Danoprevir (ITMN-191)

Catalog No.S1183

Danoprevir(ITMN-191) is a peptidomimetic inhibitor of the NS3/4A protease of hepatitis C virus (HCV) with IC50 of 0.2-3.5 nM, inhibition effect for HCV genotypes 1A/1B/4/5/6 is ~10-fold higher than 2B/3A. Phase 2.

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Danoprevir (ITMN-191) Chemical Structure

Danoprevir (ITMN-191) Chemical Structure
Molecular Weight: 731.83

Validation & Quality Control

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Product Information

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  • Danoprevir (ITMN-191) Mechanism

Product Description

Biological Activity

Description Danoprevir(ITMN-191) is a peptidomimetic inhibitor of the NS3/4A protease of hepatitis C virus (HCV) with IC50 of 0.2-3.5 nM, inhibition effect for HCV genotypes 1A/1B/4/5/6 is ~10-fold higher than 2B/3A. Phase 2.
Targets HCV NS3/4A protease [1]
IC50 0.2 nM-3.5 nM
In vitro Danoprevir (0.29 nM) inhibits the reference genotype 1 NS3/4A protease half-maximally, but a high dose of Danoprevir (10 μM) shows no appreciably inhibition in a panel of 79 proteases, ion channels, transporters, and cell surface receptors. Danoprevir remains bound to and inhibits NS3/4A for more than 5 hours after its initial association. Danoprevir (45 nM) eliminates a patient-derived HCV genotype 1b replicon from hepatocyte-derived Huh7 cells with an EC50 of 1.8 nM. [1] In HCV subgenomic replicon cell lines containing the individual mutations, V36M, R109K, and V170A substitutions confer little or no resistance to Danoprevir, but the R155K substitution confers a high level (62-fold increase) of resistance to Danoprevir. [2] In Huh7.5 cells transfected with chimeric recombinant virus, Danoprevir shows antiviral inhibition effects against HCV genotypes 1, 4 and 6 with IC50 of 2-3 nM, which are >100-fold lower than genotypes 2/3/5 (280-750 nM). [3]
In vivo Danoprevir (30 mg/kg) administered to rats or monkeys shows that its concentrations in liver 12 hours after dosing exceed the Danoprevir concentration required to eliminate replicon RNA from cells. [1]
Features A peptidomimetic inhibitor of the NS3/4A protease of hepatitis C virus (HCV).

Protocol(Only for Reference)

Kinase Assay: [1]

Continuous fluorescent resonance energy transfer (FRET) assay The assay buffer contains 25 μM NS4A peptide, 50 mM Tris-HCl, pH 7.5, 15% (vol/vol) glycerol, 0.6 mM lauryldimethylamine N-oxide, 10 mM dithiothreitol, and 0.5 μM fluorescein/QXL520-labeled FRET substrate {Ac-DE-Dap(QXL520)-EE-Abu-ψ-[COO]-AS-Cys(5-FAMsp)-NH2}. K2040 enzyme (50 pM) is added to initiate the reaction. Reactions are set up in black 96-well plates, and fluorescence data is collected. Control reactions lacking inhibitors and enzyme are included. Initial rates are calculated from the linear phase of the reaction (up to 1 hour) and are used to obtain IC50. Recovery of activity from preformed Danoprevir-NS3/4A complex is assessed by preincubating 10 nM NS3/4A with a two-fold excess of Danoprevir in 1× assay buffer for 15 min, followed by a rapid 200-fold dilution of the preformed complex into assay buffer containing substrate. A control reaction with the same final conditions without preincubation of NS3/4A and Danoprevir is initiated by the addition of enzyme to an otherwise-complete reaction mixture. Additional control reactions lack either Danoprevir or NS3. The progress of the reactions is followed over 5 hours.

Cell Assay: [1]

Cell lines Huh7 cells harboring HCV replicon
Concentrations 5 pM - 100 nM
Incubation Time 48 hours
Method Serially diluted Danoprevir is added to Huh7 cells harboring the K2040 replicon 1 day after cell plating. For antiviral assays, after a 48-hour incubation, intracellular RNA is extracted, and the level of HCV replicon RNA is quantified by reverse transcription (RT)-PCR assay with the primers (5'-CACTCCCCTGTGAGGAACTACTG-3' and 5'-AGGCTGCACGACACTCATACT-3') and a probe (5'-6-FAM-CTTCACGCAGAAAGCGTCTAGCCATGG-MGBNFQ-3' using an ABI Prism 7900 sequence detection system. Here, FAM is 6-carboxyfluorescin and MGBNFQ is a molecular-groove binding non-fluorescence quencher specific to the HCV 5' untranslated region. Single-tube reactions are performed using the TaqMan Gold RT-PCR kit. Triplicate reactions for the RNA standards and samples are performed in 50 μL with 5 μL intracellular RNA (50 ng). RT is carried out at 48 °C for 30 min followed by 10 min at 95 °C. The PCR is run as follows: 15 seconds at 95 °C and 1 min at 60 °C for 40 cycles. Each RNA concentration is determined in triplicate. The absolute concentration of replicon RNA is calculated based on its signal relative to that of a standard curve generated by known concentrations of an in vitro-transcribed RNA corresponding to a genotype 1b 5' untranslated region. Replicon levels in the presence of Danoprevir are fitted to a four-parameter logistic function to obtain EC50.

