Bicyclic pyrimidine nucleoside analogues (BCNAs) as highly selective and potent inhibitors of varicella-zoster virus replication

Jan Balzarini1,* and Christopher McGuigan2

1 Rega Institute for Medical Research, K. U. Leuven, B-3000 Leuven, Belgium; 2 Welsh School of Pharmacy, University of Wales Cardiff, Cardiff CF1 3XF, UK


    Abstract
 Top
 Abstract
 Antiviral activity of bicyclic...
 Resistance development of BCNAs...
 Molecular basis for the...
 Catabolic properties of BCNAs
 Conclusions
 References
 
Bicyclic pyrimidine nucleoside analogues (BCNAs) represent highly potent and selective inhibitors of varicella-zoster virus (VZV) replication in cell culture. The compounds inhibit a variety of clinical VZV strains, in the higher picomolar range, whilst being non-toxic at micromolar concentrations. The compounds do not inhibit the closely related simian varicella virus or any other viruses, including herpes simplex virus type 1 (HSV-1), HSV-2 and cytomegalovirus. The BCNAs owe at least part of their antiviral selectivity to a specific activation/phosphorylation by the VZV-encoded thymidine kinase (TK) and associated thymidylate kinase (dTMP-K) activity, while being not recognized by the closely related HSV-1-encoded TK/dTMP-K enzyme. In addition, the 5'-monophosphates of BCNAs are neither a substrate nor an inhibitor of the cellular dTMP-K, and are not subject of back-conversion to the corresponding nucleosides by 5'-deoxynucleotidases. In contrast to the anti-HSV-1/VZV drug (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU), the BCNAs are not catabolized by human (erythrocyte) or bacterial (Escherichia coli) thymidine phosphorylase to release the free bicyclic pyrimidine base. Also, unlike BVU (the free base of BVDU), the BCNA bases do not inhibit dihydropyrimidine dehydrogenase. Consequently, the catabolism of the anticancer drug 5-fluorouracil (5-FU) is not influenced by the BCNA base in cell-free enzyme assays or in mice that were exposed to combinations of 5-FU with BCNAs or their free base. BCNAs have a good oral bioavailability and, owing to their highly lipophilic nature, are assumed to be able to cross the blood–brain barrier efficiently. Given the above-mentioned favourable properties, BCNAs may represent a promising novel class of highly selective anti-VZV drugs that should be further pursued for clinical application.


    Antiviral activity of bicyclic pyrimidine nucleoside analogues
 Top
 Abstract
 Antiviral activity of bicyclic...
 Resistance development of BCNAs...
 Molecular basis for the...
 Catabolic properties of BCNAs
 Conclusions
 References
 
We have recently discovered that certain long-chain substituted fluorescent furano bicyclic pyrimidine nucleoside analogues (BCNAs) (Figure 1) are endowed with a potent and selective inhibitory activity against varicella-zoster virus (VZV) replication in HEL cell cultures.1 The original lead compound Cf 1368 (6-octyl-substituted BCNA) was inhibitory at an EC50 (50% effective concentration) of 0.008 µM against VZV, being non-toxic at 50 µM. Further optimization of the lead compound resulted in the synthesis of a series of 6-alkylphenyl-substituted BCNAs with the unprecedented anti-VZV activity for the 6-pentylphenyl-substituted BCNA derivative Cf 1743 (EC50 c. 0.0001 µM) and selectivity (CC50/EC50 c. 106).2 The structurally most closely related anti-VZV drug that is used in the clinic is (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU, Brivudin) (Figure 1),3,4 which is at least 10- to 20-fold less potent than the BCNA Cf 1743, whereas aciclovir (Figure 1) is almost 10 000-fold less potent an anti-VZV agent than Cf 1743. When the BCNAs (Cf 1368 and Cf 1743) were evaluated against a variety of wild-type clinical VZV isolates in a plaque reduction assay, the antiviral potencies were comparable to those obtained against the reference laboratory OKA and YS VZV strains. Indeed, Cf 1368 and Cf 1743 inhibited the replication of the clinical VZV isolates at an EC50 of 0.027 ± 0.013 and 0.0002 ± 0.00017 µM, respectively, whereas the EC50 values for BVDU and aciclovir were 0.009 ± 0.004 and 3.4 ± 0.67 µM, respectively.5 Thus, the BCNAs potently suppress clinical VZV strains at a relatively low variation between the different isolates. The BCNAs are not only the most powerful anti-VZV compounds reported so far, they also show an unprecedented specificity. Indeed, except for VZV, the BCNAs did not inhibit any other DNA virus, including the closely related herpes simplex virus type 1 (HSV-1), HSV-2, cytomegalovirus (CMV) and human herpes virus 6 (HHV-6).6 Even the simian varicella-zoster virus (SVV) proved completely unsusceptible to the inhibitory effects of a variety of BCNAs in B cell cultures under the experimental conditions used, whereas BVDU inhibits SVV at c. 0.1 µM.



