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The reaction of suitably protected nucleosides with phosphinic acid in the presence of condensing agents has been investigated and applied for the preparation of nucleoside 3'-H-phosphonothioates. Studies on synthesis and separation of diastereoisomers of nucleoside 3'-methylphosphonothioates are also discussed.
The reaction mechanism of the phosphoryl transfer catalyzed by dinucleoside diphosphate kinase from Dictyostelium discoideum is investigated by semiempirical AM1 molecular orbital computation of an active site model system on the basis of various X-ray crystallographic structures. The computational results suggest that the phosphoryl transfer from adenosine triphosphate to the His122 residue is accompanied by the simultaneous shift of a proton from the histidine residue to one of the oxygen atoms of the gamma phosphate group. This involves a doubly protonated His122 residue whilst this residue is neutral in its ternary complex with ADP and the transition state analogue AlF(3). The proposed mechanism is thus analogous to that of phosphoryl transfer by cyclic adenosine monophosphate dependent protein kinase and uridine/cytidine monophosphate kinase as found in our earlier work and clarifies the role of the ribose 3'-OH group. Furthermore, the energetics of phosphoryl transfer onto other nucleoside analogues such as 3'-azido-3'-deoxythymidine-diphosphate and 2',3'-dideoxy-2',3'-didehydro-thymidine-diphosphate are investigated. The calculated reaction barriers for the phosphorylation of the diphosphates by the enzyme are all within a range of 13.1 kJ mol(-1), which suggests that variations in the activation energies alone cannot account for the experimentally observed differences in enzymatic activity. Consequences for the design of new anti-HIV nucleoside analogues are discussed. Supporting information for this article is available on the WWW under http://www.wiley-vch.de/contents/jc_2268/2002/f360_s.pdf or from the author.
In an effort to develop more potent inhibitors of human purine nucleoside phosphorylase (PNP) as immunosuppressive and cancer chemotherapeutic agents, the affinity of the erythrocytic enzyme for 30 acyclic nucleosides, nucleotides and related compounds was determined. Among the acyclonucleosides, 2'-nordeoxyguanosine [2'NDG, 9-(1,3-dihydroxy-2-propoxymethyl)guanine] had a 3-fold greater affinity than acyclovir, and 8-amino-2'NDG was the best inhibitor with Ki = 2.6 X 10(-7) M. The ether moiety of the acyclovir and 2'NDG side-chains was not important for binding. Phosphorylated 2'NDG analogs appeared to act as multisubstrate analogs with optimal binding at low (1 mM) phosphate concentration. The 2'NDG mono- and triphosphates had higher affinities than those reported for the phosphorylated acyclovir derivatives but the diphosphate had a similar Ki value of 9 X 10(-9) M. Poor affinity, independent of phosphate concentration, was found for 9-(2-phosphonoethyl)guanine. The 3'-phosphate derivative of 8-(3-hydroxypropyl)-9-methylguanine inhibited with a Ki = 2 X 10(-5) M in 1 mM phosphate. The chemical syntheses of new analogs are described.
Mechanisms of resistance to thiopurines, 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG) were investigated in human leukemia cell lines. We developed two 6-MP- and 6-TG-resistant cell lines from the human T-lymphoblastic cell line (MOLT-4) by prolonged exposure to these drugs. The resistant cells were highly cross resistant to 6-MP and 6-TG, and exhibited marked reduction in cellular uptake of 6-MP (70% and 80%, respectively). No significant modification of the activities of hypoxanthine-guanine phosphoribosyl transferase, thiopurine methyltransferase or inosine monophosphate dehydrogenase was observed. Real-time PCR of concentrative nucleoside transporter 3 (CNT3) and equilibrative nucleoside transporter 2 (ENT2) of resistant cells showed substantial reductions in expression of messenger RNAs. Small interfering RNA designed to silence the CNT3 and ENT2 genes down-regulated the expression of these genes in leukemia cells. These decreases were accompanied by reduction of transport of 6-MP (47% and 21%, respectively) as well as its cytocidal effect (30% and 21%, respectively). Taken together these results show that CNT3 and ENT2 play a key role in the transport of 6-MP and 6-TG by leukemia cells. From a clinical point of view determination of CNT3 and ENT2 levels in leukemia cells may be useful in predicting the efficacy of thiopurine treatment.
