New in vitro method for evaluating antiviral activity of acyclic nucleoside phosphonates against plant viruses.

A new method was developed for testing antiviral compounds against plant viruses based on rapidly growing brassicas in vitro on liquid medium. This method enables exchange of media containing tested chemicals in various concentrations and simultaneous evaluation of their phytotoxicity and antiviral activity. While using ribavirin as a standard for comparison, phytotoxicity and ability of the acyclic nucleotide analogues (R)-PMPA, PMEA, PMEDAP, and (S)-HPMPC to eliminate ssRNA Turnip yellow mosaic virus (TYMV) were evaluated by this method. Double antibody sandwich ELISA and real-time PCR were used for relative quantification of viral protein and nucleic acid in plants. Ribavirin had the most powerful antiviral effect against TYMV. On the other hand, (R)-PMPA and PMEA had no antiviral effect and almost no phytotoxicity compared to the control. (S)-HPMPC and PMEDAP showed moderate antiviral effect, accompanied by higher phytotoxicity. The tested compounds can be screened within 6-9 weeks in contrast to the 6 months for traditionally used explants on solid medium. The method enables large-scale screening of potential antivirals for in vitro elimination of viruses from vegetatively propagated crops and ornamentals.

[Acyclic nucleoside phosphonates as potential antineoplastic agents]. / Acyklické nukleosidfosfonáty jako potenciální antineoplastika.

Recently, Gilead Sciences (Foster City, CA, USA) presented a potential cytostatic drug GS-9219. It is a novel lipophilic prodrug of cyprPMEDAP, in vivo releasing the active compound PMEG in a two-step process. GS-9219 has shown a substantial therapeutic potential in treatment of spontaneous non-Hodgkin's lymphoma in dogs and its utilization in the human medicine is prospective. Hence, cyprPMEDAP represents a key intermediate in the intracellular activation of GS-9219. Both acyclic nucleoside phosphonates PMEG and cyprPMEDAP, serving as the basis for development of GS-9219, were discovered and their mechanism of action was investigated in detail at the Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic. The biological studies using the rat lymphoma were carried out at the First Faculty of Medicine, Charles University.

Experimental therapy with 9-[2-(phosphonomethoxy)ethyl]-2,6-diaminopurine (PMEDAP): origin of resistance.

The role of MRP4 and MRP5 transporters in the acyclic nucleoside phosphonate PMEDAP efflux was studied in vitro (CCRF-CEM cells) and in vivo (spontaneous transplantable T-cell lymphoma of SD/Cub inbred rats). The increased resistance against the cytostatic agent PMEDAP during longterm treatment was found to be associated with overexpression of MRP4 and MRP5 genes. The course of both gene activation differs significantly. While the MRP5 function is important in the onset of PMEDAP resistance, the intensity of the relative MRP4 gene expression increases rather continuously. Our data indicate cooperative acting of both MRP4 and MRP5 genes during the PMEDAP resistance development.

Hypomethylation of CNG targets induced with dihydroxypropyladenine is rapidly reversed in the course of mitotic cell division in tobacco.

We followed the mitotic transmission of an experimentally induced hypomethylated state of several tobacco repetitive sequences in callus culture and plants. The initial hypomethylation was induced by a hypomethylation drug, dihydroxypropyladenine (DHPA), the competitive inhibitor of cellular S-adenosylhomocysteine hydrolase, which is known to preferentially inhibit methylation at CNG and non-symmetrical motifs while having a negligible effect on methylation at CG motifs. The deprivation of this drug resulted in an almost immediate remethylation of cytosines at CNG motifs ( MspI and EcoRII sites) leading us to conclude that, the hypomethylation effect of dihydroxypropyladenine is rather transient and differs from that of 5-azacytidine which often induces heritable changes in methylation patterns. The results suggest that de novo methylation of CNG motifs is a rapid and meiotically independent process on DNA sequences with pre-existing CG methylation.

Antitumour activity of a combined treatment with PMEDAP and docetaxel in the Prague inbred Sprague-Dawley/cub rat strain bearing T-cell lymphoma.

