CMX001 (1-O-Hexadecyloxypropyl-Cidofovir) Inhibits Polyomavirus JC Replication in Human Brain Progenitor-Derived Astrocytes
ABSTRACT
Polyomavirus JC (JCV) replication causes progressive multifocal leukoencephalopathy (PML), a frequently fatal brain disease in immunodeficient patients, yet antiviral drugs are lacking. We characterized the lipid conjugate 1-O-hexadecyloxypropyl-cidofovir (CMX001) regarding JCV (Mad-4) replication in human brain progenitor-derived astrocytes (PDA) and the simian virus 40 (SV40) large-T-antigen-expressing COS-7 cells up to 7 days postinfection (dpi). We examined JCV loads by PCR, the infection rate by immunofluorescence, and host cell toxicity by WST-1 and BrdU incorporation assays. Supernatants from CMX001-treated PDA demonstrated a drug concentration-dependent decrease in JCV loads and infectivity. CMX001 had only a modest effect on host cell metabolism but reduced overall BrdU incorporation. In PDA at 7 dpi, the CMX001 50% effective concentration (EC50) was 5.55 nM, the 50% cytotoxic concentration (CC50) was 184.6 nM, and the 50% selectivity index (SI50) was 33.3. The EC90 was 19.7 nM, the CC90 was 5,054 nM, and the SI90 was 256.1. In COS-7 cells, JCV replication was faster and the EC50 and EC90 were 18- and 37-fold higher than those in PDA, i.e., 0.1 μM and 0.74 μM (CC50, 0.67 μM; SI50, 6.7; CC90, 12.2 μM; SI90, 16.5) at 5 dpi. We conclude that CMX001 inhibits JCV replication at concentrations in vitro that can be attained by oral administration without significant side effects in clinical studies.
INTRODUCTION
JC virus (JCV) belongs to the genus Polyomavirus (PyV) of the Polyomaviridae family and infects about two-thirds of the adult population worldwide without obvious clinical symptoms (9, 26). Although the route of transmission is not resolved, mucosal surfaces in the oropharyngeal, respiratory, or gastrointestinal tract have been discussed as entry sites. During postulated primary viremia, many tissues might be exposed, but JCV persists in the renourinary tract, with intermittent periods of asymptomatic shedding into urine (6, 15). The key disease caused by JCV is PyV-associated progressive multifocal leukoencephalopathy (PML) (34, 42) and less frequently PyV-associated nephropathy (13). PML may arise in a wide range of patients who are characterized by a profound cellular immunodeficiency (24, 27), but the highest rates of 1 to 8% have been reported for HIV/AIDS patients before the availability of combination antiretroviral therapy (cART) (1, 3, 8, 23). Recently, PML was described as a complication in patients successfully treated for multiple sclerosis or of inflammation patients treated with the monoclonal antibody natalizumab, an anti-α4 integrin which effectively prevents homing to the sites of inflammation in the brain or the gastrointestinal tract (25, 28).
The therapeutic options for PML are rather limited at the moment and seem to depend on the underlying type of immunodeficiency and the ability to recover JCV-specific immune functions. At present, no antiviral therapy of proven efficacy is available to treat JCV replication in PML. The mainstay of current therapy is to allow specific immunity to regain control over JCV replication (14, 24). However, this approach is not uniformly successful, even in HIV/AIDS patients treated with cART (23). An antiviral drug with efficacy against JCV replication in the central nervous system (CNS) might halt the progression and extend the time window for immunological recovery. Cidofovir (CDV), an acyclic nucleotide phosphonate analogue of deoxycytosine monophosphate, has been a promising drug because of its inhibitory activity for nonhuman PyVs in vitro (2) but showed some toxicity in cells of neural origin (21). Initial studies suggested that CDV might be an effective treatment of PML (11, 16), but most current studies to date have failed to demonstrate a benefit of CDV (10, 31, 41).
