Danoprevir

Validation & Quality Control

Product Citations(3)

Curr Protoc Microbiol, 2011, 17, 17.7

Antimicrob Agents Chemother, 2012, 56(10), 5365-5373

PLoS One, 2012, 7(8):e42481

Customer Reviews(1) in vivo invitation

Quality Control & MSDS

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Danoprevir is available in the following compound libraries:

Chemical Library (96-well) Inhibitor Library (96-well)

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Danoprevir Mechanism
Danoprevir Mechanism
HCV protein part consists of protein core (C), envelope glycoproteins 1 and 2 (E1, E2), p7, and the six non-structural HCV proteins NS2, NS3, NS4A, NS4B, NS5A and NS5B. NS3 shows a serine protease activity and a helicase/NTPase activity. NS3 and its cofactor and enhancer, NS4A, form a tight complex, which contributes to proper protein folding. And the active site of the NS3/4A protease is located in a shallow groove between the two b-barrels of the protease. ^[1]
Danoprevir is a macrocyclic compound. The crystal structure indicates that Danoprevir binds non-covalently to the active site of NS34A protease variant R155K. In wild-type NS34A protease, the large P2 moiety of Danoprevir binds in the S2 subsite and extensively interact with residues Arg1155(R155), Asp1168, and Ala1156. Of which, the P1 amide nitrogen makes a hydrogen bond to the carbonyl oxygen of Arg155, the P3 carbonyl oxygen makes a hydrogen bond to the amide nitrogen of Ala157, and the third hydrogen bond is formed between the P3 amide nitrogen and the carbonyl oxygen of Ala157. The acylsulfonamide group of Danoprevir is also positioned in the oxyanion hole and forms a hydrogen bond to the backbone amide nitrogens of Gly1137. In protease variant R155K, the R155K mutation disrupts the stacking interactions with the P2 isoindoline moieties, while the flexibility of the P2 moiety of Danoprevir allows it to retain activity against two of the three major redidues, which compensates somewhat for this loss. ^[2]
References
[1] Mauss, et al. Hepatology. 2012.
[2] Romano KP, et al. PLoS Pathog. 2012, 8(7), e1002832.
Dr. Antonino Maria Sparta demonstrates a breakthrough in leukemia research by utilizing two inhibitors produced by Selleck Chemicals.

Cat.No.	Product Name	Solubility (25°C)
S1013	Bortezomib (Velcade)	<1 mg/mL	77 mg/mL	<1 mg/mL
S2619	MG-132	<<1 mg/mL	95 mg/mL	95 mg/mL
S2853	Carfilzomib (PR-171)	<1 mg/mL	50 mg/mL	<1 mg/mL
S2181	MLN9708	<1 mg/mL	103 mg/mL	<1 mg/mL
S2180	MLN2238	<1 mg/mL	72 mg/mL	9 mg/mL
S1183	Danoprevir	<1 mg/mL	144 mg/mL	144 mg/mL
S1386	Nafamostat mesylate	53 mg/mL	20 mg/mL	<1 mg/mL
S2101	Gabexate mesylate	12 mg/mL	83 mg/mL	83 mg/mL
S3017	Aspirin (Acetylsalicylic acid)	<1 mg/mL	36 mg/mL	36 mg/mL
S7049	Oprozomib (ONX 0912)	<1 mg/mL	107 mg/mL	<1 mg/mL

Product Description

Biological Activity Protocol Chemical Information Customer Reviews Product Citations Tech Support & FAQs

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.
Targets	HCV NS3/4A protease
IC50	0.2-3.5 nM ^[1]
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]
Clinical Trials	Danoprevir, associated with RO5024048, is currently under investigation in Phase II clinical trials in genotype 1 chronic hepatitis C.
Features	Danoprevir is 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.

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

1

References

Chemical Information

Download Danoprevir SDF

Molecular Weight (MW)	731.83
Formula	C₃₅H₄₆FN₅O₉S
CAS No.	850876-88-9
Synonyms	RG7227, ITMN-191, RO5190591

Solubility (25°C) DMSO 144 mg/mL Water <1 mg/mL Ethanol 144 mg/mL
Storage	2 years -20°CPowder
	2 weeks4°Cin DMSO
	6 months-80°Cin DMSO

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

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Research Area

Customer Reviews (1)

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Rating

Source

PLoS ONE 7(8), e42481. Danoprevir 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.

Product Citations (3)

Screening of hepatitis C virus inhibitors using genotype 1a HCV replicon cell lines. [Robinson M, et al. Curr Protoc Microbiol 2011;17, 17.7]
PubMed: 21805689
Efficient replication of genotype 3a and 4a hepatitis C virus replicons in human hepatoma cells. [Saeed M, et al. Antimicrob Agents Chemother 2012;56(10), 5365-5373]
PubMed: 22869572
Canine Hepacivirus NS3 Serine Protease Can Cleave the Human Adaptor Proteins MAVS and TRIF. [Parera M, et al. PLoS One 2012;7(8):e42481]
PubMed: 22870331

Tech Support & FAQs

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.

Handling Instructions

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Validation & Quality Control

Product Citations(3)

Customer Reviews(1) in vivo invitation

Quality Control & MSDS

Related Compound Libraries

Danoprevir is available in the following compound libraries:

Product Information

Compare Proteasome Inhibitors

Danoprevir Mechanism

Product Description

Biological Activity

Protocol(Only for Reference)

Kinase Assay: ^[1]

Cell Assay: ^[1]

Animal Study: ^[1]

References

Chemical Information

Research Area

Customer Reviews (1)

Product Citations (3)

Tech Support & FAQs

If you have any other enquiries, please leave a message.

Related Proteasome Inhibitors

Proteases and Microbiology Related Products

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S2619	MG-132	<<1 mg/mL	95 mg/mL	95 mg/mL
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S2180	MLN2238	<1 mg/mL	72 mg/mL	9 mg/mL
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Danoprevir

Validation & Quality Control

Product Citations(3)

Customer Reviews(1) in vivo invitation

Quality Control & MSDS

Related Compound Libraries

Danoprevir is available in the following compound libraries:

Product Information

Compare Proteasome Inhibitors

Danoprevir Mechanism

Product Description

Biological Activity

Protocol(Only for Reference)

Kinase Assay: [1]

Cell Assay: [1]

Animal Study: [1]

References

Chemical Information

Research Area

Customer Reviews (1)

Product Citations (3)

Tech Support & FAQs

If you have any other enquiries, please leave a message.

Related Proteasome Inhibitors

Proteases and Microbiology Related Products

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Kinase Assay: ^[1]

Cell Assay: ^[1]

Animal Study: ^[1]