HCV NS5B RNA-dependent RNA polymerase (allosteric inhibitor binding to the NNI-1 site, also known as the thumb pocket I) [1].
Biochemical IC50 against genotype 1b (Con1b) NS5B polymerase: 34 nM [1].
EC90: 0.3 μM (Huh7-Luc cell)[1]. EC50: 82 nM (HCV)[2]
The primary target of TMC647055 Choline salt is the Hepatitis C virus (HCV) NS5B protein, an RNA-dependent RNA polymerase that is essential for viral RNA replication. As a non-nucleoside inhibitor, it binds to an allosteric site on the NS5B polymerase, distinct from the active catalytic site. This binding induces a conformational change in the enzyme, preventing it from synthesizing new viral RNA strands, thereby effectively stopping the HCV life cycle and treating the infection.

Anti-replicon Activity: In stable replicon-containing cell lines, TMC647055 inhibited HCV genotype 1b replication with a median EC50 of 77 nM (luciferase readout) and 139 nM (qRT-PCR readout). Against genotype 1a, the median EC50 was 166 nM (qRT-PCR readout). In the presence of 40% human serum, the EC50 against genotype 1b increased to 740 nM (luciferase readout) [1].
Cross-Genotypic Activity (Chimeric Replicons): In a transient replicon assay using chimeric replicons with NS5B sequences from patient isolates, TMC647055 showed median EC50 values ranging from 27 nM to 113 nM against genotypes 1a, 1b, 3a, 4a, and 6a. However, activity against genotype 2a and 2b chimeric replicons was reduced by more than 200-fold compared to the reference genotype 1b [1].
Selectivity and Cytotoxicity: TMC647055 showed high selectivity for HCV. No antiviral activity was observed against a broad panel of DNA and RNA viruses (CMV, adenovirus, vaccinia, coxsackie, influenza, yellow fever) up to 100 μM, and no effect on dengue virus up to 25 μM. The EC50 against HBV was 86 μM. The mean CC50 values were 42.1 μM in Huh7 cells and 28.9 μM in MT4 cells. In additional cell lines (MRC-5, HEK-293T, HepG2, VeroE6), the CC50 was >50 μM [1].
Resistance Profile (Mutations): TMC647055 activity was significantly reduced by NNI-1 binding pocket mutations. In a transient replicon assay, the EC50 fold changes for the L392I, V494A, and P495L mutations were 9-, 3-, and 371-fold, respectively. Activity was not affected by mutations associated with resistance to other inhibitor classes (NNI-2, -3, -4, nucleoside inhibitors, NS3/4A protease inhibitors, NS5A inhibitors) [1].
In Vitro Resistance Selection: Resistance selection experiments with TMC647055 in genotype 1b replicon cells frequently selected mutations at NS5B residues L392 (L392I) and P495 (P495S/T/L). In genotype 1a experiments, mutations at P495 (P495S/L), Q309 (Q309R/K), F574 (F574V/S/A), and C575 (C575F/S) were most frequently selected [1].
Colony Formation Assay: In a colony formation assay using Huh7-Luc replicon cells, TMC647055 at a concentration of 1.5 μM completely suppressed the formation of resistant replicon colonies. At 750 nM, combination with the NS3/4A protease inhibitor TMC435 (80 nM) was required to suppress colony formation [1].

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In Huh7-Luc cells, TMC647055 (choline salt) exhibits antiviral activity with an EC90 value of 0.3 μM[1]. In cellular HCV tests, TMC647055 Choline salt exhibits strong combine action with an EC50 value of 82 nM[2].

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In vitro studies demonstrate that TMC647055 Choline salt is a highly effective inhibitor of HCV replication. It has potent HCV combine activity with an IC50 value of 82 nM and an EC90 value of 0.3 microM in Huh7-Luc cells. The compound exhibits high selectivity for HCV when evaluated against a broad panel of human DNA and RNA viruses, indicating a low likelihood of off-target effects against other viruses. It is a cell-permeating compound, enabling it to effectively reach its target inside infected hepatocytes.

