Herpes simplex virus type 1 (HSV 1) 1 thymidine kinase (TK) is a multifunctional enzyme that possesses kinase activities normally performed by three separate cellular enzymes. It phosphorylates thymidine (dT), which is then transformed by cellular kinases to the triphosphorylated DNA building block, and deoxyuridine (dU); both reactions are comparable to the function of human cellular TK. Further, it converts deoxycytidine (dC) to dCMP, as does human deoxycytidine kinase (dCK), and phosphorylates thymidylate (dTMP), as does human TMP kinase (TmpK) (1-3). Moreover, unlike its cellular counterpart human cellular TK, HSV 1 TK is able to phosphorylate pyrimidine, as well as purine analogs, and discloses low stereochemical demands for the ribose moiety, as it also accepts acyclic side chains as phosphoryl group acceptors e.g. (4 -6). These differences in substrate diversity are the crucial molecular basis for the selective treatment of viral infections. Nowadays, the most widely used therapeutic compounds to interfere with a severe HSV 1 infection are the purine analogs acyclovir (ACV) and penciclovir and their prodrugs valaciclovir and famciclovir, respectively. They require HSV 1 TK to be efficiently activated in order to block virus proliferation by inhibition of viral DNA polymerase. HSV 1 TK is the key enzyme in this antiviral strategy. In gene therapy of cancer (7,8) and AIDS (9), HSV 1 TK is used as a suicide enzyme in combination with the purine analog ganciclovir. Another important application is the use of HSV 1 TK as a rescue system in allogeneic bone marrow transplantation-induced graft versus host disease (10). In addition to the significance from a therapeutic point of view, HSV 1 TK seems to be important for the reactivation of the virus from lifelong latent infection in neuronal ganglia (11-13). However, there is evidence that human TK can functionally replace viral TK in terms of reactivation of the virus from latency (14).There are no recognizable sequence similarities between HSV 1 TK and human cellular TK (15). Rather, sequence alignments have detected similarities between herpesvirus TKs and human dCK (16) and to a lesser extent cellular TmpK (17). Despite the limited sequence homology with enzymes of the nucleotide kinase (NK) family, HSV 1 TK shares structural features comprising a parallel five-stranded ␤-sheet and a glycine-rich loop common to all NKs. In the crystal structure, HSV 1 TK is a homodimeric enzyme with 376 amino acids per subunit (18 -20). The two subunits are related by C2 symmetry. The active site is formed by an ATP-and a nucleoside-binding region. The visual representation of the thymidine binding site is depicted in Fig. 1, featuring a complex hydrogen bond network within the active site. The thymine ring makes pairwise hydrogen bond interaction via its 4-carbonyl and 3-NH group with the amide group of the highly conserved Gln-125 and hydrogen bonds with Arg-176 by means of two ordered water molecules. Moreover, the pyrimidine ring of thymidine is fixed between Met-128 and T...

Kinetic and crystallographic analyses of wild-type Herpes simplex virus type 1 thymidine kinase (TK(HSV1)) and its Y101F-mutant [TK(HSV1)(Y101F)] acting on the potent antiviral drug 2'-exo-methanocarba-thymidine (MCT) have been performed. The kinetic study reveals a 12-fold K(M) increase for thymidine processed with Y101F as compared to the wild-type TK(HSV1). Furthermore, MCT is a substrate for both wild-type and mutant TK(HSV1). Its binding affinity for TK(HSV1) and TK(HSV1)(Y101F), expressed as K(i), is 11 microM and 51 microM, respectively, whereas the K(i) for human cytosolic thymidine kinase is as high as 1.6 mM, rendering TK(HSV1) a selectivity filter for antiviral activity. Moreover, TK(HSV1)(Y101F) shows a decrease in the quotient of the catalytic efficiency (k(cat)/K(M)) of dT over MCT corresponding to an increased specificity for MCT when compared to the wild-type enzyme. Crystal structures of wild-type and mutant TK(HSV1) in complex with MCT have been determined to resolutions of 1.7 and 2.4 A, respectively. The thymine moiety of MCT binds like the base of dT while the conformationally restricted bicyclo[3.1.0]hexane, mimicking the sugar moiety, assumes a 2'-exo envelope conformation that is flatter than the one observed for the free compound. The hydrogen bond pattern around the sugar-like moiety differs from that of thymidine, revealing the importance of the rigid conformation of MCT with respect to hydrogen bonds. These findings make MCT a lead compound in the design of resistance-repellent drugs for antiviral therapy, and mutant Y101F, in combination with MCT, opens new possibilities for gene therapy.

