gamma-modified triphosphates of nucleoside analogues as potential antivirals and lipophilic prodrugs therof

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Nucleoside analogues have significant impact as antiviral agents in chemotherapy. However, there are also two major drawbacks associated with the use of these compounds. First, they require an intracellular metabolism to form the nucleoside triphosphate which is the ultimately active metabolite. Second, they can not only interact with viral polymerases but can also act as substrates of human polymerases which might result in severe toxic side effects. With the project presented here we aim not only to bypass the often rate-limiting metabolism in the triphosphate formation but also to develop a entirely new approach resulting potentially in an improvement in the substrate selectivity between viral and human polymerases by using gamma-modified nucleoside analogue triphosphates as putative antiviral agents. Here we would like to work on three different aspects:1. We will synthesize and study gamma-modified nucleoside analogue triphosphates as potential substrates of viral polymerases in comparison with the human polymerases. The gamma modification should be constructed as such, that this modifcation is a non-cleavable moiety, which is covalently attached to the triphosphate unit. By that, this moiety prevents also the enzymatic degradation of the triphosphate unit of the nucleoside triphosphate. In recent studies we showed that the gamma-acyloxybenzyl-modified triphosphate of the known antiviral 3-deoxy-2,3-didehydrothymidine (d4T) was acting as a substrate of HIVs reverse transcriptase which led to the incorporation of d4TMP into DNA-strands. In contrast, this gamma-modified triphosphate was not a substrate of human polymerase beta. Interestingly, the parent compound d4TTP was also a substrate of DNA-polymerase beta. Due to this difference in selectivity this offers now a completely new option for the use of gamma-modified triphosphate analogues. This is of tremendous importance in the context of possible toxic side effects which are caused by the interaction of the triphosphate with the cellular polymerases. As gamma-modifications we propose on the one hand non-cleavable acylbenzyl-(ketone) moieties which resemble closely the previously used bioreversible acyloxybenzyl-groups and on the other hand lipophilic alkyl-residues.2. It is expected that the gamma-modified triphosphates will not be up-taken by cells due to the negative charges at the triphosphate-moiety. Therefore, lipophilic prodrugs of these gamma-modified triphosphate analogues will be developed. We aim to introduce enzyme-cleavable masking groups, e.g acyloxybenzyl-groups. Studies to elucidate the hydrolysis mechanism, stability studies in biological media such as cell extracts and antiviral tests will guide us to optimized prodrug molecules.3. The cellular up-take of the new prodrugs of the gamma-modified nucleoside triphosphates will be studied by incorporating the fluorescent bicyclic nucleoside analogue (BCNA) and cell incubation assays.