Engineering of a Single Conserved Amino Acid Residue of Herpes Simplex Virus Type 1 Thymidine Kinase Allows a Predominant Shift from Pyrimidine to Purine Nucleoside Phosphorylation

Abstract

Studies of herpes simplex virus type 1 (HSV-1) thymidine (dThd) kinase (TK) crystal structures show that purine and pyrimidine bases occupy distinct positions in the active site but approximately the same geometric plane. The presence of a bulky side chain, such as tyrosine at position 167, would not be sterically favorable for pyrimidine or pyrimidine nucleoside analogue binding, whereas purine nucleoside analogues would be less affected because they are located further away from the phenylalanine side chain. Site-directed mutagenesis of the conserved Ala-167 and Ala-168 residues in HSV-1 TK resulted in a wide variety of differential affinities and catalytic activities in the presence of the natural substrate dThd and the purine nucleoside analogue drug ganciclovir (GCV), depending on the nature of the amino acid mutation. A168H-and A167F-mutated HSV-1 TK enzymes turned out to have a virtually complete knock-out of dThd kinase activity (at least ϳ4 -5 orders of magnitude lower) presumably due to a steric clash between the mutated amino acid and the dThd ring. In contrast, a full preservation of the GCV (and other purine nucleoside analogues) kinase activity was achieved for A168H TK. The enzyme mutants also markedly lost their binding capacity for dThd and showed a substantially diminished feedback inhibition by thymidine 5-triphosphate. The side chain size at position 168 seems to play a less important role regarding GCV or dThd selectivity than at position 167. Instead, the nitrogen-containing side chains from A168H and A168K seem necessary for efficient ligand discrimination. This explains why A168H-mutated HSV-1 TK fully preserves its GCV kinase activity (V max /K m 4-fold higher than wild-type HSV-1 TK), although still showing a severely compromised dThd kinase activity (V max /K m 3-4 orders of magnitude lower than wild-type HSV-1 TK).

A designed equine herpes thymidine kinase (EHV4 TK) variant improves ganciclovir-induced cell-killing

“…Recent approaches, which aim to improve transduction efficiency, to specifically target the tumor, and also to reduce immunogenicity include recombinant non-viral vectors, mesenchymal stem cells, and the use of Bifidobacterium as gene delivery vehicle [29][30][31]. Furthermore, extensive structural and kinetic studies of HSV1 TK have focused on improving the catalytic activity of HSV1 TK for ACV and GCV as substrates [18][19][20][21][22]. Our study aimed to modify the catalytic activity of another member or the herpes thymidine kinase family, the equine herpes virus-4 thymidine kinase (EHV4 TK).…”

“…Residues A167 and A168 appeared to be important in the discrimination of GCV and dT binding to HSV1 TK [18,22]. Following sequence and structural comparisons of HSV1 TK and EHV4 TK [24], we proposed that the homologous mutations A143Y and S144H would confer a shift from pyrimidine activity to predominantly purine activity.…”

“…In the case of HSV1 TK, Balzarini et al have generated HSV1 TK mutants with the aim of ablating dT kinase activity, yet preserving purine nucleoside analog activity [22]. Through a structural modeling approach, they found individual amino acid mutations, where alanine residues substituted to phenylalanine or histidine at positions 167 or 168 (A167F or A168H, respectively) successfully compromised dT activity, but preserved GCV activity.…”

“…As HSV1 TK has a highest affinity (lowest K m ) for dT, it has been proposed that enabling HSV1 TK to preferentially bind and phosphorylate GCV rather than dT, would improve the cytotoxic effect of the HSV1 TK/ GCV enzyme/prodrug combination [18][19][20][21][22]. On this basis, random mutagenesis and rational protein engineering studies have focused on generating HSV1 TK variants, which would selectively and efficiently phosphorylate GCV [18][19][20][21][22]. Several HSV1 TK mutants have been shown to have compromised dT activity while still possessing the ability to phosphorylate GCV.…”

Comparison of Bioactivities of 5-Fluoro, 5-Iodo, 5-Iodovinyl, and 5-Fluorovinyl Arabinosyl Uridines against SR-39 TK-Transfected Murine Prostate Cancer Cells

“…Among them, SR-39, 1) SR-26, 1) A-167Y, 2) A-167F 3) and A168H 3) have found to possess the highest affinity for acylic purine nucleosides e.g. Gancicrovir (GCV) or acyclovir (ACV).…”