Development of novel oxazolo[5,4- d ]pyrimidines as competitive CB 2 neutral antagonists based on scaffold hopping

A series of novel oxazolo[5,4-d]pyrimidines was designed via a scaffold hopping strategy and synthesized through a newly developed approach. All these compounds were evaluated for their biological activity toward CB1/CB2 cannabinoid receptors, their metabolic stability in mice liver microsomes and their cytotoxicity against several cell lines. Eight compounds have been identified as CB2 ligands with Ki values less than 1 μM. It is noteworthy that 2-(2-chlorophenyl)-5-methyl-7-(4-methylpiperazin-1-yl) oxazolo[5,4-d]pyrimidine 47 and 2-(2-chlorophenyl)-7-(4-ethylpiperazin-1-yl)- 5-methyloxazolo[5,4-d]pyrimidine 48 showed CB2 binding affinity in the nanomolar range and significant selectivity over CB1 receptors. Interestingly, functionality studies imply that they behave as competitive neutral antagonists. Moreover, all tested compounds are devoid of cytotoxicity toward several cell lines, including Chinese hamster ovary cells (CHO) and human colorectal adenocarcinoma cells HT29

Synthesis and biological evaluation of ferrocene-based cannabinoid receptor 2 ligands

Ferrocene analogues of known fatty acid amide hydrolase inhibitors and CB2 ligands have been synthesized and characterized spectroscopically and crystallographically. The resulting bioorganometallic isoxazoles were assayed for their effects on CB1 and CB2 receptors as well as on FAAH. None had any FAAH activity but compound 3, 5-(2-(pentyloxy)phenyl)-N-ferrocenylisoxazole- 3-carboxamide, was found to be a potent CB2 ligand (Ki = 32.5 nM)

Targeting the DNA-binding activity of the human ERG transcription factor using new heterocyclic dithiophene diamidines

Direct modulation of gene expression by targeting oncogenic transcription factors is a new area of research for cancer treatment. ERG, an ETS-family transcription factor, is commonly over-expressed or translocated in leukaemia and prostate carcinoma. In this work, we selected the di-(thiophene-phenylamidine) compound DB1255 as an ERG/DNA binding inhibitor using a screening test of synthetic inhibitors of the ERG/DNA interaction followed by electrophoretic mobility shift assays (EMSA) validation. Spectrometry, footprint and biosensor-surface plasmon resonance analyses of the DB1255/DNA interaction evidenced sequence selectivity and groove binding as dimer. Additional EMSA evidenced the precise DNA-binding sequence required for optimal DB1255/DNA binding and thus for an efficient ERG/DNA complex inhibition. We further highlighted the structure activity relationshipsfrom comparison with derivatives. In cellulo luciferase assay confirmed this modulation both with the constructed optimal sequences and the Osteopontin promoter known to be regulated by ERG and which ERG-binding site was protected from DNaseI digestion on binding of DB1255. These data showed for the first time the ERG/DNA complex modulation, both in vitro and in cells, by a heterocyclic diamidine that specifically targets a portion of the ERG DNA recognition site

Synthesis of bioorganometallic nanomolar-potent CB2agonists containing a ferrocene unit

A small library of ferrocene-containing amides has been synthesized using standard amide coupling chemistry with ferrocenylamine. Ferrocene analogues of known bioactive adamantylamides were shown to be effective cannabinoid receptor (CB1 and CB2) agonists, displaying, in many cases, single-digit nanomolar potency. Three final ferrocene-containing derivatives have been characterized in the solid state by X-ray crystallography and display intramolecular hydrogen bonding of the type NH---C═O. N-Methylation of the amide, confirmed by X-ray crystallography, leads to both loss of hydrogen bonding and biological activity

Mécanismes d'induction de l'apoptose par les glucocorticoïdes (étude à l'aide de glucocorticoïdes à effets dissociés)

