Christian Périgaud

Nucleoside Analogues as Chemotherapeutic Agents: A Review

Abstract The importance of nucleoside analogues in chemotherapy and in other potential therapeutic approaches as immunomodulation or regulation of gene expression, is reviewed.

Rational design for cytosolic delivery of nucleoside monphosphates : “SATE” and “DTE” as enzyme-labile transient phosphate protecting groups

Abstract It was demonstrated that the use of neutral 2′,3′-dideoxyuridine phosphotriesters which incorporate enzyme mediated bioreversible protection such as S-acetylthioethanol (SATE) or dithiodiethanol (DTE) resulted in intracellular delivery of the parent mononucleotide. This point was corroborated by observation of an anti-HIV effect in various cell lines and decomposition data in cell extracts.

Characterization of a gemcitabine-resistant murine leukemic cell line: reversion of in vitro resistance by a mononucleotide prodrug.

Resistance to cytotoxic nucleoside analogues is a major problem in cancer treatment. The cellular mechanisms involved in this phenomenon have been studied for several years, and some factors have been identified. Various strategies to overcome resistance have been suggested, but none has yet shown efficacy in vivo. We developed a gemcitabine-resistant cell line (L1210 10K) from the murine leukemic L1210 strain (L1210 wt) by continuous exposure to increasing concentrations of gemcitabine. L1210 10K is highly resistant to gemcitabine (14,833-fold), 1-β-d-arabinofuranosylcytosine (ara-C; 2,100-fold), troxacitabine (>200-fold), and cladribine (160-fold) and slightly resistant to trimidox (7.22-fold), but does not display cross-resistance to fludarabine or nonnucleoside anticancer drugs. Deoxycytidine kinase mRNA was not detected by quantitative real-time reverse transcription-PCR in L1210 10K cells, whereas expression of thymidine kinase 1 and ribonucleotide reductase subunit R2 gene was moderately reduced. L1210 10K cells also demonstrated in vivo resistance to nucleoside analogues: gemcitabine- or ara-C-treated mice carrying L1210 10K had significantly shorter survival than gemcitabine- or ara-C-treated mice carrying L1210 wt (P < 0.05). UA911, a mononucleotide prodrug (pronucleotide) of ara-C was found to significantly sensitize L1210 10K cells in vitro. These results suggest that reduced deoxycytidine kinase expression is a mechanism of resistance to gemcitabine that is relevant in vivo and can be circumvented by a prodrug approach.

F1-Adenosine Triphosphatase Displays Properties Characteristic of an Antigen Presentation Molecule for Vγ9Vδ2 T Cells

Human Vγ9Vδ2 T lymphocytes are activated by phosphoantigens provided exogenously or produced by tumors and infected cells. Activation requires a contact between Vγ9Vδ2 cells and neighboring cells. We previously reported a role for cell surface F1-adenosine triphosphatase (ATPase) in T cell activation by tumors and specific interactions between Vγ9Vδ2 TCRs and purified F1-ATPase. 721.221 cells do not express surface F1-ATPase and do not support phosphoantigen responses unless they are rendered apoptotic by high doses of zoledronate, a treatment that promotes F1-expression as well as endogenous phosphoantigen production. By monitoring calcium flux in single cells, we show in this study that contact of T cells with F1-ATPase on polystyrene beads can partially replace the cell-cell contact stimulus during phosphoantigen responses. Triphosphoric acid 1-adenosin-5′-yl ester 3-(3-methylbut-3-enyl) ester, an adenylated derivative of isopentenyl pyrophosphate, can stably bind to F1-ATPase–coated beads and promotes TCR aggregation, lymphokine secretion, and activation of the cytolytic process provided that nucleotide pyrophosphatase activity is present. It also acts as an allosteric activator of F1-ATPase. In the absence of Vγ9Vδ2 cells, triphosphoric acid 1-adenosin-5′-yl ester 3-(3-methylbut-3-enyl) ester immobilized on F1-ATPase is protected from nucleotide pyrophosphatase activity, as is the antigenic activity of stimulatory target cells. Our experiments support the notion that Vγ9Vδ2 T cells are dedicated to the recognition of phosphoantigens on cell membranes in the form of nucleotide derivatives that can bind to F1-ATPase acting as a presentation molecule.

Simultaneous analysis of eight nucleoside triphosphates in cell lines by liquid chromatography coupled with tandem mass spectrometry.

In this study, we developed a new method for the simultaneous determination of eight endogenous ribonucleoside triphosphates and deoxyribonucleoside triphosphates based on a combination of a selective sample preparation and an ion-pair liquid chromatography-electrospray tandem mass spectrometry. The sample preparation was based on a protein precipitation coupled with a solid phase extraction using a weak-anion-exchange cartridge. The analytical separation of the nucleotides was achieved on a porous graphitic carbon stationary phase with a binary elution gradient program employing ion-pairing reagents (diethylamine and hexylamine) and organic eluent (methanol). The triple quadrupole mass spectrometer operated in both negative and positive multiple reaction monitoring modes. The calibration assay used the stable isotope labelled analogs of each compounds as standard. Standard calibrations were from 0.25 to 10pmol injected according to deoxyribonucleotides and from 12.5 to 3000pmol injected according to ribonucleotides. The within-run precision of the assay was less than 14.5% and the between-run precision was less than 12.4% for each analytes. Assay accuracy was in the range of 92.3-107.6%. This method allows the determination of NTP and dNTP pools from lysats of several cell lines or peripheral blood mononuclear cell from patient. Assays were performed with different preparation of cells to confirm the quality and the relevance of the described method.

