Enriched PUFA environment of Leishmania infantum promastigotes promotes the accumulation of lipid mediators and favors parasite infectivity towards J774 murine macrophages.

Leishmania parasites are the causative agents of visceral or cutaneous leishmaniasis in humans and of canine leishmaniosis. The macrophage is the predilected host cell of Leishmania in which the promastigote stage is transformed into amastigote. We previously showed changes in the fatty acid composition (FA) of lipids in two strains of Leishmania donovani upon differentiation of promastigote to amastigote, including increased proportions of arachidonic acid (AA) and to a less extent of docosahexaenoic acid (DHA). Here, we carried out supplementation with AA or DHA on two Leishmania infantum strains, a visceral (MON-1) and a cutaneous (MON-24), to evaluate the role of these FA in parasite/macrophage interactions. The proportions of AA or DHA in total lipids were significantly increased in promastigotes cultured in AA- or DHA-supplemented media compared to controls. The content of FA-derived oxygenated metabolites was enhanced in supplemented strains, generating especially epoxyeicosatrienoic acids (11,12- and 14,15-EET) and hydroxyeicosatetraenoic acids (5- and 8- HETE) from AA, and hydroxydocosahexaenoic acids (14- and 17-HDoHE) from DHA. For both MON-1 and MON-24, AA-supplemented promastigotes showed higher infectivity towards J774 macrophages as evidenced by higher intracellular amastigote numbers. Higher infectivity was observed after DHA supplementation for MON-24 but not MON-1 strain. ROS production by macrophages increased upon parasite infection, but only minor change was observed between control and supplemented parasites. We propose that under high AA or DHA environment that is associated with AA or DHA enrichment of promastigote lipids, FA derivatives can accumulate in the parasite, thereby modulating parasite infectivity towards host macrophages.

Beta-hydroxyphosphonate ribonucleoside analogues derived from 4-substituted-1,2,3-triazoles as IMP/GMP mimics: synthesis and biological evaluation.

A series of seventeen ß-hydroxyphosphonate ribonucleoside analogues containing 4-substituted-1,2,3-triazoles was synthesized and fully characterized. Such compounds were designed as potential inhibitors of the cytosolic 5'-nucleotidase II (cN-II), an enzyme involved in the regulation of purine nucleotide pools. NMR and molecular modelling studies showed that a few derivatives adopted similar structural features to IMP or GMP. Five derivatives were identified as modest inhibitors with 53 to 64% of cN-II inhibition at 1 mM.

Identification of Noncompetitive Inhibitors of Cytosolic 5'-Nucleotidase II Using a Fragment-Based Approach.

We used a combined approach based on fragment-based drug design (FBDD) and in silico methods to design potential inhibitors of the cytosolic 5'-nucleotidase II (cN-II), which has been recognized as an important therapeutic target in hematological cancers. Two subgroups of small compounds (including adenine and biaryl moieties) were identified as cN-II binders and a fragment growing strategy guided by molecular docking was considered. Five compounds induced a strong inhibition of the 5'-nucleotidase activity in vitro, and the most potent ones were characterized as noncompetitive inhibitors. Biological evaluation in cancer cell lines showed synergic effect with selected anticancer drugs. Structural studies using X-ray crystallography lead to the identification of new binding sites for two derivatives and of a new crystal form showing important domain swapping. Altogether, the strategy developed herein allowed identifying new original noncompetitive inhibitors against cN-II that act in a synergistic manner with well-known antitumoral agents.

Virtual screening and biological evaluation of inhibitors targeting the XPA-ERCC1 interaction.

