Human Herpesvirus 8 Open Reading Frame 21 Is a Thymidine and Thymidylate Kinase of Narrow Substrate Specificity That Efficiently Phosphorylates Zidovudine but Not Ganciclovir (original) (raw)
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Antimicrobial Agents and Chemotherapy, 1987
The thymidine analog 5'-ethynylthymidine was a potent inhibitor of herpes simplex virus type 1 (strain K(S)-induced thymidine kinase with a Ki value of 0.09 ,uM. 5'-Ethynylthymidine was less inhibitory against herpes simplex virus type 2 (strain 333)-induced thymidine kinase with a Ki of 0.38 ,uM and showed no inhibition against human cytosolic thymidine kinase under the conditions tested. The compound was effective against the altered thymidine kinase induced by acyclovir-and bromovinyldeoxyuridine-resistant virus variants. At 100 FM 5'-ethynylthymidine, the cellular pool size of dTTP in herpes simplex virus type 1-infected cells was 5% that of infected cells receiving no drug treatment, while there was no significant effect on the pool sizes of dATP, dGTP, and dCTP. There was a positive correlation between dTTP pools and the intracellular thymidine kinase activity of herpes simplex virus type 1-infected cells. When tested alone, 5'-ethynylthymidine exhibited no antiviral activity, but it antagonized the antiviral efficacy of five compounds which require viral thymidine kinase for their action. Herpes simplex viruses type 1 (HSV-1) and type 2 (HSV-2) and varicella-zoster virus induce unique virusspecified thymidine kinases (dThd kinase) in infected cells (2, 8, 18, 20). In the past, major efforts were made to develop antiviral compounds that would serve as "selective alternative substrates" (3) for virus-specified dThd kinase. While the activity of viral dThd kinase does not appear to be critical for virus replication in cell culture systems (19), studies have suggested that it is important for virus pathogenicity and reactivation of latent virus from neural cells (9, 14, 30). In this report, we describe the effects of 5'ethynylthymidine (5'-Et-dThd) (Fig. 1) as a selective inhibitor for HSV dThd kinase and its impact on deoxynucleotide metabolism in virus-infected cells. MATERIALS AND METHODS Materials. All chemicals used were reagent grade or better. 3H-labeled deoxynucleoside 5'-triphosphates (dNTPs) were purchased from ICN Radiochemicals, Irvine, Calif. dThd, dNTPs, and calf thymus DNA were purchased from Sigma Chemical Co., St. Louis, Mo. DNA polymerase I (endonuclease-free) and hydroxyurea were purchased from Boehringer Mannheim Biochemicals, Indianapolis, Ind. RPMI 1640 medium, fetal bovine serum, and kanamycin were purchased from Hazleton Research Products, Inc., Denver, Pa. 5'-Et-dThd was synthesized by published procedures (29). Acyclovir (ACV) was a gift from Burroughs Wellcome Co., Research Triangle Park, N.C., and 9-(1,3dihydroxy-2-propoxy-methyl)guanine (DHPG) was from Syntex Co. 5'-Amino-dThd (5'-NH2-dThd) was provided by William Prusoff, Yale University, New Haven, Conn. Fluoroiodo-arabinosyl-cytosine (FIAC) and (E)-5-(2bromovinyl)-2'-deoxyuridine (BVDU) were gifts from J. J. Fox (Sloan-Kettering Cancer Institute) and G. D. Searle & Co., respectively.
