A multi-omics investigation of tacrolimus off-target effects on a proximal tubule cell-line

Introduction: Tacrolimus, an immunosuppressive drug prescribed to a majority of organ transplant recipients is nephrotoxic, through still unclear mechanisms. This study on a lineage of proximal tubular cells using a multi-omics approach aims to detect off-target pathways modulated by tacrolimus that can explain its nephrotoxicity. Methods: LLC-PK1 cells were exposed to 5 µM of tacrolimus for 24 h in order to saturate its therapeutic target FKBP12 and other high-affine FKBPs and favour its binding to less affine targets. Intracellular proteins and metabolites, and extracellular metabolites were extracted and analysed by LC-MS/MS. The transcriptional expression of the dysregulated proteins PCK-1, as well as of the other gluconeogenesis-limiting enzymes FBP1 and FBP2, was measured using RT-qPCR. Cell viability with this concentration of tacrolimus was further checked until 72 h. Results: In our cell model of acute exposure to a high concentration of tacrolimus, different metabolic pathways were impacted including those of arginine (e.g., citrulline, ornithine) (p < 0.0001), amino acids (e.g., valine, isoleucine, aspartic acid) (p < 0.0001) and pyrimidine (p < 0.01). In addition, it induced oxidative stress (p < 0.01) as shown by a decrease in total cell glutathione quantity. It impacted cell energy through an increase in Krebs cycle intermediates (e.g., citrate, aconitate, fumarate) (p < 0.01) and down-regulation of PCK-1 (p < 0.05) and FPB1 (p < 0.01), which are key enzymes in gluconeogenesis and acid-base balance control. Discussion: The variations found using a multi-omics pharmacological approach clearly point towards a dysregulation of energy production and decreased gluconeogenesis, a hallmark of chronic kidney disease which may also be an important toxicity pathway of tacrolimus

New 8-nitroquinolinone derivative displaying submicromolar in vitro activities against both Trypanosoma brucei and cruzi

International audienceAn antikinetoplastid pharmacomodulation study was conducted at position 6 of the 8-nitroquinolin-2(1H)-one pharmacophore. Fifteen new derivatives were synthesized and evaluated in vitro against L. infantum, T. brucei brucei, and T. cruzi, in parallel with a cytotoxicity assay on the human HepG2 cell line. A potent and selective 6-bromo-substituted antitrypanosomal derivative 12 was revealed, presenting EC50 values of 12 and 500 nM on T. b. brucei trypomastigotes and T. cruzi amastigotes respectively, in comparison with four reference drugs (30 nM ≤ EC50 ≤ 13 μM). Moreover, compound 12 was not genotoxic in the comet assay and showed high in vitro microsomal stability (half life >40 min) as well as favorable pharmacokinetic behavior in the mouse after oral administration. Finally, molecule 12 (E° = −0.37 V/NHE) was shown to be bioactivated by type 1 nitroreductases, in both Leishmania and Trypanosoma, and appears to be a good candidate to search for novel antitrypanosomal lead compounds

Antikinetoplastid SAR study in 3-nitroimidazopyridine series: identification of a novel non-genotoxic and potent anti-T. b. brucei hit-compound with improved pharmacokinetic properties

To study the antikinetoplastid 3-nitroimidazo[1,2-a]pyridine pharmacophore, a structure-activity relationship study was conducted through the synthesis of 26 original derivatives and their in vitro evaluation on both Leishmania spp and Trypanosoma brucei brucei. This SAR study showed that the antitrypanosomal pharmacophore was less restrictive than the antileishmanial one and highlighted positions 2, 6 and 8 of the imidazopyridine ring as key modulation points. None of the synthesized compounds allowed improvement in antileishmanial activity, compared to previous hit molecules in the series. Nevertheless, compound 8, the best antitrypanosomal molecule in this series (EC50 = 17 nM, SI = 2650 & E° = -0.6 V), was not only more active than all reference drugs and previous hit molecules in the series but also displayed improved aqueous solubility and better in vitro pharmacokinetic characteristics: good microsomal stability (T1/2 > 40 min), moderate albumin binding (77%) and moderate permeability across the blood brain barrier according to a PAMPA assay. Moreover, both micronucleus and comet assays showed that nitroaromatic molecule 8 was not genotoxic in vitro. It was evidenced that bioactivation of molecule 8 was operated by T. b. brucei type 1 nitroreductase, in the same manner as fexinidazole. Finally, a mouse pharmacokinetic study showed that 8 displayed good systemic exposure after both single and repeated oral administrations at 100 mg/kg (NOAEL) and satisfying plasmatic half-life (T1/2 = 7.7 h). Thus, molecule 8 appears as a good candidate for initiating a hit to lead drug discovery program