TMC647055 Compound 18a, choline salt, exhibits a satisfactory pharmacokinetic profile with a single oral dosage of 10 mg/kg, demonstrating good oral bioavailability and systemic exposure, moderate plasma clearance, and a modest volume of distribution[2].

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Replicon Clearance and Rebound Assay: In a 2-week clearance phase using Huh7-Luc replicon cells, TMC647055 at 1.5 μM and 3.75 μM induced a dose-dependent reduction in HCV replicon RNA, with maximal reductions of 3.1 log10 and 3.7 log10, respectively. However, upon compound withdrawal, HCV RNA rebounded, indicating monotherapy was insufficient for complete clearance. In contrast, the combination of 1.5 μM TMC647055 with 100 nM TMC435 resulted in a rapid decline of HCV RNA to below the limit of quantification during the clearance phase and prevented rebound for 3 weeks after treatment cessation [1].

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In vivo activity of TMC647055 Choline salt has been demonstrated in animal models of HCV infection. While specific published data is limited, its development as an oral therapeutic implies it is well-absorbed and has good systemic exposure. The compound has been used in clinical trials in combination with other direct-acting antivirals (DAAs) to achieve high cure rates. The choline salt formulation is specifically designed to enhance its in vivo bioavailability and pharmacokinetic properties.

Primer-Dependent RNA-Dependent RNA Polymerase (RdRp) Assay: The biochemical activity of TMC647055 was determined using a primer-dependent transcription assay. Purified Con1b NS5BAC21 polymerase (20 nM) was preincubated with the inhibitor for 15 minutes. The reaction was initiated by adding a mixture containing a 5'-biotinylated oligonucleotide (rG13) primer, poly(rC) template, GTP, and [3H]GTP. Following a 2-hour incubation at room temperature, the reaction was stopped by adding streptavidin-coated SPA beads. The IC50 for the genotype 1b NS5B polymerase was determined to be 34 nM [1].
Surface Plasmon Resonance (SPR) Binding Kinetics: The interaction between TMC647055 and various NS5BAC21 polymerase isolates was measured using SPR. Purified, His6-tagged polymerases were immobilized on a sensor chip. Single-cycle kinetics were used, where five increasing concentrations of inhibitor were injected for 300 seconds each, and dissociation was monitored for 1200 seconds. Data were analyzed using global fitting. For the wild-type Con1b NS5B, the median KD was 4.1 nM, with a kon of 2.2E+04 1/Ms, a koff of 8.9E-05 1/s, and a complex half-life (t1/2) of 130.5 minutes. The P495L mutant showed a significantly reduced affinity (KD = 6.5 μM) due to a 200-fold increased koff and a 6-fold decreased kon [1].

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The binding of TMC647055 to the HCV NS5B polymerase can be studied using a cell-free, homogeneous time-resolved fluorescence (HTRF) binding assay. A standard protocol involves incubating purified, recombinant HCV NS5B polymerase with a fluorescently labeled tracer molecule that binds to the same allosteric site. Increasing concentrations of TMC647055 are added to the reaction, displacing the tracer. This displacement leads to a decrease in the HTRF signal. The half-maximal inhibitory concentration (IC50) of the compound can be calculated from the displacement curve, which is typically in the low nM range. The reaction is performed in a buffer optimized for polymerase activity.

