Danoprevir (ITMN-191)

Catalog No.S1183 Synonyms: RG7227

Molecular Weight(MW): 731.83

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.

5 Customer Reviews

Effects of TNFα, etanercept, and danoprevir on HCV infection in HFLC. Cells (n=6 wells/condition) were treated with TNFα (20 ng/ml) ± etanercept (1 μg/ml), danoprevir (2 μM), or media control before and during infection with Jc1G. Secreted luciferase was measured 96 hours after the start of infection. Box-and-whiskers plot; whiskers represent minimum and maximum values. Values of p were calculated by one-way ANOVA. ***, p < 0.001; ****, p < 0.0001

Gastroenterology, 2017, 153(2):566-578. Danoprevir (ITMN-191) purchased from Selleck.

Western blot analysis of HCV NS3 protease cleavage of MAVS. The catalytic efficiency of four proteases (H, H-A156T, 41 and 41-A156T) from individuals H and 41 were tested in the absence or presence of an NS3 protease inhibitor (danoprevir). Expression of the HCV NS3 proteases resulted in cleavage of the lambda cI repressor with MAVS cleavage site. Expression of the protease was induced with IPTG for 3 h. The lambda cI repressor with MAVS cleavage site was not cleaved by an NS3 protease that included a substitution in catalytic residue S139A. Similarly, different catalytic efficiencies were observed with different proteases.

Mol Biol Evol 2014 31(6), 1546-53. Danoprevir (ITMN-191) purchased from Selleck.
Antiviral Res, 2018, 150:79-92. Danoprevir (ITMN-191) purchased from Selleck.

Suppression of HCV replication by anti-HCV inhibitors. HCV-Feo replicon-containing cells were seeded in 96-well plates at a density of 1 x 10₄ cells/well. Twenty-four hours later, cells were exposed to the indicated concentrations of danoprevir. After a 72-hour treatment with inhibitors, cells were harvested and firefly luciferase activity was measured. Results are the means and SDs of three independent experiments (upper panels). PrestoBlue cell viability reagent (Invitrogen, CA) was used to examine the cytotoxic effect of inhibitors according to the manufacturer's recommendations. Data are the means and SDs of three independent experiments (lower panels).

Antimicrob Agents Chemother 2012 56(10), 5365-73. Danoprevir (ITMN-191) purchased from Selleck.
Inhibition of HCV and CHV NS3/4A proteases in the presence of the protease inhibitors (A) 25a or (B) danoprevir. The graph illustrates the relative (%) lambda phage titer after selective growth of lambda in E. coli cells coexpressing the NS3/4A protease constructs and the lambda cI repressor expressing either MAVS or TRIF cleavage sites in the absence or presence of protease inhibitors (20 mM). No significant inhibition was detected with an HCV NS3/4A protease mutant carrying the substitution D168V, which confers resistance to protease inhibitors. Values are the means 6 standard deviations (error bars) of at least three experiments.

PLoS One 2012 7, e42481. Danoprevir (ITMN-191) purchased from Selleck.

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Biological Activity

Description

Features

A peptidomimetic inhibitor of the NS3/4A protease of hepatitis C virus (HCV).

Targets

HCV NS3/4A protease ^[1]

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]

Protocol

Kinase Assay:^[1]	+ Expand 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 Research:^[1]	+ Expand 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. (Only for Reference)
Animal Research:^[1]	+ Expand 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 (Only for Reference)

References

Solubility (25°C)

In vitro	DMSO	144 mg/mL (196.76 mM)
	Ethanol	144 mg/mL (196.76 mM)
	Water	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 Information Download Danoprevir (ITMN-191) SDF

Molecular Weight	731.83
Formula	C₃₅H₄₆FN₅O₉S
CAS No.	850876-88-9
Storage	3 years -20°C powder
Storage	2 years -80°C in solvent
Synonyms	RG7227

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Clinical Trial Information (data from https://clinicaltrials.gov, updated on 2018-11-16)

NCT Number	Recruitment	Conditions	Sponsor/Collaborators	Start Date	Phases
NCT03288636	Completed	HCV	Ascletis Pharmaceuticals Co. Ltd.	August 8 2017	Phase 1
NCT03362814	Active not recruiting	HCV	Ascletis Pharmaceuticals Co. Ltd.	July 1 2017	Phase 2\|Phase 3
NCT03020134	Completed	Healthy	Ascletis Pharmaceuticals Co. Ltd.	July 15 2016	Phase 1
NCT03020082	Completed	Chronic Hepatitis C	Ascletis Pharmaceuticals Co. Ltd.	June 2016	Phase 3
NCT03020004	Completed	Chronic Hepatitis C	Ascletis Pharmaceuticals Co. Ltd.	January 2016	Phase 2
NCT03019991	Completed	Healthy	Ascletis Pharmaceuticals Co. Ltd.	October 2015	Phase 1

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HCV Protease Signaling Pathway Map

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Danoprevir (ITMN-191)