Etude du mécanisme d action des composés thiényls pyrimidines et azines sur les prions et leur valorisation potentielle dans un test diagnostic des ESST

Les maladies à prions sont des affections neurodégénératives fatales, qui affectent aussi bien les hommes que les animaux. Aucun test de diagnostic précoce commercialisé, ni de traitement efficace n'existe. L'agent infectieux est composé principalement de la forme anormalement repliée, nommée PrPSc, de la protéine prion cellulaire, PrPC. Même si le cycle de réplication des prions n'est pas totalement connu, la majorité des stratégies thérapeutiques ciblent les formes monomériques PrPC, PrPSc, ainsi que les fibrilles amyloïdes. Des intermédiaires dimériques ou trimériques de PrPSc récemment décrits, seraient à l'origine d'un état préamyloïde instable. Nous proposons que des petites molécules pourraient interagir avec des intermédiaires préamyloïdes, afin de bloquer le cycle de réplication des prions. Dans ce but, nous avons utilisé une approche de criblage virtuel, suivie d'un criblage sur des cellules infectées par les prions. Une famille de composés thiényls pyrimidines et azines, capable de stabiliser des dimères de PrPSc a été identifiée. Les études in vivo indiquent que la pré-incubation des composés avec des extraits de cerveaux, réduit le titre infectieux, suggérant un potentiel thérapeutique. Le mécanisme d'action de ces molécules a été recherché et nous proposons que les composés interagissent avec les prions via un mécanisme d'agrégation. Les composés thiényls pyrimidines permettent de discriminer rapidement des homogénats infectés de ceux qui sont sains, par la présence des dimères de PrPSc en immunoblot et pourraient être valorisés dans le diagnostic des ESST.Prions diseases are fatal neurodegeneratives disorders, which affect both humans and animals. No early diagnosis test of prions as well as effective treatment exists. The infectious agent consists mainly of the abnormal form, named PrPSc of the cellular protein prion, PrPC. Even if the replication cycle of prions is not totally known, the majority of the therapeutic strategies target monomerics forms of PrPC, PrPSc as well as amyloid fibrils. Dimers or trimers of PrPSc were recently described and would be part of an unstable preamyloïde state. We hypothesize that the identification of small molecules interacting with those preamyloid intermediates would block the replication cycle of prions and thus would reduce their infectivity. For this purpose, we used an approach of virtual screening, followed by a cellular screening on prions infected-cells. We identified a family of thienyls pyrimidines and azines compounds, able of stabilizing dimers of PrPSc, and observable in denaturing conditions by immunoblot. In vivo studies indicate that the pre-incubation of compounds with infected brain homogenate, reduces the prion infectiosity of the inoculum, suggesting a potential therapeutic application. We also studied the mechanism of action of these molecules and we propose that these compounds could directly interact with prions via a mechanism of aggregation. We showed that thienyl pyrimidine compounds can discriminate the infected brain homogenates from healthy ones, by the presence of specific PrPSc dimers, suggesting a potential application for the development of a new diagnosis test for ESSTMONTPELLIER-BU Sciences (341722106) / SudocSudocFranceF

Thienyl pyrimidine derivatives with PrP(Sc) oligomer-inducing activity are a promising tool to study prions.

International audienceTransmissible spongiform encephalopathies (TSEs), also called prion diseases, are fatal, infectious, genetic or sporadic neurodegenerative disorders of humans and animals. In humans, TSEs are represented by Creutzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-Scheinker syndrome, Fatal Familial Insomnia and Kuru. In animals, the most prominent prion diseases are scrapie of sheep and goats, bovine spongiform encephalopathy (BSE) of cattle and chronic wasting disease (CWD) of deer and elk. A critical event in prion diseases is the accumulation in the central nervous system (CNS) of the abnormally folded PrP(Sc) protein that is the protease-resistant isoform of a normal cellular protein encoded by the host and called PrP(C). PrP(Sc) (also known as rPrP(Sc) or PrP27-30) represents the main marker of prion diseases and is routinely used in the reference method for the diagnosis of prion diseases. Most of the therapeutic strategies developed so far aimed at identifying compounds that diminish the levels of PrP(Sc), with variable success when tested in vivo. In this review, we present an alternative approach in which small molecules that induce PrP(Sc) oligomers are identified. By using virtual and cellular screenings, we found several thienyl pyrimidine compounds that trigger PrP(Sc) oligomerization and trap prion infectivity

Centrifugation-based assay with thienyl azine compounds to discriminate normal from prion-infected samples

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MALDI imaging mass spectrometry and chemometric tools to discriminate highly similar colorectal cancer tissues. Chimiométrie

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A Fluorescent Oligothiophene-Bis-Triazine ligand interacts with PrP fibrils and detects SDS-resistant oligomers in human prion diseases

