High Content Screening Identifies Decaprenyl-Phosphoribose 2′ Epimerase as a Target for Intracellular Antimycobacterial Inhibitors

A critical feature of Mycobacterium tuberculosis, the causative agent of human tuberculosis (TB), is its ability to survive and multiply within macrophages, making these host cells an ideal niche for persisting microbes. Killing the intracellular tubercle bacilli is a key requirement for efficient tuberculosis treatment, yet identifying potent inhibitors has been hampered by labor-intensive techniques and lack of validated targets. Here, we present the development of a phenotypic cell-based assay that uses automated confocal fluorescence microscopy for high throughput screening of chemicals that interfere with the replication of M. tuberculosis within macrophages. Screening a library of 57,000 small molecules led to the identification of 135 active compounds with potent intracellular anti-mycobacterial efficacy and no host cell toxicity. Among these, the dinitrobenzamide derivatives (DNB) showed high activity against M. tuberculosis, including extensively drug resistant (XDR) strains. More importantly, we demonstrate that incubation of M. tuberculosis with DNB inhibited the formation of both lipoarabinomannan and arabinogalactan, attributable to the inhibition of decaprenyl-phospho-arabinose synthesis catalyzed by the decaprenyl-phosphoribose 2′ epimerase DprE1/DprE2. Inhibition of this new target will likely contribute to new therapeutic solutions against emerging XDR-TB. Beyond validating the high throughput/content screening approach, our results open new avenues for finding the next generation of antimicrobials

High Content Screening Identifies Decaprenyl-Phosphoribose 2’ Epimerase as a Target for Intracellular Antimycobacterial Inhibitors

A critical feature of Mycobacterium tuberculosis, the causative agent of human tuberculosis (TB), is its ability to survive and multiply within macrophages, making these host cells an ideal niche for persisting microbes. Killing the intracellular tubercle bacilli is a key requirement for efficient tuberculosis treatment, yet identifying potent inhibitors has been hampered by labor-intensive techniques and lack of validated targets. Here, we present the development of a phenotypic cell-based assay that uses automated confocal fluorescence microscopy for high throughput screening of chemicals that interfere with the replication of M. tuberculosis within macrophages. Screening a library of 57,000 small molecules led to the identification of 135 active compounds with potent intracellular anti-mycobacterial efficacy and no host cell toxicity. Among these, the dinitrobenzamide derivatives (DNB) showed high activity against M. tuberculosis, including extensively drug resistant (XDR) strains. More importantly, we demonstrate that incubation of M. tuberculosis with DNB inhibited the formation of both lipoarabinomannan and arabinogalactan, attributable to the inhibition of decaprenyl-phospho-arabinose synthesis catalyzed by the decaprenyl-phosphoribose 2\u27 epimerase DprE1/DprE2. Inhibition of this new target will likely contribute to new therapeutic solutions against emerging XDR-TB. Beyond validating the high throughput/content screening approach, our results open new avenues for finding the next generation of antimicrobials

Discovery of Q203, a potent clinical candidate for the treatment of tuberculosis

New therapeutic strategies are needed to combat the tuberculosis pandemic and the spread of multidrug-resistant (MDR) and extensively drug-resistant (XDR) forms of the disease, which remain a serious public health challenge worldwide1, 2. The most urgent clinical need is to discover potent agents capable of reducing the duration of MDR and XDR tuberculosis therapy with a success rate comparable to that of current therapies for drug-susceptible tuberculosis. The last decade has seen the discovery of new agent classes for the management of tuberculosis3, 4, 5, several of which are currently in clinical trials6, 7, 8. However, given the high attrition rate of drug candidates during clinical development and the emergence of drug resistance, the discovery of additional clinical candidates is clearly needed. Here, we report on a promising class of imidazopyridine amide (IPA) compounds that block Mycobacterium tuberculosis growth by targeting the respiratory cytochrome bc1 complex. The optimized IPA compound Q203 inhibited the growth of MDR and XDR M. tuberculosis clinical isolates in culture broth medium in the low nanomolar range and was efficacious in a mouse model of tuberculosis at a dose less than 1 mg per kg body weight, which highlights the potency of this compound. In addition, Q203 displays pharmacokinetic and safety profiles compatible with once-daily dosing. Together, our data indicate that Q203 is a promising new clinical candidate for the treatment of tuberculosis

