title: Inhibition of large T antigen ATPase activity as a potential strategy to develop anti-polyomavirus JC drugs.
abstract: BACKGROUND
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: University of Pittsburgh, Pittsburgh, PA, USA. Electronic address: randhawapa@upmc.edu.
authors.forename: Parmjeet
authors.initials: P
authors.lastname: Randhawa
authors.affiliation: University of Pittsburgh, Pittsburgh, PA, USA.
authors.forename: G
authors.initials: G
authors.lastname: Zeng
authors.affiliation: University of Pittsburgh, Pittsburgh, PA, USA.
authors.forename: M
authors.initials: M
authors.lastname: Bueno
authors.affiliation: University of Pittsburgh, Pittsburgh, PA, USA.
authors.forename: A
authors.initials: A
authors.lastname: Salgarkar
authors.affiliation: University of Pittsburgh, Pittsburgh, PA, USA.
authors.forename: Andrew
authors.initials: A
authors.lastname: Lesniak
authors.affiliation: University of Pittsburgh, Pittsburgh, PA, USA.
authors.forename: K
authors.initials: K
authors.lastname: Isse
authors.affiliation: Laboratory for Drug Discovery in Neurodegeneration, Harvard NeuroDiscovery Center, Brigham and Women's Hospital, Harvard Medical School, Cambridge, MA, USA.
authors.forename: K
authors.initials: K
authors.lastname: Seyb
authors.affiliation: Laboratory for Drug Discovery in Neurodegeneration, Harvard NeuroDiscovery Center, Brigham and Women's Hospital, Harvard Medical School, Cambridge, MA, USA.
authors.forename: A
authors.initials: A
authors.lastname: Perry
authors.affiliation: Laboratory for Drug Discovery in Neurodegeneration, Harvard NeuroDiscovery Center, Brigham and Women's Hospital, Harvard Medical School, Cambridge, MA, USA.
authors.forename: I
authors.initials: I
authors.lastname: Charles
authors.affiliation: Laboratory for Drug Discovery in Neurodegeneration, Harvard NeuroDiscovery Center, Brigham and Women's Hospital, Harvard Medical School, Cambridge, MA, USA.
authors.forename: C
authors.initials: C
authors.lastname: Hustus
authors.affiliation: Laboratory for Drug Discovery in Neurodegeneration, Harvard NeuroDiscovery Center, Brigham and Women's Hospital, Harvard Medical School, Cambridge, MA, USA.
authors.forename: M
authors.initials: M
authors.lastname: Huang
authors.affiliation: Laboratory for Drug Discovery in Neurodegeneration, Harvard NeuroDiscovery Center, Brigham and Women's Hospital, Harvard Medical School, Cambridge, MA, USA.
authors.forename: M
authors.initials: M
authors.lastname: Smith
authors.affiliation: Laboratory for Drug Discovery in Neurodegeneration, Harvard NeuroDiscovery Center, Brigham and Women's Hospital, Harvard Medical School, Cambridge, MA, USA.
authors.forename: Marcie A
authors.initials: MA
authors.lastname: Glicksman
mesh_terms: D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors* / Q000235:genetics; D000818:Animals; D000957:Antigens, Viral, Tumor / Q000235:genetics / Q000378:metabolism*; D000998:Antiviral Agents / Q000302:isolation & purification* / Q000494:pharmacology*; D002460:Cell Line; D002522:Chlorocebus aethiops; D003124:Colorimetry / Q000379:methods; D004353:Drug Evaluation, Preclinical; D004791:Enzyme Inhibitors / Q000302:isolation & purification* / Q000494:pharmacology*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D057166:High-Throughput Screening Assays; D006801:Humans; D007577:JC Virus / Q000187:drug effects / Q000201:enzymology*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014779:Virus Replication / Q000187:drug effects
chemical_list: D000957:Antigens, Viral, Tumor; D000998:Antiviral Agents; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D000251:Adenosine Triphosphatases
keywords: ATPase; Drugs; Large T antigen; Polyomavirus JC