More recently, CMX001, the hexadecyloxypropyl lipid conjugate of CDV, was found to inhibit polyomavirus BK (BKV) replication in primary human proximal tubular epithelial cells at a 90% effective concentration (EC90) of 0.31 μM at 3 days postinfection (dpi), with a more immediate and lasting effect than CDV (4, 38). Similarly, reduced intracellular viral loads were seen in the human embryonic lung fibroblast cell line WI-38 at 7 dpi at an EC50 of 0.13 μM, 800-fold-lower than the 115.1 μM observed for CDV (37). For JCV replication, only recent in vitro data on CMX001 became available while the current work was in progress: in SVG cells, a glia-derived cell line frequently used to propagate JCV in vitro due to the favorable effect of the constitutively expressed large T antigen (LTag) of the simian polyomavirus SV40 (30), CMX001 at 0.1 μM decreased the JCV infection by 60%, with an estimated EC50 of 0.045 μM (22). Here, we report the effects of CMX001 on JCV replication in human fetal brain progenitor-derived astrocytes (17, 33) and compare the results to JCV replication in COS-7 cells bearing the same SV40 LTag-expressing vector as SVG cells.
MATERIALS AND METHODS
Cell culture.
Human fetal brain progenitor-derived astrocytes (PDA) (33) were propagated in Eagle's minimum essential medium (MEM) (M2279; Sigma, St. Louis, MO) supplemented with 10% fetal bovine serum (FBS). COS-7 cells (ATCC CRL1651) were grown in Dulbecco's modified Eagle's medium high-glucose formulation (DMEM-H) (D5671; Sigma, St. Louis, MO) containing 5% FBS (S0113; Biochrome AG, Berlin, Germany). All cultures were supplemented with 2 mM l-glutamine (K0302; Biochrome AG, Berlin, Germany).
Infection and CMX001 treatment.
JCV (Mad-4) (ATCC VR-1583) infectious supernatants harvested from COS-7 cells with a 50% tissue culture infective dose (TCID50) of 104.5 per ml were used for infection. COS-7 cells were infected, and infectious virus was harvested using 6 freeze-thaw cycles. The cleared supernatant was used to determine infectivity by infection of COS-7 cells with 10-fold serial dilutions and indicated a TCID50 of 105.4 according to immunofluorescence for VP1.
For infection of PDA or COS-7 cells, 0.2 ml of the supernatant was used to infect 5 × 104 PDA or COS-7 cells (multiplicity of infection [MOI] of approximately 1). After 2 h of incubation at 37°C, the supernatants were replaced with fresh medium with or without increasing dilutions of CMX001. CMX001 was freshly dissolved to 1 mg/ml in methanol-water-ammonium hydroxide (50/50/2) and then further diluted in the respective growth medium. JCV replication in PDA cells was followed for 7 days postinfection (dpi), whereas the COS-7 cells were followed for 5 dpi, both by real-time PCR and immunofluorescence.
Immunofluorescence.
At indicated time points, cells were fixed with 4% p-formaldehyde (PFA) in phosphate-buffered saline (PBS), pH 6.8, for 20 min and permeabilized with 0.2% Triton X-100 in PBS for 10 min. After two washes with PBS for 5 min, PFA was quenched with 0.5 M NH4Cl in PBS for 7 min followed by two washes with PBS for 5 min as described previously (17). Blocking of unspecific binding was done with 3% milk in PBS at 37°C for 15 min. Primary antibodies (polyclonal rabbit anti-JCV VP1, 1:300, or monoclonal mouse anti-SV40 large T antigen, 1:50 [DP02, Merck Darmstadt, Germany]) in 3% milk-PBS were incubated at 37°C for 45 to 60 min. Cells were washed twice with PBS on a gyratory shaker for 5 min. Secondary antibodies (goat anti-rabbit Cy3 [111-165-144; Jackson ImmunoResearch, West Grove, PA] or goat anti-mouse Cy3 [115-165-146; Jackson ImmunoResearch] [both 1:2,000]) and 5 μg/ml Hoechst 33342 dye (H21492; Invitrogen) to stain DNA in 3% milk-PBS were given to the cells and incubated at 37°C for 45 to 60 min. Cells were washed twice with PBS as before. Coverslips were mounted in N-Propyl gallate (P-3130; Sigma, St. Louis, MO) as an antifading agent. If not otherwise stated, all steps were performed at room temperature. Cells were scored as positive for viral antigens when a nuclear signal clearly distinguishable from background fluorescence was visible.
Real-time PCR.
