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 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-(2Ј-deoxy-␤-(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.

Engineering of a bacterial tyrosinase for improved catalytic efficiency towards D‐tyrosine using random and site directed mutagenesis approaches

“…The kinetic analysis of 145_V153A, showed a higher turnover number compared to the WT (126 min −1 , Table II) but a decreased affinity. Previous work involving SDM of enzymes have shown that distal mutations can have an effect on activity and substrate affinity of the enzyme (Ardissone et al, 2005; El et al, 2006; Lee and Goodey, 2011). Lee and Goodey have described possible reasons for these indirect effects based on inter molecular relationships, charge distribution and conformational motion of proteins (Lee and Goodey, 2011).…”

Single-mutation fitness landscapes for an enzyme on multiple substrates reveal specificity is globally encoded

“…For engineering enzyme specificity, it has been shown that a rational mutagenesis approach—primarily focused on residues lining a substrate binding pocket—provides greater payoffs than random mutagenesis (that is, error-prone PCR)11213. However, it is no secret that distant (>10 Å) mutations can have significant effects on catalytic function1314151617181920. For example, in a classic paper Oue et al 20.…”

In Vitro -Selected Drug-Resistant Varicella-Zoster Virus Mutants in the Thymidine Kinase and DNA Polymerase Genes Yield Novel Phenotype-Genotype Associations and Highlight Differences between Antiherpesvirus Drugs

“…Thus, the molecular basis for resistance to ACV is linked to mutations in either the viral TK or the DNA Pol gene. Herpesvirus TKs are able to transfer a ␥-phosphoryl group from ATP to the 5= hydroxyl group of thymidine (13). In addition, VZV and HSV-1 TKs possess thymidylate kinase activity, which converts dTMP to dTDP.…”