Development of a high-throughput replicon assay for the identification of respiratory syncytial virus inhibitors

Tiong-Yip, Choi-Lai; Plant, Helen; Sharpe, Paul T.; Fan, Jun; Rich, Kirsty; Gorseth, Elise; Yu, Qian

doi:10.1016/j.antiviral.2013.11.003

Cited by 14 publications

(23 citation statements)

References 14 publications

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“…The RSV replicon cell lines (Apath) were cultured as previously described (15,16). Differentiated human bronchial epithelial cell (HBEC) air-liquid interface cultures (HBEC-ALI) (MatTek) were cultured and maintained according to the manufacturer's instructions.…”

Section: Cells and Virusesmentioning

confidence: 99%

Characterization of a Respiratory Syncytial Virus L Protein Inhibitor

Tiong-Yip

Aschenbrenner

Johnson

et al. 2014

Antimicrob Agents Chemother

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The respiratory syncytial virus (RSV) L protein is a viral RNA-dependent RNA polymerase that contains multiple enzyme activities required for RSV replication. The RSV L inhibitors described in literature are limited by their cytotoxicity or the lack of RSV B subtype coverage. Here, we characterize a new RSV L inhibitor with strong antiviral activity against both RSV A and B subtypes and no detectable cytotoxicity. This compound, AZ-27, was equally active against RSV live viruses and subgenomic replicons and demonstrated advantages over other classes of RSV inhibitors in time-of-addition and cell line dependency studies. Resistance studies identified a dominant mutation in the putative capping enzyme domain of L protein, which conferred strong resistance to the AZ-27 series but not other classes of RSV inhibitors, supporting RSV L protein as the direct target for AZ-27. This novel and broad-spectrum RSV L polymerase inhibitor may pave the way toward an efficacious RSV therapeutic and provide a new tool for interrogation of the L protein function. R espiratory syncytial virus (RSV) is an enveloped, nonsegmented negative-sense RNA virus in the Paramyxoviridae family. RSV infection is ubiquitous in that virtually everyone is infected by the age of 2 years and reinfection occurs throughout all ages. It is the leading cause of acute lower respiratory tract infections in young children, the elderly, and immunosuppressed patients (1). Progress has been made toward vaccine development, but many challenges remain, as highlighted by the short-lived natural immune response against RSV with high reinfection rate, the difficulty in eliciting a protective immune response in neonates, and the unexpected enhancement of disease by RSV vaccination observed in the formalin-inactivated RSV vaccine trial (2). Immunoprophylaxis with RSV-neutralizing antibodies has been successful in protecting high-risk infants and children. However, there is no RSV-specific therapy available for postinfection treatment, and RSV continues to be the number one reason for infant hospitalization (3). The only approved treatment for RSV is ribavirin, which has limited clinical utility due to its high toxicity and controversial efficacy (4). Therefore, finding an effective treatment for RSV infection remains an important public health priority.The limited understanding of the molecular mechanisms of RSV replication and pathogenesis has hampered the development of RSV therapeutics (5). RSV replication requires the viral RNA genome, mRNAs, 11 viral proteins, and many host factors, all of which are potential targets for therapeutic intervention. Targeting host factors holds the promise of broader-spectrum coverage and a potentially higher barrier to resistance. However, there may also be on-target toxicity, the adverse pharmacologic effect of interfering with a cellular target important for host function, which would be of particular concern in treating young infants, the main population affected by severe RSV diseases. Antivirals directly targeting viral pro...