JCV loads were quantified after DNA extraction from 100 μl cell culture supernatants with the Corbett X-tractor Gene and the Corbett VX reagents (Qiagen, Hombrechtikon, Switzerland). The real-time PCR protocol for detection of JCV DNA samples targets the JCV LTag-coding sequence and has been described elsewhere (7, 13).
WST-1 assay.
The metabolic activity was monitored by the colorimetric WST-1 assay of the mitochondrial dehydrogenases according to the manufacturer's instructions (11644807001; Roche Applied Science, Rotkreuz, Switzerland). PDA or COS-7 cells were seeded in 96-well plates at 95% or 60% confluence, and CMX001 was added at appropriate concentrations. The WST-1 cleavage product was measured for both cells after 2 h at 450 nm (sample) and at 650 nm (background). WST-1 plus medium alone served as a blank, which was subtracted from all values. Percentages of WST-1 activity were calculated by the following formula: (WST-1 value/dilutent control) × 100.
BrdU assay.
DNA synthesis was quantified by the colorimetric measurement of BrdU incorporation into DNA in proliferating cells using the “Cell proliferation ELISA BrdU” kit (11 647 229 001; Roche). We validated the assay by exposing cells for different incubation times and found that COS-7 cells required 2 h whereas PDA cells required 24 h for optimal monitoring of overall replication in the presence and absence of increasing concentrations of CMX001. After seeding PDA or COS-7 cells in 96-well plates at 95% or 60% confluence and addition of the substrate, the absorbance at 450 nm (sample) and at 650 nm (background) was determined after 24 h for PDA or 2 h for COS-7 cells in the absence and in the presence of increasing concentrations of CMX001. BrdU plus medium alone served as a blank which was subtracted from all values. Calculations were as above.
Determination of the effective and cytotoxic concentrations.
Data for the extracellular JCV DNA load and BrdU incorporation in the presence of increasing CMX001 concentrations were expressed as percent inhibition for both uninfected and infected PDA and COS-7 cells. Graphs were generated by applying the best-fitting model of the software package XLfit, version 5.2.0, to calculate the EC50 and EC90 and the cytotoxic concentrations CC50 and CC90. The respective selectivity index (SI) was obtained by determining the CC50/EC50 and CC90/EC90 ratios.
RESULTS
CMX001 inhibits JCV replication in human progenitor-derived astrocytes.
To examine the effect of CMX001 on JCV progeny over time, human progenitor-derived astrocyte (PDA) cells were infected with JCV (Mad-4) for 2 h and increasing concentrations of CMX001 were added. Supernatants of CMX001-treated and untreated cells were harvested at the indicated time points and analyzed for JCV loads up to 7 dpi by quantitative real-time PCR. In untreated JCV (Mad-4)-infected PDA cells, the extracellular JCV DNA load increased by about 2 log10 genome equivalents per ml (geq/ml) over 7 days (Fig. 1A). With increasing CMX001 concentrations, we observed a gradual decrease in the JCV DNA load. At 0.075 μM CMX001, the increase in the JCV DNA load was only 0.3 log10 geq/ml (Fig. 1A). These results were confirmed in two independent repeat experiments and plotted as percent inhibition compared to CMX001-untreated JCV loads at 7 dpi after subtracting the value measured at 1 dpi. The data indicated that CMX001 at 0.01 μM reduced the JCV DNA load by more than 50% and more than 0.02 μM diminished JCV replication by more than 90% (Fig. 1B).
Fig. 1.
To investigate the effect of CMX001 on JCV gene expression in PDA cells, immunofluorescence for the JCV early gene large T antigen (LTag) was performed at 7 dpi (Fig. 2A). In untreated JCV-infected PDA cells, intranuclear LTag was detected in about 10% of the cells whereas addition of 0.038 μM CMX001 significantly reduced the number of JCV LTag-positive cells (Fig. 2A). At a concentration of 0.6 μM, essentially no LTag-positive cells could be observed (Fig. 2A). CMX001 addition was accompanied by a minor reduction in cell number at 0.038 μM, which was more pronounced at 0.6 μM (see blue nuclear staining in Fig. 2A). PDA cells were also stained for late VP1, and all cells positive for VP1 demonstrated an LTag signal (data not shown).
Fig. 2.
CMX001 inhibits JCV replication in COS-7 cells.