Antikinetoplastid SAR study in 3-nitroimidazopyridine series:identification of a novel non-genotoxic and potent anti-T. b. brucei hit-compound with improved pharmacokinetic properties.

International audienceTo study the antikinetoplastid 3-nitroimidazo[1,2-a]pyridine pharmacophore, a structure-activity relationship study was conducted through the synthesis of 26 original derivatives and their in vitro evaluation on both Leishmania spp and Trypanosoma brucei brucei. This SAR study showed that the antitrypanosomal pharmacophore was less restrictive than the antileishmanial one and highlighted positions 2, 6 and 8 of the imidazopyridine ring as key modulation points. None of the synthesized compounds allowed improvement in antileishmanial activity, compared to previous hit molecules in the series. Nevertheless, compound 8, the best antitrypanosomal molecule in this series (EC50 = 17 nM, SI = 2650 & E° = −0.6 V), was not only more active than all reference drugs and previous hit molecules in the series but also displayed improved aqueous solubility and better in vitro pharmacokinetic characteristics: good microsomal stability (T1/2 > 40 min), moderate albumin binding (77%) and moderate permeability across the blood brain barrier according to a PAMPA assay. Moreover, both micronucleus and comet assays showed that nitroaromatic molecule 8 was not genotoxic in vitro. It was evidenced that bioactivation of molecule 8 was operated by T. b. brucei type 1 nitroreductase, in the same manner as fexinidazole. Finally, a mouse pharmacokinetic study showed that 8 displayed good systemic exposure after both single and repeated oral administrations at 100 mg/kg (NOAEL) and satisfying plasmatic half-life (T1/2 = 7.7 h). Thus, molecule 8 appears as a good candidate for initiating a hit to lead drug discovery program

Identification des métabolites du Mégazol sur un modèle animal

International audienceLa Trypanosomose humaine africaine (THA), ou maladie du sommeil, est une parasitose vectorielle endémique localisée en Afrique sub-saharienne. Elle est causée par un parasite, Trypanosoma brucei, transmis par la piqûre d’une mouche, la glossine, lors de son repas sanguin. Le parasite après multiplication au niveau hémo-lympathique, est capable de franchir la barrière hémato-encéphalique et d’atteindre le système nerveux central. A ce stade, en l’absence de traitement, la mort est inéluctable. Les traitements proposés sont stades dépendants et reposent sur la pentamidine et la suramine pour le stade hémo-lymphatique, le mélarsoprol, l’eflornithine et l’association nifurtimox-eflornithine pour le stade cérébral. Mais l’apparition de résistance, la présence d’effets indésirables graves de certains de ces traitements renforcent la nécessité de trouver de nouveaux médicaments surs et efficaces à la fois chez l’Homme et le bétail, grand réservoir de cette zoonose. Le mégazol, de formule brute 2-amino-5-(1-méthyl-5-nitro-2-imidazolyl)-1-3-4-thiadiazole, dérivé nitré, possède des propriétés antiparasitaires dirigées contre ce parasite depuis longtemps démontrées, mais son intérêt a été malheureusement oublié depuis quelques années, à la suite de publications peu enclin à montrer son potentiel thérapeutique réel.Notre travail est centré sur l’étude du mégazol, afin d’identifier les métabolites qui pourraient présenter un intérêt en médecine humaine ou vétérinaire.Les métabolites ont été recherchés et caractérisés à partir d’expérimentation in vivo sur un modèle murin. Des lots de souris ont donc reçu une dose de mégazol à des temps donnés, et un recueil des urines et de plasma a été effectué en vue d’une analyse par chromatographie liquide haute performance couplée à la spectrométrie de masse (CLHP/SM), dans les différents échantillons biologiques des divers lots de souris traitées en utilisant la méthode GUS (General Unknown Screening) permettant de détecter et d’identifier sans a priori, les composés présents dans les matrices biologiques et la méthode MRM (Multiple Reaction Monitoring) permettant de quantifier une ou plusieurs molécules cibles dans un échantillon complexe. Les résultats obtenus ont permis de mettre en évidence seize ions différents, dont treize ont permis de confirmer la structure proposée lors d’une précédente étude.Il reste maintenant à caractériser par RMN notamment l’ensemble des métabolites obtenus et à tester in vitro sur des cultures de souches parasitaires différentes l’effet potentiellement trypanocide de certains métabolites synthétisés chimiquement, mais également de réévaluer la toxicité du mégazol aux doses thérapeutiques administrables à l’Homme et aux animaux en se basant sur la NOAEL (No Observable Adverse Effect Level) et la LOAEL (Lowest Observed Adverse Effect Level), et aussi de composés dérivés de ce dernier pour entrevoir un nouvel avenir thérapeutique pour les trypanosomés