Antitumour efficiency of combined therapy with N-9-[2-(phosphonomethoxy)ethyl]-2,6-diaminopurine (PMEDAP) and docetaxel (DTX) was studied in an in vivo model of s.c. transplanted Sprague-Dawley (SD/Cub) rat T-cell lymphoma (phenotype SD10/96). The effect of the combined treatment of DTX with PMEDAP was significantly higher than that of DTX or PMEDAP alone. The s.c. administration of DXT into the vicinity of growing lymphoma together with i.p. administration of PMEDAP was found to be the most efficient combination. In this case, two out of four rats did not develop any lymphoma and remained alive. An irregular expression of Bcl2 protein was found in untreated and treated lymphomas, while the expression of protein p53 as well as MDM2 was not observed. All three types of the above-mentioned treatments (PMEDAP, DXT, DXT+PMEDAP) increased significantly the number of p21-positive cells, compared with untreated tumours.

Antitumour activity of N6-substituted PMEDAP derivatives against T-cell lymphoma.

The antitumour activity of four N6-substituted PMEDAP derivatives, Me2NEt-PMEDAP, allyl-PMEDAP, Me2-PMEDAP and cypr-PMEDAP, selected on the basis of their in vitro cytostatic activity, was studied in an in vivo model of haematological malignancy of inbred Sprague-Dawley rats. These compounds are believed to serve as the prodrugs of another (phosphonomethoxy)ethyl derivative, PMEG (9-[2-phosphonomethoxy) ethyl] guanine. We compared their toxicity and ability to inhibit tumour development in two different dosage regimes with those of their parent compound PMEDAP, as well with PMEG. The study confirmed the anticancer efficacy of the parental compound PMEDAP. Unlike PMEDAP, its N6-mono- and disubstituted congeners Me2NEt-PMEDAP, allyl-PMEDAP and Me2-PMEDAP were less potent or exhibited the same antineoplastic effect as PMEDAP. cypr-PMEDAP significantly decreased the survival of lymphoma-bearing rats due to high toxicity, which was approximately the same as that of PMEG. Therefore, these acyclic nucleoside phosphonates substituted at the 6-position of 2,6-diaminopurine ring do not seem to be promising drugs for the treatment of haematological malignancies.

The supply of exogenous deoxyribonucleotides accelerates the speed of the replication fork in early S-phase.

Earlier studies have established that the average speed of a replication fork is two to three times slower in early S-phase than in late S-phase and that the intracellular 2'-deoxyribonucleoside 5'-triphosphate pools grow during S-phase. In this study, the effect of the exogenous 2'-deoxyribonucleoside 5'-triphosphate (dNTP) supply on the average replication speed in a synchronised population of human HeLa cells was tested. The speed of replication fork movement was measured on extended DNA fibers labelled with 2'-deoxythymidine analogues 5-chloro-2'-deoxyuridine and 5-iodo-2'-deoxyuridine. We show that the introduction of exogenous dNTPs accelerates the replication process at the beginning of DNA synthesis only. In late S-phase, the administration of additional dNTPs has no effect on the speed of replication forks. The availability of 2'-deoxynucleotides seems to be a rate-limiting factor for DNA replication during early S-phase.

Interaction of guanine phosphonomethoxyalkyl derivatives with GMP kinase isoenzymes.

Substrate activity and inhibitory potency of guanine phosphonomethoxyalkyl derivatives towards GMP kinase isoenzymes from L1210 cells were studied. 9-[2-(Phosphonomethoxy)ethyl]guanine (PMEG) and the (R)- and (S)-enantiomers of both 9-[3-hydroxy-2-(phosphonomethoxy)propyl]guanine (HPMPG) and 9-[2-(phosphonomethoxy)propyl]guanine (PMPG) were phosphorylated to the first step. Kinetic data showed that (R)-PMPG was a good substrate with a relative phosphorylation efficacy of 12% compared with the natural substrate GMP, whereas PMEG was a poor substrate with a relative phosphorylation efficacy of 1.1%. The structurally related 2,6-diaminopurine analogues 9-[2-(phosphonomethoxy)ethyl]-2, 6-diaminopurine (PMEDAP) and (R)- and (S)-9-[2-(phosphonomethoxy)propyl]-2,6-diaminopurine (PMPDAP) were not phosphorylated by any of the GMP kinase isoenzymes tested. The inhibitory activities of the individual compounds on GMP kinase isoenzymes decreased in the following order: (S)-HPMPG > (R)-PMPG > PMEG > (R)-HPMPG > (S)-PMPG > PMEDAP = (R)-PMPDAP = (S)-PMPDAP; each compound exerted a different type of inhibition.

