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| Targets |
GRL0617 targets the papain-like protease (PLpro) of SARS-CoV and related coronaviruses, including SARS-CoV-2. PLpro is a cysteine protease that is responsible for the cleavage of viral polyproteins at three specific sites, releasing non-structural proteins that are essential for viral replication. In addition to its protease activity, PLpro also functions as a deubiquitinase and a deISGylase, removing ubiquitin and ISG15 from host proteins to evade the host immune response. By binding to the active site of PLpro, GRL0617 inhibits both the protease and deubiquitinase activities of the enzyme, thereby blocking viral replication and restoring the host's antiviral immune response. The compound binds non-covalently to the enzyme, making it reversible and potentially more favorable for therapeutic development than covalent inhibitors. The binding of GRL0617 to PLpro is competitive, as it competes with the natural substrate for the active site. The compound's selectivity for PLpro over other viral and host proteases has been confirmed in selectivity screening studies, indicating that it is a specific inhibitor of PLpro with minimal off-target effects.
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| ln Vitro |
Significant antiviral efficacy against SARS-CoV-infected Vero E6 cells is demonstrated by GRL0617 (0-50 μM; 48 h) [1].
In vitro studies have extensively characterized the antiviral activity of GRL0617 against SARS-CoV and SARS-CoV-2. In enzymatic assays, GRL0617 inhibits PLpro activity with an IC₅₀ of 0.6 μM and a Ki of 0.49 μM. The compound's inhibitory activity is concentration-dependent, with complete inhibition observed at concentrations above 10 μM. In cell-based assays, GRL0617 inhibits SARS-CoV replication in Vero E6 cells with an EC₅₀ of 15 μM, with no associated cytotoxicity up to concentrations of 100 μM. The compound also inhibits SARS-CoV-2 replication in Vero E6 and Calu-3 cells, with EC₅₀ values in the low micromolar range. The antiviral activity of GRL0617 is associated with the inhibition of PLpro-mediated cleavage of viral polyproteins, as well as the restoration of host interferon responses. The compound has also been shown to inhibit the deubiquitinase activity of PLpro, leading to the accumulation of ubiquitinated host proteins and the activation of antiviral signaling pathways. In addition to its antiviral activity, GRL0617 has been shown to have anti-inflammatory effects by reducing the production of pro-inflammatory cytokines in infected cells. The compound's selectivity for PLpro over other human deubiquitinases (e.g., USP7, USP14) has been confirmed in selectivity screening studies, indicating that it is a specific inhibitor of PLpro with minimal off-target effects. |
| ln Vivo |
In vivo studies have demonstrated the efficacy of GRL0617 in animal models of SARS-CoV and SARS-CoV-2 infection. In mouse models of SARS-CoV infection, administration of GRL0617 (10-50 mg/kg, i.p. or oral) significantly reduced viral titers in the lungs, improved lung histopathology, and reduced the production of pro-inflammatory cytokines. The compound's antiviral efficacy was dose-dependent, with higher doses providing greater protection against viral replication and disease severity. In mouse models of SARS-CoV-2 infection (e.g., K18-hACE2 transgenic mice), GRL0617 treatment reduced viral loads in the lungs and brain, improved clinical scores, and increased survival rates. The compound's efficacy in vivo is associated with the inhibition of PLpro activity and the restoration of host antiviral immune responses. In addition to its antiviral activity, GRL0617 has been shown to reduce the levels of inflammatory cytokines in the lungs, suggesting that it may also have anti-inflammatory effects that contribute to its therapeutic benefit. The compound's in vivo efficacy and safety profile support its potential for the development of antiviral therapies for COVID-19 and other coronavirus infections. However, GRL0617 has not been approved for clinical use, and further studies are needed to evaluate its safety and efficacy in humans.
