Antitrypanosomatid Pharmacomodulation at Position 3 of the 8-Nitroquinolin-2(1H)-one Scaffold Using Palladium-Catalysed Cross-Coupling Reactions

International audienceAn antikinetoplastid pharmacomodulation study at position 3 of the recently described hit molecule 3-bromo-8-nitroquinolin-2(1H)-one was conducted. Twenty-four derivatives were synthesised using the Suzuki-Miyaura cross-coupling reaction and evaluated in vitro on both Leishmania infantum axenic amastigotes and Trypanosoma brucei brucei trypomastigotes. Introduction of a para-carboxyphenyl group at position 3 of the scaffold led to the selective antitrypanosomal hit molecule 3-(4-carboxyphenyl)-8-nitroquinolin-2(1H)-one (21) with a lower reduction potential (-0.56 V) than the initial hit (-0.45 V). Compound 21 displays micromolar antitrypanosomal activity (IC50 =1.5 μm) and low cytotoxicity on the human HepG2 cell line (CC50 =120 μm), having a higher selectivity index (SI=80) than the reference drug eflornithine. Contrary to results previously obtained in this series, hit compound 21 is inactive toward L. infantum and is not efficiently bioactivated by T. brucei brucei type I nitroreductase, which suggests the existence of an alternative mechanism of action

Novel 8-nitroquinolin-2(1H)-ones as NTR-bioactivated antikinetoplastid molecules:Synthesis, electrochemical and SAR study

International audienceTo study the antiparasitic 8-nitroquinolin-2(1H)-one pharmacophore, a series of 31 derivatives was synthesized in 1-5 steps and evaluated in vitro against both Leishmania infantum and Trypanosoma brucei brucei. In parallel, the reduction potential of all molecules was measured by cyclic voltammetry. Structure-activity relationships first indicated that antileishmanial activity depends on an intramolecular hydrogen bond (described by X-ray diffraction) between the lactam function and the nitro group, which is responsible for an important shift of the redox potential (+0.3 V in comparison with 8-nitroquinoline). With the assistance of computational chemistry, a set of derivatives presenting a large range of redox potentials (from -1.1 to -0.45 V) was designed and provided a list of suitable molecules to be synthesized and tested. This approach highlighted that, in this series, only substrates with a redox potential above -0.6 V display activity toward L. infantum. Nevertheless, such relation between redox potentials and in vitro antiparasitic activities was not observed in T. b. brucei. Compound 22 is a new hit compound in the series, displaying both antileishmanial and antitrypanosomal activity along with a low cytotoxicity on the human HepG2 cell line. Compound 22 is selectively bioactivated by the type 1 nitroreductases (NTR1) of L. donovani and T. brucei brucei. Moreover, despite being mutagenic in the Ames test, as most of nitroaromatic derivatives, compound 22 was not genotoxic in the comet assay. Preliminary in vitro pharmacokinetic parameters were finally determined and pointed out a good in vitro microsomal stability (half-life > 40 min) and a 92% binding to human albumin

From the identification of antileishmaniens compounds to the search of new therapeutic targets : optimization of the screening of synthetic compounds, and study of the cysteine-peptidase MCA and Raptor during programmed cell death in Leishmania

