Tania Ivens

In Vitro Resistance Profile of the Hepatitis C Virus NS3/4A Protease Inhibitor TMC435

ABSTRACT 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 (EC50s) observed for replicons with mutations at position 168 ranged from <10-fold for those with the D168G or D168N mutation to ∼2,000-fold for those with the D168V or D168I mutation, compared to the EC50 for the wild type. Of the positions identified, mutations at residue Q80 had the least impact on the activity of TMC435 (<10-fold change in EC50s), 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 (<3-fold change in EC50s). 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.

Indolopyridones Inhibit Human Immunodeficiency Virus Reverse Transcriptase with a Novel Mechanism of Action

ABSTRACT We have discovered a novel class of human immunodeficiency virus (HIV) reverse transcriptase (RT) inhibitors that block the polymerization reaction in a mode distinct from those of the nucleoside or nucleotide RT inhibitors (NRTIs) and nonnucleoside RT inhibitors (NNRTIs). For this class of indolopyridone compounds, steady-state kinetics revealed competitive inhibition with respect to the nucleotide substrate. Despite substantial structural differences with classical chain terminators or natural nucleotides, these data suggest that the nucleotide binding site of HIV RT may accommodate this novel class of RT inhibitors. To test this hypothesis, we have studied the mechanism of action of the prototype compound indolopyridone-1 (INDOPY-1) using a variety of complementary biochemical tools. Time course experiments with heteropolymeric templates showed “hot spots” for inhibition following the incorporation of pyrimidines (T>C). Moreover, binding studies and site-specific footprinting experiments revealed that INDOPY-1 traps the complex in the posttranslocational state, preventing binding and incorporation of the next complementary nucleotide. The novel mode of action translates into a unique resistance profile. While INDOPY-1 susceptibility is unaffected by mutations associated with NNRTI or multidrug NRTI resistance, mutations M184V and Y115F are associated with decreased susceptibility, and mutation K65R confers hypersusceptibility to INDOPY-1. This resistance profile provides additional evidence for active site binding. In conclusion, this class of indolopyridones can occupy the nucleotide binding site of HIV RT by forming a stable ternary complex whose stability is mainly dependent on the nature of the primer 3′ end.

1,5-Benzodiazepines, a Novel Class of Hepatitis C Virus Polymerase Nonnucleoside Inhibitors

ABSTRACT The exogenous control of hepatitis C virus (HCV) replication can be mediated through the inhibition of the RNA-dependent RNA polymerase (RdRp) activity of NS5B. Small-molecule inhibitors of NS5B include nucleoside and nonnucleoside analogs. Here, we report the discovery of a novel class of HCV polymerase nonnucleoside inhibitors, 1,5-benzodiazepines (1,5-BZDs), identified by high-throughput screening of a library of small molecules. A fluorescence-quenching assay and X-ray crystallography revealed that 1,5-BZD 4a bound stereospecifically to NS5B next to the catalytic site. When introduced into replicons, mutations known to confer resistance against chemotypes that bind at this site were detrimental to inhibition by 1,5-BZD 7a. Using a panel of enzyme isolates that covered genotypes 1 to 6, we showed that compound 4a inhibited genotype 1 only. In mechanistic studies, 4a was found to inhibit the RdRp activity of NS5B noncompetitively with GTP and to inhibit the formation of the first phosphodiester bond during the polymerization cycle. The specificity for the HCV target was evaluated by profiling the 1,5-BZDs against other viral and human polymerases, as well as BZD receptors.

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

The development of antiviral assays using an ATP/luminescence-based readout to profile antiviral compounds against the positive-strand RNA viruses: yellow fever virus (YFV), West Nile virus (WNV), Sindbis virus, and Coxsackie B virus, representing three virus families, is described. This assay readout is based upon the bioluminescent measurement of ATP in metabolically active cells. Antiviral efficacy was determined by measuring the ATP level in cells that were protected from the viral cytopathic effect (CPE) by the presence of antiviral compounds. The antiviral assay parameters were optimized and the assays were validated using a panel of different reference compounds to determine the intra- and inter-assay reproducibility. The signal-to-noise ratios for the yellow fever virus and West Nile virus assays were 7.5 and 36, respectively, comparing favorably with a signal-to-noise ratio of only 1.5 in the yellow fever virus neutral red dye uptake assay, an alternative readout for CPE inhibition. For Coxsackie B and Sindbis viruses, the signal-to-noise ratios were 40 and 50, respectively. These assays are robust, high-throughput, reproducible, and give much improved signal-to-noise ratios than those of dye uptake assays.

