Development of robust antiviral assays for profiling compounds against a panel of positive-strand RNA viruses using ATP/luminescence readout

ABSTRACTHepatitis C virus (HCV) infection is a major global health burden and is associated with an increased risk of liver cirrhosis and hepatocellular carcinoma. There remains an unmet medical need for efficacious and safe direct antivirals with complementary modes of action for combination in treatment regimens to deliver a high cure rate with a short duration of treatment for HCV patients. Here we report thein vitroinhibitory activity, mode of action, binding kinetics, and resistance profile of TMC647055, a novel and potent nonnucleoside inhibitor of the HCV NS5B RNA-dependent RNA polymerase.In vitrocombination studies with an HCV NS3/4A protease inhibitor demonstrated potent suppression of HCV RNA replication, confirming the potential for combination of these two classes in the treatment of chronic HCV infection. TMC647055 is a potent nonnucleoside NS5B polymerase inhibitor of HCV replication with a promisingin vitrobiochemical, kinetic, and virological profile that is currently undergoing clinical evaluation.

Section: Methodsmentioning

confidence: 99%

TMC647055, a Potent Nonnucleoside Hepatitis C Virus NS5B Polymerase Inhibitor with Cross-Genotypic Coverage

Devogelaere

Berke

Vijgen

et al. 2012

“…Antiviral activities against West Nile virus (WNV-NY99/1; Vero cells), bovine viral diarrhea virus (BVDV-NADL; MDCK cells), yellow fever virus (YFV-17D; VeroE6 cells), Sindbis virus (Sin-V-339; Vero cells), influenza virus (influenza A Virginia/88; MDCK cells), vesicular stomatitis virus (MRC-5 cells), and herpes simplex virus type 2 (G strain; E6 Vero cells) were determined with a cytopathic effect inhibition assay (14). Activity against human immunodeficiency virus type 1 (IIIB; MT-4 cells) was assessed using an enhanced green fluorescent protein-based assay (17).…”

Section: Methodsmentioning

confidence: 99%

In Vitro Activity and Preclinical Profile of TMC435350, a Potent Hepatitis C Virus Protease Inhibitor

Lin¹,

Lenz²,

Fanning³

et al. 2009

185

143

The hepatitis C virus (HCV) NS3/4A serine protease has been explored as a target for the inhibition of viral replication in preclinical models and in HCV-infected patients. TMC435350 is a highly specific and potent inhibitor of NS3/4A protease selected from a series of novel macrocyclic inhibitors. In biochemical assays using NS3/4A proteases of genotypes 1a and 1b, inhibition constants of 0.5 and 0.4 nM, respectively, were determined. TMC435350 inhibited HCV replication in a cellular assay (subgenomic 1b replicon) with a half-maximal effective concentration (EC 50 ) of 8 nM and a selectivity index of 5,875. The compound was synergistic with alpha interferon and an NS5B inhibitor in the replicon model and additive with ribavirin. In rats, TMC435350 was extensively distributed to the liver and intestinal tract (tissue/plasma area under the concentration-time curve ratios of >35), and the absolute bioavailability was 44% after a single oral administration. Compound concentrations detected in both plasma and liver at 8 h postdosing were above the EC 99 value measured in the replicon. In conclusion, given the selective and potent in vitro anti-HCV activity, the potential for combination with other anti-HCV agents, and the favorable pharmacokinetic profile, TMC435350 has been selected for clinical development.

“…In an effort to identify small molecule inhibitors of enterovirus infections, we employed a molecular screening assay described previously (16), based on protection of cells from cytotoxic effects of viral infection. HeLa-RW cells were seeded into 384-well plates containing these compounds and infected at a low multiplicity of infection with CVB3-H3, a molecular clone of coxsackievirus B3 (CVB3) (20).…”

Section: Identification Of Novel Small Molecule Inhibitors Of Cvb3 Rementioning

confidence: 99%

“…We screened for novel inhibitors of enterovirus replication using an assay to monitor cell viability and detect the enterovirus-induced cytopathic effect (CPE) by modifying the assay described by Gong et al (16). Prior to adding library compounds, 20 l culture medium per well was dispensed into 384-well microtiter plates (Greiner One) and 0.5 l of 1 mM test compound solution in dimethyl sulfoxide (DMSO) was added using a 500-nl V&P custom pin tool (San Diego, CA).…”

mentioning

confidence: 99%

Fluoxetine Is a Potent Inhibitor of Coxsackievirus Replication

Zuo

Quinn²,

Kye³

et al. 2012

ABSTRACTNo antiviral drugs currently exist for the treatment of enterovirus infections, which are often severe and potentially life threatening. Molecular screening of small molecule libraries identified fluoxetine, a selective serotonin reuptake inhibitor, as a potent inhibitor of coxsackievirus replication. Fluoxetine did not interfere with either viral entry or translation of the viral genome. Instead, fluoxetine and its metabolite norfluoxetine markedly reduced the synthesis of viral RNA and protein. In view of its favorable pharmacokinetics and safety profile, fluoxetine warrants additional study as a potential antiviral agent for enterovirus infections.