BACKGROUND: Nucleotide excision repair (NER) removes many types of DNA lesions including those induced by UV radiation and platinum-based therapy. Resistance to platinum-based therapy correlates with high expression of ERCC1, a major element of the NER machinery. The interaction between ERCC1 and XPA is essential for a successful NER function. Therefore, one way to regulate NER is by inhibiting the activity of ERCC1 and XPA. METHODOLOGY/PRINCIPAL FINDINGS: Here we continued our earlier efforts aimed at the identification and characterization of novel inhibitors of the ERCC1-XPA interaction. We used a refined virtual screening approach combined with a biochemical and biological evaluation of the compounds for their ability to interact with ERCC1 and to sensitize cells to UV radiation. Our findings reveal a new validated ERCC1-XPA inhibitor that significantly sensitized colon cancer cells to UV radiation indicating a strong inhibition of the ERCC1-XPA interaction. CONCLUSIONS: NER is a major factor in acquiring resistance to platinum-based therapy. Regulating the NER pathway has the potential of improving the efficacy of platinum treatments. One approach that we followed is to inhibit the essential interaction between the two NER elements, ERCC1 and XPA. Here, we performed virtual screening against the ERCC1-XPA interaction and identified novel inhibitors that block the XPA-ERCC1 binding. The identified inhibitors significantly sensitized colon cancer cells to UV radiation indicating a strong inhibition of the ERCC1-XPA interaction.

Structural insights into the inhibition of cytosolic 5'-nucleotidase II (cN-II) by ribonucleoside 5'-monophosphate analogues.

Cytosolic 5'-nucleotidase II (cN-II) regulates the intracellular nucleotide pools within the cell by catalyzing the dephosphorylation of 6-hydroxypurine nucleoside 5'-monophosphates. Beside this physiological function, high level of cN-II expression is correlated with abnormal patient outcome when treated with cytotoxic nucleoside analogues. To identify its specific role in the resistance phenomenon observed during cancer therapy, we screened a particular class of chemical compounds, namely ribonucleoside phosphonates to predict them as potential cN-II inhibitors. These compounds incorporate a chemically and enzymatically stable phosphorus-carbon linkage instead of a regular phosphoester bond. Amongst them, six compounds were predicted as better ligands than the natural substrate of cN-II, inosine 5'-monophosphate (IMP). The study of purine and pyrimidine containing analogues and the introduction of chemical modifications within the phosphonate chain has allowed us to define general rules governing the theoretical affinity of such ligands. The binding strength of these compounds was scrutinized in silico and explained by an impressive number of van der Waals contacts, highlighting the decisive role of three cN-II residues that are Phe 157, His 209 and Tyr 210. Docking predictions were confirmed by experimental measurements of the nucleotidase activity in the presence of the three best available phosphonate analogues. These compounds were shown to induce a total inhibition of the cN-II activity at 2 mM. Altogether, this study emphasizes the importance of the non-hydrolysable phosphonate bond in the design of new competitive cN-II inhibitors and the crucial hydrophobic stacking promoted by three protein residues.

Detecting differential allelic expression using high-resolution melting curve analysis: application to the breast cancer susceptibility gene CHEK2.

BACKGROUND: The gene CHEK2 encodes a checkpoint kinase playing a key role in the DNA damage pathway. Though CHEK2 has been identified as an intermediate breast cancer susceptibility gene, only a small proportion of high-risk families have been explained by genetic variants located in its coding region. Alteration in gene expression regulation provides a potential mechanism for generating disease susceptibility. The detection of differential allelic expression (DAE) represents a sensitive assay to direct the search for a functional sequence variant within the transcriptional regulatory elements of a candidate gene. We aimed to assess whether CHEK2 was subject to DAE in lymphoblastoid cell lines (LCLs) from high-risk breast cancer patients for whom no mutation in BRCA1 or BRCA2 had been identified. METHODS: We implemented an assay based on high-resolution melting (HRM) curve analysis and developed an analysis tool for DAE assessment. RESULTS: We observed allelic expression imbalance in 4 of the 41 LCLs examined. All four were carriers of the truncating mutation 1100delC. We confirmed previous findings that this mutation induces non-sense mediated mRNA decay. In our series, we ruled out the possibility of a functional sequence variant located in the promoter region or in a regulatory element of CHEK2 that would lead to DAE in the transcriptional regulatory milieu of freely proliferating LCLs. CONCLUSIONS: Our results support that HRM is a sensitive and accurate method for DAE assessment. This approach would be of great interest for high-throughput mutation screening projects aiming to identify genes carrying functional regulatory polymorphisms.