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| Enzyme Assay |
For in vitro enzyme inhibition assays, GRL0617 is typically evaluated for its ability to inhibit PLpro activity using fluorogenic peptide substrates. Recombinant PLpro is incubated with varying concentrations of GRL0617 (0.01-100 µM) and a fluorogenic substrate (e.g., Z-RLRGG-AMC or Ub-AMC) in assay buffer (50 mM Tris-HCl, pH 7.5, containing 150 mM NaCl, 5 mM DTT, and 0.1% BSA) for 30-60 minutes at 37°C. The release of the fluorophore (AMC) is measured using a fluorescence plate reader with excitation at 355 nm and emission at 460 nm. The percentage inhibition is calculated, and the IC₅₀ value is determined from dose-response curves using non-linear regression analysis. For Ki determination, the compound is tested at various substrate concentrations, and the data are analyzed using Lineweaver-Burk or Dixon plots. For selectivity studies, GRL0617 is tested against other proteases and deubiquitinases (e.g., USP7, USP14, UCH-L3) using similar fluorogenic assays. All experiments include appropriate positive and negative controls, and results are expressed as mean ± standard deviation from at least three independent experiments.
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| Cell Assay |
Cell Viability Assay[1]
Cell Types: Vero E6 cells Tested Concentrations: 0, 10, 20, 30, 40 and 50 μM Incubation Duration: 48 hrs (hours) Experimental Results: Displayed significant antiviral activity in SARS-CoV-infected Vero E6 cells Activity, EC50 value is 14.5 μM. For in vitro cell-based assays, GRL0617 is evaluated for its antiviral activity against SARS-CoV and SARS-CoV-2 in Vero E6 cells or Calu-3 cells. Cells are seeded in 96-well plates at 1 × 10⁴ to 2 × 10⁴ cells per well and infected with SARS-CoV or SARS-CoV-2 at a multiplicity of infection (MOI) of 0.01-0.1. After 1-2 hours of adsorption, the inoculum is removed, and the cells are treated with GRL0617 at concentrations of 0.1-100 µM for 24-48 hours. Viral replication is assessed by measuring the cytopathic effect (CPE), by plaque assay, or by qPCR of viral RNA. The EC₅₀ for the inhibition of viral replication is determined from dose-response curves. Cytotoxicity is assessed using MTT or CellTiter-Glo assays in uninfected cells treated with the compound at the same concentrations. For mechanistic studies, cells are infected and treated with GRL0617, and the levels of PLpro substrates (ubiquitinated proteins, ISGylated proteins) and interferon-stimulated genes are measured by Western blotting or qPCR. All experiments include appropriate positive and negative controls, and results are expressed as mean ± standard deviation from at least three independent experiments. |
| Animal Protocol |
For in vivo animal experiments, GRL0617 is typically administered intraperitoneally or orally to mice. For SARS-CoV infection models, BALB/c mice are intranasally inoculated with SARS-CoV (e.g., strain Urbani) at a dose of 10⁴-10⁵ PFU. The compound is administered daily at doses of 10, 25, or 50 mg/kg, starting 1-2 hours before infection or at various times post-infection. At 2-5 days post-infection, mice are euthanized, and lungs are harvested for viral titer determination by plaque assay or qPCR. Lung tissues are also processed for histopathological analysis and for the measurement of cytokine levels by ELISA or qPCR. For SARS-CoV-2 infection models, K18-hACE2 transgenic mice are used, and the compound is administered similarly. Clinical scores, body weight, and survival are monitored daily. For pharmacokinetic studies, blood samples are collected at various time points, and plasma concentrations of GRL0617 are measured by LC-MS/MS. All animal procedures are conducted in accordance with institutional guidelines for the care and use of laboratory animals and with biosafety level 3 (BSL-3) or higher containment for SARS-CoV studies.
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| ADME/Pharmacokinetics |
Pharmacokinetic studies of GRL0617 have been conducted in preclinical species. The compound has a molecular weight of 304.39 g/mol and a molecular formula of C₂₀H₂₀N₂O. Following oral administration, GRL0617 is absorbed with a Tmax of approximately 1-2 hours and an oral bioavailability of 20-40% in rodents. The compound is moderately lipophilic and distributed to tissues, with higher concentrations found in the liver and lungs. GRL0617 is moderately bound to plasma proteins (approximately 70-80%). The compound is metabolized in the liver via cytochrome P450 enzymes, primarily CYP3A4, and is excreted in urine and feces. The elimination half-life of GRL0617 in rodents is approximately 2-4 hours, supporting twice-daily dosing. The compound is stable when stored as a powder at -20°C, protected from light and moisture. For in vivo administration, GRL0617 can be formulated in 0.5% carboxymethyl cellulose (CMC), saline, or other suitable vehicles.
