ABSTRACT
Several guanosine analogues, i.e. acyclovir (and its oral prodrug valaciclovir), penciclovir (in its oral prodrug form, famciclovir) and ganciclovir, are widely used for the treatment of herpesvirus [i.e. herpes simplex virus type 1 (HSV-1), and type 2 (HSV-2), varicella-zoster virus (VZV) and/or human cytomegalovirus (HCMV)] infections. In recent years, several new guanosine analogues have been developed, including the 3-membered cyclopropylmethyl and -methenyl derivatives (A-5021 and synguanol) and the 6-membered D- and L-cyclohexenyl derivatives. The activity of the acyclic/carbocyclic guanosine analogues has been determined against a wide spectrum of viruses, including the HSV-1, HSV-2, VZV, HCMV, and also human herpesviruses type 6 (HHV-6), type 7 (HHV-7) and type 8 (HHV-8), and hepatitis B virus (HBV). The new guanosine analogues (i.e. A-5021 and D- and L-cyclohexenyl G) were found to be particularly active against those viruses (HSV-1, HSV-2, VZV) that encode for a specific thymidine kinase (TK), suggesting that their antiviral activity (at least partially) depends on phosphorylation by the virus-induced TK. Marked antiviral activity was also noted with A-5021 against HHV-6 and with D- and L-cyclohexenyl G against HCMV and HBV. The antiviral activity of the acyclic/carbocyclic nucleoside analogues could be markedly potentiated by mycophenolic acid, a potent inhibitor of inosine 5'-monophosphate (IMP) dehydrogenase. The new carbocyclic guanosine analogues (i.e. A-5021 and D- and L-cyclohexenyl G) hold great promise, not only as antiviral agents for the treatment of herpesvirus infections, but also an antitumor agents for the combined gene therapy/chemotherapy of cancer, provided that (part of) the tumor cells have been transduced by the viral (HSV-1, VZV) TK gene.
Subject(s)
Antiviral Agents/pharmacology , Guanosine/analogs & derivatives , Herpesviridae/drug effects , Animals , Guanosine/pharmacology , HumansABSTRACT
Introduction of a bulky lipophilic acyl entity at the 2'-OH position of both 1-beta-D-arabinofuranosylthymine (araT) and (E)-5-(2-bromovinyl)-1-beta-D-arabinofuranosyluracil (BVaraU), consistently resulted in a marked ( approximately 10-fold) increase in the inhibitory activity of these new arabinosyl nucleoside analogues for the mitochondrial thymidine kinase (TK-2)-catalysed conversion of 2 microM [methyl-(3)H]dThd to [methyl-(3)H]dTMP. The most potent derivatives were inhibitory to [methyl-(3)H]dThd phosphorylation by TK-2 within the lower micromolar concentration range. Substitution of the arabinosyl nucleoside derivatives with the acyl groups also dramatically increased the selectivity of these compounds. The inhibitory activity of araT and BVaraU to dThd phosphorylation by other related nucleoside kinases, including herpes simplex virus type 1 TK, varicella-zoster virus TK, and cytosolic TK-1, was completely annihilated upon 2'-O-acyl substitution (IC(50) > or = 1000 microM). Kinetic analysis revealed purely competitive inhibition of 2'-O-acyl-BVaraU against TK-2-catalysed thymidine phosphorylation (K(i)/K(m): 2.3). However, 2'-O-acyl-BVaraU was extremely poorly converted to the corresponding arabinosyl nucleoside 5'-monophosphate by TK-2 as revealed by [gamma-(32)P]phosphate transfer studies from [gamma-(32)P]ATP. Thus, the 2'-O-acyl derivatives of BVaraU did not behave as substrates, but rather as potent and highly selective inhibitors of TK-2. This is the first report on such a highly selective arabinosyl nucleoside inhibitor of mitochondrial TK-2, and opens perspectives for the rational design of selective mitochondrial TK-2 inhibitors.