Les glucocorticoïdes (GC) sont couramment utilisés en thérapeutique pour leurs propriétés anti-inflammatoires et immunosuppressives mais des effets secondaires limitent leur utilisation à long terme. Les GC exercent leur effets en se fixant à leur récepteur spécifique : le récepteur aux glucocorticoïdes (GR), capable de réguler l'expression de nombreux gènes, à la fois positivement (transactivation) et négativement (transrépression). La transactivation est le mécanisme prépondérant par lequel les GC exercent leurs effets métaboliques mais elle contribue aux effets délétères des GC. Par contre, la répression transcriptionnelle est à l'origine des propriétés thérapeutiques des GC. Il semble donc possible de dissocier les effets thérapeutiques des GC de leurs effets secondaires en utilisant des corticoïdes à activité exclusivement répressive. On parle alors de glucocorticoïdes à effets dissociés. Les GC sont également connus pour induire l'apoptose de thymocytes mais un doute subsiste quant à l'activité du GC (transrépressive ou transactivatrice) responsable de l'induction de cette mort cellulaire. Cette thèse a consisté à utiliser la particularité des glucocorticoïdes à effets dissociés en espérant clarifier le débat au sujet de l'origine du signal apoptotique. Nos résultats montrent pour la première fois que deux GC à effets dissociés, le RU24858 et le RU24782, qui conservent une activité anti-inflammatoire, sont capables d'induire l'apoptose de thymocytes murins. Le RU24782, qui ne permet pas le recrutement du coactivateur transcriptionnel SRC1 par le récepteur aux GC, montre très peu d'activité transactivatrice. De ce fait, ces données suggèrent un rôle non-génomique, cytoplasmique, du GR dans l'apoptose. De plus, des inhibiteurs de la transcription (actinomycine D) et de la traduction (cycloheximide), qui préviennent la mort cellulaire induite par les corticoïdes, interfèrent avec le récepteur des GC dans le cytoplasme même en l'absence de stéroïdes, ce qui permet d'expliquer l'inhibition de la mort cellulaire et de l'expression de gènes dans notre modèle. Nos résultats montrent également l'implication de la caspase-8 de façon précoce dans l'apoptose induite par les GC. Cette activation est suivie, de façon similaire à la voie des récepteurs de mort, par le clivage de Bid, la chute du potentiel mitochondrial DYm, le relargage du cytochrome c et l'activation des caspases. De façon inattendue, l'activité de la caspase-8 s'est également révélée indispensable à l'induction des gènes de mort, étape considérée jusqu'alors comme initiatrice du processus apoptotique induit par les GC. Parmi leurs nombreux effets secondaires, les GC entraînent un amincissement de la peau, réversible après un arrêt du traitement. Nos travaux ont aussi mis en évidence que les GC n'ont pas d'activité apoptotique directe dans les kératinocytes mais favorisent l'apoptose et la différenciation terminale induite par le calcium. Cet effet est accompagné par la relocalisation de la protéine S100A11, une nouvelle protéine régulée par les glucocorticoïdes, au sein des kératinocytes. Ces résultats suggèrent que cette activité des GC pourrait être une étape clé dans l'homéostasie de l'épiderme et les effets secondaires des GC sur ce tissu. Dans l'ensemble, nos résultats suggèrent que l'apoptose induite par les GC dans les thymocytes est non-génomique et résulte d'une activation précoce de la caspase-8. Cette caspase est essentielle à l'initiation de la cascade apoptotique mais aussi à l'expression des gènes de mort. De ce fait, l'initiation de l'apoptose ne semble pas reposer sur l'activation de gènes de mort par le GR mais plutôt une activation précoce de la caspase-8, ce qui modifie le schéma classique d'induction de l'apoptose par les GCLILLE2-BU Santé-Recherche (593502101) / SudocSudocFranceF

Proteomics unveil corticoid-induced S100A11 shuttling in keratinocyte differentiation.

