Tegobuvir (GS-9190) targets the HCV NS5B polymerase (genotype 1). [2][3]
The Y448H mutation in NS5B has been selected by GS-9190 as well as several benzothiadiazine HCV polymerase inhibitors in vitro and in vivo. [1]

In genotype 1b Huh-luc cells with a replicon encoding luciferase, Tegobuvir (GS-9190) had a mean EC50 value of 20.0 nM. [1]
Treatment of HCV subgenomic replicon cells with TGV results in a modified form of NS5B with a distinctly altered mobility on an SDS-PAGE gel, appearing as a doublet. This effect is specific to NS5B, as no aberration in protein mobility was observed for NS5A or NS3. NS5B doublet formation was not observed when replicon-containing cells were treated with other classes of inhibitors including NS5B NNIs (site II inhibitor VX-222, site III inhibitor A-782759 and site IV inhibitor HCV-796), a nucleoside chain terminator (2'CMeA), or a protease inhibitor (BILN-2061). [2]
NS5B doublet formation correlates with TGV potency. In GT1a replicon cells, the appearance of the lower band was delayed compared to GT1b replicon cells, consistent with TGV displaying sub-genotypic variations in potency (GT1a EC50 is 17-fold higher than that observed in GT1b). A 10-fold higher concentration of drug led to appearance of the lower NS5B band in GT1a replicon at an exposure time similar to GT1b. In HeLa cells carrying a GT1b replicon, TGV is vastly less potent (EC50 > 10 µM) and no doublet formation was observed even at 10 µM. In cells carrying a TGV-resistance mutation (NS5B Y448H, 36-fold less susceptible), the lower band was only faintly visible at 100 nM TGV. [2]
NS5B doublet formation does not require other viral proteins, as it was observed in Huh7 cells expressing only GT1b NS5B polymerase via a BacMam overexpression system. A more soluble deletion construct of NS5B polymerase with the membrane insertion domain removed (A21) also exhibited doublet formation following TGV treatment. [2]
Mass spectrometry analysis of NS5B from TGV-treated cells revealed a second major peak of higher molecular weight unique to the TGV-treated sample, with an observed mass change of 820.54 Da. For TGV analogs, the mass differences were 788.9 and 788.7 Da. [2]
A biotinylated TGV analog was detected in the faster migrating NS5B doublet species in both BacMam overexpressing cells and replicon cells via streptavidin detection, indicating covalent adduct formation between compound and NS5B protein. [2]
Glutathione contributes to TGV activity. Depletion of GSH with BSO (5 mM, 24-hour pretreatment) resulted in a 171-fold change in the TGV EC50 in replicon cells, and decreased doublet formation in NS5B overexpressing cells. [2]
CYP1A1 activity is necessary for the antiviral activity of TGV. SL3 (HeLa GT1b) replicon cells displayed 7- to 16-fold lower expression of CYP mRNA compared to Huh-7-based 1b-Rlu replicon cells. Overexpression of CYP1A1 in SL3 cells made them 1200-fold more sensitive to TGV (EC50 = 17 nM), reaching EC50 values similar to those observed in the Huh7 replicon system. Overexpression of CYP1A2 or CYP3A4 did not restore sensitivity. [2]
In chimeric replicon studies, Tegobuvir had EC50 values of <16 nM against genotype 1 and >100 nM for genotypes 2b, 3a, 4a, 5a, and 6a. Against 1b-con-1 replicon, tegobuvir was the most potent with EC50 approximately 10-fold lower than against 1a-H77 replicon. Treatment of 2a-JFH1 replicon and GT3a, 4a, 6a-Con, and 5a CI chimeras produced fold shifts varying from 57 to 14,700 less susceptibility to tegobuvir compared to 1b-con-1. [3]
An NS5B F445C mutation engineered into GT3a, 4a, and 6a chimeric replicons lowered the tegobuvir EC50 to levels comparable to those for genotype 1a (from >100 nM to 16 nM), but did not considerably alter the EC50 of site 2 or nucleoside analog inhibitors. This single amino acid change significantly reduced the EC50 for tegobuvir 410-fold compared to their wild-type chimeras. [3]

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In a dose-dependent manner, tigobivir quickly raised the percentage of replicons containing the Y448H mutation. Y448H was expressed by 1.2%, 6.8%, and >50% of the replicon population at 1, 10, and 20 times the 50% effective concentration of tegobuvir, respectively, following three days of therapy [1]. Through a special chemical activation and subsequent direct contact with the NS5B protein, tigobuvir demonstrates anti-HCV efficacy. When tegobuvir is administered to HCV subgenomic replicon cells, NS5B is changed and exhibits markedly altered mobility on SDS-PAGE gels [2]. Tigobuvir exhibits anti-GT1a and anti-GT1b activity, with mean EC50 values of 19.8 and 1.5 nM, correspondingly. The tegobuvir EC50 for the genotype 3a, 4a, and 6a Con chimeras was more than 100 nM. The GT3a, 4a, and 6a chimeric replicons' F445C NS5B mutation brought back tegobuvir potency to EC50 values similar to GT1a [3].