We next compared the effect of CMX001 on JCV replication in the SV40 LTag-expressing monkey kidney COS-7 cells (17). In untreated COS-7 cells, JCV replicated more rapidly and to higher supernatant viral loads than in PDA cells, with an increase of approximately 3 log10 geq/ml after 5 dpi, which successively decreased with increasing CMX001 concentrations (Fig. 1C). These results were confirmed in two independent repeat experiments to quantify CMX001 inhibition (Fig. 1D). CMX001 at 0.079 μM reduced the JCV DNA load in COS-7 cells by ∼50% and at 0.625 μM by more than 90%. We concluded that CMX001 is also effective in inhibiting JCV replication in COS-7 cells, but higher concentrations appeared to be required than for PDA cells.
To investigate the effect of CMX001 on JCV gene expression in COS-7 cells, we stained untreated COS-7 cells for the JCV late capsid protein VP1 at 5 dpi (Fig. 2B). Addition of CMX001 at 1.25 μM was associated with a significant reduction of the JCV VP1 signal but also showed a decrease in the number of cells (Fig. 2B). At 5 μM CMX001, essentially no VP1-positive cells were seen and a decrease in the number of cells was observed (Fig. 2B).
CMX001 decreases infectious JCV progeny from PDA cells.
To test the impact of increasing CMX001 concentrations on JCV progeny production, we collected cell culture supernatants of untreated or CMX001-treated JCV-infected PDA cells at 7 dpi in the presence and absence of CMX001. The supernatants were 5-fold diluted and seeded on COS-7 cells. At 5 dpi, VP1 expression was detected by immunofluorescence staining, indicating that the untreated PDA supernatant contained infectious JCV (Fig. 3). Supernatants from PDA cells treated with 0.038 μM or 0.6 μM CMX001 resulted in a significantly reduced number of JCV VP1-positive COS-7 cells compared to results for untreated JCV-PDA supernatants (Fig. 3). We concluded that CMX001 decreased the number of infectious JCV progeny in a concentration-dependent manner.
Fig. 3.
Effects of CMX001 on host cell viability.
Phase-contrast microscopy did not reveal significant signs of impaired viability of PDA or COS-7 cells during the 5- to 7-day exposure to CMX001 (data not shown), but the nuclear immunofluorescence staining suggested a decline in the total cell count. To use more sensitive and quantifiable assays, we investigated the effects on cellular metabolic activity using a WST-1 assay and that on host cell DNA replication by BrdU incorporation in both infected and uninfected PDA cells. In uninfected PDA cells, CMX001 concentrations from 0.005 to 0.6 μM decreased the metabolic activity to 67% (Fig. 4A, dark gray) and BrdU incorporation to 36% of that for the untreated control (Fig. 4A, light gray), respectively. Similar experiments were conducted with COS-7 cells at CMX001 concentrations from 0.079 to 10 μM, which decreased the metabolic activity to 52% (Fig. 4B, dark gray) and BrdU incorporation to 10% at the highest concentration (Fig. 4B, light gray). The results revealed significant side effects, but at higher concentrations than those required for JCV inhibition. Of note, PDA cells were more sensitive to CMX001 than COS-7 cells, e.g., comparing CMX001 at ∼0.6 μM regarding metabolic activity (67% versus 83%) and DNA replication (36% versus 64%), respectively. Moreover, the substrate incubation period for the BrdU assay was much longer in PDA cells than in COS-7 cells, i.e., 24 h versus 2 h, in order to reach optical densities of comparable readings.
Fig. 4.
Determination of effective concentrations and selectivity index in PDA and COS-7 cells.
To determine the effective concentrations of 50% JCV inhibition in PDA cells, we used curve fitting and identified the CMX001 EC50 as 5.55 nM (Fig. 5A). Modeling of the BrdU data identified the CC50 in uninfected PDA cells as 184.9 nM (Fig. 5B), yielding a 50% selectivity index (SI50) of 33.3. In JCV-infected PDA, the CC50 was 380.7 nM, resulting in an SI50 of 68.6. However, we stayed with the more conservative estimate in uninfected cells. The corresponding data for the JCV-infected PDA were an EC90 of 19.7, a CC90 of 5,054, and an SI90 of 256.5 (Fig. 5A and B). Fitting of the CMX001 inhibition of JCV replication and BrdU incorporation in COS-7 cells provided an EC50 of 0.1 μM, a CC50 of 0.67 μM, and an SI50 of 6.7. The corresponding CMX001 JCV COS-7 assay identified an EC90 of 0.74 μM, a CC50 of 12.2 μM, and an SI50 of 16.5 (Fig. 5C and D).