Liquid chromatographic methods for the determination of endogenous nucleotides and nucleotide analogs used in cancer therapy: a review.

Endogenous ribonucleotides and deoxyribonucleotides play a crucial role in cell function. The determination of their levels is of fundamental interest in numerous applications such as energy metabolism, biochemical processes, or in understanding the mechanism of nucleoside analog compounds. Nucleoside analogs are widely used in anticancer therapy. Their mechanisms of action are related to their structural similarity with natural nucleotides. Numerous assays have been described for the determination of endogenous nucleotides or anticancer nucleotide analogs in different matrices such as cellular cultures, tissue or peripheral blood mononuclear cells. The determination of these compounds is challenging due to the large difference of concentrations between ribonucleotides and deoxyribonucleotides, the presence of numerous endogenous interferences in complex matrices and the high polarity of the molecules due to the phosphate moiety. The extraction was generally performed at low temperature and was based on protein precipitation using acid or solvent mixture. This first phase could be coupled with extraction or cleaning step of the supernatant. Liquid chromatography coupled with UV detection and based on ion-exchange chromatography using non-volatile high salt concentrations was largely described for the quantification of nucleotides. However, the development of LC-MS and LC-MS/MS during the last ten years has constituted a sensitive and specific tool. In this case, analytical column was mostly constituted by graphite or C18 stationary phase. Mobile phase was usually based on a mixture of ammonium buffer and acetonitrile and in several assays included a volatile ion-pairing agent. Mass spectrometry detection was performed either with positive or negative electrospray mode according to compounds and mobile phase components. The purpose of the current review is to provide an overview of the most recent chromatographic assays (over the past ten years) developed for the determination of endogenous nucleotides and nucleotide analogs used in cancer therapy. We focused on sample preparation, chromatographic separation and quantitative considerations.

ADP ribosylation factor like 2 (Arl2) regulates breast tumor aggressivity in immunodeficient mice.

We have previously reported that ADP ribosylation factor like 2 (Arl2), a small GTPase, content influences microtubule dynamics and cell cycle distribution in breast tumor cells, as well as the degree and distribution of phosphorylated P53. Here we show, in two different human breast adenocarcinoma models, that Arl2 content has a major impact on breast tumor cell aggressivity both in vitro and in vivo. Cells with reduced content of Arl2 displayed reduced contact inhibition, increased clonogenic or cluster formation as well as a proliferative advantage over control cells in an in vitro competition assay. These cells also caused larger tumors in SCID mice, a phenotype which was mimicked by the in vivo administration of siRNA directed against Arl2. Cells with increased Arl2 content displayed reduced aggressivity, both in vitro and in vivo, with enhanced necrosis and were also found to contain increased PP2A phosphatase activity. A rt-PCR analysis of fresh human tumor breast samples suggested that low Arl2 expression was associated with larger tumor size and greater risk of lymph node involvement at diagnosis. These data underline the role of Arl2, a small GTPase, as an important regulator of breast tumor cell aggressivity, both in vitro and in vivo.

Special feature of mixed phosphotriester derivatives of cytarabine.

Despite the unquestionable therapeutic interest of bis(SATE) pronucleotides, a presystemic metabolism preventing the delivery of the prodrugs in target cancer cells or tumours may constitute a limitation to the in vivo development of such derivatives. In order to overcome these drawbacks several strategies have been envisaged and we report herein the application of the S-acyl-2-thioethyl (SATE) phenyl pronucleotide approach to the well-known cytotoxic nucleoside cytosine-1-beta-D-arabinofuranoside (cytarabine, araC). We describe modifications of the SATE moieties with the introduction of polar groups on the acyl residue, in order to study how these changes affect antitumoral activity and metabolic stability. Two different synthetic pathways were explored and lead to obtain the corresponding mixed derivatives in satisfactory yields. Cytotoxicity was studied in murine leukaemia cells L1210 as well as in cells derived from solid human tumours (Messa and MCF7). Biological evaluation of these compounds in cell culture experiments with nucleoside analogue-sensitive and resistant cell lines showed that the modified compounds were active at higher concentrations than unmodified cytarabine, yet were much able to partially reverse resistance due to deficient nucleoside transport or activation. These results can be correlated with an incomplete decomposition mechanism into the corresponding 5'-mononucleotide.

