HCV NS5B polymerase

For subgenomic replicons carrying HCV genotype 1b/Con1, setrobuvir (ANA598), a non-nucleoside inhibitor, binds to the palm pocket of HCV polymerase with an EC50 in the nanomolar range. Mutations that affect the activity of thumb-binding non-nucleoside inhibitors do not affect the activity of setrobuvir, which appears to limit de novo RNA synthesis and primer extension [1].

Surface plasmon resonance. [1]
All analyte binding experiments were performed with a Biacore T100 instrument using preconditioned CM5 sensor chips activated with N-hydroxysuccinimide ester and 1-ethyl-3(3-diaminopropyl) carbodiimide hydrochloride. The 1bΔ21 NS5B protein and variants were immobilized after a 5-min injection to a nominal density of about 9,000 response units. The surface was then deactivated by injection of ethanolamine for 7 min. Compounds were injected in a buffer containing 25 mM HEPES (pH 7.4), 10 mM MgCl2, 150 mM NaCl, 0.01% Tween 20, 0.05% β-mercaptoethanol, and 5% DMSO. All compounds displayed saturable 1:1 binding behavior. For competition binding, the experimental design consisted of injecting a saturating concentration of the first analyte (160 nM filibuvir) followed by immediate injection of an equimolar ratio of the analyte mixture (160 nM filibuvir plus 160 nM VX-222 or Setrobuvir (RO-5466731；ANA-598)).

EC50 determinations with HCV replicon-expressing cells. [1]
Huh7.5 cells harboring replicons were trypsinized and plated into 48-well plates at 40,000 cells/well. The next day the medium was changed and inhibitors were added to the cells at seven different concentrations, each pair of which differed by 3- or 10-fold dilutions in 200 μl complete medium with triplicates. After 48 h, total RNA was extracted from replicon cells using the TRIzol reagent, and viral RNAs were quantified by real-time reverse transcription-PCR (RT-PCR). First-strand cDNA synthesis used 1 μg of total RNA along with Moloney murine leukemia virus (NEB) and 4 μM randomized 9-nucleotide (nt) primer mix. RT-PCR used the Bio-Rad IQ SYBR green kit, and primers were HCV 5′-UTRsense (5′-AGC CAT GGC GTT AGT ATG AGT GTC-3′) and 5′-UTRanti (5′-ACA AGG CCT TTC GCG ACC CAA C-3′). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was detected using the sense and antisense oligonucleotides 5′-GAG TCA ACG GAT TTG GTC GT-3′ and 5′-TGG GAT TTC CAT TGA TGA CA-3′, respectively. All reaction mixtures were heated to 95°C for 10 min, followed by 40 cycles of PCR of 15 s at 95°C, 20 s at 55°C, and 30 s at 72°C. The fold change and percent change of each group were compared to values for controls as previously described. The effective drug concentrations that reduced HCV RNA replicon levels by 50% (EC50s) were calculated with GraphPad Prism software by nonlinear regression analysis with log curve fitting.

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Expression and purification of recombinant HCV polymerases. [1]
The HCV NS5B protein lacking the C-terminal 21 amino acids, named 1bΔ21, was cloned in pET-21b as described previously. The construct contains a C-terminal six-histidine tag to facilitate protein purification. L419M, M423T, and I482L resistance mutants were made by site-directed mutagenesis from the WT 1bΔ21 expression plasmid. Recombinant 1bΔ21 and the mutant proteins derived from 1bΔ21 were expressed and purified from Escherichia coli extract by using Talon metal affinity resin followed by a Mono S column as described by Chinnaswamy et al. Protein concentrations were quantified by use of a nanodrop spectrophotometer followed by checks for purity and protein concentration using SDS-PAGE and a titration series with bovine serum albumin.

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Gel-based RdRp assay with small RNA templates. [1]
RdRp assays were carried out in 20-μl reaction mixtures containing 20 mM sodium glutamate (pH 8.2), 12.5 mM dithiothreitol, 4 mM MgCl2, 1 mM MnCl2, 0.5% Triton X-100, 0.2 mM GTP, 0.1 mM ATP and UTP, 3.3 nM [α-32P]CTP, 40 nM recombinant RNA polymerases, 100 nM PE46, 50 nM LE19p. The reaction mixture was incubated at 30°C for 1 h, and the reaction was stopped by phenol-chloroform extraction, followed by ethanol precipitation of the RNA in the presence of 5 μg glycogen and 0.3 M NaO-acetate (pH 5.2). RNA products were separated by 7.5 M urea–20% polyacrylamide gels. The signal was detected by a PhosphorImager and quantified with the ImageQuant software. The 50% inhibitory concentration (IC50) was calculated with GraphPad Prism software by nonlinear regression analysis with log curve fitting.