The 8-aza-immucillins (8-aza-9-deazapurines linked from C9 to C1 of 1,4-dideoxy-1,4-iminoribitol) have been designed as transition-state analogues of the reactions catalyzed by purine nucleoside phosphorylase and nucleoside hydrolases. Syntheses of the 8-aza-immucillin analogues of inosine and adenosine are described. They are powerful inhibitors of the target enzymes with equilibrium dissociation constants as low as 42 pM.
The structural effects of chemical modifications upon the affinity of purine nucleosides to cytidine-transport system in Bacillus subtilis were investigated using a series of modified derivatives. The interaction involves protein molecule(s) which require the presence and proper orientation of the sugar residue and its hydroxylic functions. Moreover, a specific interaction with the heterocyclic ring system is involved in the process which results in a requirement for an aromatic pi -electron system and an absence of a polarizable function at position 6 of the purine heterocycle. The region in the protein responsible for the latter interaction is rather limited and, consequently, a proper nucleoside conformation is required.
We report the use of capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) for the determination of antiretroviral dideoxynucleosides (ddNs), their nucleotides, and a set of ribonucleosides and ribonucleotides. A CE system for separation of most commonly used antiretroviral ddNs has been developed based on a basic buffer with a volatile electrolyte suitable for ESI-MS detection in an untreated capillary column. Positive and negative ionization modes are investigated and compared for sensitive and stable electrospray performance. A 14-compound mixture of nucleosides and nucleotides is profiled in a single capillary zone electrophoresis separation with a distinct elution order: electroosmotic flow, ddNs, mononucleotides, dinucleotides, and trinucleotides in less than 18 min. The fragmentation pathways of the nucleosides and nucleotides in ESI-MS have been interpreted. Concentration limits of detection are 100 to 200 nM with an injection volume of approximately 10 nL. This technique has been used to detect naturally occurring nucleotides and to study the metabolism of lamivudine (3TC) in the human hepatoma cell line Hep G2. 3TC and its metabolites 3TC-monophosphate, 3TC-diphosphate, and 3TC-triphosphate were detected after 10 h of incubation of 3TC with the cells.
Modified urinary nucleosides are potentially invaluable in cancer diagnosis. High-performance liquid chromatography (HPLC) was combined with full scan mass spectrometry (MS), tandem mass spectrometry and MSn analysis in order to identify purine nucleosides purified from urine. UV peaks evident in the chromatogram were examined by the various mass spectrometric techniques and adenosine, 1-methyladenosine, xanthosine, N1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, N2,N2,N7-trimethylguanosine, inosine, and 1-methylinosine were each identified in the urine samples from cancer patients. The benefits of the use of LC/MS compared with HPLC alone are discussed.
Various structural analogues of cytosine and uracil nucleosides were tested as potential inducers of the nucleoside catabolizing (cyt) enzymes in Salmonella typhimurium. Some analogues, e.g. 5'-O-alkyl cytidines and uridines, resistant to catabolic enzymes, were as effective as the natural inducers cytidine and uridine; but etherification of one of the cis 2' or 3'hydroxyls fully abolished activity, pointing to a requirement of an intact ribose cis-glycol system for activity. A uridine analogue in the syn conformation, 6-methyluridine, a good substrate for uridine phosphorylase, was inactive as an inducer. The behavior of various other analogues, in relation to their structure, conformation and substrate properties, indicated the absence of any correlation between inducing activity and substrate susceptibility. The overall findings are consistent with conclusions derived from genetic experiments. The active analogues apparently act via similar pathways, and probably affect the same regulatory mechanism(s) as the natural inducers.
Transplantation of organs, tissues or cells from pigs to humans could be a potential solution to the shortage of human organs for transplantation. Porcine endogenous retroviruses (PERVs) remain a major safety concern for porcine xenotransplantation. Thus, finding drugs that could be used as virological prophylaxis (or therapy) against PERV replication would be desirable. One of the most effective ways to block retroviral multiplication is to inhibit the enzyme reverse transcriptase (RT) which catalyzes the reverse transcription of viral RNA to proviral double-stranded DNA. We report here the cloning and expression of PERV RT and its susceptibility to several inhibitors. Our data demonstrate PERV susceptibility in vitro to the triphosphorylated nucleoside analog of zidovudine (AZT) and to ddGTP and to a lesser extent to ddTTP but almost no susceptibility to the non-nucleoside RT inhibitors tested.