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Figure 1. Structural formulae of BCNAs and established anti-VZV drugs.

 

    Resistance development of BCNAs against VZV
 Top
 Abstract
 Antiviral activity of bicyclic...
 Resistance development of BCNAs...
 Molecular basis for the...
 Catabolic properties of BCNAs
 Conclusions
 References
 
A variety of VZV (OKA strain) mutant variants that had been selected for resistance in the presence of aciclovir, BVDU and BVaraU (Sorivudine) (Figure 1) are available in our laboratory. They have been shown to be resistant to all anti-VZV drugs that depend on metabolic activation by the VZV-encoded thymidine kinase (TK), but remain active against the VZV TK-independent foscarnet and against acyclic nucleoside phosphonates (ANPs) such as adefovir and cidofovir. The BCNAs were shown to be unable to suppress the drug-resistant TK mutant VZV strains, as has also been observed for aciclovir and BVDU.5 Similar patterns of drug susceptibility/resistance were observed when VZV strains selected for resistance against BCNAs were evaluated for their susceptibility to BVDU, aciclovir, foscarnet and ANPs.6 Therefore, it could be concluded that development of BCNA resistance in HEL cell cultures is probably due to the appearance of mutations in the VZV TK gene, resulting in complete annihilation of the antiviral activity of the BCNAs. It will be of particular interest to reveal the nature of the mutations that may have appeared in the VZV TK to cause BCNA resistance.


    Molecular basis for the anti-VZV specificity of BCNAs
 Top
 Abstract
 Antiviral activity of bicyclic...
 Resistance development of BCNAs...
 Molecular basis for the...
 Catabolic properties of BCNAs
 Conclusions
 References
 
It is puzzling why the BCNAs are so specific in their anti-VZV action. All other anti-VZV drugs known so far have shown at least measurable or marked antiviral activity against other herpesviruses (i.e. HSV-1) as well. Our recent pharmacological studies revealed that the BCNAs are recognized as a substrate for phosphorylation by recombinant purified VZV TK.7 As is also the case for BVDU, the BCNAs are not only converted to their 5'-monophosphates but also to their 5'-diphosphate derivatives by the intrinsic thymidylate (dTMP) kinase activity of VZV TK (Figure 2). However, in contrast to BVDU, purified HSV-1 and HSV-2 TK do not recognize the BCNAs as substrates for phosphorylation, and the BCNA-5'-monophosphates do not act as substrates for the dTMP kinase activity of HSV-1 TK. It should also be noted that BCNAs are not recognized by cytosolic TK-1 and mitochondrial TK-2, and their monophosphates are not recognized by cytosolic dTMP kinase. Therefore, it could be concluded that the unique recognition of the BCNAs by VZV TK, but not by HSV TK or any other cellular TK, forms the basis of the specificity of the BCNAs as anti-VZV agents (Figure 2).



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Figure 2. Anabolic and catabolic pathways of BCNAs. TK, thymidine kinase; dTMP-K, thymidylate kinase; NDP-K, nucleoside 5'-diphosphate kinase; DNA pol, DNA polymerase; TPase, thymidine phosphorylase; DPD, dihydropyrimidine dehydrogenase.

 
The three-dimensional structure of VZV TK is not known. However, the structure of HSV-1 TK complexed with its natural substrate thymidine (dThd) or with several antiherpes inhibitors (i.e. aciclovir, BVDU) is available. When the BCNAs are modelled in the active site of HSV-1 TK, superposing the deoxyribose of the BCNAs with the deoxyribose of dThd, there is a severe steric clash of the 6-substituent of the BCNAs with the main chain amino acids 167 and 168 in the HSV-1 TK (Figure 3). These data may clarify why the BCNAs are not recognized by HSV-1 TK. It would be interesting to clarify whether, and where, there is a lipophilic pocket or cavity in VZV TK to host the lipophilic alkylphenyl side chain. Crystallographic studies on VZV TK–BVDU and VZV TK–BCNA complexes are ongoing.



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Figure 3. Modelling of the BCNA Cf 1743 into HSV-1 TK, based on the coordinates of HSV-1 TK–ganciclovir and dThd complexes (courtesy of R. Esnouf, Division of Structural Biology, The Henry Wellcome Building for Genomic Medicine, Oxford, UK).