Age at onset as stratifier in idiopathic Parkinson’s disease – effect of ageing and polygenic risk score on clinical phenotypes

Several phenotypic differences observed in Parkinson’s disease (PD) patients have been linked to age at onset (AAO). We endeavoured to find out whether these differences are due to the ageing process itself by using a combined dataset of idiopathic PD (n = 430) and healthy controls (HC; n = 556) excluding carriers of known PD-linked genetic mutations in both groups. We found several significant effects of AAO on motor and non-motor symptoms in PD, but when comparing the effects of age on these symptoms with HC (using age at assessment, AAA), only positive associations of AAA with burden of motor symptoms and cognitive impairment were significantly different between PD vs HC. Furthermore, we explored a potential effect of polygenic risk score (PRS) on clinical phenotype and identified a significant inverse correlation of AAO and PRS in PD. No significant association between PRS and severity of clinical symptoms was found. We conclude that the observed non-motor phenotypic differences in PD based on AAO are largely driven by the ageing process itself and not by a specific profile of neurodegeneration linked to AAO in the idiopathic PD patients

Implications fonctionnelles de l organisation de la chromatine (Rôles du pore nucléaire chez saccharomyces cerevisiae)

Dans le noyau des cellules eucaryotes, la chromatine ainsi que les processus métaboliques qui l accompagnent ne sont pas distribués de manière aléatoire. Au cours de ma thèse, je me suis intéressé au rôle que pouvait jouer cette organisation dans la régulation de la réparation de l ADN et de la transcription. J ai participé à la mise au point de méthodes permettant de localiser in vivo un locus chromosomique unique et de le suivre au court du temps chez la levure Saccharomyces cerevisiae. J ai ainsi pu montrer que les gènes GAL sont confinés dans des territoires bien définis et que leur mobilité est très contrainte. Par ailleurs, lorsqu ils sont transcrits, les mouvements de ces gènes sont confinés le long de l enveloppe nucléaire. Mes travaux ont également montré que le complexe d initiation de la transcription SAGA interagissant avec le promoteur et les pores nucléaires (NPC) via la machinerie d export des ARNm, était responsable de l ancrage des gènes GAL à l enveloppe nucléaire. Ces résultats constituent la première description du mécanisme de "gene gating". L utilisation des techniques d imagerie cellulaire, nous a aussi permis de montrer que la réparation des cassures de l ADN dans les régions subtélomériques dépendait de leur ancrage aux NPC. Enfin, l implication des pores dans le contrôle de la réparation de l ADN a été confortée par l identification d interactions fonctionnelles entre certaines nucléoporines, et des protéines impliquées dans la réparation de l ADN. Ensembles mes travaux démontrent l importance des NPC dans l organisation fonctionnelle de la chromatine chez S. cerevisiae.In the nucleus of eukaryotic cells, chromatin and nuclear processes are not randomly distributed. During my PhD thesis, I have focused on the role of nuclear organization may play in regulating transcriptional regulation and DNA metabolism. To investigate this assumption, I developed an experimental system able to monitor the movement and sub-nuclear position of a single tagged genetic locus in the yeast Saccharomyces cerevisiae. When tracked in the nuclear volume over time, I found chromatin to undergo very constrained movement. Interestingly, I show that transcriptional activation of the GAL genes leads to the confinement of their motility towards the nuclear periphery. I further demonstrate that members of the SAGA transcription initiation complex and mRNA export factors mediate this recruitment by physically linking the activated GAL genes to nuclear pore complexes (NPC). These results prove for the first time that the gene gating mechanism occurs in living cells. Additionally, I participated in a study showing that binding of chromosome ends to the NPC is essential for efficient DNA double strand break repair in subtelomeric region. I also performed a genetic screen revealing an exciting genetic interaction network of nucleoporins with the DNA repair machinery and chromatin remodeling complexes. Altogether the studies I carried out during my PhD uncover the role of the NPC in chromatin organization and consequently in regulating nuclear processes.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Quality control of messenger ribonucleoprotein particles in the nucleus and at the pore.