Reverse transcription takes place in the cytoplasm of infected cells, although it has been demonstrated that retroviruses can also initiate reverse transcription prior to infection of target cells. In addition to partial reverse transcripts, full-length proviral molecules have been detected in the plasma and seminal fluid of HIV-1 seropositive patients. Intravirion endogenous reverse transcription appears to be directly correlated with an increased level of infectivity. Therefore, the ability of an inhibitor to reach and inhibit the replication complex in the core of the free-virion may constitute an important part of its capacity to suppress viral infection. In this work we tested the ability of some reverse transcriptase inhibitors to decrease viral infectivity in pretreated highly purified virions. Our results showed that Curie pyridinone [Dollé et al. (1995), J Med Chem 38: 4,679-4,686], a non nucleoside RT inhibitor, strongly inhibited the infectivity of extracellular HIV-1 particles. Other non nucleoside inhibitors (TIBO R82913, HEPT, nevirapine) tested in these conditions were unable to do so. Our data indicate that the effect of Curie pyridinone on intact virions may be related to its capacity to tightly bind the target RT. This approach may lead to the design and synthesis of new drugs able to interact with the retroviral enzyme inside the viral core.
In contrast to their effects on mammalian cells, the nucleoside transport inhibitors nitrobenzylthioinosine 5'-monophosphate (NBMPR-P) dilazep, benzylacyclouridine (BAU), and to a lesser extent, dipyridamole have no significant effect on the in vitro uptake of adenosine analogues by Schistosoma mansoni [el Kouni and Cha, Biochem. Pharmac. 36, 1099 (1987)]. Coadministration of either NMBPR-P or dilazep with potentially lethal doses of tubercidin (7-deazaadenosine), nebularine or 9-deazaadenosine protected mice from the toxicity of these adenosine analogues. Dipyridamole caused partial protection, whereas BAU did not protect the animals from this toxicity. Toyocamycin caused delayed mortality (after 16 weeks) which could not be prevented by coadministration of NBMPR-P. In S. mansoni infected mice, treated with the combination of NBMPR-P and 9-deazaadenosine was not effective against the parasite. On the other hand, the combinations of NBMPR-P or dilazep with either tubercidin or nebularine were highly toxic to the parasite but not the host. Combination therapy caused a marked reduction in the number of pairing of worms. Effectiveness of combination therapy could also be noted by a drastic decrease in the number of eggs in the liver and small intestine. All eggs found were dead, indicating a direct effect on ovigenesis. Although dipyridamole was less effective than NBMPR-P or dilazep in protecting the host from the toxicity of tubercidin or nebularine, the combinations with dipyridamole produced similar significant therapeutic effects in animals that survived. Mice receiving the combination of tubercidin (or nebularine) plus NBMPR-P or dilazep, as well as those that survived the combination with dipyridamole, appeared healthy and were found to have normal size livers and spleens. These results suggest that highly selective toxicity against schistosomes can be achieved by coadministration of various nucleoside transport inhibitors with adenosine analogues.
The combination of L(-)-2',3'-dideoxy-3'-thiacytidine (L(-)SddC, 3TC), L(-)-2',3'-dideoxy-5-fluorocytidine (L(-)FddC), or L(-)-2',3'-dideoxy-5-fluoro-3'-thiacytidine (L(-)(FTC) with 3'-azido-3'-deoxythymidine (AZT) synergistically inhibited replication of human immunodeficiency virus (HIV) in vitro. Similar synergistic activity was also obtained when these compounds were used in combination with 2',3'-didehyro-2',3'-dideoxythymidine (D4T). In terms of 2',3'- dideoxyinosime (ddI) and 2',3'-dideoxycytidine (ddC), only additive anti-HIV activity was observed. None of the beta-L(-) nucleoside analogues had additive toxicity in cell culture, and they could protect against the delayed mitochondrial toxicity associated with AZT, D4T, ddC, and ddI in drug-treated cells. Thus, combinations of beta-L(-) nucleoside analogues with any of the approved anti-HIV drugs could have a potentially beneficial outcome.
