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Introduction This study evaluates polyomavirus JC (JCV) large T antigen (LTA) as a potential target for drug development. LTA is a hexameric protein with a helicase activity that is powered by ATP binding and hydrolysis. The helicase and ATPase function is critical for viral replication. Methods Recombinant JCV LTA was produced in an Escherichia coli based expression plasmid. ATPase activity was measured using the malachite green assay. A high throughput screen was completed using a brain-biased library of 75,000 drug-like compounds selected for physicochemical properties consistent with blood brain barrier permeability. Results Five compounds showed non-competitive inhibition of ATPase activity with an EC50 ≤ 15 μM. Modest antiviral activity was demonstrated in an immunofluorescence assay for JCV VP-1 expression in COS7 cells (EC50 15, 18, 20, 27, and 52 μM respectively). The compounds also inhibited viral replication in a real time PCR assay at comparable concentrations. LD50 in the MTS96 and Cell TiterGlo assays was >100 μM for all compounds in COS7 as well as HEK293 cells. However, two compounds inhibited cell proliferation in culture with IC50 values of 43 and 34 μM respectively. Despite substantial amino acid similarity between polyomavirus JC, BK and SV40 proteins, these compounds differ from those previously reported to inhibit SV40 LTA ATPase in chemical structure as well as a non-competitive mechanism of inhibition. Conclusion LTA ATPase is a valid target for discovery. Additional screening and chemical optimization is needed to develop clinically useful compounds with less toxicity, which should be measured by metabolic as well as cell proliferation assays.