Methods: The anti-viral activity of PC786 was assessed in in vitro cytopathic effect (CPE) assays using laboratory adapted RSV (RSVA2, RSVB Washington (WST)) and low passage clinical isolates with genotypic diversity including ten isolates (five iolates each) of RSV A and B (Tennessee) in HEp2 cells. Cellular RSV replication/transcription and protein expression were also investigated by PCR in BEAS2B cells, Western blotting and a minigenome-based reporter assay in HEp2 cells. Effects of PC786 were also investigated in air liquid interface cultured fully differentiated bronchial epithelial cells (ALI cells) infected with RSV A2 at an MOI of 0.01.
Results: PC786 potently inhibited CPE induced by RSVA2 (IC50,IC90: 0.40 nM, 1.0 nM) and B WST strain (IC50,IC90: 1.3nM, 1.8nM), respectively, which were ≈420-49,000 times more potent than ribavirin. PC786 also exhibited potent inhibition of CPE induced by various low passage RSV clinical isolates (x5 RSV A: IC50 (range) :< 0.09 to 0.71nM, IC90:0.57 -3.77 nM and x5 RSV B: IC50: 13.4 nM to 50.6nM, IC90:27.4 -821 nM). PC786 showed no activity against PIV3, measles, influenza A (H1N1), rhinovirus 16, HIV-1 and Herpes virus at 14µM, suggesting selective index is >20000 for RSV A and >277 for RSV B. PC786 also inhibited RSV virus gene transcription evaluated by RT-PCR (>90% inhibition at 1.4nM) in BEAS2B cells and minigenome assay (IC50 value: 0.476 nM) in HEp2 cells. Furthermore, PC786 (14nM) inhibited RSV protein (G, N, NS-1, F, P, M, M2-1) expression below detection levels at 48 hrs post infection. When PC786 (0.02, 0.1 and 0.5µg/mL: 28, 140 and 700nM equivalent) was added in apical compartment of ALI cells 1hr post infection with further daily treatment for 5 days, a concentration-dependent inhibition of RSV viral load (Plaque assay) in daily apical wash occurred, particularly inhibition below detectable levels occurring at 0.5µg/mL.
Conclusion: PC786 is a potent selective inhibitor of both RSV A and B replication, including low passage clinical isolates. PC786 therefore has the potential to be a novel therapy for the treatment of RSV infections in humans.
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