Fig. 5.
DISCUSSION
Antivirals for treatment of JCV-mediated PML are lacking, and the difficulty of perpetuating this virus in relevant in vitro cell culture has hampered a better characterization of potential candidate drugs. Our data demonstrate that CMX001 inhibits JCV replication in human fetal brain progenitor-derived astrocytes (PDA) and in COS-7 cells. CMX001 reduced not only the progeny viral loads in the cell culture supernatants but also the number of JCV LTag- and VP1-expressing cells, as well as the number of infectious progeny virus when supernatants were titrated on COS-7 as indicator cells.
In PDA cells, the CMX001 EC50 was as low as 5.55 nM and the EC90 as low as 19.7 nM (Fig. 5A). When attempting to characterize side effects of CMX001, we noted that crude viability visualized by phase contrast or by dye exclusion was little affected. However, similar to our studies on CMX001 and CDV inhibition of BKV replication in primary tubular epithelial cells (4, 38), the overall metabolic activity of PDA cells in the WST-1 assay was little affected up to CMX001 concentrations of 0.6 μM, where a reduction to 70% of the activity of untreated PDA was noted. Using estimates in this range would increase the corresponding selectivity index (SI50) to more than 100. However, given the presumed key mechanism of CMX001 as a cytosine phosphonate analogue, dye exclusion and WST-1 might underestimate toxic effects on the host cells. Using BrdU incorporation, we noted a more-pronounced inhibitory effect of CMX001, yielding a CC50 of 184.6 nM and a CC90 of 5054 nM, with a corresponding SI50 and SI90 of 33.3 and 256.1, respectively. Although these estimates appeared more conservative, they still provide good support for bringing CMX001 to clinical trials. Single doses of 2 mg/kg of body weight were well tolerated in a phase 1 clinical study conducted with healthy volunteers, producing peak concentrations in excess of 0.6 μM per week without significant side effects (39).
Our results in COS-7 cells support the inhibitory activity of CMX001 on JCV replication in a cell line known to significantly facilitate JCV replication by activating JCV early gene expression through constitutively expressed SV40 LTag (17). Compared to PDA cells, COS-7 cells required 19- and 37-fold-higher CMX001 concentrations for a JCV EC50 and EC90 of 0.1 μM and 0.74 μM, respectively (Fig. 5B). BrdU incorporation was more affected than the metabolic WST-1 activity, yielding a CMX001 CC50 of 0.67 μM (SI50, 6.7) and a CC90 of 12.2 μM (SI90, 16.5), respectively.
The difference between PDA and COS-7 cells with a lower EC50 and EC90 and a higher SI50 and SI90 suggests an enhanced effectiveness of CMX001 in primary human target cells. This may result from the cell type (primary human brain-derived astrocytes versus monkey kidney-derived cell line). Progeny virus in untreated PDA increased by approximately 2 log10 geq/ml at 7 dpi compared to a more than 3-log10 geq/ml rise of that in COS-7 cells at 5 dpi (Fig. 1), indicating that the JCV replication cycle is significantly accelerated in COS-7 cells compared to that in PDA cells. Our earlier data indicate that this difference in JCV replication can be attributed in part to the trans-activating effect of SV40 LTag in COS-7 cells in comparison to results in CV-1 precursor cells (17). Recently, CMX001 at a 0.1 μM concentration was reported to reduce the number of JCV-positive cells by 60% (estimated EC50 of 0.045 μM) in SVG cells, a human glia-derived cell line also expressing SV40 LTag (22). Although no data were reported for the CMX001 CC50 (SI50) or the EC90 (CC90, SI90) values of JCV replication in SVG cells, thus precluding a more detailed comparison, the EC50 for JCV in SVG cells were close to our results in COS-7 cells, suggesting a key role for LTag expression.