Au cours de ce travail de thèse, les deux approches permettant la caractérisation de nouveaux agents antileishmaniens ont été suivies : d’une part identification de molécules de synthèse originales et actives, par des méthodes de criblage, et d’autre part recherche de nouvelles cibles thérapeutiques leishmaniennes, faisant appel à des outils de biologie moléculaire, et de protéomique.Dans une première partie consacrée à l’optimisation du criblage antileishmanien de molécules de synthèse, nous avons notamment mis au point et validé une nouvelle méthode applicable aux formes promastigotes. Cette méthode reposant sur le principe de la bioluminescence, s’est avérée précise, rapide, répétable, sensible, automatisable et applicable à des isolats cliniques. Nous nous sommes également intéressés à la recherche d’une nouvelle méthode de criblage sur les formes amastigotes intracellulaires remplissant ces mêmes critères.Dans une seconde partie consacrée au lien entre la mort cellulaire programmée et les cystéines peptidases (métacaspase et Raptor) chez Leishmania, nous avons mis en évidence un lien possible entre métacaspase et autophagie chez L. infantum. De plus, nous avons identifié, in vivo, plusieurs substrats protéiques potentiels de ces peptidases : HSP70, ARN-Hélicase ATP-Dépendante et Lmjf09.1010 pour la métacaspase ; NDPKb et la protéine associée à l’ADN du kinétoplaste pour Raptor. Ces différents résultats ont permis de proposer un modèle conciliant les rôles possibles des cystéine-Peptidases métacaspase et Raptor au cours de l’autophagie et de l’apoptose chez Leishmania, rôles potentiellement dus au clivage de substrats protéiques spécifiques.During this thesis work, two approaches affording the characterization of new antileishmanial agents were followed: on the one hand, the identification of new original antileishmanial synthetic drugs, through screening assays and on the other hand, research of new parasitic therapeutic targets by using molecular biology and proteomic tools. In the first part, dedicated to the optimization of the antileishmanial screening of synthetic compounds, we set up and validated a new bioluminescence-Based screening method for studying anti-Promastigote compounds. This method appears accurate, rapid, repeatable, sensitive and is also transposable to automats and usable on clinical isolates. In parallel, we investigated new screening protocols aiming at improving the screening in intracellular amastigotes which could meet the same criteria. In the second part focusing on the link between programmed cell death and cystein peptidases (metacaspase and Raptor) in Leishmania, our study first highlighted a possible link between metacaspase and autophagy in L. infantum. Moreover, we identified in vivo several potential subtrates for both peptidases: HSP70, ATP-Dependent RNA-Helicase and Lmjf09.1010 for the metacaspase; NDPKb and kinetoplast-DNA-Associated-Protein for Raptor. These results allowed us to propose a model reconciling the possible roles of cystein peptidases metacaspase and Raptor during autophagy and apoptosis in Leishmania, roles potentially due to the cleavage of specific proteic subtrates

Résistance de l'agent du paludisme, Plasmodium falciparum aux combinaisons thérapeutiques à base d'artémisinine (ACTs) : Craintes d'une chimiorésistance généralisée

International audienceMalaria treatment with ACTs (Artemisinin-based Combination Therapies), combining a chemical derivative of artemisinin, and a partner drug has, for more than 15 years, produced a notable decrease in the mortality in tropical and subtropical areas. However, since 2008, a serious threat has emerged in western Cambodia, where the clinical efficacy of artemisinins has significantly declined, with a delayed parasite clearance rate and high recrudescence rates in the following weeks. Resistance of Plasmodium to artemisinins is now reported in several countries in South-East Asia. ACTs remain effective as long as the partner drug retains its activity but more and more clinical failures related to parasite resistance to both artemisinin and the partner molecule are reported. A major concern is the diffusion of these resistant parasites in sub-Saharan Africa, the continent most affected by malaria, as was the case in the past with other antimalarial treatments. It is therefore essential to better understand, from phenotypic and genotypic points of view, the mechanisms of resistance developed by the parasite Plasmodium falciparum face artemisinin and its derivatives in order to offer new therapeutic tools.L’utilisation, depuis plus de 15 ans, dans le traitement du paludisme de combinaisons thérapeutiques associant un dérivé de l’artémisinine avec une molécule partenaire (dénommées ACTs pour Artemisinin-based Combination Therapies), a permis une diminution notable de la mortalité dans les régions tropicales et subtropicales. Cependant ces progrès sont gravement menacés par la diminution de l’efficacité clinique des artémisinines caractérisée par une clairance parasitaire retardée et un taux de recrudescence élevé, et rapportée dès 2008 dans l’Ouest du Cambodge. Cette résistance de Plasmodium aux artémisinines s’est déjà étendue à plusieurs pays du Sud-est Asiatique. Cependant les ACTs restent efficaces tant que la molécule partenaire garde son activité, mais de plus en plus d’échecscliniques sont aujourd’hui corrélés à la résistance du parasite à la fois à l’artémisinine et à la molécule associée. Une des craintes majeures est la diffusion de ces parasites aux multiples résistances en Afrique subsaharienne, continent le plus touché par le paludisme, comme cela fut le cas par le passé avec d’autres traitements antipaludiques. Il est donc indispensable de mieux comprendre, d’un point de vue phénotypique et génotypique, la résistance du parasite Plasmodium falciparum à l’artémisinine et à ses dérivés afin de proposer de nouveaux outils thérapeutiques

Synthesis, characterization, and antileishmanial activities of gold(I) complexes involving quinoline functionalized N-heterocyclic carbenes