Subject(s)
Arabinofuranosyluracil/analogs & derivatives , Arabinofuranosyluracil/pharmacology , Arabinonucleosides/pharmacology , Mitochondria/drug effects , Thymidine Kinase/antagonists & inhibitors , Thymidine/analogs & derivatives , Thymidine/pharmacology , Adenosine Triphosphate/metabolism , Antineoplastic Agents/pharmacology , Antiviral Agents/pharmacology , Arabinofuranosyluracil/chemistry , Arabinonucleosides/chemistry , Binding, Competitive , Humans , Kinetics , Mitochondria/enzymology , Mitochondria/metabolism , Phosphorus Radioisotopes , Phosphorylation/drug effects , Thymidine/chemistry , Thymidine/metabolism , Thymidine Kinase/metabolism , TritiumABSTRACT
The broad substrate specificity of herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) has provided the basis for selective antiherpetic therapy and, more recently, suicide gene therapy for the treatment of cancer. We have now constructed an HSV-1 TK mutant enzyme, in which an asparagine (N) residue is substituted for glutamine (Q) at position 125, and have evaluated the effect of this amino acid change on enzymatic activity. In marked contrast with wild-type HSV-1 TK, which displays both thymidine kinase and thymidylate kinase activities, the HSV-1 TK(Q125N) mutant was unable to phosphorylate pyrimidine nucleoside monophosphates but retained significant phosphorylation activity for thymidine and a series of antiherpetic pyrimidine and purine nucleoside analogs. The abrogation of HSV-1 TK-associated thymidylate kinase activity resulted in a 100-fold accumulation of the monophosphate form of (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) in osteosarcoma cells transfected with the HSV-1 TK(Q125N) gene compared with osteosarcoma cells expressing wild-type HSV-1 TK. BVDU monophosphate accumulation gave rise to a much greater inhibition of cellular thymidylate synthase in HSV-1 TK(Q125N) gene-transfected cells than wild-type HSV-1 TK gene-transfected osteosarcoma tumor cells without significantly changing the cytostatic potency of BVDU for the HSV-1 TK gene-transfected tumor cells. Accordingly, the presence of the Q125N mutation in HSV-1 TK gene-transfected tumor cells was found to result in a multilog decrease in the cytostatic activity of those pyrimidine nucleoside analogs that in their monophosphate form do not have marked affinity for thymidylate synthase [i.e., 1-beta-D-arabinofuranosylthymine and (E)-5-(2-bromovinyl)-1-beta-D-arabinofuranosyluracil].
Subject(s)
Herpesvirus 1, Human/enzymology , Nucleoside-Phosphate Kinase/metabolism , Thymidine Kinase/metabolism , Amino Acid Substitution , Antimetabolites/pharmacology , Antiviral Agents/pharmacology , Asparagine/genetics , Bromodeoxyuridine/analogs & derivatives , Bromodeoxyuridine/pharmacology , Cell Cycle/drug effects , Ganciclovir/pharmacology , Glutamine/genetics , Herpesvirus 1, Human/drug effects , Humans , Mutation , Nucleoside-Phosphate Kinase/genetics , Osteosarcoma , Thiouridine/analogs & derivatives , Thiouridine/pharmacology , Thymidine Kinase/genetics , Transfection , Tumor Cells, CulturedABSTRACT
Herpes simplex virus type 1 (HSV-1) encodes a thymidine kinase (TK) that markedly differs from mammalian nucleoside kinases in terms of substrate specificity. It recognizes both pyrimidine 2'-deoxynucleosides and a variety of purine nucleoside analogs. Based on a computer modeling study and in an attempt to modify this specificity, an HSV-1 TK mutant enzyme containing an alanine-to-tyrosine mutation at amino acid position 167 was constructed. Compared with wild-type HSV-1 TK, the purified mutant HSV-1 TK(A167Y) enzyme was heavily compromised in phosphorylating pyrimidine nucleosides such as (E)-5-(2-bromovinyl)-2'-deoxyuridine and the natural substrate dThd, whereas its ability to phosphorylate the purine nucleoside analogs ganciclovir (GCV) and lobucavir was only reduced approximately 2-fold. Moreover, a markedly decreased competition of natural pyrimidine nucleosides (i.e., thymidine) with purine nucleoside analogs for phosphorylation by HSV-1 TK(A167Y) was observed. Human osteosarcoma cells transduced with the wild-type HSV-1 TK gene were extremely sensitive to the cytostatic effects of antiherpetic pyrimidine [i.e., (E)-5-(2-bromovinyl)-2'-deoxyuridine] and purine (i.e., GCV) nucleoside analogs. Transduction with the HSV-1 TK(A167Y) gene sensitized the osteosarcoma cells to a variety of purine nucleoside analogs, whereas there was no measurable cytostatic activity of pyrimidine nucleoside analogs. The unique properties of the A167Y mutant HSV-1 TK may give this enzyme a therapeutic advantage in an in vivo setting due to the markedly reduced dThd competition with GCV for phosphorylation by the HSV-1 TK.