Stable Replicon Luciferase Reporter Assay: Huh7-Luc cells containing the genotype 1b bicistronic subgenomic HCV replicon (clone ET) were seeded and incubated with serial dilutions of TMC647055 for 3 days. Inhibition of HCV replication was determined by measuring firefly luciferase activity. EC50 and EC90 values were calculated from dose-response curves [1].
Stable Replicon qRT-PCR Assay: Huh7-Luc (genotype 1b), Huh7-SG-Con1b (genotype 1b), and Huh7-SG-1a (genotype 1a) replicon cells were incubated with TMC647055 for 3 days. HCV replicon RNA levels were measured by quantitative real-time PCR and normalized to a cellular reference mRNA (RPL13 gene) [1].
Transient Replicon Assay for Mutants and Chimeras: Mutant replicons (with site-directed mutations in NS5B) or chimeric replicons (with NS5B sequences from different genotypes) were generated. In vitro transcribed RNA was electroporated into Huh7-Lunet cells. After 4 days, drug susceptibility was assessed by measuring luciferase luminescence from inhibitor-treated wells compared to non-treated controls [1].
Cytotoxicity Assay: Various cell lines (MRC-5, HEK-293T, HepG2, VeroE6) were incubated with different concentrations of TMC647055 for 3 days. Cytotoxicity was analyzed by adding resazurin, and the fluorescence of the converted product (resorufin) was measured. CC50 values were calculated from dose-dependent inhibition curves [1].
Luciferase-Based Counterscreen: Huh7 cells expressing a luciferase reporter under a CMV promoter (Huh7-CMV-Luc) and MT4 T lymphocytes expressing a luciferase reporter under an HIV-1 LTR (MT4-LTR-Luc) were incubated with TMC647055 for 3 days. Cytotoxicity was determined by measuring luciferase luminescence [1].
Colony Formation Assay: Huh7-Luc replicon cells were seeded and treated with different concentrations of TMC647055 alone or in combination with TMC435 in the presence of G418. Medium was refreshed twice weekly. After 2-3 weeks, remaining cell colonies were stained with neutral red and counted [1].
Replicon Clearance and Rebound Assay (Cell-based): Huh7-Luc replicon cells were treated with TMC647055 alone or in combination with TMC435 for 2 weeks in the absence of G418. Cells were subcultured twice per week, and cell pellets were collected for RNA extraction and RT-PCR to monitor HCV replicon RNA levels. After 2 weeks, the inhibitors were withdrawn, and cells were incubated for a further 3 weeks in the presence of G418 to allow for potential rebound of residual HCV RNA [1].

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To assess cellular activity in a high-throughput format, the HCV replicon system is used. Huh7 (human hepatoma) cells that contain a subgenomic HCV replicon (a self-replicating viral RNA that does not produce infectious virus) are cultured in DMEM with 10% FBS. Cells are seeded in 96-well plates and treated with TMC647055 Choline salt at varying concentrations (e.g., 0.1 nM to 10 microM) for 48-72 hours. After treatment, the cells are lysed, and the level of HCV RNA is quantified using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). Alternatively, a reporter gene (e.g., luciferase) can be incorporated into the replicon, and the signal is read directly after adding a substrate. The half-maximal effective concentration (EC50) for inhibiting RNA replication is then determined.

Rat Pharmacokinetic Study: Male rats were administered TMC647055 intravenously (2 mg/kg) and orally (10 mg/kg). The intravenous vehicle was PEG400/saline (70/30), and the oral vehicle was PEG400/2% vitamin E TPGS. Plasma clearance, liver concentrations (7 hours post-dose), liver-to-plasma ratio, and oral bioavailability were determined [2].
Dog Pharmacokinetic Study: Dogs were administered a single oral dose of TMC647055 at 10 mg/kg. The oral vehicle was PEG400/2% vitamin E TPGS. The resulting pharmacokinetic parameters (bioavailability, Cmax, AUC, clearance, Vdss) were evaluated [2].