Specific Requirements for Vγ9Vδ2 T Cell Stimulation by a Natural Adenylated Phosphoantigen

Human Vγ9Vδ2 T lymphocytes recognize phosphorylated alkyl Ags. Isopentenyl pyrophosphate (IPP) was previously proposed as the main Ag responsible for Vγ9Vδ2 T cell activation by cancer cells. However, triphosphoric acid 1-adenosin-5′-yl ester 3-(3-methylbut-3-enyl) ester (ApppI), a metabolite in which the isopentenyl moiety is linked to ATP, was reported in cells activated with aminobisphosphonates. The contribution of this compound to tumor-stimulatory activity was thus examined. ApppI induces selective expansion of Vγ9Vδ2 T cells from PBMCs. In the absence of APCs, however, ApppI has little stimulatory activity on Vγ9Vδ2 T cells, and optimal activation with ApppI requires addition of a nucleotide pyrophosphatase releasing IPP plus AMP. Thus, ApppI has no intrinsic stimulatory activity. Nevertheless, stimulation by ApppI is strengthened by the presence of APCs. Moreover, in contrast to IPP, ApppI can be efficiently pulsed on dendritic cells as well as on nonprofessional APCs. Pulsed APCs display stable and phosphatase-resistant stimulatory activity, indicative of Ag modification. HPLC analysis of tumor cell extracts indicates that latent phosphoantigenic activity is stored intracellularly in the Vγ9Vδ2 cell-sensitive tumor Daudi and can be activated by a nucleotide pyrophosphatase activity. The presence of ApppI in Daudi cell extracts was demonstrated by mass spectrometry. Nucleotidic Ags such as ApppI are thus a storage form of phosphoantigen which may represent a major source of phosphoantigenic activity in tumor cells. The unique properties of ApppI may be important for the design of Ags used in anticancer immunotherapeutic protocols using Vγ9Vδ2 cells.

Increase of the anti-HIV activity of D4T in human T-cell culture by the use of the sate pronucleotide approach

Abstract The bis( S -acetyl-2-thioethyl) phosphotriester derivative of 2′,3′-didehydro-2′,3′-dideoxythymidine has been synthesized and evaluated for its inhibitory effects on the replication of HIV-1 in several cell culture systems. This pronucleotide showed potent anti-HIV activity and proved to be significantly superior to the parent nucleoside with regard to the antiviral efficiency.

Comments on nucleotide delivery forms

Publisher Summary A multitude of factors affect the biological activity of nucleoside analogs (Nu), which must be phosphorylated intracellularly by cellular or viral enzymes before exerting their effects. It follows that the intracellular metabolism of nucleosides is a key event for the appearance of their biological response. In all the nucleoside series with the same sugar modification, the biological responses differ greatly according to the type of nucleobase. Nucleosides may be first converted to their corresponding 5-mononucleotides (NuMP) by cellular or viral enzymes. Some nucleosides do not show a biological response only, because they are not enzymatically transformed to the corresponding NuMP. The presence and activity of the intracellular enzymes necessary for activation of nucleoside analogs are highly dependent on host species, cell type, and stage in the cell cycle. The emergence of resistance to nucleoside analogs has been frequently attributed to a decrease or loss of activity of the first phosphorylating enzyme. Nucleotides are readily dephosphorylated in extracellular fluids and on cell surfaces by nonspecific phosphohydrolases.

Development of a sensitive and selective LC/MS/MS method for the simultaneous determination of intracellular 1-beta-d-arabinofuranosylcytosine triphosphate (araCTP), cytidine triphosphate (CTP) and deoxycytidine triphosphate (dCTP) in a human follicular lymphoma cell line

A method was developed for the quantification of araCTP, CTP and dCTP in a human follicular lymphoma cell line. This method involves solid phase extraction (SPE) using a weak anion-exchanger (WAX) cartridge, a porous graphitic carbon high-performance liquid chromatography (HPLC) column separation, and tandem mass spectrometry (MS/MS) detection. By using a triple quadrupole mass spectrometer operating in negative ion multiple reaction monitoring (MRM) mode, the method was able to achieve a lower limit of quantification (LLOQ) of 0.1 microg mL(-1) for araCTP and of 0.01 microg mL(-1) for both CTP and dCTP. The method was validated and used to determine the amount of araCTP, CTP and dCTP formed after incubation of araC and an araCMP prodrug in the human follicular lymphoma cell line RL.

Aptamer-modified micellar electrokinetic chromatography for the enantioseparation of nucleotides.

In this paper, a new aptamer-based capillary electrophoresis (CE) method, which was able to separate the enantiomers of an anionic target (adenosine monophosphate, AMP) displaying the same electrophoretic mobility as that of the oligonucleotidic chiral selector, is reported. The design of the aptamer-modified micellar electrokinetic chromatography (MEKC) mode consisted of nonionic micelles which acted as a pseudostationary phase and a hydrophobic cholesteryl group-tagged aptamer (Chol-Apt) which partitioned into the uncharged micellar phase. Under partial-filling format and suppressed electroosmotic flow conditions, the strong mobility alteration of Chol-Apt permitted AMP enantiomers to pass through the micelle-anchored aptamer zone and promoted the target enantioseparation. The influence of several electrophoretic parameters (such as concentration and nature of the nonionic surfactant, preincubation of the Chol-Apt and surfactant, capillary temperature, and applied voltage) on the AMP enantiomer migration was investigated in order to define the utilization conditions of the aptamer-modified MEKC mode. The chiral resolution, in a single run, of three adenine nucleotides, i.e., AMP, ADP (adenosine diphosphate), and ATP (adenosine triphosphate), was further accomplished using such methodology. This approach demonstrates the possibility to extend the CE applicability of aptamer chiral selectors to potentially any target, without restriction on its charge-to-mass ratio.