Identification and characterization of mefloquine efficacy against JC virus in vitro - PubMed (original) (raw)
Identification and characterization of mefloquine efficacy against JC virus in vitro
Margot Brickelmaier et al. Antimicrob Agents Chemother. 2009 May.
Abstract
Progressive multifocal leukoencephalopathy (PML) is a rare but frequently fatal disease caused by the uncontrolled replication of JC virus (JCV), a polyomavirus, in the brains of some immunocompromised individuals. Currently, no effective antiviral treatment for this disease has been identified. As a first step in the identification of such therapy, we screened the Spectrum collection of 2,000 approved drugs and biologically active molecules for their anti-JCV activities in an in vitro infection assay. We identified a number of different drugs and compounds that had significant anti-JCV activities at micromolar concentrations and lacked cellular toxicity. Of the compounds with anti-JCV activities, only mefloquine, an antimalarial agent, has been reported to show sufficiently high penetration into the central nervous system such that it would be predicted to achieve efficacious concentrations in the brain. Additional in vitro experiments demonstrated that mefloquine inhibits the viral infection rates of three different JCV isolates, JCV(Mad1), JCV(Mad4), and JCV(M1/SVEDelta), and does so in three different cell types, transformed human glial (SVG-A) cells, primary human fetal glial cells, and primary human astrocytes. Using quantitative PCR to quantify the number of viral copies in cultured cells, we have also shown that mefloquine inhibits viral DNA replication. Finally, we demonstrated that mefloquine does not block viral cell entry; rather, it inhibits viral replication in cells after viral entry. Although no suitable animal model of PML or JCV infection is available for the testing of mefloquine in vivo, our in vitro results, combined with biodistribution data published in the literature, suggest that mefloquine could be an effective therapy for PML.
Figures
FIG. 1.
Detection and measurement of JCV infection. (A) SVG-A cells infected with JCV(M1/SVEΔ) were fixed and stained 3 days postinfection with murine monoclonal antibodies specific for VP1 protein (green staining). All cells present in the culture were visualized with DAPI DNA nuclear staining (blue). The picture was taken with a Cellomics ArrayScan camera. Magnification, ×200. (B) The number of infected cells (i.e., VP1-positive cells) per group is plotted against the dilution factor of the viral stock used to infect the cells (mean ± standard deviation [n = 2]; blue line). The total numbers of cells (yellow bars) were similar for all groups. Cells were infected in the presence of various dilutions of JCV-neutralizing antiserum (C) or cidofovir (D). Cells were fixed and stained at 3 days postinfection, and the total numbers of VP1-positive and DAPI-positive events per treatment group were determined with a Cellomics ArrayScan imager. Data are presented as percent inhibition relative to that for the no-drug control of the number of JCV-positive (JCV+) cells, the total number of cells, or the number of JCV-positive cells normalized by total cell number (percent JCV-positive cells). Data from a representative experiment of at least three performed are shown.
FIG. 2.
Flowchart describing the steps used to screen the compounds in the Spectrum collection. Primary screening employed the SVG-A cell line and the virus JCV(M1/SVEΔ), and the assay was performed as described in the legend to Fig. 1. TC50, drug concentration for inhibition of total cell numbers by 50%.
FIG. 3.
Characterization of mefloquine anti-JCV effect. (A and B) To further characterize the anti-JCV effect of mefloquine in different cell types and against different JCV isolates, viral infections were performed over the range of mefloquine concentrations in SVG-A cells with JCV(M1/SVEΔ) (n = 12) (A), in primary human fetal astrocytes with JCV(M1/SVEΔ) (B), or in SVG-A cells with JCV(Mad4) (n = 5) (C). (D and E) Characterization of the effect of mefloquine on the inhibition of JCV DNA replication. (D) Viral T-antigen DNA was quantified in the presence of various drug concentrations by qPCR in SVG-A cells infected with JCV(M1/SVEΔ); the inhibition of JCV DNA copy number and the inhibition of JCV-positive (JCV+) cells were measured in a replicate plates, the results of one representative experiment of two performed are shown. (E) By using the same qPCR assay, mefloquine's ability to inhibit JCV(Mad1) DNA replication in PFHG cells was measured over a range of drug concentrations at days 7 and 10 postinfection. The graph represents the average percent JCV DNA inhibition for three independent experiments with duplicate samples per time point. (F) The effect of the delay of mefloquine addition was measured in cultures of primary human fetal astrocytes infected with JCV(M1/SVEΔ). Cells were exposed to various concentrations of mefloquine at the same time (T) as virus addition or at 3 h or 24 h after virus addition. Ten days after infection with virus, cells were fixed and stained and the number of virus-infected cells was determined. The results of a representative experiment of five experiments conducted with either primary astrocytes or SVG-A cells is shown. The method used for the calculation of percent JCV inhibition is described in Materials and Methods. Inhibition of total cell numbers (i.e., DAPI-positive events) was less than 20% for all drug concentrations plotted. Unless otherwise noted, only one representative graph is shown, but the EC50 is calculated as an average of all experiments.
FIG. 4.
Human CSF does not interfere with mefloquine's anti-JCV activity. SVG-A cells were infected with JCV(M1/SVEΔ) over a range of mefloquine concentrations in the presence of 2%, 10%, or 20% human CSF. Three days later the cells were fixed and stained and the total numbers of VP1-positive cells and DAPI-positive events per treatment group were determined with a Cellomics ArrayScan imager. The results from one representative experiment (of a total of two independent experiments) are shown. The method used for the calculation of the percent JCV inhibition is described in Materials and Methods.
FIG. 5.
Anti-JCV activities of various forms of mefloquine. The (R,S)-mefloquine (A) and (S,R)-mefloquine (B) enantiomers were separated from a mefloquine drug racemate by chiral chromatography; the racemate of (S,S)- and (R,R) enantiomers of mefloquine (C) or (2,8-bis-trifluoromethyl-quinolin-4-yl)-pyridin-2-yl-methanol (D) were added to SVG-A cells simultaneously with JCV(M1/SVEΔ). The cells were fixed and stained at 3 days postinfection, and the total numbers of VP1-positive cells and DAPI-positive events per treatment group were determined with a Cellomics ArrayScan imager. The results of one representative experiment of a total of six (A and B) or two (C and D) performed are shown. EC50s are the means of all experiments performed. Ten micromolar was the highest concentration tested; TI, therapeutic index (TC50/EC50).
FIG. 6.
Structure-activity relationship of the arylanthranilic and arylalkanoic acid JCV inhibitors. Viral inhibition was measured by an infectivity assay with SVG-A cells and JCV(M1/SVEΔ). The EC50 data represent averages calculated from two or more experiments, and the therapeutic index (TC50/EC50) was >3 for all compounds shown.
FIG. 7.
Shape similarity among chemically diverse JCV inhibitors. The shapes and chemical features of mefloquine (magenta), mefenamic acid (yellow), indomethacin (gray), and 8-chloroadenosine 3′,5′-monophosphate (green) are compared. The overlays were achieved with the ROCS program and were visualized by the use of PyMOL software.
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