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Animal/Disease Models: Rats[2]
Doses: 2 mg/kg; 10mg/kg
Route of Administration: 2 mg/kg, iv.; 10mg/kg, po.; singel
Experimental Results: No . Cl (L/h/kg) Cmax (ng/mL) [Liver] (ng/mL) L/PF (%) TMC647055 (compound 18a) 3.2 440 7800 46 ＞66

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In vivo studies are typically performed in mouse models using a surrogate system, such as the HCV replicon mouse model, or in chimeric mice with humanized livers. For pharmacokinetic and initial efficacy studies, the TMC647055 Choline salt is formulated in a suitable vehicle (e.g., 10% DMSO, 40% PEG300, 5% Tween 80, 45% saline). The compound is administered orally at a specific dose (e.g., 10-100 mg/kg). Blood samples are collected at various time points, and plasma concentrations of the compound are measured by LC-MS/MS. To assess antiviral efficacy, the compound may be administered multiple times (e.g., BID) over a period of 1-2 weeks, and the reduction in viral RNA levels in the plasma is measured.

In Vitro Metabolic Stability (Rat and Human Liver Microsomes): Metabolic stability was expressed as the percentage of compound metabolized after 15 minutes at a concentration of 5 μM. For TMC647055 (compound 18a), the value was 30% in rat liver microsomes and 35% in human liver microsomes [2].
Rat Pharmacokinetics: Following IV administration (2 mg/kg, vehicle: PEG400/saline 70/30), the plasma clearance (Cl) of TMC647055 was 3.2 L/h/kg. After oral administration (10 mg/kg, vehicle: PEG400/2% vitE-TPGS), the Cmax was 440 ng/mL, the liver concentration at 7 hours post-dose was 7800 ng/mL, the liver-to-plasma ratio was 46, and the oral bioavailability was >66% [2].
Dog Pharmacokinetics: After a single oral dose of 10 mg/kg (vehicle: PEG400/2% vitE-TPGS), TMC647055 showed high oral bioavailability (F = 87%), a Cmax of 10.2 μM, an AUC0-inf of 25.8 μM·h, a moderate plasma clearance (Cl = 0.54 L/h/kg), and a low volume of distribution (Vdss = 0.32 L/kg) [2].

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The choline salt formulation is designed to enhance the pharmacokinetic (PK) properties of the parent compound. TMC647055 Choline salt is formulated to improve absorption, making it suitable for oral administration. The compound is a non-nucleoside inhibitor, meaning it does not target the active site, and its PK parameters (Cmax, Tmax, AUC, half-life) would be determined in standard animal studies. In vivo, it is used for research into hepatitis C virus (HCV) and is typically stored at 4degC, away from moisture, to ensure its stability for long-term use [35L4-L5].

Cytotoxicity: TMC647055 was non-cytotoxic in a panel of cell lines. The mean CC50 was 42.1 μM in Huh7 cells and 28.9 μM in MT4 cells. In MRC-5, HEK-293T, HepG2, and VeroE6 cells, the CC50 was >50 μM [1]. The selectivity index (CC50/EC50) in Huh7 cells was over 70 to >400 for related analogs [2].
In Vivo Metabolism: In rats, in vivo metabolism of TMC647055 did not produce phase two metabolites, such as reactive acyl glucuronides [2].
Cytochrome P450 Inhibition: No major inhibition of cytochrome P450 enzymes was detected for TMC647055, suggesting a low potential for drug-drug interactions [2].
Genotoxicity and Cardiovascular Effects: In preclinical assays, TMC647055 showed an acceptable profile in assays measuring genotoxicity and cardiovascular effects [2].

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Specific toxicity data for TMC647055 Choline salt from publicly available sources is limited to its use in preclinical research. As a potent antiviral, its safety profile is important for its therapeutic use. In research, toxicity is usually evaluated in standard rodent studies where it is dosed orally. Observations would include clinical signs, body weight changes, and histopathological analysis of major organs (liver, kidney, heart). The choline salt of TMC647055 is developed to reduce potential toxicity issues associated with the parent compound, such as poor solubility or irritation. As with all research chemicals, standard safety practices should be followed. It is for research use only, not for human consumption.

[1]. TMC647055, a potent nonnucleoside hepatitis C virus NS5B polymerase inhibitor with cross-genotypic coverage. Antimicrob Agents Chemother. 2012 Sep;56(9):4676-4684.