Expression and Purification of Dengue Virus NS5 Polymerase and Development of a High-Throughput Enzymatic Assay for Screening Inhibitors of Dengue 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.

Development of a homogeneous screening assay for automated detection of antiviral agents active against severe acute respiratory syndrome-associated coronavirus.

Abstract The severity and global spread of the 2003 outbreak of the severe acute respiratory syndrome-associated coronavirus (SARS-CoV) highlighted the risks to human health posed by emerging viral diseases and emphasized the need for specific therapeutic agents instead of relying on existing broadly active antiviral compounds. The development of rapid screening assays is essential for antiviral drug discovery. Thus, a screening system for anti-SARS-CoV agents was developed, which evaluated compound potency, specificity and cytotoxicity at the initial screening phase. Cell lines were engineered to constitutively express an enhanced green fluorescent protein (EGFP) and used to detect (1) antiviral potency in SARS-CoV infection tests; (2) antiviral specificity in tests using the porcine coronavirus transmissible gastroenteritis virus (TGEV); and (3) cytotoxicity in the same assays without virus challenge. The assay system involves minimal manipulation after assay set-up, facilitates automated read-out and minimizes risks associated with hazardous viruses. The suitability of this assay system in drug discovery was demonstrated by screening of 3388 small molecule compounds. The results show that these assays can be applied to high-throughput screening for identification of inhibitors selectively active against SARS-CoV.

Cell-Based Antiviral Assays for Screening and Profiling Inhibitors Against Dengue Virus

Dengue, a mosquito-borne virus of the Flaviviridae family, is reemerging as one of the most important human pathogens in tropical and subtropical regions of the world. It is estimated that 2.5 billion people live in areas at risk for transmission of dengue virus (DENV). Furthermore, it causes significant morbidity and mortality with 50-100 million infections per year. Currently, there are no vaccines commercially available and no effective antiviral drugs for treatment of DENV infections. In this chapter, we describe a plaque reduction assay and a cell-based high-throughput antiviral assay for identifying inhibitors against DENV. The latter is a homogeneous high-throughput assay that has been fully validated according to the Food and Drug Administration (FDA) guidelines for assay validation and can be used for screening compound libraries.

Development of a high-throughput antiviral assay for screening inhibitors of chikungunya virus and generation of drug-resistant mutations in cultured cells.

Chikungunya virus (CHIKV) is a mosquito-borne Alphavirus that has already infected millions of people in recent large-scale epidemics in Africa, the islands of the Indian Ocean, South and Southeast Asia, and northern Italy. The infection is still ongoing in many countries, such as India. Although the fatal rate is approximately 0.1% in the La Réunion outbreak, it causes painful arthritis-like symptoms that can last for months or even years. Currently, neither vaccine nor approved antiviral therapy exists to protect humans from chikungunya infection. Therefore, there is an urgent unmet medical need for the development of antiviral drugs for pre-exposure prophylaxis and/or treatment of chikungunya infections. In this chapter, we describe a fully validated ATP/luminescence assay that is effective for high-throughput screening of CHIKV inhibitors. Protocols for growing CHIKV stocks and generating drug-resistant viral variants for modes of action studies of compounds are also described.

Abstract 4213: BRAF V600 mutation testing on FFPE samples using a novel fully integrated molecular diagnostics platform: A concordance study with reference methods.

Introduction Activating mutations in the BRAF gene are common in skin and colorectal tumors. In more than 90% of the cases, these mutations are located in the BRAF V600 codon (V600E and V600K). Targeted therapies like vemurafenib and dabrafenib have shown objective response rates in up to half of metastatic melanomas harboring these BRAF V600 mutations. Tumor mutation status is usually assessed starting from formalin-fixed, paraffin-embedded (FFPE) tumor tissue shavings. Typically, the test involves shipment of the paraffin block from the local pathology lab to a specialized molecular lab, where several steps need to be performed. Methods The Biocartis molecular diagnostics (MDx) prototype platform is a novel, random access, sample-in result-out automated qPCR system. It uses a disposable cartridge which can simultaneously detect and quantify up to 30 molecular markers from a wide range of solid and liquid sample types, including blood, feces, and sputum. For FFPE-based assays, a single curl or shaving is directly placed into the cartridge. The complete process time for sample preparation, PCR and reporting is less than 90 min, with Results One sample was not eligible for Cobas testing because of low tumor content. Three other samples failed on both platforms. For the remaining 60 samples, results on the Biocartis MDx platform and Cobas were concordant in 58 (96.7%) samples (44 BRAF V600 WT and 14 BRAF V600 mutant samples, of which 36 samples were randomly selected and confirmed by deep sequencing). In 2 (3.3%) samples, no BRAF V600 mutation was detected by Cobas, while a BRAF V600 mutation was detected by both the Biocartis MDx platform and by deep sequencing. Both were melanoma samples (one of which was highly pigmented), excised in 1993 and 2005, and contained 4.6% V600E and 5.6% V600K (as determined by deep sequencing), respectively. Conclusions The new MDx platform is a fast and reliable method for BRAF V600 mutation testing directly on FFPE tumor shavings with superior analytical sensitivity, ease of use, and turnaround time compared to existing diagnostic tests. The BRAF V600 Mutation prototype assay provided excellent concordance with both Cobas (96.7%) and deep sequencing (100%). Citation Format: Benoit Devogelaere, Koen Van Acker, Inky De Baere, Pascale Holemans, Tania Ivens, Bart Claes, Evelien Rondelez, Genevieve Vandercruyssen, Marijke Van der Auwera, Mark Kockx, Isabelle Vanden Bempt, Ina Vandenbroucke, Geert Maertens, Erwin Sablon. BRAF V600 mutation testing on FFPE samples using a novel fully integrated molecular diagnostics platform: A concordance study with reference methods. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4213. doi:10.1158/1538-7445.AM2013-4213

