Home Proteases HCV Protease inhibitor - COVID-19 inhibitor Danoprevir For research use only.

Danoprevir

Synonyms: ITMN-191, RG7227

Danoprevir 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.

Danoprevir Chemical Structure

Danoprevir Chemical Structure

CAS No. 850876-88-9

Purity & Quality Control

Batch: S118301 DMSO]144 mg/mL]false]Ethanol]144 mg/mL]false]Water]Insoluble]false Purity: 99.94%
99.94

Danoprevir Related Products

Signaling Pathway

HCV Protease Signaling Pathways

HCV Protease Signaling Pathway

Biological Activity

Description Danoprevir 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.
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
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]
Kinase Assay 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, 10 mM dithiothreitol, and 0.5 μM 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 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.
In Vivo
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]
Animal Research Animal Models Sprague-Dawley rats, Cynomolgus monkeys
Dosages 30 mg/kg
Administration Oral gavage
NCT Number Recruitment Conditions Sponsor/Collaborators Start Date Phases
NCT01714154 Completed
Healthy Volunteer
Hoffmann-La Roche
 November 2012 Phase 1
NCT01592318 Completed
Healthy Volunteer
Hoffmann-La Roche
 May 2012 Phase 1
NCT01588002 Completed
Healthy Volunteer
Hoffmann-La Roche
 April 2012 Phase 1
NCT01519336 Completed
Healthy Volunteer
Hoffmann-La Roche
 February 2012 Phase 1

Chemical Information & Solubility

Molecular Weight 731.83 Formula

C35H46FN5O9S
CAS No. 850876-88-9 SDF Download Danoprevir SDF
Smiles CC(C)(C)OC(=O)NC1CCCCCC=CC2CC2(NC(=O)C3CC(CN3C1=O)OC(=O)N4CC5=C(C4)C(=CC=C5)F)C(=O)NS(=O)(=O)C6CC6
Storage (From the date of receipt)

In vitro
Batch:

DMSO : 144 mg/mL ( (196.76 mM) Moisture-absorbing DMSO reduces solubility. Please use fresh DMSO.)

Ethanol : 144 mg/mL

Water : Insoluble


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Tech Support

Answers to questions you may have can be found in the inhibitor handling instructions. Topics include how to prepare stock solutions, how to store inhibitors, and issues that need special attention for cell-based assays and animal experiments.

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