5-Methyluridine was produced continuously from thymine and inosine by immobilized enzymes, which consisted of thermostable purine nucleoside phosphorylase and thermostable pyrimidine nucleoside phosphorylase obtained from Bacillus stearothermophilus JTS 859. The process was carried out in a column reactor at 60 degrees C for 17 d without any bacterial contamination under non-aseptical conditions. Half-lives of the activity of the immobilized enzymes were 47 d and 4.5 d at 60 degrees C and 70 degrees C, respectively, although half-life of the crude enzyme was only 14 h at 70 degrees C.
Synthesis and base-pairing studies of two 2'-deoxyribonucleosides, containing a common heterocyclic base, 7(4)-amino-5(6)H-imidazo[4,5-d]pyridazin-4(7)one (1 and 2), have been reported. The synthesis was accomplished by base-promoted deoxyribosylation of ethyl 5(4)-cyanoimidazole-4(5)-carboxylate (6), followed by ring-closure with hydrazine hydrate. The 1H NMR-based base-pair studies were conducted using DMF-d7 as a solvent by measuring changes in chemical shifts of the amino, hydrazide, imidazole H-2, and the sugar H-1' protons of the nucleosides with variations in concentrations and temperatures. Large downfield chemical shifts were observed for the NH, NH2, and to a lesser extent for the H-1' protons when the temperature was lowered from 25 to 0 degrees C, and then further down to -50 degrees C in 10 degree intervals. The observed experimental data are consistent with the results of molecular modeling studies. Nucleoside 2 exhibited low level antiviral activity against HIV-1 in CEM-SS cells with an IC50 of 89.2 microM. No cellular toxicity was observed at the highest concentration of the compound tested.
A new class of acyclic nucleoside phosphonates, the 5-phosphono-pent-2-en-1-yl nucleosides and their hexadecyloxypropyl esters, were synthesized from butyn-1-ol. Only the hexadecyloxypropyl esters showed antiviral activity against herpes simplex virus type 1, in vitro. Hexadecyloxypropyl 1-(5-phosphono-pent-2-en-1-yl)-thymine was the most active and selective compound among the synthesized nucleotides with an EC50 value of 0.90 microM.
Electrospray tandem mass spectrometry (ESI-MS/MS) is used to evaluate the assembling of cytosine and thymine nucleosides in the gas phase, through the formation of hydrogen bonded supermolecules. Mixtures of cytidine analogues and homologues deliver in the gas phase proton-bound heterodimers stabilized by multiple interactions, as proven by the kinetics of their dissociation into the corresponding protonated monomers. Theoretical calculations, performed on initial structures of methylcytosine homodimers available in the literature, converged to a minimized structure whereby the two pyrimidine rings interact through the formation of three hydrogen bonds of similar energy. The crystallographic data here reported show the equivalency of the two interacting pyrimidines which is attributable to the presence of an inversion center. Thymine and uracil pyrimidyl nucleosides form, by ESI, gaseous proton-bound dimers. The kinetic of their dissociation into the related protonated monomers shows that the nucleobases are weekly interacting through a single hydrogen bond. The minimized structure of the protonated heterodimer formed by thymine and N-1-methylthymine confirmed the existence of mainly one hydrogen bond which links the two nucleobases through the O4 oxygens. No crystallographic data exists on thymine proton-bound species, nor have we been able to obtain these aggregates in the solid phase. The gaseous phase, under high vacuum conditions, seems therefore a suitable environment where vanishing structures produced by ESI can be studied with a good degree of approximation.
Most nucleoside-derived anticancer drugs are taken up by the high-affinity Na-dependent nucleoside transporter CNT1. Since such drugs are to some extent cell-cycle-dependent in their cytotoxic action, we examined the relationship between CNT1 expression and cell-cycle progression in the rat hepatoma cell line FAO. Cell cultures were synchronized either at late G1 or early S stages by combining mimosin treatment with either previous synchronization or not by serum starvation. Cell-cycle progression was then assessed by measuring [methyl-3H]thymidine incorporation into DNA and monitoring cyclin E and A protein levels. In these conditions, CNT1 protein amounts increase at the G1-S transition. When cells were synchronized using hydroxyurea (HU), which directly interacts with nucleotide metabolism by inhibiting ribonucleotide reductase, CNT1 protein amounts increased in synchronized cells and remained high during cell-cycle progression. These data indicate that CNT1 adapts to cell-cycle progression and responds to nucleos(t)ide metabolism status, a feature that might contribute to the cytotoxic action of cell-cycle-dependent anticancer drugs.