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Section: Cells and Virusesmentioning

confidence: 99%

Characterization of a Respiratory Syncytial Virus L Protein Inhibitor

Tiong-Yip

Aschenbrenner

Johnson

et al. 2014

Antimicrob Agents Chemother

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“…10 Briefly, the enrichment of the repliconexpressing APC126 cells to 40% of the total cell population resulted in a 4-fold increase in assay signal-to-background ratio, a reduced coefficient of variation (15%), and a significantly improved Z′ from 0.42 to 0.55 (p < 0.005). To pharmacologically validate the replicon assay, we tested an analogue of YM-53403, obtaining an EC 50 of 0.16 µM, which is in line with published data 11 (Fig.…”

Section: Rsv Replicon Primary Htsmentioning

confidence: 99%

“…10 The use of an immortalized replicon cell line to prosecute multiple target classes was highly desirable. It gave us the advantages associated with a robust phenotypic cellular assay without the challenge of the biological safety requirements associated with pathogen screening at scale.…”

Section: High-throughput Hit Screening Cascade To Identify Respiratormentioning

confidence: 99%

“…To test the validity of the replicon assay in CR format, we used the validation data 10 to select a set of 47 reference compounds with a range of potencies. These were tested twice in CR format in the replicon assay.…”

Section: Ec 50 Screening Phasementioning

confidence: 99%

“…Luminescence was measured using an Ultra plate reader (Tecan, Männedorf, Switzerland). Screening assays using cryopreserved APC126-E (campaign 2) were performed as described previously 10 by dispensing cells into ARPs at a density of 3000 cells/well in 60 µL assay media. After a 48 h incubation under standard culture conditions, assay media were removed to leave a final volume of 10 µL, to which 20 µL of 25 µM EnduRen substrate was added.…”

Section: Rsv Replicon Hts Assaysmentioning

confidence: 99%

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High-Throughput Hit Screening Cascade to Identify Respiratory Syncytial Virus (RSV) Inhibitors

et al. 2015

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Respiratory syncytial virus (RSV) infects 99% of children by age 2 years and is a leading cause of serious lower respiratory tract infection (LRTI) and infant hospitalization in the United Kingdom. Identification of efficacious RSV therapeutics has been hindered by the lack of a robust and appropriate primary assay for high-throughput screening (HTS). Here we report an HTS cascade that identified inhibitors of RSV replication using a robust RSV replicon luminescence-reporter assay for the primary campaign. The performance of the assay was consistent and reliable at scale, with Z′ of 0.55 ± 0.08 across 150 assay plates and signal-to-background ratios >40. The HTS assay was used to screen the AstraZeneca compound library of 1 million compounds at a single concentration of 10 µM. Hits specifically targeting the RSV replicon were determined using a series of hit generation assays. Compounds nonspecifically causing cell toxicity were removed, and hits were confirmed in live viral inhibition assays exhibiting greater physiological relevance than the primary assay. In summary, we developed a robust screening cascade that identified hit molecules that specifically targeted RSV replication.

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Recent Advances in Developing Antiviral Therapies for Respiratory Syncytial Virus

Shook

Lin

2017

Top Curr Chem (Z)

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Respiratory syncytial virus (RSV) infection presents a significant health challenge in small children, the elderly and immunocompromised patients. There is still a high unmet medical need among patients with RSV infection, and no specific antiviral therapy is available. The only approved agents are palivizumab, which has to be given prophylactically, mainly in high-risk infants, and ribavirin, which is rarely used due to toxicity concerns and questionable benefits. Efforts to develop new antiviral agents have been hampered by the perceived need for high safety hurdles in pediatric patient populations use and the lack of well-characterized druggable targets. Despite these challenges, significant progress has been made in discovering multiple classes of inhibitors targeting various stages of the viral life cycle. Several recent proof-of-concept human studies with specific antivirals have shown promising results and completely reinvigorated the field. In this review we discuss the current status of RSV drug development, remaining challenges and future directions.

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Development of a high-throughput replicon assay for the identification of respiratory syncytial virus inhibitors

Cited by 14 publications

References 14 publications

Characterization of a Respiratory Syncytial Virus L Protein Inhibitor

Characterization of a Respiratory Syncytial Virus L Protein Inhibitor

High-Throughput Hit Screening Cascade to Identify Respiratory Syncytial Virus (RSV) Inhibitors

Recent Advances in Developing Antiviral Therapies for Respiratory Syncytial Virus

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