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Antimicrobial Agents and Chemotherapy, December 2007, p. 4492-4494, Vol. 51, No. 12
0066-4804/07/$08.00+0     doi:10.1128/AAC.00843-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

BK Virus Replication In Vitro: Limited Effect of Drugs Interfering with Viral Uptake and Intracellular Transport{triangledown}

Parmjeet Randhawa,* Noush Afarin Farasati, and Yuchen Huang

Division of Transplantation Pathology, Department of Pathology, University of Pittsburgh Medical Center, UPMC Montefiore Hospital, Room E733, Pittsburgh, PA 15213

Received 28 June 2007/ Returned for modification 9 August 2007/ Accepted 11 September 2007


   ABSTRACT
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 ABSTRACT
TEXT
 REFERENCES
 
BK virus is an important pathogen in kidney transplant recipients. In vitro studies demonstrated slight antiviral activity for chloroquine and nystatin. A sialic acid derivative, BTB11968, was identified as a lead compound for further development.


   TEXT
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 ABSTRACT
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REFERENCES
 
The polyomaviruses are small double-stranded DNA viruses, of which the most important species are BK virus (BKV) and JC virus. BKV reactivation occurs in 10 to 60% of kidney transplant recipients, while 1 to 10% develop viral nephropathy (3, 14). No effective drugs are currently available for the treatment of these patients. This article describes the in vitro anti-BKV activity of compounds selected for their potential ability to interfere with mechanisms of virus uptake and intracellular transport. The antiviral drug sensitivity assay used is based on direct measurement of BKV (Gardner strain, ATCC VR837) replication in WI-38 human embryonic fibroblast cells (ATCC CCL-75) using a 7-day real-time PCR assay. Cells are pretreated with the drug of interest for 2 h, and then virus infection is allowed to occur for an additional 2 h. Quantitation of a cell gene, the aspartoacylase gene, provides a measure of cell number and cytotoxicity. Drug cytotoxicity is also evaluated by the conventional neutral red assay. Technical details of these methods have been published previously (7, 13, 15). The results are summarized in Table 1 and are further discussed below.


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  		TABLE 1. Summary of antiviral activities of tested compoundsa

 
The first therapeutic target to be explored was the viral receptor on host cells. The primary receptor binding determinant on the BKV capsid is the VP-1 protein. The corresponding host ligand is not completely characterized, but it appears to contain a terminal {alpha}-2,3-linked sialic acid associated with type II gangliosides (4, 11). Tunicamycin, a compound known to inhibit synthesis of {alpha}-2,3-linked sialic acids (4), was not effective. We then tested nine sialic acid derivatives that were selected from the Maybridge HitFinder collection based on favorable log P values and absence of reactive groups (Fig. 1).


Figure 1
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  		FIG. 1. Chemical structures of sialic acid derivatives tested for anti-BKV activity. 1, BTB11968 [2-(hydroxymethyl)-6-(nonyloxy)tetrahydro-2H-pyran-3,4,5-triol]; 2, BTB11967 [2-(hydroxymethyl)-6-(octyloxy)tetrahydro-2H-pyran-3,4,5-triol]; 3, RJC01353 [(2R,3S,4R,5S)-3,4,5-triacetoxy-2-(acetoxymethyl)tetrahydropyran]; 4, RJC01352 [(2R,3R,4R,5S)-3,4,5-triacetoxy-2-(acetoxymethyl)tetrahydropyran]; 5, RJC00828 (methyl 3,4,5,6-tetrahydroxytetrahydro-2H-pyran-2-carboxylate); 6, NRB05166 [2-(hydroxymethyl)-6-methoxytetrahydro-2H-pyran-3,4,5-triol]; 7, NRB05157 [methyl 3,4,5,6-tetra(acetyloxy)tetrahydro-2H-pyran-2-carboxylate]; 8, JFD03599 (3,4,5,6-tetrahydroxytetrahydro-2H-pyran-2-carboxylic acid); 9, NRB05186 [5-(1,2-dihydroxyethyl)-3,4-dihydroxytetrahydrofuran-2-one]. All structures were obtained from Maybridge, Thermo Fisher Scientific.

 
BTB11968 or [2-(hydroxymethyl)-6-(nonyloxy)tetrahydro-2H-pyran-3,4,5-triol] (Fig. 1, compound 1), showed antiviral activity with a 50% effective concentration (EC50) of 6.35 ± 2.17, a 50% cytotoxic concentration (CC50) of 126.37 ± 7.65, and a neutral red selectivity index (SI) of 30.51 ± 2.02. The PCR assay gave an SI within twofold of the SI determined by the neutral red assay. A decrease in antiviral efficacy was observed if the standard 2-hour preincubation of cells with the drug was omitted (EC50 = 98.93 ±18.02, CC50 = 114.5 ± 27.46, and SI = 1.25 ± 0.51), consistent with its presumptive mechanism of interfering with the virus-receptor interaction. All other sialic acid derivatives tested were inactive.