Synthesis and cytostatic activity of substituted 6-phenylpurine bases and nucleosides: application of the Suzuki-Miyaura cross-coupling reactions of 6-chloropurine derivatives with phenylboronic acids.

The Suzuki-Miyaura reaction of protected 6-chloropurine and 2-amino-6-chloropurine bases and nucleosides with substituted phenylboronic acids led to the corresponding protected 6-(substituted phenyl)purine derivatives 6-9. Their deprotection yielded a series of substituted 6-phenylpurine bases and nucleosides 10-13. Significant cytostatic activity (IC(50) 0.25-20 micromol/L) in CCRF-CEM, HeLa, and L1210 cell lines was found for several 6-(4-X-substituted phenyl)purine ribonucleosides 12 (X = H, F, Cl, and OR), while the 6-phenylpurine and 2-amino-6-phenylpurine bases 10 and 11, as well as 2-amino-6-phenylpurine ribosides 13, were entirely inactive against these cell lines.

Anticancer effect of PMEDAP--monitoring of apoptosis.

Antitumor effect of N-9-[2-(phosphonomethoxy) ethyl]-2,6-diaminopurine (PMEDAP) was studied in an in vivo model of s.c. transplanted Sprague-Dawley (SD/cub) rat T-cell lymphomas. Three individual SD/cub neoplasias (SD10/96, SD14/97, SD1/90) of different phenotypes were used. During the treatment, survival of the rats, increase of lymphoma mass, and DNA fragmentation detected by APO/BRDU kit, as well as Bcl2 and p53 protein expression, were followed. The study gives evidence of the positive therapeutic effect of PMEDAP in two of the three tested lymphomas, SD10/96 and SD14/97. Slowly growing SD1/90 lymphoma differs from the others in a uniform karyotype with trisomy of chromosome 11, CD4- immunophenotype, heterogeneous cellular morphology and constitutive expression of p53 protein found in some neoplastic cells. Thus, the diverse anticancer efficacy of PMEDAP treatment among SD/cub lymphomas could be associated with the different phenotypes of individual neoplasias.

9-[2-(Phosphonomethoxy)ethyl]adenine diphosphate (PMEApp) as a substrate toward replicative DNA polymerases alpha, delta, epsilon, and epsilon*.

The diphosphoryl derivative of the acyclic nucleotide phosphonate analog 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA), found previously to weakly inhibit DNA pol delta/proliferating cell nuclear antigen, was studied as a substrate for pol alpha, delta, epsilon, and epsilon*. A comparison of the Vmax and Km for this derivative (PMEApp) and dATP demonstrated that the relative efficiency of the incorporation of this analog into the DNA chain is decreasing in the following order: pol delta approximately equal to pol epsilon approximately equal to pol epsilon* > pol alpha. Under the reaction conditions, this incorporation amounted to 4.4 to 0.7% of dAMP molecules. Similar Km values for PMEApp and dATP in pol epsilon and pol epsilon* catalyzed reactions revealed that proteolysis of the enzyme probably does not affect the dNTP binding site. The DNA polymerases tested were inhibited by the reaction product (PMEA terminated DNA chain) with similar Ki/Km ratios (pol alpha 0.2; pol delta, 0.1; pol epsilon 0.05; and pol epsilon*, 0.06). The associated 3'-5'-exonuclease activity of pol delta, epsilon, and epsilon* was able to excise PMEA from the 3'-OH end of DNA with a rate one order of magnitude lower than that of the dAMP residue.

Acyclic nucleotide analogues suppress growth and induce apoptosis in human leukemia cell lines.

