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Research Area
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Description
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Hepatitis C |
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Biological Activity
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Description
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Danoprevir is a peptidomimetic inhibitor of the NS3/4A protease of hepatitis C virus (HCV) with IC50 of 0.2-3.5 nM. |
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Targets
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HCV NS3/4A protease |
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IC50 |
0.2-3.5 nM [1] |
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In Vitro
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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] |
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In Vivo
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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] |
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Clinical Trials
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Danoprevir, associated with RO5024048, is currently under investigation in Phase II clinical trials in genotype 1 chronic hepatitis C. |
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Features
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Danoprevir is a peptidomimetic inhibitor of the NS3/4A protease of hepatitis C virus (HCV). |
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Protocol
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Kinase Assay
[1]
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| 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. |
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Cell Assay
[1]
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| Cell Lines |
Huh7 cells harboring HCV replicon |
| Concentrations |
5 pM - 100 nM |
| Incubation Time |
48 hours |
| Methods |
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. |
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Animal Study
[1]
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| Animal Models |
Sprague-Dawley rats, Cynomolgus monkeys |
| Formulation |
Dissolved in water. 6 mg/mL for rats and 3 mg/mL for monkeys |
| Doses |
30 mg/kg |
| Administration |
Oral gavage |
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References |
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[1] Seiwert SD, et al. Antimicrob Agents Chemother, 2008, 52(12), 4432-4441.
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[2] Bartels DJ, et al. J Infect Dis, 2008, 198(6), 800-807.
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[3] Imhof I, et al. Hepatology, 2011, 53(4), 1090-1099.
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