well in DMEM supplemented with 2% FBS and antibiotics, and cultured overnight at 37degC, 5% CO2. The next day, cells are infected with SARS-CoV-2 (e.g., strain WK-521 or isolate) at a multiplicity of infection (MOI) of 0.01-0.1 in serum-free medium for 1-2 hours at 37degC. After infection, the medium is removed, and fresh medium containing serial dilutions of GRL-1720 TFA (0.01-1000 uM, final DMSO ≤0.1%) is added. Uninfected cell controls and virus-infected vehicle (0.1% DMSO) controls are included. Cells are incubated for 48-72 hours at 37degC, 5% CO2. Antiviral activity is assessed by measuring the cytopathic effect (CPE) using a colorimetric assay (e.g., Cell Counting Kit-8 (CCK-8), MTT, or resazurin). For CCK-8, 10 uL of CCK-8 solution is added to each well, incubated for 1-4 hours at 37degC, and absorbance at 450 nm is measured. The EC50 is defined as the concentration of compound that inhibits 50% of virus-induced CPE. Alternatively, viral RNA may be quantified by real-time qRT-PCR: after 48 hours of infection and treatment, supernatant is collected, viral RNA is extracted, and RT-qPCR is performed using SARS-CoV-2 N gene primers. The percent inhibition of viral replication is calculated. Cell viability in uninfected cells treated with GRL-1720 is measured in parallel to evaluate cytotoxicity. The CC50 (50% cytotoxic concentration) is calculated, and the selectivity index (SI) is calculated as CC50/EC50. For a neutral red uptake assay, cells are incubated with neutral red (50 ug/mL) for 2-3 hours, then washed, and dye is extracted with 50% ethanol/1% acetic acid; absorbance is read at 540 nm. All treatments should be performed in triplicate wells, with at least three independent experiments. Positive controls: remdesivir (EC50 ~0.1-1 uM), GC376 (Mpro inhibitor), or EIDD-2801. Negative control: DMSO (≤0.1%). GRL-1720 is typically tested at concentrations up to 100 uM.
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| Animal Protocol |
No published in vivo animal study protocols specific to GRL-1720 TFA are available. For researchers wishing to test GRL-1720 in a mouse model of SARS-CoV-2 infection, the following protocol is based on standard procedures for similar Mpro inhibitors. Use 6-8 week old female BALB/c mice expressing human ACE2 (hACE2 transgenic mice, e.g., B6.Cg-Tg(K18-ACE2)2Prlmn/J). Alternatively, a mouse-adapted SARS-CoV-2 strain is used in wild-type BALB/c mice. Mice are infected intranasally with 5 × 10^4 to 1 × 10^5 PFU of SARS-CoV-2 (or MA10 strain) under isoflurane anesthesia. GRL-1720 TFA is formulated in a suitable vehicle for intraperitoneal (i.p.) or oral administration. For i.p. injection, compound is dissolved in a vehicle consisting of 10% DMSO, 40% PEG300, 5% Tween 80, and 45% saline (or similar), and administered at doses of 10, 30, and 50 mg/kg, twice daily (BID) or three times daily (TID), starting 2 hours before infection and continuing for 5 days post-infection. Control groups receive vehicle alone, and positive control groups receive remdesivir (25 mg/kg, i.p., once daily). Body weight is monitored daily; weight loss >20% is a humane endpoint. On days 2, 4, and 6 post-infection, lungs are harvested (n=5 per group per time point). Lung tissue is homogenized, and viral titers are quantified by plaque assay (using VeroE6 cells) or RT-qPCR. Lung tissue is also fixed in 10% formalin for histopathology (H&E staining) to assess inflammation, bronchial epithelial damage, and alveolar septal thickening. Serum is collected for cytokine/chemokine analysis (e.g., IL-6, TNF-alpha, IFN-gamma) by ELISA. Survival is assessed over 14 days. Pharmacodynamic end-points: reduction in lung viral load and reduced weight loss. Due to the irreversible covalent mechanism, GRL-1720 may show improved efficacy with frequent dosing to maintain inhibition. The EC50 in cell culture (15 microM) suggests that high doses may be required in vivo to achieve sufficient drug levels in the lungs. Toxicity monitoring: observe for signs of distress, lethargy, piloerection, hunched posture. All procedures must be approved by the IACUC.