Subject(s)
Herpesvirus 1, Human/enzymology , Thymidine Kinase/metabolism , Alanine/genetics , Alanine/metabolism , Amino Acid Substitution , Antiviral Agents/metabolism , Antiviral Agents/pharmacology , Bone Neoplasms , Cell Division/drug effects , Ganciclovir/metabolism , Ganciclovir/pharmacology , Herpesvirus 1, Human/drug effects , Herpesvirus 1, Human/genetics , Humans , Osteosarcoma , Phosphorylation , Point Mutation , Purine Nucleosides/metabolism , Pyrimidine Nucleosides/metabolism , Thymidine Kinase/genetics , Transduction, Genetic , Tumor Cells, Cultured , Tyrosine/genetics , Tyrosine/metabolismABSTRACT
A kinetic analysis was performed for the novel 1-(8-phosphonooctyl)-6-amino-5-bromouracil and 1-(8-phosphonooctyl)-7-deazaxanthine inhibitors of Escherichia coli thymidine (dThd) phosphorylase (TPase). The structure of the compounds was rationally designed based on the available crystal structure coordinates of bacterial TPase. These inhibitors reversibly inhibited TPase. Kinetic analysis revealed that the compounds inhibited TPase in a purely competitive or mixed fashion not only when dThd, but also when inorganic phosphate (Pi), was used as the variable substrate. In contrast, the free bases 6-amino-5-bromouracil and 7-deazaxanthine behaved as non-competitive inhibitors of the enzyme in the presence of variable Pi concentrations while being competitive or mixed with respect to thymine as the natural substrate. Our kinetic data thus revealed that the novel 1-(8-phosphonooctyl)pyrimidine/purine derivatives are able to function as multisubstrate inhibitors of TPase, interfering at two different sites (dThd(Thy)- and phosphate-binding site) of the enzyme. To our knowledge, the described compounds represent the first type of such multisubstrate analogue inhibitors of TPase; they should be considered as lead compounds for the development of mechanistically novel type of TPase inhibitors.
Subject(s)
Bromouracil/analogs & derivatives , Enzyme Inhibitors/pharmacology , Thymidine Phosphorylase/antagonists & inhibitors , Bromouracil/chemistry , Bromouracil/pharmacology , Enzyme Inhibitors/chemistry , Escherichia coli/enzymology , Kinetics , Models, Molecular , Structure-Activity Relationship , Substrate Specificity , Thymidine/metabolism , Thymidine Phosphorylase/chemistry , Thymidine Phosphorylase/metabolism , Thymine/metabolism , Xanthines/chemistry , Xanthines/pharmacologyABSTRACT
We evaluated the molecular mechanism of resistance in herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) gene-transfected murine mammary carcinoma (FM3ATK-/HSV-1 TK+) cells, that were selected for resistance against (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) and ganciclovir (GCV) by prolonged exposure of the cell cultures to dose-escalating concentrations of these compounds. Drug-resistant FM3ATK-/HSV-1 TK+ cells showed marked differences in their sensitivity spectrum to a series of antiherpetic nucleoside analogues. BVDU-resistant FM3ATK-/HSV-1 TK+ cells displayed the same sensitivity profile as wild-type FM3A/0 cells. In contrast, GCV-resistant FM3ATK-/HSV-1 TK+ cells were still sensitive to BVDU, (E)5-(2-iodovinyl)-2'-deoxyuridine (IVDU) and (E)-5-(2bromovinyl)-2'-deoxycytidine (BVDC), a typical feature of FM3A TK cells lacking cytosolic TK. Southern blot and PCR analysis revealed that HSV-1 TK genes were not deleted from the genome of the drug-resistant FM3ATK-/HSV-1 TK+ cells. However, the TK genes in drug-resistant FM3ATK-/HSV-1 TK+ cells were shown to be heavily methylated. Accordingly, RT-PCR demonstrated the complete abrogation of TK mRNA production resulting in a complete loss of TK enzyme activity in drug-resistant FM3A TK-/HSV-1 TK+ cells.
Subject(s)
Antiviral Agents/pharmacology , Bromodeoxyuridine/analogs & derivatives , Ganciclovir/pharmacology , Herpesvirus 1, Human/enzymology , Mammary Neoplasms, Experimental/genetics , Thymidine Kinase/genetics , Animals , Antiviral Agents/chemistry , Blotting, Southern , Bromodeoxyuridine/chemistry , Bromodeoxyuridine/pharmacology , Cell Division/drug effects , DNA, Viral/analysis , Drug Resistance, Neoplasm/genetics , Female , Ganciclovir/chemistry , Mice , Reverse Transcriptase Polymerase Chain Reaction , Thymidine Kinase/analysis , Transfection , Tumor Cells, CulturedABSTRACT
The inhibitory effects of (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) and its arabinosyl derivative (E)-5-(2-bromovinyl)-1-beta-D-arabinofuranosyluracil (BVaraU) on the growth of both MDA-MB-435 human breast carcinoma and 9L rat gliosarcoma cells expressing the thymidine kinase (tk)-encoding gene of the Varicella zoster virus (VZV) or the Herpes simplex virus (HSV) were evaluated. In vitro, BVDU and BVaraU effectively killed both cell types expressing VZVtk, with 50% inhibitory concentration values ranging from 0.06 to 0.4 microM, whereas ganciclovir (GCV) lacked activity. On HSVtk+ cells, BVDU had high cytotoxic activity, with 50% inhibitory concentration values that were similar to those of GCV, whereas BVaraU was inactive. In vivo, BVDU applied intraperitoneally caused a 50% tumor growth inhibition in nude mice inoculated subcutaneously with VZVtk+ as well as HSVtk+ mammary tumor cells. In mice and at variance with the in vitro results, BVaraU had very little activity against the VZVtk+ mammary cells; GCV had the highest activity on the HSVtk+ cells, resulting in a 50% eradication of the tumors. With the 9L rat gliosarcoma model, the VZVtk/BVDU system completely failed to inhibit the development of VZVtk+ glioma tumors induced subcutaneously in syngeneic rats, although BVDU had a similar 45-minute half-life in both rats and mice. Factors other than degradation of the prodrug and related to the mode of action of these analogs are possibly involved in the observed discrepancies between the in vitro and in vivo results.
