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TMC435 is a small-molecule inhibitor of the NS3/4A serine protease of hepatitis C virus (HCV) currently in phase 2 development. The in vitro resistance profile of TMC435 was characterized by selection experiments with HCV genotype 1 replicon cells and the genotype 2a JFH-1 system. In 80% (86/109) of the sequences from genotype 1 replicon cells analyzed, a mutation at NS3 residue D168 was observed, with changes to V or A being the most frequent. Mutations at NS3 positions 43, 80, 155, and 156, alone or in combination, were also identified. A transient replicon assay confirmed the relevance of these positions for TMC435 inhibitory activity. The change in the 50% effective concentrations (EC 50 s) observed for replicons with mutations at position 168 ranged from 50 for the wild type. Of the positions identified, mutations at residue Q80 had the least impact on the activity of TMC435 (50 s), while greater effects were observed for some replicons with mutations at positions 43, 155, and 156. TMC435 remained active against replicons with the specific mutations observed after in vitro or in vivo exposure to telaprevir or boceprevir, including most replicons with changes at positions 36, 54, and 170 (50 s). Replicons carrying mutations affecting the activity of TMC435 remained fully susceptible to alpha interferon and NS5A and NS5B inhibitors. Finally, combinations of TMC435 with alpha interferon and NS5B polymerase inhibitors prevented the formation of drug-resistant replicon colonies.

The RNA-dependent RNA polymerase (NS5B) of hepatitis C virus (HCV) is an unusually attractive target for drug discovery since it contains five distinct drugable sites. The success of novel antiviral therapies will require nonnucleoside inhibitors to be active in at least patients infected with HCV of subtypes 1a and 1b. Therefore, the genotypic assessment of these agents against clinical isolates derived from genotype 1-infected patients is an important prerequisite for the selection of suitable candidates for clinical development. Here we report the 1a/1b subtype profiling of polymerase inhibitors that bind at each of the four known nonnucleoside binding sites. We show that inhibition of all of the clinical isolates tested is maintained, except for inhibitors that bind at the palm-1 binding site. Subtype coverage varies across chemotypes within this class of inhibitors, and inhibition of genotype 1a improves when hydrophobic contact with the polymerase is increased. We investigated if the polymorphism of the palm-1 binding site is the sole cause of the reduced susceptibility of subtype 1a to inhibition by 1,5-benzodiazepines by using reverse genetics, X-ray crystallography, and surface plasmon resonance studies. We showed Y415F to be a key determinant in conferring resistance on subtype 1a, with this effect being mediated through an inhibitor- and enzyme-bound water molecule. Binding studies revealed that the mechanism of subtype 1a resistance is faster dissociation of the inhibitor from the enzyme.

Optimization through parallel synthesis of a novel series of hepatitis C virus (HCV) NS5B polymerase inhibitors led to the identification of ( R )-11-(4-benzyloxy-2-fluorophenyl)-6-hydroxy-3,3-dimethyl-10-(6-methylpyridine-2-carbonyl)-2,3,4,5,10,11-hexahydro-dibenzo[ b,e ][1,4]diazepin-1-one 11zc and ( R )-11-(4-benzyloxy-2-fluorophenyl)-6-hydroxy-3,3-dimethyl-10-(2,5-dimethyloxazol-4-carbonyl)-2,3,4,5,10,11-hexahydro-dibenzo[ b,e ][1,4]diazepin-1-one 11zk as potent (replicon EC 50 = 400 nM and 270 nM, respectively) and selective (CC 50 > 20 μM) inhibitors of HCV replication. These data warrant further lead-optimization efforts.

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.

Screening of a focused library of TGF beta kinase inhibitors in the cellular HCV replicon model with luciferase read out yielded a number of low micromolar HCV inhibitors. Medicinal chemistry driven optimization resulted in the discovery of 4-[2-(5-bromo-2-fluoro-phenyl)pteridin-4-ylamino]-N-[3-(2- oxopyrrolidin-1-yl)propyl]nicotinamide 36 with a replicon EC50 of 64 nM, associated with a selective kinase inhibitory profile for human JNK kinases 2 and 3 as well as VEGFR-1, 2, and 3 kinases. Moreover, 36 showed an advantageous PK profile in mice. Experiments performed using different replicon constructs suggest that this series of kinase inhibitors might mediate their effect through the HCV non-structural protein 5A (NS5A).