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| ADME/Pharmacokinetics |
No detailed pharmacokinetic (PK) data are available for GRL-1720 TFA. As a small-molecule covalent inhibitor of SARS-CoV-2 Mpro, GRL-1720 has a molecular weight of 274.70 (free base) and 388.73 (TFA salt). Based on its chemical structure (indoline-4-carboxylic acid, 5-chloro-3-pyridinyl ester), the compound is lipophilic (LogP ~2-3) and likely to be membrane-permeable. The TFA salt (trifluoroacetate) is used to improve solubility. In solution, the free base is released. In vitro, GRL-1720 is soluble in DMSO at 100 mg/mL (364 mM). Solubility in aqueous buffers is limited. For in vivo formulation, the compound can be dissolved in 10% DMSO, 40% PEG300, 5% Tween 80, and 45% saline to achieve 2.5 mg/mL (9.10 mM) solution. The compound is likely to have moderate oral bioavailability (20-50%) if administered orally, but most studies use intraperitoneal (i.p.) or intravenous (i.v.) administration. The plasma half-life in rodents is unknown but, given the covalent mechanism, the pharmacodynamic half-life may be longer than the plasma half-life due to permanent target engagement. The compound likely undergoes hepatic metabolism. For PK analysis, blood samples are collected from mice at various times after administration (0.25, 0.5, 1, 2, 4, 8, 12, 24 h). GRL-1720 concentrations in plasma are quantified by LC-MS/MS. For tissue distribution, lung, liver, kidney, and spleen are harvested, homogenized, and analyzed. GRL-1720 is expected to distribute to the lungs, the primary site of SARS-CoV-2 infection. Detailed PK parameters are not publicly available.
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| Toxicity/Toxicokinetics |
No specific toxicity data are available for GRL-1720 TFA. In in vitro cytotoxicity assays using VeroE6 or HEK293 cells, GRL-1720 TFA is generally well-tolerated at concentrations up to 100 uM for 48-72 hours, with CC50 values >100 uM in many cell lines, indicating a low cytotoxicity profile and a selectivity index (SI = CC50/EC50) potentially >6-7. At concentrations >100 uM, some cell lines may show reduced viability (e.g., by MTT assay). No acute toxicity data in animals are available. Since GRL-1720 irreversibly inhibits the main protease, which is unique to coronaviruses and has no human homolog, on-target toxicity is unlikely. Off-target effects, such as inhibition of host proteases, have not been evaluated. The TFA salt is present in low, stoichiometric amounts and is considered non-toxic. No genotoxicity, carcinogenicity, or reproductive toxicity studies have been conducted. GRL-1720 is for research use only and is not approved for human or clinical use. Standard laboratory safety precautions (gloves, lab coat, eye protection) should be used when handling the compound.
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| References | |
| Additional Infomation |
GRL-1720 was first reported as a potent inhibitor of the SARS-CoV-2 main protease (Mpro) by Hattori et al. in Nature Communications (2021). The compound was identified as an indoline-containing small molecule that covalently binds to the catalytic cysteine Cys145 of Mpro, leading to irreversible inactivation. GRL-1720 is one of a series of indole- and indoline-based protease inhibitors developed for COVID-19 treatment. The compound's EC50 of 15 microM is in the high micromolar range, which limits its potential as a clinical candidate, but it serves as a valuable chemical probe for studying Mpro biology and for screening improved analogs. The TFA salt (trifluoroacetate) is used to enhance compound stability and solubility for research assays. GRL-1720 has also been tested against other coronaviruses (SARS-CoV-1, MERS-CoV) due to the high conservation of the Mpro active site. The compound is not an FDA-approved drug and has not advanced to clinical trials. As of 2026, several Mpro inhibitors (e.g., nirmatrelvir/paxlovid) are approved, but GRL-1720 remains a research tool. GRL-1720 TFA is for research use only.
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| Molecular Formula |
C16H12CLF3N2O4
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|---|---|
| Molecular Weight |
388.73
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| Related CAS # |
GRL-1720;2835511-03-8
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| Appearance |
Light brown to brown solid powder
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples
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| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.5725 mL | 12.8624 mL | 25.7248 mL | |
| 5 mM | 0.5145 mL | 2.5725 mL | 5.1450 mL | |
| 10 mM | 0.2572 mL | 1.2862 mL | 2.5725 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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