ABSTRACT
The crystal structures of the full-length Herpes simplex virus type 1 thymidine kinase in its unligated form and in a complex with an adenine analogue have been determined at 1.9 A resolution. The unligated enzyme contains four water molecules in the thymidine pocket and reveals a small induced fit on substrate binding. The structure of the ligated enzyme shows for the first time a bound adenine analogue after numerous complexes with thymine and guanine analogues have been reported. The adenine analogue constitutes a new lead compound for enzyme-prodrug gene therapy. In addition, the structure of mutant Q125N modifying the binding site of the natural substrate thymidine in complex with this substrate has been established at 2.5 A resolution. It reveals that neither the binding mode of thymidine nor the polypeptide backbone conformation is altered, except that the two major hydrogen bonds to thymidine are replaced by a single water-mediated hydrogen bond, which improves the relative acceptance of the prodrugs aciclovir and ganciclovir compared with the natural substrate. Accordingly, the mutant structure represents a first step toward improving the virus-directed enzyme-prodrug gene therapy by enzyme engineering.
Subject(s)
Adenine/analogs & derivatives , Herpesvirus 1, Human/chemistry , Nucleosides/metabolism , Organophosphonates , Thymidine Kinase/chemistry , Adenine/chemistry , Adenine/metabolism , Adenosine/analogs & derivatives , Adenosine/chemistry , Adenosine/metabolism , Adenosine Monophosphate/analogs & derivatives , Adenosine Monophosphate/chemistry , Adenosine Monophosphate/metabolism , Amino Acid Substitution , Antiviral Agents/chemistry , Antiviral Agents/metabolism , Binding Sites , Catalytic Domain , Crystallography, X-Ray , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/metabolism , Herpesvirus 1, Human/isolation & purification , Herpesvirus 1, Human/metabolism , Mutation , Nucleosides/chemistry , Prodrugs/chemistry , Prodrugs/metabolism , Protein Structure, Tertiary , Stereoisomerism , Substrate Specificity , Thymidine/chemistry , Thymidine/metabolism , Thymidine Kinase/antagonists & inhibitors , Thymidine Kinase/metabolism , Viral Proteins/chemistry , Viral Proteins/isolation & purification , Viral Proteins/metabolism , Water/metabolismABSTRACT
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.
Subject(s)
Bridged Bicyclo Compounds/chemistry , Herpesvirus 1, Human/enzymology , Mutagenesis, Site-Directed , Phenylalanine/genetics , Thymidine Kinase/chemistry , Thymidine Kinase/genetics , Thymidine/analogs & derivatives , Thymidine/chemistry , Tyrosine/genetics , Amino Acid Substitution/genetics , Binding, Competitive/genetics , Crystallization , Crystallography, X-Ray , Enzyme Inhibitors/chemistry , Herpesvirus 1, Human/genetics , Humans , Kinetics , Macromolecular Substances , Models, Molecular , Molecular Sequence Data , Phosphorylation , Protein Conformation , Thymidine Kinase/antagonists & inhibitorsABSTRACT
In rational drug design the study of protein-ligand-interactions is one of the most important approaches to get knowledge of SAR. On this study N2-Phenylthioguanines were synthesized by Schiemann reaction from thioguanine followed by a substitution of the fluorine by aniline-derivatives. The activity of these HSV1 TK inhibitors was determined by kinetic measurements of thymidine phosphorylation. The N2-Phenylthioguanines gave the same activity as the oxoanalogues. Interaction energies between thymidine and HSV1 TK were measured by microcalorimetry. Results of the measurement showed negative delta G and delta H values which indicates that the binding of the natural substrate occurs spontaneously and is enthalpy driven.