Abstract 5584: BRAF mutation testing in cell-free DNA from plasma of patients with advanced cancers using a novel, rapid, automated molecular diagnostics prototype platform (IdyllaTM)

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: Cell-free (cf) DNA from the plasma of cancer patients offers an easily obtainable, low-risk, inexpensive and repeatedly applicable source of biologic material for mutation analysis of druggable targets and monitoring molecular changes in tumor(s) during and after therapeutic interventions. Novel, fast, and accurate diagnostic systems are needed for further development of plasma cfDNA testing in personalized therapy. Methods: cfDNA from plasma samples of patients with advanced cancers who progressed on systemic therapy was purified and 100 ng was used for testing for V600 BRAF mutations using the prototype molecular diagnostics (IdyllaTM) fully integrated real-time PCR-based prototype platform (Biocartis, Mechelen, Belgium) with a quick turnaround time (< 90 minutes). The IdyllaTM platform and the BRAF V600 mutation prototype assay were used for research purposes only. Results were compared to mutation analysis of archival primary or metastatic tumor tissue from a CLIA-certified laboratory if available. Results: cfDNA was extracted from plasma samples of 81 patients with advanced cancers (colorectal, n=37; melanoma, n=16; non-small cell lung, n=10; breast, n=4, Erdheim-Chester histiocytosis, n=3; appendiceal, n=3; other cancers, n=8). BRAF mutations were detected in 31% (25/81) of plasma samples and in 42% (30/72) of available archival tumor samples, resulting in concordance for patients who had plasma and tissue tested (N=72) in 90% (65/72) of cases (kappa=0.80, 95% confidence interval 0.65- 0.94). In 6 of 7 discrepant cases identical plasma cfDNA samples were tested using an alternative cfDNA BRAF mutation PCR-based method (BEAMing, Sysmex Inostics, Baltimore, MD), which yielded 100% agreement. Longitudinally collected plasma samples were available in 3 patients (appendiceal, melanoma and papillary thyroid cancer) treated with BRAF targeting combinations and changes in the amount of BRAF-mutant cfDNA corresponded with changes in tumor markers or subsequent responses to therapy visualized via imaging. Conclusions: Detecting V600 BRAF mutations in cfDNA from plasma using the IdyllaTM platform is a fast and noninvasive alternative to mutation testing of tumor tissue with an acceptable level of concordance and sensitivity, and should be investigated further for testing and monitoring of BRAF mutation status in patients with cancer. Citation Format: Helen J. Huang, Bart Claes, Gerald S. Falchook, Veronica R. Holley, Aung Naing, Sarina A. Piha-Paul, Apostolia M. Tsimberidou, Ralph G. Zinner, Daniel D. Karp, Siqing Fu, Vivek Subbiah, David S. Hong, Jennifer J. Wheler, Vanda M. Stepanek, Goran Cabrilo, Rajyalakshmi Luthra, Agop Y. Bedikian, Bryan K. Kee, Cathy Eng, Michael J. Overman, Kevin B. Kim, Tania Ivens, Erwin Sablon, Geert Maertens, Razelle Kurzrock, Funda Meric-Bernstam, Filip Janku. BRAF mutation testing in cell-free DNA from plasma of patients with advanced cancers using a novel, rapid, automated molecular diagnostics prototype platform (IdyllaTM). [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 5584. doi:10.1158/1538-7445.AM2014-5584