Biochemistry, 2000
The herpes simplex virus type 1 thymidine kinase (HSV-1 TK) is the major anti-herpes virus pharmacological target, and it is being utilized in combination with the prodrug ganciclovir as a toxin gene therapeutic for cancer. One active-site amino acid, glutamine-125 (Gln-125), has been shown to form hydrogen bonds with bound thymidine, thymidylate, and ganciclovir in multiple X-ray crystal structures. To examine the role of Gln-125 in HSV-1 TK activity, three site-specific mutations of this residue to an aspartic acid, an asparagine, or a glutamic acid were introduced. These three mutants and wild-type HSV-1 TK were expressed in E. coli and partially purified and their enzymatic properties compared. In comparison to the Gln-125 HSV-1 TK, thymidylate kinase activity of all three mutants was decreased by over 90%. For thymidine kinase activity relative to Gln-125 enzyme, the K m of thymidine increased from 0.9 µM for the parent Gln-125 enzyme to 3 µM for the Glu-125 mutant, to 6000 µM for the Asp-125 mutant, and to 20 µM for the Asn-125 mutant. In contrast, the K m of ganciclovir decreased from 69 µM for the parent Gln-125 enzyme to 50 µM for the Asn-125 mutant and increased to 473 µM for the Glu-125 mutant. The Asp-125 enzyme was able to poorly phosphorylate ganciclovir, but with nonlinear kinetics. Molecular simulations of the wild-type and mutant HSV-1 TK active sites predict that the observed activities are due to loss of hydrogen bonding between thymidine and the mutant amino acids, while the potential for hydrogen bonding remains intact for ganciclovir binding. When expressed in two mammalian cell lines, the Glu-125 mutant led to GCV-mediated killing of one cell line, while the Asn-125 mutant was equally as effective as wild-type HSV-1 TK in metabolizing GCV and causing cell death in both cell lines.
Proceedings of the National Academy of Sciences, 1977
Transformation of human cells from a thymidine kinase (ATP:thymidine 5'-phosphotransferase, EC 2.7.1.75)-negative to a thymidine kinase-positive phenotype has been achieved by using purified DNA from herpes simplex virus type 2. The specific activity of the DNA was in the range 0.5 to 2.0 transformants per microng and the efficiency of gene transfer was up to 1 transformant per 10(5) recipient cells. Several transformed lines able to grow continuously in medium selective for thymidine kinase-positive cells have been established. All of these lines express a thymidine kinase activity of viral origin but they differ from each other in the stability of enzyme expression. Subclones derived from a given transformed line inherited the degree of stability of the parental line.
Protein Engineering Design and Selection, 2002
Understanding the functional and mechanistic properties of the multi-substrate herpes simplex virus type-1 thymidine kinase (HSV-1 TK) remains critical to defining its role as a major pharmacological target in herpesvirus and gene therapies for cancer. An inherent limitation of the activity of HSV-TK is the >70-fold difference in the K m s for phosphorylation of thymidine over the pro-drug ganciclovir (GCV). To engineer an HSV-1 TK isoform that is specific for GCV as the preferred substrate, 16 site-specific mutants were generated. The mutations were concentrated at conserved residues involved in nucleoside base binding, Gln125 and near sites 3 and 4 involved in catalysis and substrate binding. The substrate preferences of each mutant enzyme were compared with wild-type HSV-1 TK. One mutant, termed Q7530 TK, had a lower K m for GCV than thymidine. Expression of the Q7530 TK in tumor cells indicated comparable metabolism to and improved sensitivity to GCV over wild-type HSV-1 TK, with minimal thymidine phosphorylation activity. A molecular modeling simulation of the different HSV-1 TK active-sites was done for GCV and thymidine binding. It was concluded that mutations at Gln125 and near site 4, especially at Ala168, were responsible for loss of deoxypyrimidine substrate binding.
Biochemistry, 2000
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′-exomethanocarba-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 µM and 51 µM, 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 Å, 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.
Infection and Immunity, 1977
Thymidine kinase (TK) from herpes simplex virus type 1 (HSV-1) biochemically transformed HeLa cells, purified by affinity chromatography, has been characterized with respect to its electrophoretic mobility, molecular weight, activation energy, substrate specificity, and immunological specificity. TK purified from HSV-1-transformed HeLa cells has the same electrophoretic mobility as TK purified from HeLa cells lytically infected with HSV-1. The sedimentation velocity of purified TK from transformed cells was similar to that previously reported for the lytic enzyme, and its molecular weight was estimated to be 70,000. The activation energy of purified transformed-cell TK was 18.3 kcal/mol. Antiserum prepared against purified HSV-1 TK, although it showed some crossreactivity, preferentially neutralized homologous TK. The transformed-cell TK antiserum also neutralized the deoxycytidine kinase activity of HSV-1-infected cell extracts but had no effect on deoxycytidine kinase activity of HSV-2-infected cell extract. These results further support the notion that TK acquired by HeLa cells transformed by HSV-1 is of viral and not of cellular origin.