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

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.

Gallic acid-based small-molecule inhibitors of JC and BK polyomaviral infection.

JCPyV and BKPyV are common human polyomaviruses that cause lifelong asymptomatic persistent infections in their hosts. In immunosuppressed individuals, increased replication of JCPyV and BKPyV cause significant disease. JCPyV causes a fatal and rapidly progressing demyelinating disease known as progressive multifocal leukoencephalopathy. BKPyV causes hemorrhagic cystitis and polyomavirus associated nephropathy in bone marrow transplant recipients and in renal transplant recipients respectively. There are no specific anti-viral therapies to treat polyomavirus induced diseases. Based on detailed studies of the structures of these viruses bound to their receptors we screened several compounds that possessed similar chemical space as sialic acid for their ability to bind the virus. Positive hits in the assay were restricted to gallic acid based compounds that mimic the viruses known cellular glycan receptors. Pre-treatment of virions with these inhibitors reduced virus infection in cell culture and as such may form the basis for the development of virion specific antagonists to treat these infections.

Human polyomavirus JC reactivation and pathogenetic mechanisms of progressive multifocal leukoencephalopathy and cancer in the era of monoclonal antibody therapies.

Progressive multifocal leukoencephalopathy (PML) is a demyelinating disease of the central nervous system caused by the neurotropic human polyomavirus JC (JCV) lytic infection of oligodendrocytes. PML was first described as a complication of lymphoproliferative disorders more than 50 years ago and emerged as a major complication of human immunodeficiency virus (HIV) infection in the 1980s. Despite the ubiquity of this virus, PML is rare and always seen in association with underlying immunosuppressive condition, such as HIV infection, autoimmune diseases, cancer, and organ transplantation. JCV remains quiescent in the kidneys, where it displays a stable archetypal non-coding control region (NCCR). Conversely, rearranged JCV NCCR, including tandem repeat patterns found in the brain of PML patients, have been associated with neurovirulence. The specific site and mechanism of JCV NCCR transformation is unknown. According to one model, during the course of immunosuppression, JCV departs from its latent state and after entering the brain, productively infects and destroys oligodendrocytes. Although the majority of PML cases occur in severely immunesuppressed individuals, PML has been increasingly diagnosed in patients treated with biological therapies such as monoclonal antibodies (mAbs) that modulate immune system functions: in fact, CD4+ and CD8+ T lymphopenia, resulting from this immunomodulatory therapy, are the primary risk factor. Furthermore, JCV reactivation in nonpermissive cells after treatment with mAbs, such as intestinal epithelial cells in Crohn's disease patients, in association with other host tumor-inducing factors, could provide valid information on the role of JCV in several malignancies, such as colorectal cancer.

Topotecan and PML: the limits of pharmaceutical industry research.

AIDS: Topotecan, a drug typically used to treat cancer, has shown promising in vitro results against the JC virus. The JC virus causes progressive multifocal leukoencephalopathy (PML). SmithKline Beecham is planning to announce phase II placebo-controlled trials for PML. There is currently no known treatment for PML, although it sometimes responds to anti-HIV drugs, alpha-interferon, and peptide T. AIDS advocates are questioning why SmithKline Beecham did not perform animal and pre-clinical studies to see if topotecan would be effective and tolerable among HIV/AIDS patients. Topotecan treatment has resulted in minimum success fighting ovarian cancer, however, its toxic effects are dangerous and powerful. Advocates advise that any patient considering a trial of topotecan have their blood monitored very carefully, particularly for neutropenia. Participants should consider pre- and post-treatment with G-CSF (Neupogen) to boost white blood cells.^ieng