Inhibition and enhancement of Respiratory Syncytial Virus replication by nucleoside analogues and bis(indole) compounds

Abstract

Respiratory syncytial virus (RSV) is an Orthopneumovirus that infects the epithelium of the airways. Severe RSV infection of the lower respiratory tract in infants is a leading cause of pediatric hospitalizations. RSV also causes substantial morbidity in immunocompromised and elderly populations. Palivizumab, a humanized monoclonal antibody, is available for the prophylactic treatment of high-risk infants. However, this intervention is expensive and has a limited impact on annual hospitalization rates caused by RSV. No vaccine is available to prevent RSV infection and no efficacious antivirals are available to treat active infection. To address the burden of disease imposed by RSV, this project sought to develop and implement a screening assay to identify compounds with antiviral activity against RSV. Different screening protocols were examined as platforms for testing antiviral activity. The first protocol quantified changes to RSV replication complex morphology during antiviral treatment. Through confocal microscopy, changes to replication complex morphology were identified as early as six hours post infection. However, this assay was hindered by the low signal intensity produced by replication complexes. Therefore, alternative RSV-antiviral screening protocols were investigated. Subsequent protocols quantified initial monolayer infection, or quantified RSV progeny production, via colourimetric or immunofluorescence (IF) staining. Automated detection of IF-stained RSV-infected cells was conducted using a high content imaging system. This protocol (referred to as ‘the IF protocol’) offered the highest throughput screening capacity and most reliable detection of RSV infection versus the other methods that were tested. Using the IF protocol, the chemotherapeutic nucleoside analogue cytarabine was investigated and antiviral activity against RSV was observed. The IF protocol was then used to screen a series of bis(indole) compounds for antiviral activity against RSV. Bis(indole) compounds were hypothesized to have antiviral activity as they were derived from Isatisine A, a naturally occurring compound with modest antiviral activity. Bis(indole) compounds with antiviral activity against RSV were identified; the IF protocol was then used to guide the synthesis of novel bis(indole) compounds with improved cytotoxicity profiles. The Toll-like receptor 7 (TLR7) agonist loxoribine was investigated for antiviral activity against RSV, however, enhancement of RSV replication during loxoribine treatment was observed. This observation was unexpected as TLR7 is a pattern recognition receptor which contributes to the identification of pathogens by the innate immune system and TLR7 stimulation typically elicits an antiviral immune response. Furthermore, TLR7 agonists are undergoing clinical investigations to examine their potential as immunomodulatory treatments for airway diseases. As enhancing the severity of RSV infections in this population could be hazardous, it was considered essential to further characterize the relationship of loxoribine with RSV replication. Loxoribine-mediated enhancement of RSV replication in human airway epithelial cells was determined to be concentration-dependent and this effect was reproducible with the distinct TLR7 agonist CL097. Inhibition of TLR7 stimulation by the antagonist IRS-661, or by siRNA knock down of TLR7, prevented enhancement of RSV replication by loxoribine. Finally, TLR7-mediated enhancement of RSV replication was determined to be dependent on extracellular signal-regulated kinase activation. These results support the novel conclusion that exogenous stimulation of TLR7 benefits RSV replication. These results also suggest caution is warranted during the ongoing development of TLR7-based therapeutics, especially for therapeutics being developed for the treatment of airway diseases.