HCV NS5B polymerase; Setrobuvir targets the HCV NS5B RNA-dependent RNA polymerase, an essential enzyme for viral RNA replication . As a non-nucleoside inhibitor (NNI), it binds to the palm region I of the NS5B polymerase, specifically near the Tyr448 residue . Setrobuvir inhibits both de novo RNA synthesis and primer extension, with IC50 values between 4 and 5 nM in cell-free enzymatic assays . The compound shows differential potency across HCV subtypes: it is more potent against genotype 1b than genotype 1a .

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].

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In cell-free enzymatic assays, setrobuvir exhibits potent inhibition of recombinant HCV NS5B polymerases from genotypes 1a and 1b, with sub-nanomolar IC50 values . In Huh-7 cell-based HCV replicon assays, setrobuvir demonstrates EC50 values of 3 nM against genotype 1b (Con1 strain) and 52 nM against genotype 1a (H77 strain), with no appreciable cytotoxicity (CC50 ~100 μM) . The compound retains full activity against mutations that confer resistance to protease inhibitors, nucleoside polymerase inhibitors, and other non-nucleoside inhibitors that bind at distinct sites . Setrobuvir also exhibits synergy in vitro with interferon-alpha and with representative HCV protease and polymerase inhibitors .

In a Phase I monotherapy study, setrobuvir administered for 3 days at doses of 200 mg, 400 mg, and 800 mg twice daily produced rapid and sustained reductions in HCV RNA . Viral kinetic modeling revealed that setrobuvir's EC50 and viral clearance rate differ significantly between patients infected with HCV subtypes 1b and 1a, leading to an increased viral load decline in genotype 1b-infected patients . In a Phase IIb study, setrobuvir added to pegylated interferon and ribavirin achieved complete early virologic response (cEVR) rates of 78% in treatment-naïve patients and 76% in prior partial responders/relapsers, compared to 56% and 44% respectively for placebo plus standard of care . Notably, 29% of prior null responders achieved cEVR with setrobuvir plus standard of care .

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)).

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The inhibitory activity of setrobuvir against HCV NS5B polymerase was assessed using cell-free biochemical enzymatic assays with recombinant enzymes. Recombinant NS5B polymerases derived from HCV genotype 1a and 1b clinical isolates were expressed and purified. Polymerase activity was measured by monitoring the incorporation of radiolabeled nucleotides into RNA templates. Setrobuvir was tested at various concentrations, and IC50 values were calculated from concentration-response curves using nonlinear regression analysis. Setrobuvir was found to inhibit both de novo RNA synthesis and primer extension, with IC50 values between 4 and 5 nM .

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.

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The antiviral activity of setrobuvir in cell culture was assessed using HCV subgenomic replicon systems. Huh-7 cells (human hepatoma cells) harboring subgenomic HCV replicons of genotype 1b (Con1 strain) or genotype 1a (H77 strain) were seeded in assay plates. Cells were incubated with serially diluted setrobuvir at 37°C with 5% CO₂ for 3 days. After incubation, HCV replication was quantified by measuring luciferase activity or by real-time PCR. EC50 values (the concentration required to reduce HCV RNA replication by 50%) were calculated from concentration-response curves. Cytotoxicity was assessed in parallel using CellTiter-Glo or MTT assays to determine CC50 values. Setrobuvir demonstrated EC50 values of 3 nM (genotype 1b) and 52 nM (genotype 1a), with a CC50 of approximately 100 μM .

Preclinical toxicology studies of setrobuvir were conducted in rats and monkeys to support clinical development. Anadys Pharmaceuticals completed long-term, chronic toxicology studies of 26 weeks duration in rats and 39 weeks duration in monkeys, with favorable results that supported dosing for as long as a year in clinical studies . Comprehensive details regarding specific dosing regimens, animal models, and endpoints are not available in the provided search results.