The role of LTag expression and CMX001 susceptibility is of interest since it may pertain to the prophylactic use of CMX001 preventing de novo cell infection and to its use in patients with established PML and ongoing high-level replication of JCV variants with a rearranged noncoding control region (NCCR) and correspondingly increased cellular levels of LTag expression (17, 29). For BKV replication in primary human renal tubular epithelial cells, the CMX001 EC90 was determined to be 310 nM (38), and the EC50 estimates were approximately 0.04 μM (H. H. Hirsch, unpublished data) using a BKV strain (Dunlop) adapted to tissue culture bearing a highly rearranged NCCR with significantly increased viral early gene expression (18, 38). Similarly, higher CMX001 EC50s have been reported by Randhawa et al. using rearranged BKV strains in a human lung fibroblast cell line, WI-98 (37). The JCV Mad-4 strain was isolated from the brain of a PML patient and is considered representative of other PML isolates (12, 32). In fact, rearranged NCCRs are a hallmark of practically all JCV variants identified in patients with PML today. We have recently demonstrated that compared to the archetype JCV NCCR, these naturally occurring rearranged JCV NCCRs show an increased viral early gene expression and a viral replication rate similar to that of the Mad-4 isolate (17). Therefore, we consider Mad-4 an acceptable model strain for measuring antiviral activities for the clinically relevant pathology of PML and expect equal or higher potency of CMX001 against slowly replicating JCV isolates with archetype NCCRs.
Upon uptake into the cell, the lipid phosphate ester linkage of CMX001 is cleaved by cellular phospholipases to release CDV that is converted to CDV diphosphate (CDV-pp) by cellular kinases (20). CDV-pp is a potent inhibitor of DNA synthesis catalyzed by herpesvirus and adenovirus DNA polymerases (19, 40). However, polyomaviruses do not encode a viral DNA polymerase. Therefore, the sensitivity of JCV replication in PDA might be related to a more favorable uptake of CMX001, with subsequent enhanced conversion of CDV to active CDV-pp in de novo infected and LTag-expressing cells. In COS-7 or SVG cells chronically expressing LTag, any of these steps could be altered, including the membrane lipid composition, the phospholipase localization and activity, and the intracellular nucleotide pools, which are possibly more rapidly turned over and replenished, together leading to a less-active drug and less inhibition.
Antiviral agents, and in particular CDV, have been used to treat PML patients but with little success (10, 31, 41). Two recent studies of JCV in SVG cells indicated that CDV may not be very effective (5, 22). Instead, our study supports and extend the results of Jiang et al. (22) and provide the most complete data on the inhibitory characteristics of CMX001 on JCV replication in primary human glia-derived astrocytes with regard to the effective concentrations, cytotoxic concentrations, and selectivity indices. The data indicate that CMX001 has a favorable inhibitory and toxicity profile in this relevant in vitro model of JCV replication. While data for humans are currently being collected, studies using [14C]CMX001 in mice reported potent inhibition of experimental herpes simplex virus encephalitis, reaching drug concentrations of 0.01 to 0.02 mg equivalents per gram of spinal cord and brain (36). Together with the oral bioavailability and the lack of nephrotoxicity from CMX001 observed to date in preclinical and clinical studies (20, 35), our results provide an important rational basis for further exploring the antiviral potential of CMX001 in clinical studies of JCV-mediated PML.
ACKNOWLEDGMENTS
We thank Séverine Louvel for help with the XLfit software program.
This work was supported by an unrestricted grant from Chimerix Inc. to H.H.H.
We declare that there is no conflict of interest regarding the role of sponsor for this research.
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Information & Contributors
Information
Published In
Antimicrobial Agents and Chemotherapy
Volume 55 • Number 5 • May 2011
Pages: 2129 - 2136
PubMed: 21402853
Copyright
© 2011 American Society for Microbiology.
History
Received: 12 January 2011
Returned for modification: 9 February 2011
Accepted: 2 March 2011
Published online: 28 April 2011
Contributors
Metrics & Citations
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Citations
Citation
2011.
CMX001 (1-O-Hexadecyloxypropyl-Cidofovir) Inhibits Polyomavirus JC Replication in Human Brain Progenitor-Derived Astrocytes. Antimicrob Agents Chemother
55:.
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