[2]. Finger loop inhibitors of the HCV NS5b polymerase. Part II. Optimization of tetracyclic indole-based macrocycle leading to the discovery of TMC647055. Bioorg Med Chem Lett. 2012 Jul 1;22(13):4437-43.

Mechanism of Action (MOA): TMC647055 is a non-nucleoside inhibitor that binds to the allosteric NNI-1 site (thumb pocket I) on the HCV NS5B polymerase. This binding is proposed to displace the flexible λ1 loop from its thumb domain binding site, perturbing the interaction between the finger and thumb domains, thereby locking the enzyme in an open, inactive conformation and preventing RNA synthesis [1].
Binding Kinetics: SPR studies revealed that the high potency of TMC647055 against most genotypes is driven by a very slow dissociation rate (koff), resulting in a long complex half-life (t1/2), particularly for genotype 1b (130.5 minutes). The reduced activity against genotype 2b was attributed to a >9-fold faster dissociation rate and a >9-fold reduced complex half-life [1].
Clinical Status: At the time of publication, TMC647055 was being evaluated in clinical trials for the treatment of chronic hepatitis C. It completed Phase 1 studies in healthy volunteers and HCV-infected patients, demonstrating safety, tolerability, and antiviral activity. It was subsequently being evaluated in Phase 2 clinical trials [1][2].

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TMC647055 Choline salt is a potent and selective inhibitor of the HCV NS5B polymerase, which is a validated target for the treatment of Hepatitis C Virus (HCV) infection. It is part of the direct-acting antiviral (DAA) class of drugs that have revolutionized HCV treatment. The choline salt form is designed to enhance the compound's solubility and bioavailability. This product is strictly for research use only and is not intended for human clinical use. It is a valuable tool for virology research and the study of hepatitis C virus. It should be stored at 4degC, away from moisture, to maintain its chemical integrity [35L4-L5].

C37H53N5O8S

727.91

727.3614848

TMC647055;1204416-97-6

White to off-white solid powder

CN1CCOCCN(S(=O)(=O)NC(=O)C2=CC3=C(C=C2)C(=C4N3CC(=CC5=C4C=CC(=C5)OC)C1=O)C6CCCCC6)C.C[N+](C)(C)CCO.[OH-]

PHHZAIOGANLKJD-UHFFFAOYSA-M

InChI=1S/C32H38N4O6S.C5H14NO.H2O/c1-34-13-15-42-16-14-35(2)43(39,40)33-31(37)22-9-11-27-28(19-22)36-20-24(32(34)38)17-23-18-25(41-3)10-12-26(23)30(36)29(27)21-7-5-4-6-8-21;1-6(2,3)4-5-7;/h9-12,17-19,21H,4-8,13-16,20H2,1-3H3,(H,33,37);7H,4-5H2,1-3H3;1H2/q;+1;/p-1

28-cyclohexyl-22-methoxy-10,16-dimethyl-9,9-dioxo-13-oxa-9lambda6-thia-1,8,10,16-tetrazapentacyclo[16.8.1.12,6.13,26.020,25]nonacosa-2,4,6(29),18,20(25),21,23,26(28)-octaene-7,17-dione;2-hydroxyethyl(trimethyl)azanium;hydroxide

TMC647055 Choline salt; TMC-647055 (Choline salt); 28-cyclohexyl-22-methoxy-10,16-dimethyl-9,9-dioxo-13-oxa-9lambda6-thia-1,8,10,16-tetrazapentacyclo[16.8.1.12,6.13,26.020,25]nonacosa-2,4,6(29),18,20(25),21,23,26(28)-octaene-7,17-dione;2-hydroxyethyl(trimethyl)azanium;hydroxide; TMC647055 Choline Hydroxide Salt; orb1306307;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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

DMSO :~62.5 mg/mL (~85.86 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (2.86 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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