Received for publication February 14, 2006.
Revised March 22, 2006.
Accepted for publication March 22, 2006.

Mutations distal to the substrate site can affect Varicella Zoster Virus Thymidine Kinase activity: implications for drug design

Abstract

Varicella Zoster Virus encodes a thymidine kinase responsible for activation of antiherpetic nucleoside prodrugs such as acyclovir. Additionally, herpes virus thymidine kinases are being explored in gene/chemotherapy strategies aimed at developing novel anti-tumour therapies. In order to investigate and improve compound selectivity, we report here structure-based site-directed mutagenesis studies of VZVTK. Earlier reports showed that mutating residues at the core of the VZVTK active site invariably destroyed activity, hence we targeted more distal residues. Based on the VZVTK crystal structure, we have constructed six mutants (E59S, R84V, H97Y/A, Y21H/E), and tested substrate activity and competitive inhibition for several compound series. All VZVTK mutants tested retained significant phosphorylation activity with dThd as substrate, apart from Y21E (350-fold diminution in the k_cat/K_m). Some mutations give slightly improved affinities: bicyclic nucleoside analogs (BCNAs) with a p-alkyl-substituted phenyl group appear to require aromatic ring stacking interactions with residue 97 for optimal inhibitory effect. Mutation Y21E decreased the IC₅₀ for the BCNA Cf1368 4-fold, whereas mutation Y21H increased the IC₅₀ by more than 15-fold. These results suggest that residue 21 is important for BCNA selectivity, and might explain why HSV1TK is unable to bind BCNAs. Other mutants such as the E59S and R84V TKs, which in wild-type VZVTK stabilise the dimer interface, give opposite results regarding the level of sensitivity to BCNAs. The work described here shows that distal mutations which affect the VZVTK active-site may help in the design of more selective substrates for gene suicide therapy or as anti-varicella-zoster virus drugs.

Key words: Mutagenesis/Chimeric approaches, Antiviral drugs, Nucleoside/Nucleotide derivatives