The nonstructural protein 5 (NS5) of dengue virus (DENV) plays a central role in the virus replication. It functions as a methyltransferase and an RNA-dependent RNA polymerase. As such, it is a promising target for antiviral drug development. To develop a high-throughput biochemical assay for screening compound libraries, we expressed and purified the polymerase domain of the dengue NS5 protein in bacterial cells. The polymerase activity is measured using a scintillation proximity assay. This homogeneous and high--throughput assay enables screening of compound libraries for identifying polymerase inhibitors against DENV. In this chapter we describe the methods to express and purify the dengue NS5 polymerase from E. coli and a validated high-throughput enzymatic assay for screening inhibitors of NS5 polymerase.

The nonstructural protein 5 (NS5) of dengue virus (DENV) plays a central role in the virus replication. It functions as a methyltransferase and an RNA-dependent RNA polymerase. As such, it is a promising target for antiviral drug development. To develop a high-throughput biochemical assay for screening compound libraries, we expressed and purified the polymerase domain of the dengue NS5 protein in bacterial cells. The polymerase activity is measured using a scintillation proximity assay. This homogeneous and high--throughput assay enables screening of compound libraries for identifying polymerase inhibitors against DENV. In this chapter we describe the methods to express and purify the dengue NS5 polymerase from E. coli and a validated high-throughput enzymatic assay for screening inhibitors of NS5 polymerase.

Introduction The MAPK/ERK pathway is a complex signaling cascade involved in many cancer types. KRAS and BRAF gene mutations are present in a number of cancers, including colon, lung and pancreas, and identification of mutations in these genes is of great importance in clinical diagnostics. Moreover, there is a growing demand for performing multiple tests simultaneously on a single sample and there is an increased need to provide these answers to oncologists in a short timeframe. Methods IdyllaTM is a fully integrated and automated molecular diagnostics platform (1) that combines speed and ease of use with high sensitivity and high multiplexing capabilities. Moreover, it overcomes the current time-consuming step of processing formalin-fixed paraffin-embedded tissue (FFPE) samples. After insertion of a single FFPE slice into the cartridge, the complete process of sample preparation, real-time PCR and reporting is fully automated and takes less than 2 hours. We present here a KRAS-BRAF mutations prototype assay that allows the sensitive detection of 13 KRAS mutations and 5 BRAF mutations in one single assay. The assay discriminates the individual mutations at codons 12, 13 and 61 of KRAS and codon 600 of BRAF using novel “Primer Assisted Sequence Switching” (PASS) primers along with “Multi-component Nucleic Acid enzyme” (MNAzyme) detection. These technologies confer advantages for multiplex mutation analysis; PASS primers selectively amplify the target sequences of interest resulting in enhanced specificity between wild type (WT) and mutant, and between mutants, and MNAzymes allow for efficient detection and discrimination of multiple mutations simultaneously. Results Several performance characteristics of the IdyllaTM KRAS-BRAF prototype assay were examined: specificity, cross-reactivity, sensitivity and performance on clinical samples. Specificity of mutant versus WT as well as cross-reactivity between individual mutations at each codon was evaluated. Results demonstrated excellent specificity and cross-reactivity for all individual targets, with delta Cq values of >7 between mutants and >12 between mutant and WT. Sensitivity was assessed using cell lines embedded in FFPE containing defined ratios of mutants and dilutions of these in FFPE WT background. The results indicated sensitivities of 96 % concordance. Conclusion The new and fully integrated IdyllaTM KRAS-BRAF prototype assay combines extended multiplexing capabilities with excellent specificity, high sensitivity, ease of use, and short turnaround time for mutation analysis on FFPE samples. (1) For research use only Citation Format: Ina Vandenbroucke, Katrien Vermeiren, Elisa Mokany, Lit Yeen Tan, Nicole Lima, Samantha Walker, Geneviève Vandercruyssen, Bart Claes, Inky De Baere, Pascale Holemans, Evelien Rondelez, Alison Todd, Geert Maertens, Erwin Sablon. A rapid and fully automated multiplex assay for KRAS-BRAF mutations with high mutation sensitivity using novel selective amplification and detection technologies. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1502. doi:10.1158/1538-7445.AM2014-1502