Understanding the Binding of 5-Substituted 2‘-Deoxyuridine Substrates to Thymidine Kinase of Herpes Simplex Virus Type-1

Abstract

Thymidine kinase from HSV-1 (HSV-1 TK) is a key enzyme in the metabolic activation of antiviral nucleosides. High affinity of such compounds for the enzyme is required for efficient phosphorylation. In this study, affinity data from a series of 5-substituted 2'-deoxyuridine substrates in combination with the crystal structure of the viral enzyme were used to investigate the structural factors influencing the affinity of these compounds for the enzyme. Calculations showed that the binding energetics and conformations of thymidine and the 5-substituted 2'-uridine analogues are similar. The major part of the binding energy arises from interactions involving sugar and base moieties. Small differences in affinity for the enzyme are explained by the hydrophobicity of the 5-substituent or by its energetic complementarity with the active site pocket. In designing high-affinity nucleoside substrates of HSV-1 TK, care should be taken to maintain the geometry of the base moiety and sugar hydroxyl functionalities. Substitutions at the 5-position of the nucleobase should be lipophilic and characterized by well-defined geometrical properties. The present study represents a first quantitative explanation for HSV-1 TK affinity of 5-substituted 2'-deoxyuridines which are historically the first group of selective antivirals. The results may be used to design new and more potent compounds.

Herpesviral thymidine kinases: laxity and resistance by design.

“…Consider, for example, the pairing of Glu-83 and Pro-84 (totally conserved in all herpesviral TK sequences), where the glutamate side-chain forms a hydrogen bond with the 5h-hydroxyl group of the sugar of thymidine (Brown et al, 1995 ;Wild et al, 1997) or the surrogate group in drug analogues (De Winter & Herdewijn, 1996). This hydroxyl group is the nucleophile that interacts with the phosphoryl group transferred to it from ATP or other nucleotide triphosphate.…”

“…Some herpesviral TK can also carry out the monophosphorylation of deoxycytidine, though usually not as well, perhaps because of greater difficulty in forming two hydrogen bonds between conserved Gln-125 and the base during the catalytic cycle. The Gln-125 side-chain is observed to be rotated 180m when TK structures with bound thymidine (a pyrimidine) and bound ganciclovir (a pseudopurine nucleoside) are compared (Brown et al, 1995 ;De Winter & Herdewijn, 1996). Some considerable flexibility of conformation for Gln-125 may be envisioned to be required for accommodating deoxycytidine because of the difference in disposition of hydrogen bonding donors and acceptors around the heterocyclic rings of these two pyrimidines.…”

“…The possibility exists that the more highly branched side-chain of serine or threonine either takes up too much space or alters the otherwise hydrophobic character of the binding pocket for the 5-substituent. The reported ease of accommodating five-membered heterocyclic substituents (De Winter & Herdewijn, 1996) suggests that the residue type has a more important effect than a modest increase in residue size in this context. While this line of reasoning fails to account for the ability of VZV TK to phosphorylate BVdU, the effects of the further Leu169Thr substitution for VZV TK in comparison to HSV-1 TK could compensate for the effects of the Ala168Ser substitution given the flexible ligand associations characteristic of the substrate pocket.…”

“…Levine!bham.ac.uk using knowledge of primary sequence and predictive models for secondary structure. A recent interpretation of the binding of a range of nucleoside analogues to the TK of HSV-1 by De Winter & Herdewijn (1996) exemplifies such interest following closely upon the appearance of reports of partial structure determinations for HSV-1 TK complexed with natural and synthetic substrates (Brown et al, 1995 ;Wild et al, 1995).…”

Enzymatic and Structural Analysis of Inhibitors Designed against Mycobacterium tuberculosis Thymidylate Kinase

“…Acyclovir is phosphorylated by several viral or host kinases into acyclovir triphosphate, which terminates DNA synthesis when incorporated into the viral DNA (5,6). The comparative x-ray structures of different enzyme-ligand complexes of HSV type 1 thymidine kinase (7-10) revealed a number of interesting structural features and paved the way for rational structure-based drug design of antiviral compounds (11,12). A similar approach might lead to potent antituberculosis agents.…”