A Parallel Phenotypic Versus Target-Based Screening Strategy for RNA-Dependent RNA Polymerase Inhibitors of the Influenza A Virus

Abstract

Influenza A virus infections cause significant morbidity and mortality, and novel antivirals are urgently needed. Influenza RNA-dependent RNA polymerase (RdRp) activity has been acknowledged as a promising target for novel antivirals. In this study, a phenotypic versus target-based screening strategy was established to identify the influenza A virus inhibitors targeting the virus RNA transcription/replication steps by sequentially using an RdRp-targeted screen and a replication-competent reporter virus-based approach using the same compounds. To demonstrate the utility of this approach, a pilot screen of a library of 891 compounds derived from natural products was carried out. Quality control analysis indicates that the primary screen was robust for identification of influenza A virus inhibitors targeting RdRp activity. Finally, two hit candidates were identified, and one was validated as a putative RdRp inhibitor. This strategy can greatly reduce the number of false positives and improve the accuracy and efficacy of primary screening, thereby providing a powerful tool for antiviral discovery.

Screening for Anti-Influenza Actives of Prefractionated Traditional Chinese Medicines

“…Previously, we generated a recombinant IAV expressing Gaussia luciferase, based on which a phenotypic high-throughput screening approach was subsequently established, providing a powerful tool for antiviral discovery [ 11 , 12 , 15 ]. A phenotypic screening was therefore carried out against the prefractionated TCM library for anti-influenza actives.…”

“…Madin–Darby canine kidney (MDCK) epithelial cells were grown in Dulbecco's modified Eagle's medium (DMEM; Cellgro, Manassas, VA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Carlsbad, CA, USA), 1,000 units/mL penicillin and 100 μ g/mL of streptomycin (Invitrogen, Carlsbad, CA, USA). The replication-competent reporter influenza A virus carrying the Gaussia luciferase gene (PR8-PB2-Gluc) and wildtype influenza A/Puerto Rico/8/34 (H1N1, PR8) were propagated as previously described [ 11 , 12 , 15 ]. Infections were performed in Opti-MEM containing 1.5 µ g/mL N-tosyl-L-phenylalanine chloromethyl ketone (TPCK)-trypsin (Sigma-Aldrich, St. Louis, MO, USA).…”

“…A phenotypic screening for anti-influenza actives were carried out as previously described [ 11 , 16 ]. In brief, MDCK cells growing in white, flat-bottom, 96-well culture plates (PerkinElmer, Waltham, MA) were infected with PR8-PB2-Gluc virus at 0.01 multiplicities of infection (MOI) in the presence of test samples of 20 μ g/mL.…”

“…Titer reduction assay was performed as previously described [ 11 ]. In brief, MDCK cells grown in 24-well plates were inoculated with the influenza PR8 virus at an MOI of 0.01.…”

“…In the present study, 100 medicinal plants which have been included in TCM prescriptions for antiviral treatment were selected and prefractionated into 5 fractions each. The library consisting of 500 prefractionated TCM extracts was subsequently subjected to a phenotypic screening for anti-influenza actives [ 11 , 12 ]. As a result, ten TCM fractions were identified to have antiviral potency against IAV, deserving further analysis for novel anti-influenza lead drugs.…”