[1]. Biochemical study of the comparative inhibition of hepatitis C virus RNA polymerase by VX-222 and filibuvir. Antimicrob Agents Chemother. 2012;56(2):830‐837.

[2]. Elfiky AA. SARS-CoV-2 RNA dependent RNA polymerase (RdRp) targeting: an in silicoperspective [published online ahead of print, 2020 May 6]. J Biomol Struct Dyn. 2020;1‐9.

Setrobuvir has been used in trials studying the treatment of Hepatitis C, Chronic.

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Filibuvir and VX-222 are nonnucleoside inhibitors (NNIs) that bind to the thumb II allosteric pocket of the hepatitis C virus (HCV) RNA-dependent RNA polymerase. Both compounds have shown significant promise in clinical trials and, therefore, it is relevant to better understand their mechanisms of inhibition. In our study, filibuvir and VX-222 inhibited the 1b/Con1 HCV subgenomic replicon, with 50% effective concentrations (EC(50)s) of 70 nM and 5 nM, respectively. Using several RNA templates in biochemical assays, we found that both compounds preferentially inhibited primer-dependent RNA synthesis but had either no or only modest effects on de novo-initiated RNA synthesis. Filibuvir and VX-222 bind to the HCV polymerase with dissociation constants of 29 and 17 nM, respectively. Three potential resistance mutations in the thumb II pocket were analyzed for effects on inhibition by the two compounds. The M423T substitution in the RNA polymerase was at least 100-fold more resistant to filibuvir in the subgenomic replicon and in the enzymatic assays. This resistance was the result of a 250-fold loss in the binding affinity (K(d)) of the mutated enzyme to filibuvir. In contrast, the inhibitory activity of VX-222 was only modestly affected by the M423T substitution but more significantly affected by an I482L substitution.[1]

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New treatment against SARS-CoV-2 now is a must. Nowadays, the world encounters a huge health crisis by the COVID-19 viral infection. Nucleotide inhibitors gave a lot of promising results in terms of its efficacy against different viral infections. In this work, molecular modeling, docking, and dynamics simulations are used to build a model for the viral protein RNA-dependent RNA polymerase (RdRp) and test its binding affinity to some clinically approved drugs and drug candidates. Molecular dynamics is used to equilibrate the system upon binding calculations to ensure the successful reproduction of previous results, to include the dynamics of the RdRp, and to understand how it affects the binding. The results show the effectiveness of Sofosbuvir, Ribavirin, Galidesivir, Remdesivir, Favipiravir, Cefuroxime, Tenofovir, and Hydroxychloroquine, in binding to SARS-CoV-2 RdRp. Additionally, Setrobuvir, YAK, and IDX-184, show better results, while four novel IDX-184 derivatives show promising results in attaching to the SARS-CoV-2 RdRp. There is an urgent need to specify drugs that can selectively bind and subsequently inhibit SARS-CoV-2 proteins. The availability of a punch of FDA-approved anti-viral drugs can help us in this mission, aiming to reduce the danger of COVID-19. The compounds 2 and 3 may tightly bind to the SARS-CoV-2 RdRp and so may be successful in the treatment of COVID-19.[2]

C25H25FN4O6S2

560.6154

560.12

C, 53.56; H, 4.50; F, 3.39; N, 9.99; O, 17.12; S, 11.44

1071517-39-9

135565932

Typically exists as solid at room temperature

4.725

3

9

5

38

1270

4

FC1C=CC(CN2C3C(C4CC3CC4)C(O)=C(C3=NC4C=CC(NS(C)(=O)=O)=CC=4S(=O)(=O)N3)C2=O)=CC=1

DEKOYVOWOVJMPM-RLHIPHHXSA-N

InChI=1S/C25H25FN4O6S2/c1-37(33,34)28-17-8-9-18-19(11-17)38(35,36)29-24(27-18)21-23(31)20-14-4-5-15(10-14)22(20)30(25(21)32)12-13-2-6-16(26)7-3-13/h2-3,6-9,11,14-15,20,22,28,31H,4-5,10,12H2,1H3,(H,27,29)/t14-,15+,20+,22-/m0/s1

N-[3-[(1R,2S,7R,8S)-3-[(4-fluorophenyl)methyl]-6-hydroxy-4-oxo-3-azatricyclo[6.2.1.02,7]undec-5-en-5-yl]-1,1-dioxo-4H-1λ6,2,4-benzothiadiazin-7-yl]methanesulfonamide

RG-7790; ANA-598; RG7790; ANA598; RO5466731; RO-5466731; RO 5466731; 1071517-39-9; Setrobuvir [USAN]; Setrobuvir [USAN:INN]; Setrobuvir [INN]; Setrobuvir (USAN); T5B2GI8F84; Setrobuvir

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*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).

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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)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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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).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)