Subject(s)
Antineoplastic Agents/toxicity , Antiviral Agents/toxicity , Arabinofuranosyluracil/analogs & derivatives , Bromodeoxyuridine/analogs & derivatives , Herpesvirus 3, Human/drug effects , Simplexvirus/drug effects , Thymidine Kinase/biosynthesis , Animals , Antineoplastic Agents/metabolism , Antiviral Agents/metabolism , Arabinofuranosyluracil/toxicity , Breast Neoplasms/drug therapy , Breast Neoplasms/pathology , Bromodeoxyuridine/metabolism , Bromodeoxyuridine/toxicity , Female , Genetic Vectors , Herpesvirus 3, Human/enzymology , Herpesvirus 3, Human/genetics , Humans , Male , Mice , Mice, Nude , Rats , Rats, Inbred F344 , Simplexvirus/enzymology , Simplexvirus/genetics , Thymidine Kinase/genetics , Tumor Cells, CulturedABSTRACT
The antiherpetic agent (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) was found to be an efficient substrate for recombinant Drosophila melanogaster-deoxyribonucleoside kinase with a K(m) of 4.5 microM and a V(max) of 400 nmol/microg protein/h compared with 1.3 microM and 62.5 nmol/microg protein/h, respectively, for the natural substrate thymidine. Mammalian cytosolic thymidine kinase-1 does not recognize BVDU as a substrate. In sharp contrast to mammalian cells, the insect D. melanogaster and Spodoptera frugiperda (Sf) embryonic cells proved highly sensitive to the cytostatic action of BVDU. BVDU was efficiently metabolized to its 5'-mono-, 5'-di- and 5'-triphosphate derivatives in the insect cell cultures and abundantly incorporated into the insect cell DNA. BVDU prevented the D. melanogaster cells to initiate the S phase of their cell cycle, and exposure of S. frugiperda cells to BVDU led to a dose-dependent retardation of the insect cells in the S phase of their cell cycle. Both inhibition of nucleic acid synthesis (through the 5'-triphosphate of BVDU) and inhibition of thymidylate synthase (through the 5'-monophosphate of BVDU) would account for the cytostatic activity of BVDU against the insect cells. Because of the virtual lack of cytotoxicity of BVDU against mammalian cells, the drug should be considered highly selective in its cytostatic action against the insect cells. When added to the food of S. frugiperda larvae, BVDU caused a remarkable decrease in the weight gain of the larvae and heavily compromised the transformation of the larvae to the pupae and their subsequent adult (moth) phase. Our data indicate that insect multifunctional deoxyribonucleoside kinase should be considered an entirely novel and attractive target in the development of new nucleoside types of highly selective insecticidal drugs.
Subject(s)
Drosophila melanogaster/enzymology , Insecticides/pharmacology , Phosphotransferases (Alcohol Group Acceptor)/drug effects , Animals , Bromodeoxyuridine/analogs & derivatives , Bromodeoxyuridine/metabolism , Bromodeoxyuridine/pharmacology , Cell Cycle/drug effects , Cell Division/drug effects , Deoxycytidine/metabolism , Drosophila melanogaster/cytology , Insecticides/metabolism , Mitochondria/drug effects , Mitochondria/enzymology , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Thymidine/pharmacology , Thymidine Kinase/drug effects , Thymidine Kinase/metabolism , Thymidylate Synthase/metabolism , TritiumABSTRACT
We present a fast, convenient and inexpensive method that allows the automated, large-scale screening of chemical libraries for compounds that are converted by the herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) into inhibitors of cell growth. The method is based on the use of budding yeast (Saccharomyces cerevisiae) transformed with the HSV-1 TK gene on a multicopy plasmid. Eight nucleoside analogs (acyclovir, ganciclovir, penciclovir, lobucavir, brivudin, sorivudine, IVDU and ara-T), for which the cytostatic action against mammalian cells expressing the HSV-1 TK gene has been well documented, were studied for their inhibitory effect on the growth of yeast expressing the viral TK. These nucleoside analogs had little or no inhibitory effect on the growth of yeasts transformed with the empty vector, but inhibited to a significant extent the growth of yeast expressing the viral TK. Use of HSV-1 TK-expressing yeast allows quick screening in multi-well plate format for compounds with potential use in HSV-1 TK suicide gene therapy. The method may also be used as a tool to selectively suppress or arrest the growth of one population of yeast out of mixed yeast cell cultures.