A high-throughput molecular docking approach was successfully applied for the selection of potential inhibitors of the Influenza RNA-polymerase which act by targeting the PA-PB1 protein-protein interaction. Commercially available compounds were purchased and biologically evaluated in vitro using an ELISA-based assay. As a result, some compounds possessing a 3-cyano-4,6-diphenyl-pyridine nucleus emerged as effective inhibitors with the best ones showing IC50 values in the micromolar range.

Several drug-resistant strains of herpes simplex virus type 1 (HSV1) isolated in vivo or from tissue culture, have exhibited a mutated thymidine kinase (TK). Moreover, various site-directed-mutagenesis experiments conducted on HSV1 TK allowed the assignment of specific amino acid residues to specific functional properties. From this, a range of hypotheses was generated related to substrate binding of TK at the molecular level. A site-directed-mutagenesis study on Q125 was performed to clarify the contribution of this residue to the binding of thymidine or aciclovir beyond the hydrogen-bonding pattern observed in the crystal structure. While Q125L is only able to phosphorylate thymidine, Q125N accepts thymidine and aciclovir as substrates. Q125E shows no phosphorylation activity. Several mutations identified previously as relevant in drug resistance were studied in an attempt to further understand their role in these processes. Four amino acid positions are described (T63, A168, R176 and C336) that confer drug resistance when mutated ; however, the molecular mechanisms are considerably different in each case. Analysis of the crystal structures and the molecular modeling presented in this paper suggest that T63 is essential for the binding of Mg 2ϩ and thus the catalytic activity of the enzyme, while A168 limits steric accessibility and if mutated to a bulkier residue will exclude binding of larger substrate analogues. R176 appears to be essential for electrostatic balance within the active site, and C336, which is located at the surface of TK and directed toward the ATP-binding site, disrupts the three-dimensional structure of the whole active site by shifting the LID-domain. The present work contributes to a detailed understanding of nucleoside binding to TK, thereby facilitating the rational design of substrates for HSV1 TK and of drug-specific TK for gene therapy.Keywords : herpes simplex virus ; thymidine kinase ; mutation; aciclovir ; resistance.Thymidine kinase (TK) is a key enzyme in the pyrimidine-herpesviral TK isoenzymes is the molecular basis of selective salvage pathway catalyzing the transfer of γ-phosphate from antiviral therapy. Furthermore, HSV1 TK was used recently in ATP to thymidine, to synthesize thymidine monophosphate combination with aciclovir or gangciclovir as suicide enzyme in (dTMP). Herpes viruses encode their own thymidine kinases gene therapy of cancer [2Ϫ4] and AIDS [5]. Therefore, further which differ considerably from the human host cell isoenzyme. understanding of the molecular mechanism of HSV1 TK and its In particular, viral TKs are able to phosphorylate a broad drug-resistant mutants could lead to rational design of antiviral spectrum of pyrimidine and purine substrate analogues including drugs and to customized drug-specific TK for gene therapy. those carrying an acyclic sugar moiety such as the antiviral There are four classes of single mutations of the HSV gedrugs aciclovir and ganciclovir [1]. Compounds such as nome that confer drug resistance. Three of these affect the th...