Novel Chemical Scaffolds for Inhibition of Rifamycin-Resistant RNA Polymerase Discovered from High-Throughput Screening

The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) plays an essential role in the replication of HCV and is a key target for novel antiviral therapies. Several RdRp inhibitors are in clinical trials and have increased response rates when combined with current interferon-based therapies for genotype 1 (G1) HCV patients. These inhibitors, however, show poor efficacy against non-G1 genotypes, including G3a, which represents ~20% of HCV cases globally. Here, we used a commercially available fluorescent dye to characterize G3a HCV RdRp in vitro. RdRp activity was assessed via synthesis of double-stranded RNA from the single-stranded RNA poly(C) template. The assay was miniaturized to a 384-well microplate format and a pilot high-throughput screen was conducted using 10,208 “lead-like” compounds, randomly selected to identify inhibitors of HCV G3a RdRp. Of 150 compounds demonstrating greatest inhibition, 10 were confirmed using both fluorescent and radioactive assays. The top two inhibitors (HAC001 and HAC002) demonstrated specific activity, with an IC50 of 12.7 µM and 1.0 µM, respectively. In conclusion, we describe simple, fluorescent-based high-throughput screening (HTS) for the identification of inhibitors of de novo RdRp activity, using HCV G3a RdRp as the target. The HTS system could be used against any positive-sense RNA virus that cannot be cultured.

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A simple cell-based high throughput screening (HTS) assay for inhibitors of Salmonella enterica RNA polymerase containing the general stress response regulator RpoS (σ S )

Journal of Microbiological Methods ◽

10.1016/j.mimet.2018.05.009 ◽

2018 ◽

Vol 150 ◽

pp. 1-4 ◽

Cited By ~ 1

Author(s):

Javier Campos-Gomez ◽

Jorge A. Benitez

Keyword(s):

Stress Response ◽

Rna Polymerase ◽

High Throughput ◽

Salmonella Enterica ◽

High Throughput Screening ◽

Response Regulator ◽

Simple Cell ◽

General Stress Response ◽

General Stress

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High-throughput screening identification of poliovirus RNA-dependent RNA polymerase inhibitors

Antiviral Research ◽

10.1016/j.antiviral.2011.06.006 ◽

2011 ◽

Vol 91 (3) ◽

pp. 241-251 ◽

Cited By ~ 31

Author(s):

Grace Campagnola ◽

Peng Gong ◽

Olve B. Peersen

Keyword(s):

Rna Polymerase ◽

High Throughput ◽

High Throughput Screening ◽

Rna Dependent Rna Polymerase ◽

Polymerase Inhibitors

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Luminescence Resonance Energy Transfer-Based High-Throughput Screening Assay for Inhibitors of Essential Protein-Protein Interactions in Bacterial RNA Polymerase

Applied and Environmental Microbiology ◽

10.1128/aem.69.3.1492-1498.2003 ◽

2003 ◽

Vol 69 (3) ◽

pp. 1492-1498 ◽

Cited By ~ 27

Author(s):

Veit Bergendahl ◽

Tomasz Heyduk ◽

Richard R. Burgess

Keyword(s):

Energy Transfer ◽

Drug Discovery ◽

Rna Polymerase ◽

High Throughput ◽

High Throughput Screening ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Screening Assay ◽

Essential Protein ◽

High Throughput Screening Assay

ABSTRACT The binding of sigma factors to core RNA polymerase is essential for the specific initiation of transcription in eubacteria and is thus critical for cell growth. Since the responsible protein-binding regions are highly conserved among all eubacteria but differ significantly from eukaryotic RNA polymerases, sigma factor binding is a promising target for drug discovery. A homogeneous assay for sigma binding to RNA polymerase (Escherichia coli) based on luminescence resonance energy transfer (LRET) was developed by using a europium-labeled σ70 and an IC5-labeled fragment of the β′ subunit of RNA polymerase (amino acid residues 100 through 309). Inhibition of sigma binding was measured by the loss of LRET through a decrease in IC5 emission. The technical advances offered by LRET resulted in a very robust assay suitable for high-throughput screening, and LRET was successfully used to screen a crude natural-product library. We illustrate this method as a powerful tool to investigate any essential protein-protein interaction for basic research and drug discovery.

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High-Throughput Screening of RNA Polymerase Inhibitors Using a Fluorescent UTP Analog

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057106291978 ◽

2006 ◽

Vol 11 (8) ◽

pp. 968-976 ◽

Cited By ~ 18

Author(s):

Jyothi Bhat ◽

Rajendra Rane ◽

Suresh M. Solapure ◽

Dhiman Sarkar ◽

Umender Sharma ◽

...

Keyword(s):

Rna Polymerase ◽

High Throughput ◽

Mycobacterium Smegmatis ◽

High Throughput Screening ◽

E Coli ◽

Sequence Identity ◽

Primary Screening ◽

Naphthalene Sulfonic Acid ◽

Comparable Activity ◽

Radioactive Assay

RNA polymerase (RNAP) is a well-validated target for the development of antibacterial and antituberculosis agents. Because the purification of large quantities of native RNA polymerase from pathogenic mycobacteria is hazardous and cumbersome, the primary screening was carried out using Escherichia coli RNAP. The authors have developed a high-throughput screening (HTS) assay to screen for novel inhibitors of RNAP. In this assay, a fluorescent analog of UTP, gamma-amino naphthalene sulfonic acid (γ-AmNS) UTP, was used as one of the nucleotide substrates. Incorporation of UMP in RNA results in the release of γ-AmNS-PPi, which has higher intrinsic fluorescence than (γ-AmNS) UTP. The assay was optimized in a 384-well format and used to screen 670,000 compounds at a concentration of 10 μM. About 0.1% of the compounds showed more than 60% inhibition in the primary HTS. All the primary actives tested for dose response using the same assay had an EC50 below 100 μM. Eighty percent of the primary HTS actives obtained using E. coli RNAP showed comparable activity against Mycobacterium smegmatis RNAP in the conventional radioactive assay. Activity of hits selected for the hit-to-lead optimization was also confirmed against Mycobacterium bovis RNAP which has >99% sequence identity with Mycobacterium tuberculosis RNAP subunits.

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