 
The structure–activity relationship of the BCNAs for antiviral activity proved clearly different from that of VZV TK enzyme affinity.6 These findings indicate that VZV TK acts as the activating (phosphorylating) enzyme, whereas the eventual target of the BCNAs for antiviral activity represents another enzyme/protein or function of VZV with a different structure–activity relationship.

Interestingly, when human erythrocyte NDP kinase was added to the reaction mixture containing BVDU or BCNAs and VZV TK, a considerable amount of BVDU-triphosphate was formed, whereas no trace of BCNA-triphosphate could be detected.7 Although we cannot exclude the possibility that BCNA-diphosphate can be recognized by other NDP kinase isoenzymes or other cellular enzymes, our observations may suggest that the mechanism of anti-VZV activity of the BCNAs does not occur through the 5'-triphosphate derivatives, but through the 5'-monophosphate or 5'-diphosphate derivative, virtually excluding viral DNA polymerase as the eventual target for anti-VZV activity. The mechanism of antiviral action of the BCNAs is currently under further investigation in our laboratories.


    Catabolic properties of BCNAs
 Top
 Abstract
 Antiviral activity of bicyclic...
 Resistance development of BCNAs...
 Molecular basis for the...
 Catabolic properties of BCNAs
 Conclusions
 References
 
Role of thymidine phosphorylase

Pyrimidine nucleoside analogues are often prone to hydrolysis to their free base by the pyrimidine nucleoside catabolic enzyme uridine phosphorylase (UPase) or thymidine phosphorylase (TPase). The free bases are, as a rule, devoid of any therapeutic activity. Thus, the UPase and TPase enzymes may inactivate the antiviral pyrimidine nucleoside analogues. BVDU is a well-known example of an antiherpes virus drug that is highly susceptible to hydrolytic cleavage by both human and Escherichia coli TPase.8,9 Interestingly, the BCNAs were found to be entirely resistant to the phosphorolytic cleavage by TPase, and thus are expected to be relatively stable in biological fluids.10 Human plasma does indeed not catabolize BCNAs to their free bases. When modelled in the active site of E. coli TPase,11,12 it became clear why the BCNA Cf 1743 did not function as a substrate for TPase. First of all, the BCNAs have no free carbonyl at the corresponding position (C-4) in the pyrimidine ring, but, more importantly, the BCNA base has no hydrogen available on the original N-3 position of the pyrimidine ring that forms a hydrogen bond with Ser-186 in the active site of TPase. Moreover, the presence of a bulky 6-alkylphenyl side chain on the fused bicyclic pyrimidine ring clashes with the main chain residues (176 and 177) of E. coli TPase, which must prevent proper positioning of Cf 1743 in the active site of the enzyme. Our observation that Cf 2002, which represents a BCNA without the 6-substituent on the fused pyrimidine ring, also lacks substrate activity for human and E. coli TPase indicates that lacking one hydrogen bond between the BCNA and the enzyme active site may be sufficient to annihilate substrate activity of the BCNAs for TPase.

Role of dihydropyrimidine dehydrogenase

The free nucleobase of BVDU, (E)-5-(2-bromovinyl)uracil (BVU), and related compounds such as 5-propynyl-uracil have proven to be efficient inhibitors of human dihydropyrimidine dehydrogenase (DPD).13 DPD is a key catabolic enzyme in the degradation of natural pyrimidines and pyrimidine analogues such as 5-fluorouracil (5-FU). It has been shown that the combination of BVDU (upon conversion to BVU by TPase) with 5-FU results in a marked potentiation of the toxicity and/or antitumour activity of 5-FU in a variety of cell cultures and animal models.1416 Indeed, co-administration of oral sorivudine (BVaraU) with 5-FU in cancer patients suffering from a VZV infection has led to the appearance of severe toxicity of 5-FU, resulting in a number of deaths.1618 Presumably, degradation of sorivudine by intestinal prokaryotic TPase(s) had released BVU from sorivudine,17 which then blocked the catabolic inactivation of 5-FU by DPD resulting in unacceptably high plasma levels of 5-FU.18 Further clinical trials with sorivudine were suspended. Given the potential above-mentioned complications of co-administration of BVU (as BVDU or BVaraU) or BVU analogues and 5-FU to patients, it is of clinical importance to reveal whether the free base of the BCNAs also inhibits DPD. Indeed, in marked contrast to BVU, which inhibited human liver DPD activity at an IC50 of c. 10 µM,10 several free BCNA bases were found to be completely ineffective in inhibiting the DPD enzyme at 100–250 µM. In addition, when 5-FU was administered to mice in combination with free BCNA bases or with BCNA Cf 1368, no effect of BCNAs on 5-FU plasma levels was observed under the experimental conditions at which BVDU markedly raised and prolonged the plasma levels of 5-FU.10 Thus, it could be concluded that the BCNAs may be expected not to affect 5-FU plasma levels in patients treated with 5-FU for cancer that would concomitantly be treated with the BCNAs for a concurrent VZV infection.