Subject(s)
Antiviral Agents/pharmacology , Herpesvirus 1, Human/genetics , Saccharomyces cerevisiae/drug effects , Saccharomyces cerevisiae/genetics , Thymidine Kinase/genetics , Acyclovir/pharmacology , Gene Expression , Genetic Therapy , Genetic Vectors , Plasmids/genetics , Promoter Regions, Genetic , Saccharomyces cerevisiae/growth & development , TransfectionABSTRACT
We have reported previously that the herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) fused with green fluorescent protein (GFP) is localized in the nucleus of HSV-1 TK-GFP gene-transfected cells (Degrève et al. (1998) J. Virol. 72, 9535-9543). Deletion of the N-terminal 34 amino acids or selective mutation of the nonapeptide (25)RRTALRPRR(33), located in the N-terminal region of HSV-1 TK, resulted in the loss of the specific nuclear localization of HSV-1 TK. Utilizing information on the crystallographic structure of HSV-1 TK, we have now identified three additional putative nuclear localization signals and evaluated their potential role in the nuclear trafficking of HSV-1 TK by site-directed mutagenesis. We found that the sites containing the amino acids R236-R237 and K317-R318 are absolutely required for specific nuclear targeting of HSV-1 TK. The K317-R318 region, located at the interface between the two monomers in the dimeric HSV-1 TK structure, could act as a nuclear localization signal for monomeric HSV-1 TK. Alternatively, crystallographic data indicate that R318 might be essential for the formation of the TK dimer, and therefore it is required if HSV-1 TK is transported as a dimer.
Subject(s)
Cell Nucleus/virology , Herpesvirus 1, Human/enzymology , Thymidine Kinase/genetics , Amino Acid Sequence , Binding Sites , Cell Nucleus/metabolism , Cytosol/virology , Green Fluorescent Proteins , Herpesvirus 1, Human/genetics , Humans , Luminescent Proteins/analysis , Luminescent Proteins/genetics , Molecular Sequence Data , Mutagenesis, Site-Directed , Mutation , Plasmids , Recombinant Fusion Proteins/analysis , Thymidine Kinase/chemistry , Thymidine Kinase/metabolism , Transfection , Tumor Cells, CulturedABSTRACT
A Drosophila melanogaster deoxyribonucleoside kinase (Dm-dNK) was reported to phosphorylate all four natural deoxyribonucleosides as well as several nucleoside analogs (Munch-Petersen, B., Piskur, J., and Sondergaard, L. (1998) J. Biol. Chem. 273, 3926-3931). The broad substrate specificity of this enzyme together with a high catalytic rate makes it unique among the nucleoside kinases. We have in the present study cloned the Dm-dNK cDNA, expressed the 29-kDa protein in Escherichia coli, and characterized the recombinant enzyme for the phosphorylation of nucleosides and clinically important nucleoside analogs. The recombinant enzyme preferentially phosphorylated the pyrimidine nucleosides dThd, dCyd, and dUrd, but phosphorylation of the purine nucleosides dAdo and dGuo was also efficiently catalyzed. Dm-dNK is closely related to human and herpes simplex virus deoxyribonucleoside kinases. The highest level of sequence similarity was noted with human mitochondrial thymidine kinase 2, and these enzymes also share many substrates. The cDNA cloning and characterization of Dm-dNK will be the basis for studies on the use of this multisubstrate nucleoside kinase as a suicide gene in combined gene/chemotherapy of cancer.
Subject(s)
Drosophila melanogaster/enzymology , Phosphotransferases (Alcohol Group Acceptor)/genetics , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , DNA, Complementary/chemistry , Genetic Therapy , Molecular Sequence Data , Neoplasms/therapy , Phosphorylation , Phosphotransferases (Alcohol Group Acceptor)/chemistry , Recombinant Proteins/chemistry , Sequence AlignmentABSTRACT
Introduction of the herpes simplex virus type 1 thymidine kinase gene into tumor cells, followed by the administration of the antiherpes nucleoside analogue ganciclovir has been demonstrated to be effective in eliminating solid tumors in animals. The success of this combination treatment largely depends on the bystander effect, i.e. the killing of nontransfected tumor cells by activated drug carried over from the nearby herpes thymidine kinase (tk) gene-transfected cells. We evaluated the in vitro bystander effect of several antiherpes purine and pyrimidine nucleoside analogues, using a colorimetric assay. All pyrimidine nucleoside analogues, including (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU), showed low, if any, bystander killing effect. In contrast, purine nucleoside analogues, such as ganciclovir, were endowed with a pronounced bystander killer effect. Lobucavir (Cyclobut-G), a ganciclovir analogue, displayed a two- to three-fold more pronounced bystander killer effect than ganciclovir, eliminating, at a concentration of 10 microM, 75% and 90% of a cell population that contained 5% and 10% tk gene-transfected cells, respectively. These findings were corroborated by autoradiographic analysis that showed that 2'-3H-BVDU metabolites formed in the herpes tk gene-transfected tumor cells were much less efficiently incorporated in the DNA of bystander cells than 8-3H-GCV. This indicates that, under the same experimental conditions, BVDU metabolites are less prone to pass the gap junctions than GCV metabolites.
