ABSTRACT
The recent development of a Hepatitis C virus (HCV) infectious virus cell culture model system has facilitated the development of whole-virus screening assays which can be used to interrogate the entire virus life cycle. Here, we describe the development of an HCV growth assay capable of identifying inhibitors against all stages of the virus life cycle with assay throughput suitable for rapid screening of large-scale chemical libraries. Novel features include, 1) the use of an efficiently-spreading, full-length, intergenotypic chimeric reporter virus with genotype 1 structural proteins, 2) a homogenous assay format compatible with miniaturization and automated liquid-handling, and 3) flexible assay end-points using either chemiluminescence (high-throughput screening) or Cellomics ArrayScan™ technology (high-content screening). The assay was validated using known HCV antivirals and through a large-scale, high-throughput screening campaign that identified novel and selective entry, replication and late-stage inhibitors. Selection and characterization of resistant viruses provided information regarding inhibitor target and mechanism. Leveraging results from this robust whole-virus assay represents a critical first step towards identifying inhibitors of novel targets to broaden the spectrum of antivirals for the treatment of HCV.
Subject(s)
Antiviral Agents/analysis , Antiviral Agents/pharmacology , Hepacivirus/drug effects , Hepacivirus/growth & development , High-Throughput Screening Assays/methods , Drug Resistance, Viral/drug effects , Genome, Viral/genetics , Hepacivirus/genetics , Humans , Reproducibility of Results , Virus Replication/drug effectsABSTRACT
Receptor activator of nuclear factor-kappaB (NF-kappaB) (RANK) plays a key role in the differentiation, activation, and survival of osteoclasts. Upon activation of RANK with RANK ligand (RANKL), osteoclast precursor cells differentiate into tartrate-resistant acid phosphatase (TRAP)-positive, multinucleated osteoclasts. To identify compounds that block osteoclastogenesis, a cell-based assay was developed using RAW264.7 cells stably transfected with a TRAP promoter-dependent reporter gene as a surrogate readout for differentiation. Described herein is the strategy for high throughput screening and subsequent secondary biological assays for hit triage, which resulted in the identification of compound 1, a 4-nitroimidazole derivative, that specifically inhibited RANKL-induced TRAP gene and protein expression. Compound 1 did not affect the tumor necrosis factor-alpha- or lipopolysaccharide-induced TRAP-luciferase response, suggesting selective inhibition of the RANKL-induced pathway. Reverse transcription polymerase chain reaction analysis confirmed the inhibition of expression of osteoclast marker genes, such as TRAP, cathepsin K, and carbonic anhydrase type II. Compound 1 did not inhibit the RANKL-induced activation of a NF-kappaB reporter gene, or p38 kinase activity, suggesting a mechanism of action downstream of NF-kappaB. Together, these results suggest that we have identified a RANK pathway-specific inhibitor able to block the RANKL-induced osteoclast differentiation process. The hit identification strategy described here can be applied to other cell-based assays using an indirect surrogate readout to improve success rates.
Subject(s)
Nitroimidazoles/pharmacology , Osteoprotegerin/pharmacology , RANK Ligand/metabolism , Signal Transduction/drug effects , Acid Phosphatase/genetics , Acid Phosphatase/metabolism , Animals , Cell Line , Cell Line, Tumor , Cell Survival/drug effects , Dimethyl Sulfoxide/pharmacology , Dose-Response Relationship, Drug , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Gene Expression/drug effects , Humans , Imidazoles/chemistry , Imidazoles/pharmacology , Isoenzymes/genetics , Isoenzymes/metabolism , Lipopolysaccharides/pharmacology , Luciferases/genetics , Luciferases/metabolism , Nitroimidazoles/chemistry , Osteoclasts/cytology , Osteoclasts/drug effects , Osteoclasts/metabolism , Osteoprotegerin/chemistry , Pyridines/chemistry , Pyridines/pharmacology , RANK Ligand/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reproducibility of Results , Reverse Transcriptase Polymerase Chain Reaction , Tartrate-Resistant Acid Phosphatase , Tumor Necrosis Factor-alpha/metabolism , Tumor Necrosis Factor-alpha/pharmacologyABSTRACT
The identification of a new series of selective nonsteroidal progesterone receptor (PR) agonists is reported. Using a high-throughput screening assay based on the measurement of transactivation of a mouse mammary tumor virus promoter-driven luciferase reporter (MMTV-Luc) in human breast cancer T47D cells, a benzimidazole-2-thione analog was identified. Compound 1 showed an apparent EC50 of 53 nM and efficacy of 93% with respect to progesterone. It binds to PR with high affinity (Ki nM), but had no or very low affinity for other steroid hormone receptors. Structure-activity relationship studies of a series of benzimidazole-2-thione analogs revealed critical positions for high PR binding affinity and transactivation potency as well as receptor selectivity, as exemplified by 25. Compound 25 binds to human PR with high affinity (Ki nM) and had at least > 1000-fold selectivity for PR versus other steroid receptors. Molecular modeling studies suggested that these agonists overlap favorably with progesterone in the ligand-binding domain of PR. In T47D cells, compound 25 acted as a full agonist in the MMTV-Luc reporter assay, as well as in the induction of endogenous alkaline phosphatase activity with apparent EC50 values of 4 and 9 nM, respectively. In the immature rat model, compound 25 provided a significant suppression of estrogen-induced endometrium hypertrophy as measured by luminal epithelial height. In contrast, compound 25 was inactive in the luteinizing hormone release assay in young ovariectomized rats. These benzimidazole-2-thione analogs constitute a new series of nonsteroidal PR agonists with an excellent steroid receptor selectivity profile. The differential activities observed in the in vivo progestogenic assays in rat models suggest that these analogs can act as selective PR modulators.