In a study, plasma from 65 treatment-naïve HCV-infected patients (42 and 23 with genotype 1a and 1b, respectively) was tested for the presence of Y448H by AS-PCR and population sequencing. All patient samples were wild type at NS5B Y448 by population sequencing. AS-PCR detected low levels of Y448H ranging from 0.5% to 3.0% in 5/62 (8%) treatment-naïve patient samples. [1]
In patients following 8 days of monotherapy with GS-9190, AS-PCR detected subpopulations with the Y448H mutation at 2.0% and 3.3% in two genotype 1a-infected patients at day 8, while population sequencing still showed wild-type. Single-genome sequencing identified the Y448H mutant from 1/16 (6.3%) and 5/51 (9.8%) clones from these patients. In a genotype 1b-infected patient at day 8, population sequencing identified mixtures at position Y448 (H/Y/C/R), AS-PCR detected >50% Y448H mutants, and SGS detected 3 amino acids: Y (6/13), H (6/13), and N (1/13). [1]

The mechanism of action of Tegobuvir is unique, as it does not inhibit the enzymatic activity of the recombinant NS5B protein in cell-free systems. Studies indicate that this compound requires intracellular biotransformation to become active . Therefore, standard in vitro enzyme assays (using purified NS5B protein, RNA template, and nucleotide substrates) are not suitable for evaluating Tegobuvir. Its activity is primarily assessed using cell-based replicon assays, which indirectly reflect the compound's antiviral potency by measuring HCV RNA replication levels within cells. This dependence on metabolic activation is a key characteristic of this compound .

For determination of 50% effective concentration (EC50) in replicon assays, replicon cells were seeded into 96-well plates and GS-9256 was serially diluted in DMSO at 200x final concentrations. Luciferase expression was quantified after 3 days in luciferase-encoding replicon cell lines using a commercial luciferase assay. Data were fit to the logistic dose response equation and EC50 values were calculated. [1]
For replicon EC50 determinations of Tegobuvir, replicon-containing cells were trypsinized and seeded in cell culture media without G418 in white 96-well plates. Stable replicon carrying cell lines were seeded at a density of 5,000 cells per well. Serial threefold dilutions (10 concentrations) of compounds were performed in DMSO followed by further dilution in cell culture media and subsequent addition to cell plates. Compound-treated cells were incubated 72 hours at 37°C in a 5% CO2 incubator. For luciferase-encoding replicons, the luciferase signal was quantified using a commercially available assay system. Curve fitting and EC50 values were derived using non-linear regression analysis. [2]
For Western blot analysis, cells were washed with PBS followed by lysis with RIPA buffer containing protease and phosphatase inhibitors. Samples were placed on ice for 15 minutes then pelleted. Supernatants were run on Tris-Bis NuPage gels in MOPS buffer at 200 V for 2 hours under reducing conditions. Gels were transferred to nitrocellulose. Mouse anti-NS5B antibody, mouse anti-NS5A antibody, and mouse anti-NS3 antibody were used as primary antibodies for detection. For chemiluminescence analysis, HRP anti-mouse secondary antibody was used. For infrared readout, LiCor IR Dye Goat-anti-mouse-800CW was used as a secondary antibody. For detection of biotin, IR Dye 700-linked streptavidin was used. [2]
For BacMam NS5B overexpression, Huh7-Lunet cells were plated in DMEM/10% FBS/NEAA and infected with BacMam virus reagent. Three hours later, compound was added to the desired concentration as well as sodium butyrate to a final concentration of 10 mM. Flasks were incubated overnight. Cells were then washed, trypsinized, pelleted, flash frozen on dry ice and stored at -80°C until purification. [2]
For NS5B purification for mass spectrometry, cell pellets were resuspended and lysed in lysis buffer. Following lysis, debris was pelleted by centrifugation. Supernatant was incubated overnight with protein A Dynabeads previously conjugated to anti-NS5B antibody. Beads were washed three times with lysis buffer. Captured protein was eluted and immediately analyzed by mass spectrometry. [2]
For cytochrome P450 overexpression in SL3 cells, cells were plated at a density of 1.5×10^6 cells, 24 hours prior to transfection. Each dish of cells was transfected with 15 µg CYP1A1, CYP1A2 or CYP3A4 DNA using transfection reagent. Transfection efficiency was determined using RT-PCR to detect expression levels 48 hours post-transfection. [2]
For chimeric replicon assays, stable cell lines harboring chimeric replicons were treated with tegobuvir and other NS5B inhibitors. EC50 values were determined. [3]

For replicon studies, GS-9190 was used to treat replicon cells at levels 1×, 5×, 10×, and 20× the EC50. A total of 10^6 replicon cells were placed in each T75 flask with various concentrations of GS-9190. After 3 to 5 days, when the cells reached 90% confluence, 10^6 cells were replated per T75 flask. At each passage, cells were also collected for RNA extraction. [1]

Tegobuvir has been shown to be metabolically very stable; the elimination half-life of TGV in humans is 10-15 hours. [2]
Preclinical metabolite identification studies revealed small amounts of GSH adducts with oxidized versions of TGV. [2]

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Tegobuvir is a prodrug that requires intracellular metabolic activation. Its activation is dependent on the metabolic action of CYP1A enzymes, followed by the formation of a glutathione conjugate, which is considered to be the active antiviral species . Experiments have shown that the antiviral potency of Tegobuvir is reduced when combined with a CYP1A inhibitor, confirming its activation-dependent mechanism of action .

