L-Thymidine is phosphorylated by herpes simplex virus type 1 thymidine kinase and inhibits viral growth (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.
ChemMedChem, 2008
The stereoselective syntheses of the (+)-D and (−)-L enantiomers of racemic isomethanocarbathymidine (iso-MCT) was achieved through two independent linear approaches that converged on two antipodal enantiomers, common to a key precursor utilized in the synthesis of racemic iso-MCT. In this study we identified (+)-3 [D-iso-(+)-MCT] as the active enantiomer that was exclusively recognized by the herpes 1 thymidine kinase (HSV1-tk) as was predicted by molecular modeling. For this purpose, a human osteosarcoma (HOS) cell line modified to contain, and express, HSV1-tk from herpes simplex virus (HSV1) was utilized to determine the cytotoxicity of the compounds via an assay that measures the level of ATP in the cells. The work demonstrates that changes in the substitution pattern of rigid bicyclo[3.1.0]hexane nucleosides, which relative to normal nucleosides appear unconventional, can lead to the spatial optimization of pharmacophores and a vastly improved substrate recognition.
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.
it can cause rapidly ascending encephalitis that is often fatal. To understand the low susceptibility of BV to the acyclonucleosides, we have cloned, expressed, and characterized the BV thymidine kinase (TK), an enzyme that is expected to "activate" nucleoside analogs. This enzyme is similar in sequence and properties to the TK of herpes simplex virus (HSV), i.e., it has a broad substrate range and low enantioselectivity and is sensitive to inhibitors of HSV TKs. The BV enzyme phosphorylates some modified nucleosides and acyclonucleosides and L enantiomers of thymidine and related antiherpetic analogs. However, the potent anti-HSV drugs acyclovir (ACV), ganciclovir (GCV), and 5-bromovinyldeoxyuridine were poorly or not phosphorylated by the BV enzyme under the experimental conditions. The antiviral activities of a number of marketed antiherpes drugs and experimental compounds were compared against BV strains and, for comparison, HSV type 1 (HSV-1) in Vero cell cultures. For most compounds tested, BV was found to be about as sensitive as HSV-1 was. However, BV was less sensitive to ACV and GCV than HSV-1 was. The abilities of thymidine analogs and acyclonucleosides to inhibit replication of BV in Vero cell culture were not always proportional to their substrate properties for BV TK. Our studies characterize BV TK for the first time and suggest new lead compounds, e.g., 5-ethyldeoxyuridine and pencyclovir, which may be superior to ACV or GCV as treatment for this emerging infectious disease.
Nucleoside binding site ofHerpes simplex type 1 thymidine kinase analyzed by X-ray crystallography
Proteins: Structure, Function, and Genetics, 2000
The crystal structures of the fulllength 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 Å 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 Å 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. Proteins 2000;41:545-553.
Journal of Medicinal Chemistry, 2005
Derivatives of the herpes simplex thymidine kinase inhibitor HBPG (2-phenylamino-9-(4hydroxybutyl)-6-oxopurine) have been synthesized, and tested for inhibitory activity against recombinant enzymes (TK) from herpes simplex types 1 and 2 (HSV-1, HSV-2). The compounds inhibited phosphorylation of [ 3 H]thymidine by both enzymes, but potencies differed quantitatively from those of HBPG, and were generally greater for HSV-2 than HSV-1 TKs. Changes in inhibitory potency were generally consistent with the inhibitor/substrate binding site structure based on published x-ray structures of HSV-1 TK. In particular, several 9-(4-aminobutyl) analogs with bulky tertiary amino substituents, were among the most potent inhibitors. Variable substrate assays showed that the most potent compound, 2-phenylamino-9-[4-(1-decahydroquinolyl)butyl]-6-oxopurine, was a competitive inhibitor, with K i values of 0.03 and 0.005 gM against HSV-1 and HSV-2 TKs, respectively. The parent compound HBPG was uniquely active in viral infection models in mice, both against ocular HSV-2 reactivation and against HSV-1 and HSV-2 encephalitis. In assays lacking [ 3 H]thymidine, HBPG was found to be an efficient substrate for the enzymes. The ability of the TKs to phosphorylate HBPG may relate to its antiherpetic activity in vivo.
Journal of Virology, 2000
Human herpesvirus 8 (HHV8) open reading frame (ORF) 21 is predicted to encode a protein similar to the thymidine kinase (TK) enzyme of other herpesviruses. Expressed in mammalian cells, ORF 21 was found to have low TK activity, based on poor growth in media containing hypoxanthine-aminopterin-thymidine (HAT) and low incorporation of [ 3 H]thymidine into high-molecular-weight DNA. Kinetic analysis using HHV8 TK as a purified glutathione S -transferase (GST) fusion protein showed that the enzyme has a comparatively high K m for thymidine (dThd) of ∼33.2 μM. Nearly 50% of the phosphorylated product of the reaction with dThd was thymidylate. This monophosphate kinase activity was more pronounced with 3′-azido-3′-deoxythymidine (AZT), in which 78% of the reaction product was AZT diphosphate. Thymidine analogs competitively inhibited dThd phosphorylation by HHV8 TK, while 2′-deoxyguanosine, 2′-deoxyadenosine, 2′-deoxycytidine, and corresponding analogs did not. Further competition experim...