Setrobuvir is orally bioavailable and demonstrates good in vitro metabolic stability, with a half-life exceeding 60 minutes in human and monkey liver microsomes at 0.5 mg/mL microsomal protein . In clinical studies, setrobuvir was administered at doses of 200 mg, 400 mg, and 800 mg twice daily . Pharmacokinetic data from healthy volunteers and HCV-infected patients were used to develop a two-compartment PK model with first-order absorption and lag-time . The compound's molecular weight is 560.62 g/mol, with molecular formula C25H25FN4O6S2 . The calculated ALogP is 2.98, indicating moderate lipophilicity .

Setrobuvir has demonstrated a favorable safety profile in clinical studies. In a Phase IIb study involving 283 patients, the rate of treatment discontinuation due to adverse events was similar between patients receiving setrobuvir plus pegylated interferon/ribavirin (5.6%) and those receiving placebo plus standard of care (5.9%) . The profile of adverse events was similar between groups, with reported events being typical for patients treated with interferon and ribavirin alone. Rash occurred in 39% of patients in the setrobuvir group (98% mild or moderate) compared to 22% in the control group . No Grade 4 rashes were reported in either group. Preclinical toxicology studies in rats (26 weeks) and monkeys (39 weeks) showed favorable results supporting long-term dosing . According to the Material Safety Data Sheet, setrobuvir is classified as not a hazardous substance or mixture; no carcinogenicity has been identified by NTP, IARC, OSHA, or ACGIH .

[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]. SARS-CoV-2 RNA dependent RNA polymerase (RdRp) targeting: an in silico perspective. J Biomol Struct Dyn. 2021 Jun;39(9):3204-3212.

Cetrobuvir has been used in clinical trials to treat chronic hepatitis C. Filibuvir and VX-222 are non-nucleoside inhibitors (NNIs) that bind to the thumb II allosteric pocket of hepatitis C virus (HCV) RNA-dependent RNA polymerase. Both compounds have shown significant efficacy in clinical trials, making a deeper understanding of their inhibitory mechanisms crucial. In this study, fenibvir and VX-222 inhibited the HCV 1b/Con1 subgenomic replicon with half-maximal effective concentrations (EC50) of 70 nM and 5 nM, respectively. Biochemical analysis using various RNA templates revealed that these two compounds preferentially inhibit primer-dependent RNA synthesis, with little or no effect on de novo RNA synthesis. The dissociation constants of fenibvir and VX-222 with HCV polymerase were 29 nM and 17 nM, respectively. The researchers analyzed the impact of three potential resistance mutations in the thumb II pocket on the inhibitory effects of these two compounds. In subgenomic replicon and enzyme activity assays, the M423T mutation in RNA polymerase increased resistance to fenibvir by at least 100-fold. This resistance was due to a 250-fold decrease in the binding affinity (K_d) of the mutant enzyme to fenibvir. In contrast, the inhibitory activity of VX-222 was only slightly affected by the M423T mutation, but was more significantly affected by the I482L mutation. [1]

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New therapies for SARS-CoV-2 are urgently needed. Today, COVID-19 infection is causing a huge health crisis in the world. Nucleotide inhibitors have shown good efficacy in combating a variety of viral infections. In this study, a model of viral protein RNA-dependent RNA polymerase (RdRp) was constructed using molecular modeling, molecular docking and kinetic simulation methods, and its binding affinity with some clinically approved drugs and candidate drugs was tested. Molecular dynamics simulations were used to balance the system after binding calculations to ensure successful reproduction of previous results and to incorporate the kinetics of RdRp to understand how it affects binding. The results showed that sofosbuvir, ribavirin, galidesivir, remdesivir, favipiravir, cefuroxime, tenofovir, and hydroxychloroquine could all effectively bind to SARS-CoV-2 RdRp. In addition, cetrabuvir, YAK, and IDX-184 showed better binding effects, and four novel IDX-184 derivatives also showed good promise in binding to SARS-CoV-2 RdRp. There is an urgent need to develop drugs that can selectively bind to and inhibit SARS-CoV-2 proteins. There are currently several FDA-approved antiviral drugs that can help us accomplish this task aimed at reducing the risk of COVID-19. Compounds 2 and 3 may bind tightly to SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) and therefore may be effective in treating 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.)