International audienceBACKGROUND : Prion diseases are characterized by the accumulation in the central nervous system of an abnormally folded isoform of the prion protein, named PrP(Sc). Aggregation of PrP(Sc) into oligomers and fibrils is critically involved in the pathogenesis of prion diseases. Oligomers are supposed to be the key neurotoxic agents in prion disease, so modulation of prion aggregation pathways with small molecules can be a valuable strategy for studying prion pathogenicity and for developing new diagnostic and therapeutic approaches. We previously identified thienyl pyrimidine compounds that induce SDS-resistant PrP(Sc) (rSDS-PrP(Sc)) oligomers in prion-infected samples.RESULTS : Due to the low effective doses of the thienyl pyrimidine hits, we synthesized a quaterthiophene-bis-triazine compound, called MR100 to better evaluate their diagnostic and therapeutic potentials. This molecule exhibits a powerful activity inducing rSDS-PrP(Sc) oligomers at nanomolar concentrations in prion-infected cells. Fluorescence interaction studies of MR100 with mouse PrP fibrils showed substantial modification of the spectrum, and the interaction was confirmed in vitro by production of rSDS-oligomer species upon incubation of MR100 with fibrils in SDS-PAGE gel. We further explored whether MR100 compound has a potential to be used in the diagnosis of prion diseases. Our results showed that: (i) MR100 can detect rSDS-oligomers in prion-infected brain homogenates of various species, including human samples from CJD patients; (ii) A protocol, called "Rapid Centrifugation Assay" (RCA), was developed based on MR100 property of inducing rSDS-PrP(Sc) oligomers only in prion-infected samples, and avoiding the protease digestion step. RCA allows the detection of both PK-sensitive and PK-resistant PrP(Sc) species in rodents samples but also from patients with different CJD forms (sporadic and new variant); (iii) A correlation could be established between the amount of rSDS-PrP(Sc) oligomers revealed by MR100 and the duration of the symptomatic phase of the disease in CJD patients; and (iv) Bioassay experiments showed that MR100 can trap prion infectivity more efficiently than P30 drug.CONCLUSIONS : MR100 is a powerful tool not only for studying the prion aggregation pathways regarding oligomeric and sPrP(Sc) species, but also for developing alternative methods for the detection of prion-infected samples. Considering our bioassay results, MR100 is a promising molecule for the development of prion decontamination approaches

Rational development of synergistic combinations of chemotherapy and molecular targeted agents for colorectal cancer treatment

Abstract Background The irinotecan-induced phosphokinome changes in colorectal cancer (CRC) cells were used to guide the selection of targeted agents to be tested in combination with irinotecan. Methods Phosphokinome profiling with peptide arrays of tumour samples from nude mice xenografted with HT29 cells and treated or not with an effective dose of irinotecan was used to identify signalling pathways activated by irinotecan treatment. Then, drugs targeting these pathways were combined in vitro with irinotecan to test potential synergistic effect. The interactions between these drug combinations were assessed by a dose matrix approach. Confirmation of the most potential combination has been confirmed in vivo in xenografted mice. Results Irinotecan induced in vivo the activation of AKT and MEK1 phosphorylation. The dose matrix approach showed that BKM120 (PI3K inhibitor) and MEK162 (MEK inhibitor) are synergistic in vitro and in vivo with a cytostatic and cytotoxic effect, while combination of BKM120 and irinotecan or MEK162 and irinotecan are only additive or even antagonistic. However, the triple combination of SN38, BKM120 and MEK162 showed a better synergistic effect that BKM120 and MEK162, indicating that the cells need to inhibit both AKT and ERK pathways to become more sensitive to irinotecan-based chemotherapies. Conclusion Analysis of chemotherapy-induced phosphokinome changes helps to elucidate the mechanisms of drug resistance and to guide the selection of targets for combination therapies with synergistic activity

Additional file 1: Table S1. of A Fluorescent Oligothiophene-Bis-Triazine ligand interacts with PrP fibrils and detects SDS-resistant oligomers in human prion diseases

Densitometry analysis of monomers in the pellet (P) and in the supernatant (S) fractions to determine ratios (P/S). (PDF 54 kb

The multifunctional protein E4F1 links P53 to lipid metabolism in adipocytes

International audienceGrowing evidence supports the importance of the p53 tumor suppressor in metabolism but the mechanisms underlying p53-mediated control of metabolism remain poorly understood. Here, we identify the multifunctional E4F1 protein as a key regulator of p53 metabolic functions in adipocytes. While E4F1 expression is upregulated during obesity, E4f1 inactivation in mouse adipose tissue results in a lean phenotype associated with insulin resistance and protection against induced obesity. Adipocytes lacking E4F1 activate a p53-dependent transcriptional program involved in lipid metabolism. The direct interaction between E4F1 and p53 and their co-recruitment to the Steaoryl-CoA Desaturase-1 locus play an important role to regulate monounsaturated fatty acids synthesis in adipocytes. Consistent with the role of this E4F1-p53-Steaoryl-CoA Desaturase-1 axis in adipocytes, p53 inactivation or diet complementation with oleate partly restore adiposity and improve insulin sensitivity in E4F1-deficient mice. Altogether, our findings identify a crosstalk between E4F1 and p53 in the control of lipid metabolism in adipocytes that is relevant to obesity and insulin resistance