Animal Study: [1]

Animal Models Sprague-Dawley rats, Cynomolgus monkeys
Formulation Dissolved in water. 6 mg/mL for rats and 3 mg/mL for monkeys
Dosages 30 mg/kg
Administration Oral gavage

Conversion of different model animals based on BSA (Value based on data from FDA Draft Guidelines)

SpeciesMouseRatRabbitGuinea pigHamsterDog
Weight (kg)0.020.151.80.40.0810
Body Surface Area (m2)0.0070.0250.150.050.020.5
Km factor36128520
Animal A (mg/kg) = Animal B (mg/kg) multiplied by  Animal B Km
Animal A Km

For example, to modify the dose of resveratrol used for a mouse (22.4 mg/kg) to a dose based on the BSA for a rat, multiply 22.4 mg/kg by the Km factor for a mouse and then divide by the Km factor for a rat. This calculation results in a rat equivalent dose for resveratrol of 11.2 mg/kg.

Rat dose (mg/kg) = mouse dose (22.4 mg/kg) ×  mouse Km(3)  = 11.2 mg/kg
rat Km(6)
1

References

[1] Seiwert SD, et al. Antimicrob Agents Chemother, 2008, 52(12), 4432-4441.

[2] Bartels DJ, et al. J Infect Dis, 2008, 198(6), 800-807.

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Clinical Trial Information( data from http://clinicaltrials.gov, updated on 2016-05-07)

NCT Number Recruitment Conditions Sponsor
/Collaborators
Start Date Phases
NCT01749150 Completed Hepatitis C, Chronic Hoffmann-La Roche April 2013 Phase 2
NCT01714154 Completed Healthy Volunteer Hoffmann-La Roche November 2012 Phase 1
NCT01654211 Completed Healthy Volunteer Hoffmann-La Roche July 2012 Phase 1
NCT01579019 Withdrawn Hepatitis C, Chronic Hoffmann-La Roche July 2012 Phase 2
NCT01592318 Completed Healthy Volunteer Hoffmann-La Roche May 2012 Phase 1

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Chemical Information

Download Danoprevir (ITMN-191) SDF
Molecular Weight (MW) 731.83
Formula

C35H46FN5O9S

CAS No. 850876-88-9
Storage 3 years -20℃powder
6 months-80℃in solvent
Synonyms RG7227
Solubility (25°C) * In vitro DMSO 144 mg/mL (196.76 mM)
Ethanol 144 mg/mL (196.76 mM)
Water <1 mg/mL (<1 mM)
* <1 mg/ml means slightly soluble or insoluble.
* Please note that Selleck tests the solubility of all compounds in-house, and the actual solubility may differ slightly from published values. This is normal and is due to slight batch-to-batch variations.
Chemical Name 2H-Isoindole-2-carboxylic acid, 4-fluoro-1,3-dihydro-, (2R,6S,13aS,14aR,16aS)-14a-[[(cyclopropylsulfonyl)amino]carbonyl]-6-[[(1,1-dimethylethoxy)carbonyl]amino]-1,2,3,5,6,7,8,9,10,11,13a,14,14a,15,16,16a-hexadecahydro-5,16-dioxocyclopropa[e]pyrrolo[1,2-a][1,4]diazacyclopentadecin-2-yl ester

Customer Product Validation (3)


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Rating
Source Mol Biol Evol 2014 31(6), 1546-53. Danoprevir (ITMN-191) purchased from Selleck
Method Western blot
Cell Lines
Concentrations 20 uM
Incubation Time 3 h
Results Using site-directed mutagenesis, we introduced the A156T substitution in the dominant NS3 protease variant of each individual. The catalytic efficiency of the 56 wild-type and 56 mutated proteases was assayed via MAVS cleavage using a bacteriophage λ genetic screen. The enzymatic activities of variant proteases were evaluated by engineering the MAVS cleavage site into the λ cI repressor. The enzymatic activity was related to the activity of a protease variant carrying the lethal substitution S139A. The S139 residue is part of the catalytic triad of the enzyme. As it have previously demonstrated, the expression of the HCV NS3 protease resulted in cleavage of the lambda cI repressor with MAVS cleavage site. Expression of an NS3 protease that included a substitution in catalytic residue S139 completely abolished the cleavage of lambda cI repressor.

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Rating
Source Antimicrob Agents Chemother 2012 56(10), 5365-73. Danoprevir (ITMN-191) purchased from Selleck
Method Firefly luciferase activity assay
Cell Lines HCV-Feo replicon-containing cells
Concentrations 900 nM
Incubation Time 72 h
Results To examine the inhibitory effect of direct-acting antivirals (DAAs), we exposed the replicons to various concentrations of danoprevir, an NS3 protease inhibitor. Danoprevir are highly potent against genotype 1b replicons, with reported 50% effective concentrations (EC50s) of 1.8 nM and 0.2 to 0.5 uM. Results showed that genotype 2a and 4a replicons, with average EC50s of 65 and 1.2 nM, respectively, were sensitive to danoprevir, whereas the EC50 for genotype 3a replicons was 900 nM, suggesting a lower inhibitory effect of danoprevir against genotype 3a.

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Rating
Source PLoS One 2012 7, e42481. Danoprevir (ITMN-191) purchased from Selleck
Method Virus Titring
Cell Lines E.Coli cells
Concentrations 10 μM
Incubation Time 4 h
Results Danoprevir inhibited HCV NS3/4A protease.

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