International audienceA series of new mononuclear cationic or neutral gold(I) complexes containing quinoline functionalized N-heterocyclic carbene(s) (NHC(s)) were synthesized and fully characterized by spectroscopic methods. The X-ray structures of two key compounds are presented. Proligands and their corresponding gold(I) complexes together with previously described silver(I) and gold(I) bis(NHC-quinoline) and gold(I) bis(NHC-methylbipyridine) complexes were evaluated in vitro towards Leishmania infantum. In parallel, the in vitro cytotoxicity of these molecules was assessed on the murine macrophages J774A.1. All gold(I) compounds show potent antileishmanial activity against L. infantum promastigotes and three of them are also efficient against L infantum intracellular amastigotes. Structure-activity and toxicity relationships enables to evidence a lead-compound (6) displaying both a high activity and a good selectivity index

resistance to artemisinin-based combination therapies: A sword of Damocles in the path toward malaria elimination

The use of artemisinin-based combination therapies (ACTs), which combine an artemisinin derivative with a partner drug, in the treatment of uncomplicated malaria has largely been responsible for the significant reduction in malaria-related mortality in tropical and subtropical regions. ACTs have also played a significant role in the 18% decline in the incidence of malaria cases from 2010 to 2016. However, this progress is seriously threatened by the reduced clinical efficacy of artemisinins, which is characterised by delayed parasitic clearance and a high rate of recrudescence, as reported in 2008 in Western Cambodia. Resistance to artemisinins has already spread to several countries in Southeast Asia. Furthermore, resistance to partner drugs has been shown in some instances to be facilitated by pre-existing decreased susceptibility to the artemisinin component of the ACT. A major concern is not only the spread of these multidrug-resistant parasites to the rest of Asia but also their possible appearance in Sub-Saharan Africa, the continent most affected by malaria, as has been the case in the past with parasite resistance to other antimalarial treatments. It is therefore essential to understand the acquisition of resistance to artemisinins by Plasmodium falciparum to adapt malaria treatment policies and to propose new therapeutic solutions

Plasmodium falciparum resistance to artemisinin-based combination therapies: A sword of Damocles in the path toward malaria elimination

The use of artemisinin-based combination therapies (ACTs), which combine an artemisinin derivative with a partner drug, in the treatment of uncomplicated malaria has largely been responsible for the significant reduction in malaria-related mortality in tropical and subtropical regions. ACTs have also played a significant role in the 18% decline in the incidence of malaria cases from 2010 to 2016. However, this progress is seriously threatened by the reduced clinical efficacy of artemisinins, which is characterised by delayed parasitic clearance and a high rate of recrudescence, as reported in 2008 in Western Cambodia. Resistance to artemisinins has already spread to several countries in Southeast Asia. Furthermore, resistance to partner drugs has been shown in some instances to be facilitated by pre-existing decreased susceptibility to the artemisinin component of the ACT. A major concern is not only the spread of these multidrug-resistant parasites to the rest of Asia but also their possible appearance in Sub-Saharan Africa, the continent most affected by malaria, as has been the case in the past with parasite resistance to other antimalarial treatments. It is therefore essential to understand the acquisition of resistance to artemisinins by Plasmodium falciparum to adapt malaria treatment policies and to propose new therapeutic solutions

A new, rapid and sensitive bioluminescence assay for drug screening on Leishmania

International audienceWe validated anew method, based on luciferine/luciferase bioluminescence, for drug screening on promastigotes of different Leishmania species. Results obtained with this new, rapid, reproducible, and reliable method are in good accordance with results obtained by the conventional MTT assay. This bioluminescence assay has a lower detection limit. (C) 2013 Elsevier B.V. All rights reserved

Epigenetic regulation as a therapeutic target in the malaria parasite Plasmodium falciparum

Abstract Over the past thirty years, epigenetic regulation of gene expression has gained increasing interest as it was shown to be implicated in illnesses ranging from cancers to parasitic diseases. In the malaria parasite, epigenetics was shown to be involved in several key steps of the complex life cycle of Plasmodium, among which asexual development and sexual commitment, but also in major biological processes like immune evasion, response to environmental changes or DNA repair. Because epigenetics plays such paramount roles in the Plasmodium parasite, enzymes involved in these regulating pathways represent a reservoir of potential therapeutic targets. This review focuses on epigenetic regulatory processes and their effectors in the malaria parasite, as well as the inhibitors of epigenetic pathways and their potential as new anti-malarial drugs. Such types of drugs could be formidable tools that may contribute to malaria eradication in a context of widespread resistance to conventional anti-malarials