International audienceUnlike classical protein extraction techniques, proteomic mapping using a selective subcellular extraction kit revealed S100A11 as a new member of the S100 protein family modulated by glucocorticoids in keratinocytes. Glucocorticoids (GC)-induced S100A11 redistribution in the "organelles and membranes" compartment. Microscopic examination indicated that glucocorticoids specifically routed cytoplasmic S100A11 toward perinuclear compartment. Calcium, a key component of skin terminal differentiation, directed S100A11 to the plasma membrane as previously reported. When calcium was added to glucocorticoids, minor change was observed at the proteomic level while confocal microscopy revealed a rapid and dramatic translocation of S100A11 toward plasma membrane. This effect was accompanied by strong nuclear condensation, loss of mitochondrial potential and DNA content, and increased high molecular weight S100A11 immunoreactivity, suggesting corticoids accelerate calcium-induced terminal differentiation. Finally, our results suggest GC-induced S100A11 relocalization could be a key step in both keratinocyte homeostasis and glucocorticoids side effects in human epidermis

Inflammation and ER stress differentially regulate STAMP2 expression and localization in adipocytes

Background Chronic ER stress and dysfunction is a hallmark of obesity and a critical contributor to metaflammation, abnormal hormone action and altered substrate metabolism in metabolic tissues, such as liver and adipocytes. Lack of STAMP2 in lean mice induces inflammation and insulin resistance on a regular diet, and it is dysregulated in the adipose tissue of obese mice and humans. We hypothesized that the regulation of STAMP2 is disrupted by ER stress. Methods 3T3-L1 and MEF adipocytes were treated with ER stress inducers thapsigargin and tunicamycin, and inflammation inducer TNFα. The treatments effect on STAMP2 expression and enzymatic function was assessed. In addition, 3T3-L1 adipocytes and HEK cells were utilized for Stamp2 promoter activity investigation performed with luciferase and ChIP assays. Results ER stress significantly reduced both STAMP2 mRNA and protein expression in cultured adipocytes whereas TNFα had the opposite effect. Concomitant with loss of STAMP2 expression during ER stress, intracellular localization of STAMP2 was altered and total iron reductase activity was reduced. Stamp2 promoter analysis by reporter assays and chromatin immunoprecipitation, showed that induction of ER stress disrupts C/EBPα-mediated STAMP2 expression. Conclusion These data suggest a clear link between ER stress and quantitative and functional STAMP2-deficiency

Distinctly Different Dynamics and Kinetics of Two Steroid Receptors at the Same Response Elements in Living Cells

<div><p>Closely related transcription factors (TFs) can bind to the same response elements (REs) with similar affinities and activate transcription. However, it is unknown whether transcription is similarly orchestrated by different TFs bound at the same RE. Here we have compared the recovery half time (t_1/2), binding site occupancy and the resulting temporal changes in transcription upon binding of two closely related steroid receptors, the androgen and glucocorticoid receptors (AR and GR), to their common hormone REs (HREs). We show that there are significant differences at all of these levels between AR and GR at the MMTV HRE when activated by their ligands. These data show that two TFs bound at the same RE can have significantly different modes of action that can affect their responses to environmental cues.</p></div

Flow cytometry: An accurate tool for screening P2RX7 modulators

International audienceThe P2X purinergic receptor 7 (P2RX7) is a poorly selective ATP‐gated ion channel. Although P2RX7 binds ATP with relatively low affinity, prolonged activation can lead to nonselective membrane pore formation. Indeed, brief exposure to ATP triggers a rapid Ca2+ influx, whereas prolonged exposure to high ATP concentrations results in the passage of larger organic molecules. P2RX7 is involved in the physiopathology of a number of diseases and has notably emerged as a potential therapeutic target in inflammation, neuropathic pain, Alzheimer's disease, and cancer—prompting growing interest in the synthesis of novel P2RX7 modulators and the development of reliable, stringent screening methods. In the present study, we developed methods based on conventional flow cytometry, imaging flow cytometry and spectral flow cytometry and used them to measure P2RX7's activity upon activation by 3'‐O‐(4‐benzoyl)benzoyl ATP. We also demonstrated the use of the highly sensitive DNA‐intercalating dye TO‐PRO‐3 to determine P2RX7's large pore activity. The simultaneous quantification of calcium influx (Fluo‐3 AM), large pore opening (TO‐PRO‐3), and viability (propidium iodide) is a very efficient method for low‐ to medium‐throughput screening of P2RX7 modulators. Agonist and antagonist potencies can be accurately evaluated. Spectral cytometry notably enabled us to assay several biological activities while correcting for the intrinsic fluorescence of the screened compounds—otherwise a well‐known limitation of fluorescence‐based screening. Hence, spectral cytometry appears to be a useful, novel tool for drug candidate screening