Acyclic nucleotide analogues perturb DNA replication by terminating the growing DNA chain. The analogues selected for testing on human leukemia cell lines, namely 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA), 9-[2-(phosphonomethoxy)ethyl]-2,6-diaminopurine (PMEDAP), and 9-[2-(phosphonomethoxy)ethyl]guanine (PMEG) exhibited growth-inhibiting activity at low concentrations, and apoptosis-inducing activity at high concentrations. A common feature was a reduction of the proportion of G1 cell cycle phase. Activities of the analogues increased in the order PMEA

Inhibitory potency of 5-benzyluracil, 5-phenylcytosine and 5-phenylpyrimidin-2-one nucleosides against uridine phosphorylase from mouse leukemic L1210 cells.

The inhibitory activity of a series of novel sugar-modified nucleosides derived from 5-benzyluracil, 5-phenylcytosine and 5-phenylpyrimidin-2-one against uridine phosphorylase purified from mouse leukemic L-1210 cells was investigated. Significant activity was encountered with O2,2'-anhydro-5-benzylcytidine hydrochloride, 2',3'-dideoxy-5-benzyluridine, 2',3'-dideoxy-4-thiouridine and alpha- and beta-anomers of 5-benzyl-1-(2-deoxy-D-arabino-hexopyranosyl)uracil.

9-[2-(phosphonomethoxy)ethyl]-2,6-diaminopurine (PMEDAP)--a potential drug against hematological malignancies--induces apoptosis.

Antitumor activity of the acyclic nucleotide analogs PMEDAP, PMEA, and PMEG was studied on a model of a spontaneous T-cell lymphoma in inbred SD/cub rats. Significant therapeutic effects were recorded after a treatment with 16 daily doses of PMEDAP at 5 mg/kg applied to the vicinity of the growing lymphoma. Identical administration of PMEA, or PMEG at a daily dose of 0.1 mg/kg did not affect the survival of lymphoma-bearing animals compared with untreated controls. A decrease in the lymphoma weight during PMEDAP administration was accompanied by the suppression of mitotic activity in neoplastic cells and increased chromatin condensation as witnessed by karyological examinations. Electron-microscopy showed the morphology of apoptotic cells (shrunken cells with condensed chromatin, apoptotic bodies) in lymphoma cell suspensions. An increase of nuclear DNA fragmentation was found during PMEDAP administration compared with spontaneous DNA fragmentation of untreated control lymphomas. These results indicate that PMEDAP application induces apoptosis in in vivo growing lymphomas. The antitumor effect of PMEDAP lasts only during the administration of the drug. After its cessation progression of neoplasia was reestablished.

Therapeutic effect of heat shock on T-cell lymphoma in inbred Sprague-Dawley rat.

Anticancer effect of heat shock, either alone or in combination with the drug PMEDAP, and cold water immersion stress were studied in an in vivo model of s.c. transplanted rat T-cell lymphomas in an inbred Sprague-Dawley rat line (SD/cub). Significant anticancer effect was induced by repeated sessions of heat shock; decrease of s.c. lymphoma weight and prolongation of survival time of treated rats was found to be dependent on the number of HS sessions. Much stronger therapeutic effect was observed after repeated heat shock in combination with PMEDAP administration. Light and electron microscopy studies were performed to characterize the alterations within the lymphomas. Morphologically, cellular alterations corresponding with apoptosis were observed in lymphoma cells after repeated heat shock. Indirect immunoperoxidase technique was used to detect HSP 72/73 protein(s), p53 and Bcl2 proteins in lymphomas heated directly or indirectly. The induction of HSP 72/73 protein(s) was found in the lymphoma tissues from autopsied animals exposed to heat shock; the intensity of its expression was dependent on the experimental design. The expression of p53 and BcL2 proteins was not changed in lymphoma cells of HS treated animals as compared to that of untreated lymphoma bearing controls; the Bcl2 protein was present in both treated and untreated lymphomas, and the p53 protein remained undetectable in all samples. Contrary to the heat shock, the cold stress did not suppress growth of lymphomas and, furthermore, accelerated the infiltration of parenchymatous organs with lymphoma cells.