Relative quantitative proteomic analysis of CNI-induced toxicity in a porcine proximal tubule cell line using iTRAQ technique

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Identification of Megazol metabolites from 2 models

International audienceAfrican Trypanosomiasis is endemic in nearly 30 countries in sub-Saharan Africa. The parasite responsible for this infection is Trypanosoma brucei, transmitted by the bite of glossina or tsetse fly. This parasitosis affects both animals (AAT - Animal African Trypanosomosis) and humans (HAT - Human African Trypanosomosis) and is fatal in absence of treatment. Its therapeutic care is complex and depends on trypanosome species, host and stage of the disease. Therefore, it is essential to find new treatments. Megazol, a molecule previously descripted with a trypanocid effect, has interesting derivates that are investigated in our work.The aim of our work is to highlight trypanocidal metabolites derived from megazol by two approaches: 1) an in vivo study on murine model then, 2) an approach of bioconversion by microorganisms.1) Lots of mice received a dose of megazol at given times and a urine collection was carried out for analysis by high-performance liquid chromatography coupled with mass spectrometry(HPLC/MS) using GUS (General Unknown Screening) and MRM (Multiple Reaction Monitoring) methods. The results revealed 16 different ions.2) The bioconversion approach by selecting microorganisms capable of transforming megazol by a combinatorial approach, after LC/MS-MS analysis, made it possible to show the formation of metabolites identical to those in the in vivo study. This detection confirms the fact that some fungal species are capable of mimicking mammalian metabolism. The production and purification of metabolites are underway to identify and study their activity and toxicity

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI–TOF–MS) coupled to XAD fractionation: Method to algal organic matter characterization

International audienceThis workisfocusedonthedevelopmentofananalyticalprocedurefortheimprovementoftheOrganic Matter structurecharacterization,particularlythealgalmatter.Twofractionsofalgalorganicmatter from laboratoryculturesofalgae(Euglena gracilis) andcyanobacteria(Microcystis aeruginosa) were extractedwithXADresins.Thefractionswerestudiedusinglaserdesorptionionization(LDI)and Matrix-Assisted laserdesorptionionizationtime-of-flight massspectrometry(MALDI–TOF). Acompar- ison withthenaturalorganicmattercharacteristicsfromcommercialhumicacidsandfulvicacids extractedfromSuwanneeRiverwasperformed. Resultsshowthatalgalandnaturalorganicmattershaveuniquequasi-polymericstructures. Significant repeatingpatternswereidentified. Differentfractionsextractedfromorganicmatterwith common originhadcommonstructures.Thus,44,114and169Dapeaksseparationforfractionsfrom E. gracilis organic matterand28,58and100Dafor M. aeruginosa ones wereclearlyobserved.Using thedevelopedprotocol,astructuralschemeandorganicmattercompositionwereobtained.Therange 600–2000Dacontainedmorearchitecturalcompositiondifferencesthantherange100–600Da,suggest- ingthatorganicmatteriscomposedofanassemblyofcommonsmallmolecules.Associatedtospecific monomers,particularpatternswerecommontoallsamplesbutassemblyandresultingstructurewere uniqueforeachorganicmatter.Thus,XADfractionationcoupledtomassspectroscopyalloweddetermin- ingaspecific fingerprintforeachorganicmatter

