| Size | Price | |
|---|---|---|
| Other Sizes |
| Targets |
Dengue virus NS2B/3 serine proteinase (inhibitor) [1]
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| ln Vitro |
At 100 μM, 1,8-Dihydroxy-4,5-dinitroanthraquinone (50 μM-2 mM; 90 min) significantly affects BHK-21 cells by inhibiting the NS2B/3 site [1].
In a cell-free assay using a minimal NS2B/3 proteinase construct (NS2B/3pro) produced in rabbit reticulocyte lysate and labeled with ³⁵S-methionine, ARDP0006 inhibited intramolecular cleavage at the NS2B-3 junction in a concentration-dependent manner. The IC₅₀ for inhibition of this self-cleavage was determined to be approximately 620 μM [1]. In pulse-chase experiments with the NS2B/3pro construct, treatment with 100 μM ARDP0006 resulted in a modest but statistically significant reduction in the cleavage rate, with the half-life of the precursor increasing from 10.8 minutes to 14.9 minutes [1]. Using an elongated NS2B/3/4A proteinase construct containing the NS3int and NS3-4A cleavage sites, ARDP0006 (100 μM) dramatically inhibited processing when the construct was capable of cleavage at all three sites (NS2B-3, NS3int, and NS3-4A), with the half-life increasing from 70 minutes to >10¹⁵ minutes [1]. For a construct (G459L) that is not cleavable at the internal NS3 site but can be cleaved at NS2B-3 and NS3-4A, ARDP0006 (100 μM) slowed processing to a similar degree as for the minimal NS2B/3pro construct, with the half-life increasing from 19 minutes to 25 minutes [1]. For a construct (S1L) that is cleavable at NS2B-3 and NS3int but not at NS3-4A, ARDP0006 (100 μM) significantly inhibited processing, with the half-life increasing from 40 minutes to 66 minutes [1]. |
| Enzyme Assay |
The study references previous work where ARDP0006 was identified as an inhibitor of intermolecular NS2B/3 proteinase activity using a minimal proteinase construct (NS2B residues 49-96 joined to NS3 residues 1-185 via a flexible linker) and fluorogenic substrates. The IC₅₀ for intermolecular cleavage in solution was previously reported as 432 μM [1].
An in vitro assay was developed in this study to measure inhibition of intramolecular cleavage at the NS2B-3 junction. Minimal proteinase (NS2B/3pro) was produced in rabbit reticulocyte lysate in the presence of increasing concentrations of ARDP0006 (50 μM to 2 mM). After incubation, proteins were separated by SDS-PAGE, and the remaining precursor was quantified to determine the IC₅₀ of self-cleavage (approximately 620 μM) [1]. Pulse-chase assays were performed to monitor the kinetics of NS2B/3pro cleavage. Protein was translated with ³⁵S-methionine for 30 minutes, followed by addition of excess unlabeled methionine and either 100 μM ARDP0006 or DMSO. Aliquots were taken at various time points, and precursor loss was quantified to calculate cleavage rates [1]. |
| Cell Assay |
Viral Growth Inhibition Assay: BHK-21 cells were infected with dengue virus (multiplicity of infection, 0.1 PFU per cell) and treated with 25 μM ARDP0006 or DMSO control. Extracellular and intracellular virus were collected at various time points post-infection and quantified by plaque assay. Treatment with ARDP0006 reduced the yield of extracellular and intracellular virus by approximately 100-fold during a single 24-hour infectious cycle [1].
Cytotoxicity Assay: BHK-21 cells were treated with 25 μM ARDP0006, and negligible cytotoxicity was observed at this concentration (referenced to SI Appendix, Fig. S1) [1]. Drug Resistance Selection: Dengue virus was passaged in cultured cells in the presence of ARDP0006 to select for resistant variants. From two independent selection pools, no individual or shared mutations were found in NS2B/3. A mutation A21V in NS4A arose, but when reconstructed in isolation, it did not confer specific resistance to ARDP0006 but rather increased the rate of viral growth in both the presence and absence of inhibitor (SI Appendix, Fig. S2) [1]. |
| References | |
| Additional Infomation |
1,8-Dihydroxy-4,5-dinitroanthraquinone is a brownish-gold solid. (NTP, 1992)
ARDP0006 (1,8-dinitro-4,5-dihydroanthraquinone) is an inhibitor of the dengue virus NS2B/3 serine proteinase that was previously identified in a high-throughput screen [1]. The compound is one of the most potent NS2B/3-targeted inhibitors of dengue viral growth, with a previously reported IC₅₀ of 4.2 μM during infection, despite a much higher IC₅₀ (432 μM) for inhibition of intermolecular cleavage in solution [1]. This study demonstrates that the NS3 internal cleavage site (NS3int) is highly susceptible to inhibition by ARDP0006, with sensitivity comparable to that of the virus itself [1]. The study proposes that the antiviral efficacy of ARDP0006 may be explained by the accumulation of toxic precursors due to inhibition of specific intramolecular cleavages, particularly at the NS3int site, rather than general inhibition of proteolytic activity [1]. A high barrier to resistance against ARDP0006 was observed, with no resistance mutations identified in NS2B/3 after selection [1]. Another NS2B/3-targeted molecule, NSC135618, was tested in parallel and showed similar discrepancies between viral inhibition IC₅₀ (1.7 μM) and intramolecular cleavage IC₅₀ (490 μM), suggesting that this mechanism may be common among certain protease inhibitors [1]. |
| Molecular Formula |
C14H6N2O8
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|---|---|
| Molecular Weight |
330.2
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| Exact Mass |
330.012
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| CAS # |
81-55-0
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| PubChem CID |
3378440
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| Appearance |
Yellow to brown solid powder
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| Density |
1.8±0.1 g/cm3
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| Boiling Point |
669.4±55.0 °C at 760 mmHg
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| Melting Point |
225 °C (dec.)(lit.)
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| Flash Point |
280.8±20.0 °C
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| Vapour Pressure |
0.0±2.1 mmHg at 25°C
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| Index of Refraction |
1.782
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| LogP |
4.94
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
8
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| Rotatable Bond Count |
0
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| Heavy Atom Count |
24
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| Complexity |
549
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
GJCHQJDEYFYWER-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C14H6N2O8/c17-7-3-1-5(15(21)22)9-11(7)14(20)12-8(18)4-2-6(16(23)24)10(12)13(9)19/h1-4,17-18H
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| Chemical Name |
1,8-dihydroxy-4,5-dinitroanthracene-9,10-dione
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| Synonyms |
ARDP0006 ARDP 0006 ARDP-0006
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO : ~41.67 mg/mL (~126.19 mM)
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|---|---|
| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.0285 mL | 15.1423 mL | 30.2847 mL | |
| 5 mM | 0.6057 mL | 3.0285 mL | 6.0569 mL | |
| 10 mM | 0.3028 mL | 1.5142 mL | 3.0285 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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