Structure-activity relationship of the affinity of 5-substituted uracil nucleoside analogues for varicella-zoster virus thymidine kinase and their activity against varicella-zoster virus
Introduction
Varicella-zoster virus (VZV) encodes a thymidine kinase (TK) having a biochemical behavior similar to that induced by the herpes simplex virus types 1 (HSV-1) and 2 (HSV-2) (Ogino et al., 1977, Cheng et al., 1979) and VZV TK has amino acid sequence homology in the substrate binding regions with HSV TKs (Balasubramanian et al., 1990). These herpesvirus-induced kinases have broad substrate specificities and phosphorylate a number of selective antiviral nucleoside analogues which are scarcely or cannot be phosphorylated by cellular kinase(s). Indeed, the sufficiency of nucleoside analogues as substrates for viral TK correlates with their anti-herpesvirus activity and the critical role of viral TKs in their activation is the rationale for the development of selective anti-herpesvirus nucleoside analogues as demonstrated by the phosphorylation and anti-HSV-1 activity of acyclic purine nucleoside analogues and 5-(2-halogenovinyl)-2′-deoxyuridines (Keller et al., 1981, Cheng et al., 1981). On the other hand, marked differences have been shown in the antiviral spectra of nucleoside analogues: 1-β-d-arabinofuranosyl-5-(E-2-bromovinyl)uracil (BV-araU) and 5-(E-2-bromovinyl)-2′-deoxyuridine (BVDU) exhibt particularly potent antiviral activity against VZV but have little activity against HSV-2, while acyclovir is much less active against VZV than against HSV-1 and HSV-2 (Machida, 1986, Machida, 1990, Shigeta et al., 1983). The 5-prop-1-ynyl derivative of 1-β-d-arabinofuranosyluracil (araU) also exhibits significant anti-VZV activity without activity against HSV-1 (Rahim et al., 1992). There are some differences in the substrate specificities of the viral TKs despite of the similarity in structural and biochemical features and the differences in the antiviral spectra may reflect, at least in part, differences in the substrate specificities of the viral kinases. The thymidine analogues that are particularly selective against VZV have been considered as candidates of novel anti-VZV agents. In clinical trials, oral BV-araU effectively reduced the time of vesicle formation, erythema and pain in immunocompromised patients with herpes zoster (Hiraoka et al., 1991) and BVDU was found to be effective in the treatment of VZV infection in immuno-compromised children (Heidl et al., 1991).
To study structure-activity relationships, we tested 5-substituted uracil nucleoside analogues for their selective antiviral activity against VZV in comparison with anti-HSV-1 and HSV-2 activities and their anti-proliferative activity using human lymphoblastoid leukemia cells. We also measured the inhibition constants of the nucleoside analogues for VZV TK using bacterially expressed enzyme.
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Cells and viruses
A human embryonic lung (HEL) cell line, HAIN-55, a gift from Dr Okumura, (National Institute of Health of Japan, Tokyo) and the VZV Oka strain, the HSV-1 VR-3 strain and the HSV-2 MS strain were used for the antiviral activity tests. The origin of viruses has been described previously (Machida, 1990). Human T-cell acute lymphoblastoid leukemia cells, CCRF-HSB-2, were used for the anti-cell growth activity test.
Compounds
The following pyrimidine nucleoside analogues were synthesized at the Chemistry
Anti-herpesvirus activities
As shown in Table 1, 5-substituted 2′-deoxyuridine, araU and 2′-deoxy-2′-fluoroarabinofuranosyluracil (F-araU)-analogues all showed potent to modest anti-VZV effects, except for Et-dU and Et-araU. These compounds, other than Prpy-araU and DiBrV-araU, also showed antiviral activities against HSV. 5-(2-Halogenovinyl) and 5-alkynyl uracil nucleosides exhibited more potent activity against VZV than against HSV-1 (ED50 for HSV-1 divided by ED50 for VZV was almost ten or greater) and 5-methyluracil
Discussion
A number of 5-substituted uracil nucleoside analogues act as thymidine analogues and show selective anti-herpesvirus effects. Phosphorylation by herpesvirus-induced TKs is essential for their intracellular activation and the exhibition of antiviral actions. The present study reveals that many 5-substituted uracil nucleosides have no or weak inhibitory effects on cell growth. These results, taken together with the results of the anti-VZV activity tests, confirm that the anti-VZV activity of the
Acknowledgements
We gratefully thank Dr A. Kuninaka (Yamasa Corporation) for supporting this study and Miss M. Nishitani for her technical assistance in the plaque reduction and MTT assays.
References (24)
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