Subject(s)
Antiviral Agents/therapeutic use , Genetic Therapy/methods , Osteosarcoma/therapy , Purine Nucleosides/chemistry , Thymidine Kinase/genetics , Antiviral Agents/chemistry , Autoradiography , Ganciclovir/chemistry , Ganciclovir/therapeutic use , Guanine/analogs & derivatives , Guanine/chemistry , Guanine/therapeutic use , Humans , Purine Nucleosides/therapeutic use , Pyrimidine Nucleosides/chemistry , Pyrimidine Nucleosides/therapeutic use , Transfection , Tumor Cells, CulturedABSTRACT
The thymidine kinases (TKs) of herpes simplex virus type 1 (HSV-1), HSV-2, and varicella-zoster virus (VZV) were expressed in human osteosarcoma cells as fusion proteins with the green fluorescent protein (GFP), and their intracellular localizations were determined. The three TK-GFP fusion products were localized in different subcellular compartments of the transfected tumor cells. HSV-1 TK-GFP was localized exclusively in the nucleus, HSV-2 TK-GFP was predominantly found in the cytosol, while VZV TK-GFP was localized in both the nucleus and the cytosol. In support of these findings, we identified a nuclear localization signal (NLS) in the N-terminal arginine-rich region of HSV-1 TK that was absent in HSV-2 and VZV TK. The first 34 amino acids proved necessary for the specific nuclear localization of HSV-1 TK and, when added to the VZV TK-GFP gene construct, also sufficed to specifically target VZV TK-GFP to the nucleus. Further analysis of this NLS through site-directed mutagenesis revealed that the basic amino acid-rich nonapeptide 25R-R-T-A-L-R-P-R-R33 is of crucial importance in the nuclear targeting of HSV-1 TK. In particular, we revealed that the presence of the arginine residues at positions 25, 26, 30, 32, and 33 is obligatory for efficient NLS functioning, whereas arginine and histidine residues outside of the nonapeptide (i.e., residues R18, R20, and H22) did not change the functional properties of the NLS.
Subject(s)
Genes, Viral , Herpesviridae/enzymology , Herpesviridae/genetics , Herpesvirus 1, Human/enzymology , Herpesvirus 1, Human/genetics , Nuclear Localization Signals , Thymidine Kinase/genetics , Amino Acid Sequence , Base Sequence , Cell Compartmentation , DNA Primers/genetics , Gene Expression , Genetic Complementation Test , Green Fluorescent Proteins , Herpesvirus 2, Human/enzymology , Herpesvirus 2, Human/genetics , Herpesvirus 3, Human/enzymology , Herpesvirus 3, Human/genetics , Humans , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Molecular Sequence Data , Mutagenesis, Site-Directed , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sequence Deletion , Thymidine Kinase/metabolism , Transfection , Tumor Cells, CulturedABSTRACT
Ganciclovir (GCV) and its lipophilic elaidic acid ester prodrug E-GCV were evaluated for their antiherpetic, cytostatic and metabolic properties, E-GCV proved exquisitely inhibitory to the replication of herpes simplex virus type 1 (HSV-1) and HSV-2 in cell cultures (50% effective concentration (EC50): 0.002 microM). It was five- to 10-fold more effective than its parent drug GCV. E-GCV was at least 2000-fold more cytostatic to HSV-1 or HSV-2 thymidine kinase (tk) gene-transfected mammary carcinoma FM3A tk-/HSVtk+ tumor cells than to the corresponding nontransfected tumor cells. The cytostatic activity of E-GCV to the HSVtk gene-transfected tumor cells was far superior to that of GCV. Metabolic studies revealed that both GCV and E-GCV were converted to the mono-, di- and tri-phosphate derivatives of GCV to a markedly higher extent in FM3Atk-/HSV-1 tk+ cells than in wild-type FM3A/0 cells. Strikingly, mono-, di- and tri-phosphate metabolites of GCV were retained for a substantially longer time in E-GCV-treated cells (half-life approximately 50 h) than in GCV-treated cells (half-life approximately 20 h). The longer retention time of the GCV metabolites most likely explains why E-GCV is superior to GCV against herpes simplex virus replication and HSVtk gene-transfected tumor cell proliferation. Taking into account the marked stability of E-GCV in human plasma and its much higher lipophilicity than GCV, E-GCV should be considered as an effective lipophilic prodrug of GCV with a markedly enhanced cytostatic activity in HSVtk gene-transfected tumor cells compared with parental ganciclovir. Its usefulness in the combined gene/chemotherapy of HSVtk gene-transfected tumors should be further pursued.