While the sialic acid composition of the BKV receptor has now been partly characterized, the associated protein remains undefined. The polyomavirus JC virus receptor, however, has recently been shown to contain a 5-hydroxytryptamine receptor 2A (5HT2AR) protein component (6). Receptors for 5HT2A have been described for kidney-derived cell lines such as HEK293 cells (9) and for the rat kidney (2), where they modulate cell proliferation. Accordingly, we tested the anti-BKV activities of several known 5HT2AR inhibitors, namely, chlorpromazine, clozapine, olanzapine, and ziprasidone. The results were not encouraging and indicate that 5HT2AR-associated pathways do not play a major role in the cellular uptake and intracellular transport of BKV.

Next, we explored the idea of testing compounds that might interfere with intracellular transport of BKV. Viral entry into host cells is mediated by vesicles that have been identified as caveolae (1). The route taken by the virus from the cell membrane to the nucleus includes the endoplasmic reticulum and microtubules (5, 11). Participation of the Golgi apparatus, other cytoskeletal elements such as actin, and microfilaments has been shown for other polyomaviruses (8). BKV transport into the nucleus is facilitated by nuclear localization signals located on BKV proteins VP-2 and VP-3. Since these localization signals are not normally exposed on the surface of the virus, partial disassembly of the protein capsid must occur in the host cytoplasm prior to viral entry in the nucleus. This disassembly occurs, at least in part, within the acidic environment of endosomes and lysosomes.

A number of FDA-approved compounds with a potential to disrupt intracellular pathways of BKV transport were evaluated. Chloroquine was tested because it raises the lysosomal pH and shows activity against a number of other viruses (10, 12, 16-21). A slight anti-BKV effect was found (SI = 5.32). Notably, the EC50 was comparable to that reported by Vincent et al. (21) for coronaviruses. That study also showed that chloroquine inhibits glycosylation of the severe acute respiratory syndrome coronavirus receptor. However, in our culture system, drug treatment of cells prior to BKV infection showed no evidence for blockade of the viral receptor (data not shown).

Nystatin, an FDA-approved antifungal agent was tested for anti-BKV activity because of its ability to disrupt caveolae (1). The SI was 5.13 ± 0.9. Amphotericin B, a water-soluble functional analog of nystatin, yielded an SI of 2.99. There was a very low margin of safety, as the drug concentration just one step higher than the CC50 showed severe cytotoxicity on light microscopic examination. Finally, an SI of 3.63 ± 1.33 was obtained for colchicine, a compound that disrupts microtubules. The EC50 (0.022 µg/ml) is substantially higher than therapeutic concentrations of the drug in patients treated with this compound for gout (0.00075 to 0.0063 µM at a daily dose of 1 mg colchicine).

In summary, our testing has identified a compound, BTB11968, which appears worth of further evaluation and optimization as an anti-BKV agent (SI = 30.5). Of the FDA-approved compounds tested, chloroquine has an SI of 5.32 and nystatin an SI of 5.13. Generally speaking, only drugs with SIs of >10 are pursued by the pharmaceutical industry for clinical development. However, nystatin and chloroquine are already being used clinically for fungal infections and malaria, respectively, with an acceptable toxicity profile. Hence, it could be argued that until better drugs become available, these compounds could be empirically tried in patients with progressive BKV nephropathy. The in vitro EC50 of chloroquine for several viruses matches the blood concentrations in human subjects under acute antimalarial treatment (1 to 15 µM) (10, 12, 16-21). Whereas chloroquine can be administered systemically, nystatin is suitable only for local use, possibly as a bladder wash for hemorrhagic cystitis after bone marrow transplantation.


   ACKNOWLEDGMENTS
 
This work was supported by NIH grants AI51227-01 and AI63360 and NIAID contract no. N01-AI-30044.

We thank Darko Butina for retrieving sialic acid analogs from the Maybridge collection.


   FOOTNOTES
 
* Corresponding author. Mailing address: Division of Transplantation Pathology, Department of Pathology, University of Pittsburgh, Room E737, UPMC-Montefiore Hospital, 3459 Fifth Ave., Pittsburgh, PA 15213. Phone: (412) 647-7646. Fax: (412) 647-5237. E-mail: randhawapa{at}upmc.edu Back

{triangledown} Published ahead of print on 24 September 2007. Back


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Antimicrobial Agents and Chemotherapy, December 2007, p. 4492-4494, Vol. 51, No. 12
0066-4804/07/$08.00+0     doi:10.1128/AAC.00843-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.




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