Mutations distal to the substrate site can affect varicella zoster virus thymidine kinase activity: implications for drug design.
Varicella zoster virus encodes a thymidine kinase responsible for the activation of antiherpetic nucleoside prodrugs such as acyclovir. In addition, herpes virus thymidine kinases are being explored in gene/chemotherapy strategies aimed at developing novel antitumor therapies. To investigate and improve compound selectivity, we report here structure-based site-directed mutagenesis studies of varicella zoster virus thymidine kinase (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 constructed six mutants (E59S, R84V, H97Y/A, and 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 seem to require aromatic ring stacking interactions with residue 97 for optimal inhibitory effect. Mutation Y21E decreased the IC(50) value for the BCNA 3-(2'-deoxy-beta-D-ribofuranosyl)-6-octyl-2,3-dihydrofuro[2,3-d]pyrimidin-2-one (Cf1368) 4-fold, whereas mutation Y21H increased the IC(50) value 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 thymidine kinases, which in wild-type VZVTK stabilize the dimer interface, give opposite results regarding the level of sensitivity to BCNAs. The work described here shows that distal mutations that 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.
Division of Structural Biology, The Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford, OX3 7BN, UK.
Kamel
K
El Omari
Sandra
S
Liekens
Louise E
LE
Bird
Jan
J
Balzarini
David K
DK
Stammers
D000998:Antiviral Agents / Q000737:chemistry*; D001665:Binding Sites / Q000235:genetics; D019281:Dimerization; D015195:Drug Design; D004791:Enzyme Inhibitors / Q000737:chemistry; D014645:Herpesvirus 3, Human / Q000201:enzymology*; D006574:Heterocyclic Compounds, 2-Ring / Q000737:chemistry; D020128:Inhibitory Concentration 50; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009705:Nucleosides / Q000737:chemistry*; D011355:Prodrugs / Q000737:chemistry; D011487:Protein Conformation; D013379:Substrate Specificity; D013937:Thymidine Kinase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics*; D014764:Viral Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics*
D000998:Antiviral Agents; C446025:Cf 1368; D004791:Enzyme Inhibitors; D006574:Heterocyclic Compounds, 2-Ring; D009705:Nucleosides; D011355:Prodrugs; D014764:Viral Proteins; D013937:Thymidine Kinase