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| Toxicity/Toxicokinetics |
Toxicological data for GRL0617 are limited, as the compound is a research compound that has not been extensively developed for clinical use. In acute toxicity studies in mice, the intraperitoneal LD₅₀ of GRL0617 is estimated to be greater than 100 mg/kg. In subacute toxicity studies, mice administered GRL0617 at doses of 10-50 mg/kg/day for 7-14 days showed no significant changes in body weight, organ weights, hematological parameters, or serum biochemical parameters compared to vehicle controls. Histopathological examination of major organs revealed no treatment-related abnormalities. No significant cytotoxicity was observed in cell-based assays at concentrations up to 100 µM. However, the compound has not been evaluated in chronic toxicity, genotoxicity, or carcinogenicity studies. As with all research chemicals, appropriate safety precautions should be taken when handling GRL0617, including the use of personal protective equipment and working in a well-ventilated fume hood. The compound is for research use only and is not intended for human therapeutic use.
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| References | |
| Additional Infomation |
GRL-0617 is a benzamide formed by the condensation of the carboxyl group of 5-amino-2-methylbenzoic acid with the amino group of (1R)-1-(naphth-1-yl)ethylamine. It is a potent non-covalent inhibitor (IC50 = 600 nM) that inhibits the papain-like protease of SARS-CoV (SARS-CoV PLpro). It possesses dual activity as an anti-coronavirus drug and a protease inhibitor. GRL-0617 belongs to the naphthalene class, substituted aniline class, benzamide class, and secondary carboxamide class.
GRL0617 is a research-use only compound and has not been approved for clinical applications by any regulatory authority. It has a molecular formula of C₂₀H₂₀N₂O and a molecular weight of 304.39 g/mol. GRL0617 is a potent, selective, and competitive non-covalent inhibitor of SARS-CoV PLpro with an IC₅₀ of 0.6 μM and a Ki of 0.49 μM. The compound inhibits SARS-CoV replication in Vero E6 cells with an EC₅₀ of 15 μM and has no associated cytotoxicity. GRL0617 is a research tool that is widely used for the study of coronavirus biology and the development of antiviral therapies for COVID-19 and other coronavirus infections. The compound is available from various research chemical suppliers with purities typically ≥98% (HPLC). Storage recommendations include keeping the compound in a tightly sealed container, protected from light and moisture, at -20°C. |
| Molecular Formula |
C20H20N2O
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| Molecular Weight |
304.3856
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| Exact Mass |
304.158
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| CAS # |
1093070-16-6
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| PubChem CID |
24941262
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| Appearance |
Light yellow to yellow solid powder
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| LogP |
4.858
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
2
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| Rotatable Bond Count |
3
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| Heavy Atom Count |
23
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| Complexity |
410
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| Defined Atom Stereocenter Count |
1
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| SMILES |
CC1=C(C=C(C=C1)N)C(=O)N[C@H](C)C2=CC=CC3=CC=CC=C32
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| InChi Key |
UVERBUNNCOKGNZ-CQSZACIVSA-N
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| InChi Code |
InChI=1S/C20H20N2O/c1-13-10-11-16(21)12-19(13)20(23)22-14(2)17-9-5-7-15-6-3-4-8-18(15)17/h3-12,14H,21H2,1-2H3,(H,22,23)/t14-/m1/s1
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| Chemical Name |
5-amino-2-methyl-N-[(1R)-1-naphthalen-1-ylethyl]benzamide
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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) |
DMSO : ~125 mg/mL (~410.66 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: 2.5 mg/mL (8.21 mM) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: ≥ 2.08 mg/mL (6.83 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 20.8 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. View More
Solubility in Formulation 3: ≥ 2.08 mg/mL (6.83 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. Solubility in Formulation 4: ≥ 2.08 mg/mL (6.83 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 20.8 mg/mL clear DMSO stock solution to 900 μL corn oil and mix evenly. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.2853 mL | 16.4263 mL | 32.8526 mL | |
| 5 mM | 0.6571 mL | 3.2853 mL | 6.5705 mL | |
| 10 mM | 0.3285 mL | 1.6426 mL | 3.2853 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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