Identification of Metabolites Derived from the Anti-trypanosomal Drug Megazol

Background: African Trypanosomiasis is an endemic vector-borne parasitic disease in sub-Saharan Africa. It is caused by different parasites of the genus Trypanosoma and is transmitted through a tsetse fly (Glossina sp.) bite during a blood meal. This neglected tropical disease remains difficult to control due to the complexity of treatment protocols and use of toxic drugs. Over the decades, nitroimidazole compounds have been promising molecules for anti-parasite therapy. One of them, megazol, has proven to be an effective anti-trypanosomal drug, but interest dropped after reports were published concerning its mutagenic properties. Objectived: We therefore decided to characterize and identify megazol metabolites, with the hypothesis that they could be less toxic. Methods: We treated groups of mice with different derivatives and then detected metabolites by high performance liquid chromatography combined with mass spectrometry in urine, feces, and plasma samples from mice. Results: In vivo results showed that eleven metabolites were detected in urine (M1 to M11); six metabolites were detected in plasma (M1a/b, M2a/b, M5, M7a/b M9, andM10a/b) and in feces, only two (M1 a/b and M5) were found. Conclusions: The structures of metabolites were deduced using chromatograms and mass spectra data combined with usual metabolic patterns.

Recherche de Métabolites du Mégazol par bioconversion.

International audienceLa Trypanosomose Africaine est une des 18 Maladies Tropicales Négligées endémique localisée dans près de 30 pays d’Afrique sub-saharienne. Le parasite responsable de cette infection est Trypanosoma brucei, vectorisé par la piqûre d’une mouche, la glossine ou mouche tsé-tsé, lors de son repas sanguin. Cette parasitose touche aussi bien les animaux (TAA - Trypanosomose Animale Africaine) que les Hommes (THA - Trypanosomose Humaine Africaine) et est d’évolution fatale en l’absence de traitement. Les impacts économiques et environnementaux sont encore importants pour les populations. Sa prise en charge thérapeutique est complexe et est fonction de l’espèce du trypanosome, de l’hôte et du stade de la maladie. A toutes ces contraintes, s’ajoutent l’apparition de résistance, et la présence d’effets indésirables graves. Il est donc primordial de trouver de nouveaux traitements sûrs et efficaces pour l’Homme mais aussi pour l’Animal afin d’assainir les potentiels réservoirs de ce parasite. C’est l’objectif que nous nous sommes fixés en reprenant les travaux d’une molécule trypanocide : le mégazol. Ce dérivé nitré de formule brute 2-amino-5-(1-méthyl-5-nitro-2-imidazolyl)-1-3-4-thiadiazole, a des propriétés trypanocides reconnues mais est inutilisable chez l’Homme en raison de risques toxiques trop importants.Le but de notre travail est de mettre en évidence des métabolites trypanocides dérivés du mégazol par une approche innovante de bioconversion par des microorganismes. Le but étant de proposer de nouvelles molécules utilisables à la fois par la médecine humaine et vétérinaire.Des micro-organismes capables de transformer le mégazol ont été sélectionnés par une approche combinatoire(1). Le suivi des biotransformations a été effectué par analyse LC/MS-MS, et les résultats obtenus ont permis de mettre en évidence la formation de métabolites identiques à ceux d’une étude précédente, obtenus par biotransformation avec des souris traitées au mégazol. Cette détection confirme le fait que certaines espèces fongiques sont capables de mimer le métabolisme des mammifères, et pourraient donc être un meilleur modèle de synthèse de molécules, notamment dans notre cas pour sélectionner un ou plusieursmétabolites d’intérêt. La production et la purification des métabolites sont en cours pour leur identification et l’étude de leur activité et de leur toxicité