Subject(s)
Antiviral Agents/pharmacology , Ganciclovir/analogs & derivatives , Herpesvirus 1, Human/drug effects , Mammary Neoplasms, Experimental/metabolism , Prodrugs/pharmacology , Animals , Antiviral Agents/metabolism , Cell Division/drug effects , Female , Ganciclovir/metabolism , Ganciclovir/pharmacology , Genetic Therapy , Half-Life , Herpesvirus 1, Human/physiology , Humans , Mammary Neoplasms, Experimental/pathology , Mice , Prodrugs/metabolism , Thymidine Kinase/genetics , Transfection , Tumor Cells, Cultured , Virus Replication/drug effectsABSTRACT
The acyclic nucleoside phosphonate 9-(2-phosphonylmethoxyethyl)adenine (PMEA) has previously been shown to be a strong inducer of differentiation in several tumor cell lines. We have now investigated the in vitro differentiation-inducing and the in vivo antitumor, properties of PMEA in a rat choriocarcinoma tumor cell model. PMEA at 2 to 50 microM induced choriocarcinoma RCHO cell differentiation in vitro in a concentration-dependent manner, as monitored by morphological changes, induction of alkaline phosphatase and production and secretion of progesterone. Likewise, a clear dose-response relationship was established for the in vivo antitumor activity of PMEA in choriocarcinoma-bearing rats. (R)-PMPA, a structural analogue of PMEA which is much less effective than PMEA in inducing differentiation in vitro did not demonstrate any in vivo antitumor activity. This observation points to the specificity of the differentiation-inducing potential of PMEA.
Subject(s)
Adenine/analogs & derivatives , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Choriocarcinoma/drug therapy , Organophosphonates , Adenine/pharmacology , Adenine/therapeutic use , Alkaline Phosphatase/metabolism , Animals , Cell Differentiation/drug effects , Cell Division/drug effects , Choriocarcinoma/pathology , Methotrexate/pharmacology , Progesterone/metabolism , Rats , Rats, Inbred Strains , StereoisomerismABSTRACT
The acyclic nucleoside phosphonate 9-(2-phosphonylmethoxyethyl)adenine (PMEA) is a potent and selective antiretroviral agent which is currently evaluated in its oral prodrug form, bis(POM)PMEA (adefovir dipivoxil), in phase II and III clinical trials in human hepatitis B virus (HBV)- and human immunodeficiency virus (HIV)-infected individuals, respectively. We have now found that PMEA is also a potent inhibitor of growth of the highly aggressive choriocarcinoma tumor arising from rat choriocarcinoma RCHO cells grafted under the kidney capsule of syngeneic WKA/H rats. In untreated rats, massive invasive RCHO tumors, covering the whole surface of the kidney and resulting in a marked enlargement of the kidney, were observed at day 10 after tumor cell grafting. Daily treatment with PMEA at 25 mg/kg/day afforded a marked reduction in tumor size (i.e., smaller tumors and slight, if any, enlargement of the kidney). Increasing the PMEA dose to 50, 100 or 250 mg/kglday resulted in a gradual increase of the antitumor effect of the compound. At the highest dose tested, i.e., 250 mg/kg/day, PMEA completely suppressed tumor growth. The antitumor activity of PMEA persisted for at least 10 days after termination of drug treatment. In addition, delayed treatment with PMEA at a dose of 200 mg/kg/day, started at a time point where choriocarcinoma tumors had already developed, stopped further growth and even induced regression of the tumors. PMPA, a closely related structural analogue of PMEA, failed to inhibit choriocarcinoma tumor growth. This observation points to the specificity of PMEA as an antitumor agent. In view of our findings, the therapeutic potential of PMEA for the treatment of neoplastic diseases appears to merit further investigation.