Pharmacokinetics of BCNAs in vivo

It is important that antiviral drugs have favourable pharmacokinetics and a good oral bioavailability. The BCNAs with optimal antiviral activity have a pronounced lipophilicity with optimal calculated octanol:water log P values ranking between 2.5 and 3.5. The high log P values correlate with a low water solubility (<1 mg/L) of the BCNAs. This property presents a challenge for an appropriate oral formulation. We made a reasonable solution of 2 mg/mL Cf 1368 in 11% dimethyl sulphoxide, 22% cremophore and 67% phosphate-buffered saline and administered the drug (in 200 µL) to adult NMRI mice by oral gavage. Drug plasma levels were determined at a variety of time points after drug administration and were compared with the plasma levels of drug given by intravenous route. Preliminary data revealed a very high oral bioavailability (>50%) of the BCNA Cf 1368 in mice (R. Sienaert, L. Naesens and J. Balzarini, unpublished results), which is a strong beneficial property from a clinical viewpoint.


    Conclusions
 Top
 Abstract
 Antiviral activity of bicyclic...
 Resistance development of BCNAs...
 Molecular basis for the...
 Catabolic properties of BCNAs
 Conclusions
 References
 
The BCNAs represent a unique class of exceptionally potent and specific anti-VZV agents endowed with an unprecedented selectivity. They are characterized by a number of favourable properties in terms of good stability (lack of susceptibility to a hydrolytic cleavage by thymidine phosphorylase) and pronounced oral bioavailability. The drugs retain high antiviral activity against a variety of clinical VZV isolates in cell culture. Given also their ease of synthesis, further (pre-)clinical evaluations of BCNAs as anti-VZV agents are highly warranted.


    Footnotes
 
* Corresponding author. Tel: +32-16-337-352; Fax: +32-16-337-340; E-mail: Jan.Balzarini{at}rega.kuleuven.ac.be Back


    References
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 Abstract
 Antiviral activity of bicyclic...
 Resistance development of BCNAs...
 Molecular basis for the...
 Catabolic properties of BCNAs
 Conclusions
 References
 
1 . McGuigan, C., Yarnold, C. J., Jones, G., Velazquez, S., Barucki, H., Brancale, A. et al. (1999). Potent and selective inhibition of varicella-zoster virus (VZV) by nucleoside analogues with an unusual bicyclic base. Journal of Medicinal Chemistry 42, 4479–84.[ISI][Medline]

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6. Balzarini, J. & McGuigan, C. (2001). Chemotherapy of varicella-zoster virus (VZV) by a novel class of highly specific anti-VZV bicyclic pyrimidine nucleosides. Biochimica et Biophysica Acta, in press.

7. Sienaert, R., Naesens, L., Brancale, A., De Clercq, E., McGuigan, C. & Balzarini, J. (2002). Specific recognition of the bicyclic pyrimidine nucleoside analogues, a new class of highly potent and selective inhibitors of varicella-zoster virus (VZV), by the VZV-encoded thymidine kinase. Molecular Pharmacology 61, 249–54.[Abstract/Free Full Text]

8. Desgranges, C., Razaka, G., Rabaud, M., Bricaud, H., Balzarini, J. & De Clercq, E. (1983). Phosphorolysis of (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) and other 5-substituted 2'-deoxyuridines by purified human thymidine phosphorylase and intact blood platelets. Biochemical Pharmacology 32, 3583–90.[ISI][Medline]

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15 . Keizer, H. J., Pauwels, R., Landuyt, W., Balzarini, J., Van der Schueren, E. & De Clercq, E. (1988). Combined effects of bromovinyldeoxyuridine and fractionated or continuous administration of 5-fluorouracil in P388 leukemia-bearing mice. Cancer Letters 39, 217–23.[ISI][Medline]

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17 . Nakayama, H., Kinouchi, T., Kataoka, K., Akimoto, S., Matsuda, Y. & Ohnishi, Y. (1997). Intestinal anaerobic bacteria hydrolyse sorivudine, producing the high blood concentration of 5-(E)-5-(2-bromovinyl)uracil that increases the level and toxicity of 5-fluorouracil. Pharmacogenetics 7, 5–43.

18 . Okuda, H., Ogura, K., Kato, A., Takubo, H. & Watabe, T. (1998). A possible mechanism of eighteen patient deaths caused by interactions of sorivudine, a new antiviral drug, with oral 5-fluorouracil prodrugs. Journal of Pharmacology and Experimental Therapeutics 287, 791–9.[Abstract/Free Full Text]