Inhibition of large T antigen ATPase activity as a potential strategy to develop anti-polyomavirus JC drugs

Abstract

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.

Merkel Cell Polyomavirus and Merkel Cell Carcinoma

“…No specific inhibitors of PyV LT or sT exist, although small molecules that inhibit the ATPase activity of SV40, BKPyV and JCPyV have been described [ 257 , 258 ]. Since MCC tumors express tLT devoided of the ATPase domain, these drugs are not applicable for VP-MCC.…”

Fragment-Based Discovery of Dual JC Virus and BK Virus Helicase Inhibitors

“…(4-Amino-3-pyridyl) N,N-Diisopropylcarbamodithioate (10). A mixture of 4-pivalamidopyridin-3-yl diisopropylcarbamodithioate (9) (5.43 g, 13.8 mmol) and sodium hydroxide (1.10 g, 27.6 mmol) was reacted at room temperature in MeOH (100 mL) overnight.…”

“…They contain a SF3 hexameric helicase domain that uses energy derived from ATP hydrolysis to unwind duplex DNA. Because LTAgs are solely viral proteins with no human counterpart, they represent attractive targets for therapeutic intervention. , As a result, early proof of concept screens for the modulation of LTAgs ATPase activity have recently been reported for polyomaviruses BKV, , JCV, and SV40 . To the best of our knowledge, these efforts have not been pursued past the stage of hit identification.…”

Efficient propagation of archetype JC polyomavirus in COS-7 cells: evaluation of rearrangements within the NCCR structural organization after transfection

“…Previous reports have demonstrated that archetype JCPyV replicates efficiently in simian-kidney-derived COS-7 cells expressing simian virus 40 (SV40) T antigen and in COS-7 cells expressing HIV-1 Tat [ 20 , 21 ]. It is also important to know that other JCPyV strains, such as Mad-4, replicate efficiently in simian-kidney-derived COS-7 cells [ 22 ], and other cell lines, such as the human embryonic kidney (HEK) cell line 293TT [ 23 ] and the human fetal glial cell line SVG, have been employed to study the virus [ 24 ]. Although clearly different from primary oligodendrocytes, these cell lines support rapid replication of JCPyV.…”