Subject(s)
Adenine/analogs & derivatives , Antiviral Agents/therapeutic use , Choriocarcinoma/drug therapy , Organophosphonates , Adenine/administration & dosage , Adenine/therapeutic use , Animals , Drug Administration Schedule , Neoplasm Transplantation , Prodrugs/therapeutic use , Rats , Rats, Inbred Strains , Tumor Cells, CulturedSubject(s)
Zidovudine/metabolism , Zidovudine/pharmacology , Animals , HIV Reverse Transcriptase/antagonists & inhibitors , Humans , Mice , Nucleoside-Phosphate Kinase/genetics , Nucleoside-Phosphate Kinase/metabolism , Phosphates/metabolism , Phosphorylation , Thymidine Kinase/antagonists & inhibitors , Zidovudine/chemistryABSTRACT
The sulfonic acid polymers poly(2-acrylamido-2-methyl-1-propanesulfonic acid) (PAMPS), poly(4-styrenesulfonic acid) (PSS), and poly(anetholesulfonic acid) (PAS) proved to be highly potent inhibitors of angiogenesis in the chick chorioallantoic membrane (CAM) assay. PAMPS was found to achieve a dose-dependent inhibition of microvessel formation in the CAM assay ranging from 57 +/- 16% inhibition at 10 micrograms/disc to 72 +/- 15% at 150 micrograms/ disc. Also, PSS and PAS caused a strong inhibition of angiogenesis (55 +/- 19% and 48 +/- 16%, respectively, at 50 micrograms/disc), whereas poly(vinylsulfonic acid) (PVS) was found to be inactive at this dose. The compounds proved to be nontoxic for the developing chick embryo at these doses. Suramin, which was included as a reference compound, caused only a slight inhibition of vascular density, at a dose of 150 micrograms/disc, whereas pentosan polysulfate (PPS) was found to be toxic. PAMPS, PAS, and PSS, but not PVS, inhibited microvessel formation in the rat aorta-ring assay. In addition, the increased [3H-methyl]dThd uptake in endothelial cells in vitro upon stimulation with basic fibroblast growth factor (bFGF) was inhibited by PAMPS, PAS, and PSS at 20 micrograms/ml. A strong correlation (r = 0.95) was found between the antiangiogenic effect of the sulfonic acid polymers in the CAM assay and their inhibition of the bFGF-induced mitogenic response, indicating that bFGF is the target for these sulfonic acid polymers. These results suggest that sulfonic acid polymers, and in particular PAMPS, may be considered as specific, nontoxic angiogenesis inhibitors.
Subject(s)
Neovascularization, Physiologic/drug effects , Polymers/pharmacology , Sulfonic Acids/pharmacology , Animals , Biological Assay , Cell Division/drug effects , Cells, Cultured , Chick Embryo , Endothelium, Vascular/cytology , Fibroblast Growth Factor 2/pharmacology , Growth Inhibitors/pharmacology , Humans , Rats , Structure-Activity RelationshipABSTRACT
Ten pyrimidine nucleoside analogues, including (B)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) and closely related analogues, were evaluated for their cytostatic activity against human osteosarcoma cells transfected with the varicella-zoster virus (VZV) thymidine kinase (tk) (ATP:thymidine 5' phosphotransferase, EC 2.7.2.21) gene. (E)-5-(2-bromovinyl)-1-beta-D-arabinofuranosyluracil (BVaraU), (E)-5-(2-iodovinyl)-2'-deoxy-2'-fluoro-1-beta-D-arabinofuranosyluracil (IVFAU) and (E)-5-(2-bromovinyl)-2'-deoxy-4'-thiouridine (S-BVDU) were among the most potent inhibitors of VZVtk gene-transfected cell proliferation. They displayed an inhibitory activity at drug concentrations that were up to four orders of magnitude lower than those required to inhibit the corresponding nontransfected tumor cells. Inhibition of cellular DNA polymerase and/or incorporation of the drugs into cellular DNA may be a likely target for the cytostatic activity of the BVDU derivatives against the VZVtk gene-transfected tumor cells. These compounds were approximately 40- to 80-fold more potent cytostatic agents in VZVtk gene-transfected cells than the anti-VZV compound 6-methoxy-9-beta-D-arabinofuranosylpurine (araM), and at least five- to 50-fold more cytostatic than ganciclovir in HSV-1tk gene-transfected murine mammary carcinoma FM3A cells. In addition, the intrinsic resistance of BVaraU, IVFAU and S-BVDU to glycosidic bond cleavage by mammalian dThd phosphorylases makes them promising candidate compounds for the treatment of VZVtk gene-transfected tumors in vivo.
Subject(s)
Antiviral Agents/pharmacology , Genetic Therapy , Herpesvirus 3, Human/enzymology , Osteosarcoma/therapy , Pyrimidine Nucleosides/pharmacology , Thymidine Kinase/genetics , Cell Division/drug effects , Humans , Osteosarcoma/genetics , Osteosarcoma/virology , Transfection , Tumor Cells, Cultured/drug effectsABSTRACT
The authors report the case of an epidermoid carcinoma, originating from the maxillary sinus, with orbital extension. The first harmless symptoms can mislead the diagnosis. Other signs will appear a few weeks later leading to a tumoral process. The further examinations will confirm the fears.