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].

In Huh7-Luc cells, TMC647055 exhibits antiviral activity with an EC90 value of 0.3 μM [1]. With an EC50 value of 82 nM, TMC647055 exhibits strong combinatorial action in cellular HCV detection [2].

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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].

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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TMC647055 (Compound 18a) (2 mg/kg IV; 10 mg/kg PO) demonstrated acceptable PK profiles characterized by high oral bioavailability following a single oral dose of 10 mg/kg combined with moderate doses and high systemic exposure. Plasma clearance and volume of distribution are low [2].

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].

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].

Animal/Disease Models: Rat[2]
Doses: 2 mg/kg; 10mg/kg
Route of Administration: 2mg/kg, intravenous (iv) (iv)injection; 10mg/kg, oral administration; Single
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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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].

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].

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].

[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 has been used in research trials for the treatment of hepatitis C, chronic hepatitis C, and chronic hepatitis C virus.

C32H38N4O6S

606.732326984406

606.251

C, 63.35; H, 6.31; N, 9.23; O, 15.82; S, 5.28

1204416-97-6

TMC647055 Choline salt

44556044

Typically exists as solid at room temperature

1.4±0.1 g/cm3

1.669

3.77

1

7

2

43

1170

0

S1(N(C)CCOCCN(C)C(C2=CC3C=C(C=CC=3C3=C(C4C=CC(C(N1)=O)=CC=4N3C2)C1CCCCC1)OC)=O)(=O)=O

UOBYJVFBFSLCTQ-UHFFFAOYSA-N

InChI=1S/C32H38N4O6S/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/h9-12,17-19,21H,4-8,13-16,20H2,1-3H3,(H,33,37)

28-cyclohexyl-22-methoxy-10,16-dimethyl-9,9-dioxo-13-oxa-9λ6-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

TMC647055; TMC-647055; TMC 647055; UNII-11BD024G7J; 11BD024G7J;

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.)