Preparation, characterization and in vitro evaluation of a new nucleotide analogue prodrug cyclodextrin inclusion complexes.

Bis(tbutyl-S-acyl-2-thioethyl)-cytidine monophosophate is a new cytotoxic mononucleotide prodrug which have been developed to reverse the cellular resistance to nucleoside analogues. Unfortunately, its in vivo utilisation was hampered by its poor water solubility, raising the need of a molecular vector capable to mask its physicochemical characteristics although without affecting its cytotoxic activity. Hydroxypropyl-beta-cyclodextrin was used to prepare the prodrug inclusion complexes, allowing it to be solubilized in water and hence to be used for in vitro and in vivo experiments. A molar ratio of the cyclodextrin: prodrug of 3 was sufficient to obtain complete solubilization of the prodrug. The inclusion complex was characterized by differential scanning calorimetry, which revealed the disappearance of the melting peak of the prodrug suggesting the formation of inclusion complex. Proton Nuclear Magnetic Resonance spectroscopy provided a definitive proof of the inclusion complex formation, which was evidenced by the large chemical shift displacements observed for protons located in the interior of the hydrophobic cyclodextrin cavity. The complex retained its cytotoxic activity as shown by in vitro cell survival assays on murine leukemia cells. These results provided a basis for potential therapeutic applications of co-formulation of this new nucleotide analogue with hydroxypropyl-beta-CD in cancer therapy.

F-ara-AMP is a substrate of cytoplasmic 5'-nucleotidase II (cN-II): HPLC and NMR studies of enzymatic dephosphorylation.

Intracellular accumulation of triphosphorylated derivatives is essential for the cytotoxic activity of nucleoside analogues. Different mechanisms opposing this accumulation have been described. We have investigated the dephosphorylation of monophosphorylated fludarabine (F-ara-AMP) by the purified cytoplasmic 5'-nucleotidase cN-II using HPLC and NMR. These studies clearly showed that cN-II was able to convert F-ara-AMP into its non phosphorylated form, F-ara-A, with a Km in the millimolar range and Vmax = 35 nmol/min/mg, with both methods. Cytoplasmic 5'-nucleotidase cN-II can degrade this clinically useful cytotoxic nucleoside analogue and its overexpression is thus likely to be involved in resistance to this compound.

Recent developments to improve the efficacy of cytotoxic nucleoside analogues.

Cytotoxic nucleoside analogues are clinically important anticancer drugs. The newer member of this family, gemcitabine, has shown great activity in solid tumors and thus enlarged the spectra of malignancies treated by this family of molecules. However, the clinical use of nucleoside analogues is limited by important side-effects and primary or acquired drug resistance, and there is an unmet medical need for the development of new molecules and technologies allowing a suitable treatment of cancer patients with these agents. In this article we will review literature data and patents concerning (i) the recent development of some novel nucleoside analogues, (ii) oral formulations for cytotoxic nucleoside analogues, (iii) strategies to circumvent acquired resistance to nucleoside analogues, and (iv) gene therapy strategies used to increase cytotoxicity of nucleoside analogues.

[Metabolism, mechanism of action and resistance to cytotoxic nucleoside analogues]. / Métabolisme, mécanisme d'action et résistance aux analogues de nucléosides cytotoxiques.

Cytotoxic nucleoside analogues are widely used in treatment of patients with hematological malignancies as well as for some solid tumors. Resistance developed against these molecules limit their clinical use. Many studies on cell models and clinical samples have identified cellular mechanisms involved in this phenomenon. Here, we describe the available data concerning the proteins involved in the metabolism and the mechanism of action of nucleoside analogues, as well as the clinical studies showing their implication in the resistance to these drugs.