Danoprevir

製品コードS1183 バッチS118301

化学情報

	Synonyms	ITMN-191, RG7227	Storage (From the date of receipt)	3 years -20°C powder 1 years -80°C in solvent
	化学式	C₃₅H₄₆FN₅O₉S
	分子量	731.83	CAS No.	850876-88-9
Solubility (25°C)*	体外	DMSO	144 mg/mL (196.76 mM)
		Ethanol	144 mg/mL (196.76 mM)
		Water	Insoluble
* <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.

溶剤液（一定の濃度）を調合する

生物活性

製品説明	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.
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]
特徴	A peptidomimetic inhibitor of the NS3/4A protease of hepatitis C virus (HCV).

プロトコル(参考用のみ)

キナーゼアッセイ	Continuous fluorescent resonance energy transfer (FRET) assay
キナーゼアッセイ	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.
細胞アッセイ	細胞株	Huh7 cells harboring HCV replicon
	濃度	5 pM - 100 nM
	反応時間	48 hours
	実験の流れ	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.
動物実験	動物モデル	Sprague-Dawley rats, Cynomolgus monkeys
	投薬量	30 mg/kg
	投与方法	Oral gavage

参考

カスタマーフィードバック

: Data from [Data independently produced by Mol Biol Evol, 2014, 31(6), 1546-53]

: Data from [Data independently produced by Antimicrob Agents Chemother, 2012, 56(10), 5365-73]

: Data from [PLoS One, 2012, 7, e42481]

Selleckの高級品が、幾つかの出版された研究調査結果（以下を含む）で使われた：

Preclinical characterization of a non-peptidomimetic HIV protease inhibitor with improved metabolic stability [ Antimicrob Agents Chemother, 2024, 68(4):e0137323.]	PubMed: 38380945
Mechanisms of Action of the Host-Targeting Agent Cyclosporin A and Direct-Acting Antiviral Agents against Hepatitis C Virus [ Viruses, 2023, 15(4)981]	PubMed: 37112961
RECOVER identifies synergistic drug combinations in vitro through sequential model optimization [ Cell Rep Methods, 2023, 3(10):100599]	PubMed: 37797618
Integrative multiomics and in silico analysis revealed the role of ARHGEF1 and its screened antagonist in mild and severe COVID-19 patients [ J Cell Biochem, 2022, 10.1002/jcb.30213]	PubMed: 35037717
Hepatitis C virus drugs that inhibit SARS-CoV-2 papain-like protease synergize with remdesivir to suppress viral replication in cell culture [ Cell Rep, 2021, 35(7):109133]	PubMed: 33984267
Characterization of fluorescent probe substrates to develop an efficient high-throughput assay for neonatal hepatic CYP3A7 inhibition screening [ Sci Rep, 2021, 11(1):19443]	PubMed: 34593846
Visualization of Positive and Negative Sense Viral RNA for Probing the Mechanism of Direct-Acting Antivirals against Hepatitis C Virus. [ Viruses, 2019, 11(11)]	PubMed: 31717338
Modular cell-based platform for high throughput identification of compounds that inhibit a viral interferon antagonist of choice [Vasou A, et al. Antiviral Res, 2018, 150:79-92]	PubMed: 29037975
Tumor Necrosis Factor Inhibits Spread of Hepatitis C Virus Among Liver Cells, Independent from Interferons. [Laidlaw SM, et al. Gastroenterology, 2017, 10.1053/j.gastro.2017.04.021]	PubMed: 28456632
Quantifying antiviral activity optimizes drug combinations against hepatitis C virus infection. [ Proc Natl Acad Sci U S A, 2017, 114(8):1922-1927]	PubMed: 28174263

長期の保管のために-20°Cの下で製品を保ってください。

人間や獣医の診断であるか治療的な使用のためにでない。

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