When given with pegylated interferon-alfa+RBV or in combination with other direct-acting antivirals, TGV's covalent MOA has not been an impediment for TGV to be generally safe, well-tolerated and associated with potent antiviral activity. [2]

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Tegobuvir is classified as a compound for research use only and is not intended for human therapeutic use . The toxicological information varies across different safety data sheets: one source classifies it as "Not a hazardous substance or mixture", while another labels it as "Toxic" and a pharmaceutical active ingredient requiring strict handling precautions . This source also indicates that prolonged exposure may cause serious damage to health, with potential risks including impaired fertility and harm to the unborn child .

[1]. Allele-specific real-time PCR system for detection of subpopulations of genotype 1a and 1b hepatitis C NS5B Y448H mutant viruses in clinical samples. J Clin Microbiol. 2011 Sep;49(9):3168-74.

[2]. The HCV non-nucleoside inhibitor Tegobuvir utilizes a novel mechanism of action to inhibit NS5B polymerase function. PLoS One. 2012;7(6):e39163.

[3]. Tegobuvir (GS-9190) potency against HCV chimeric replicons derived from consensus NS5B sequences from genotypes 2b, 3a, 4a, 5a, and 6a. Virology. 2012 Jul 20;429(1):57-62.

The Y448H mutation in NS5B has been selected by GS-9190 as well as several benzothiadiazine HCV polymerase inhibitors in vitro and in vivo. The NS5B Y448H mutant remained sensitive to interferon, ribavirin, and inhibitors of HCV NS3 protease, NS5A, and NS5B (site II non-nucleoside and nucleoside). [1]
Tegobuvir (TGV, GS-9190) is an analog of a novel class of imidazopyridine inhibitors selectively targeting HCV. TGV demonstrated anti-HCV potency both in vitro and in vivo. In a recent exploratory study using TGV in combination with the NS3 protease inhibitor GS-9256 and PEG/RBV, 100% of patients had HCV RNA levels below the lower limit of quantification after only 28 days of treatment. [2]
The mechanism of action of TGV involves oxidative metabolic activation. TGV is likely oxidized to an epoxide intermediate which can eliminate the fluoride ion yielding a ketone. This creates possibilities for a nucleophilic attack by GSH or a reactive cysteine residue. Following CYP-mediated oxidative metabolic activation of the fluorophenyl moiety and assistance of GSH, TGV binds covalently to NS5B, thereby disrupting viral replication. [2]
In chimeric replicon studies, an NS5B F445C mutation engineered into GT3a, 4a, and 6a chimeric replicons lowered the tegobuvir EC50 to levels comparable to those for genotype 1a. A query of the EU HCV sequence database for GT3a, 4a, and 6a at NS5B position 445 shows phenylalanine as 100% conserved in those genotypes. [3]

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Tegobuvir has been investigated for the treatment of chronic hepatitis C.

C25H14F7N5

517.400989055634

517.113

C, 58.03; H, 2.73; F, 25.70; N, 13.54

1000787-75-6

23649154

White to off-white solid powder

1.5±0.1 g/cm3

558.0±60.0 °C at 760 mmHg

291.3±32.9 °C

0.0±1.5 mmHg at 25°C

1.605

4.07

0

11

4

37

765

0

FC(C1C=C(C(F)(F)F)C=CC=1C1=CC=C(CN2C=CC3C(=C2)N=C(C2C=CC=CC=2F)N=3)N=N1)(F)F

XBEQSQDCBSKCHJ-UHFFFAOYSA-N

InChI=1S/C25H14F7N5/c26-19-4-2-1-3-17(19)23-33-21-9-10-37(13-22(21)34-23)12-15-6-8-20(36-35-15)16-7-5-14(24(27,28)29)11-18(16)25(30,31)32/h1-11,13H,12H2

5-[[6-[2,4-bis(trifluoromethyl)phenyl]pyridazin-3-yl]methyl]-2-(2-fluorophenyl)imidazo[4,5-c]pyridine

GS9190; GS-333126; Tegobuvir; 1000787-75-6; 5-((6-(2,4-bis(Trifluoromethyl)phenyl)pyridazin-3-yl)methyl)-2-(2-fluorophenyl)-5H-imidazo[4,5-c]pyridine; GS-9,190; GS-333,126; GS 9190; GS333126; GS-9190

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)

DMSO : ≥ 50 mg/mL (~96.64 mM)

Solubility in Formulation 1: ≥ 2.75 mg/mL (5.32 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 27.5 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.75 mg/mL (5.32 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 27.5 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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