Evaluation of the impact of S-adenosylhomocysteine metabolic pools on cytosine methylation of the tobacco genome.

We explored the possibility that the cytosine DNA methylation might be regulated by S-adenosyl-L-methionine (AdoMet) and S-adenosyl-L-homocysteine (AdoHcy) pools in plant cells. In order to change the AdoHcy/AdoMet ratio (methylation index), (S)-9-(2,3-dihydroxypropyl)adenine was employed, a selective reversible inhibitor of cellular S-adenosyl-L-homocysteine hydrolase. Micromolar concentrations of the inhibitor increased dramatically (more than 1000-fold) intracellular AdoHcy levels (and concominantly the AdoHcy/AdoMet ratio) in tobacco TBY-2 cells. No toxic effect of the drug was observed and the cells displayed only marginal inhibition of growth at high AdoHcy levels. At near equal intracellular concentrations of AdoHcy and AdoMet, a significant reduction of cytosine methylation in transcribed (5SrDNA) and non-transcribed (HRS60, NTRS) sequences was observed. Interestingly, the CpCpG and CpApG trinucleotide targets appeared to be most sensitive to changes in the methylation index. Methylation of cytosine residues at CpG sites was not affected even at AdoHcy/AdoMet ratio of > 10. These results support the possible regulation of DNA methylation via AdoHcy/AdoMet metabolic pathways in plant cells.

Modulation of cell cycle progression and of antibody production in mouse hybridomas by a nucleotide analogue.

The nucleotide analogue 9-[2-(phosphonomethoxy)ethyl]guanine (PMEG) has been identified as a powerful antiproliferative substance when acting on hybridoma cells. In the range of 10 nM to 100 nM concentrations this agent reduces cell growth rate, while its apoptosis-inducing activity is marginal. Marked induction of apoptosis can be observed at micromolar and higher order concentrations. In PMEG-supplemented media the cell cycle progression is perturbed, the flow-cytometric DNA profile shows a higher proportion of cells in the S and G2/M phases of the cell cycle. Concomitantly with the reduction of the growth rate, the specific monoclonal antibody production rate may rise by 20-27%. Addition of PMEG at the end of the exponential phase of a batch culture results in an enhancement of the final monoclonal antibody concentration.

Antitumor activity of novel purine acyclic nucleotide analogs PMEA and PMEDAP.

PMEDAP and/or PMEA treatment of SD rat lymphomas significantly prolonged the mean survival time of tumor-bearing animals. Dose-dependent genotoxicity of both PMEDAP and PMEA was not observed in in vitro tests on stabilized diploid MRC-5 cell line. The mitotic activity of MRC-5 cells was completely inhibited after 48 hours exposure in culture medium containing PMEDAP (10 micrograms/ml), or PMEA (25 micrograms/ml), respectively. Significant concentration dependent inhibition of cell proliferation caused by PMEDAP and/or PMEA was also observed in murine splenocytes. The analogs specifically inhibit proliferation of mitogen-activated T-lymphocytes. Modulation of subpopulations of peripheral blood cells under in vivo conditions was found in inbred SD animals. Intraperitoneal administration of PMEDAP to young healthy SD animals induced the decrease of the CD4+/CD8+ value from 1.3-1.6 to 0.72 while i.p. application of PMEA caused a decrease of the same ratio to 0.62.

Genotoxicity of purine acyclic nucleotide analogs.

The genotoxic and embryotoxic effects of phosphonomethoxyalkylpurines, a new group of antiviral agents, decrease in the following order: PMEG > PMEthioG > PMEDAP > PMEA > (R)-PMPDAP = (R)-PMPA. Results of the present study are fully consistent with the previously found efficacy of their diphosphates to inhibit the replicative DNA polymerases. The marked genotoxicity of PMEG and PMEthioG is comparable to that of mitomycin C, whereas the moderate genotoxicity of PMEA is comparable to that of AZT. (R)-PMPDAP and (R)-PMPA did not induce any structural aberrations of chromosomes under the experimental conditions.