International audienceThe spatial separation of nuclear transcription and cytoplasmic translation in eukaryotic cells implies that mRNAs have to travel. On their journey, proteins involved in the various steps of transcript formation, processing and transport dynamically interact with mRNAs to form diverse messenger ribonucleoprotein complexes (mRNPs). Increasing evidence indicates that the protein complexes involved in distinct phases of manufacturing a bona fide mRNA in the nucleus are tightly coupled. Moreover, the recent demonstration that active genes migrate into preassembled, shared nuclear sub-compartments suggests that mRNAs are churned out in large 'transcription factories' with distinct but interconnected divisions. Nuclear factors have now been identified that specifically control the quality of mRNAs without affecting mRNP biogenesis or export

Analysing the role of a cellular mechanism in HIV infection and evaluation of novel antiviral compounds

VIH est responsable du syndrome d'immunodéficience acquise (SIDA). L'étape fondamentale pour découvrir de nouvelles stratégies antivirales reste la compréhension de la pathogénie du SIDA ainsi que les mécanismes moléculaires de l'interaction du virus avec l'hôte.Une caractéristique des lentivirus est le transport actif du complexe de pré-intégration (PIC) dans le noyau de la cellule hôte. Le mécanisme de ce transport n'est pas connu, mais Vpr (viral protein R) pourrait être impliqué dans ce processus. Vpr reste associée au PIC jusqu'à la translocation du génome à travers la membrane nucléaire. Par ailleurs, il a été montré que Vpr est capable d'intéragir avec le complexe du pore nucléaire (NPC). Cela laisse soupçonner que cette interaction est à la base de l'import nucléaire du PIC. Il a également été montré que le cytosquelette joue un rôle prépondérant dans le transport de particules virales. Dans cette perspective, nous avons observé que la membrane nucléaire est entourée d'actine. Nous avons mis au point une nouvelle approche pour visualiser l'actine dans les cellules vivantes, ce qui nous a permis de visualiser un réservoir d'actine qui polymérise autour de l'enveloppe nucléaire ainsi que dans les invaginations de cette membrane. Nous avons montré que des enveloppes nucléaires isolées suffisent à initier la polymérisation de filaments d'actine in vitro. Nos résultats démontrent que la dynamique de l'actine périnucléaire est orchestrée par l'enveloppe nucléaire elle-même. C'est donc via la liaison de Vpr au NPC que le virus pourrait exploiter la présence de l'actine comme stratégie pour pénétrer dans le noyau en dépit de la barrière que constitue l'enveloppe.HIV is the causative agent of the acquired immunodeficiency syndrome (AIDS). For the development of novel antiviral compounds, understanding of the lifecycle as well as the molecular mechanisms of host pathogen interactions are a prerequisite.A particular feature of lentiviruses is the active transport of the pre-integration complex (PIC) into the nucleus. The mechanism remains elusive, yet in the case of HIV Vpr is one candidate possibly implicated in the process. The protein stays associated to the PIC until nuclear translocation. It has been shown, that Vpr binds to the nuclear pore complex. This interaction might therefore be the link mediating import of the PIC. Moreover the cytoskeleton has been shown to play a major role in cytoplasmic transport of viral particles. In this context, we observed the nuclear envelope (NE) to be embedded in a perinuclear actin shell and displaying highly dynamic nuclear invaginations. We developed a novel approach to visualize actin inside living cells and thus we could show a high turnover actin pool around the NE and inside NE invaginations of living cells. Although reported in different cell types, the possible role of perinuclear actin filaments in the dynamic structural plasticity of the NE remains unresolved. We could show that NE-membranes alone are sufficient to nucleate polymerizing actin filaments in vitro, involving both actin recruitment to their surface, and filament growth. Accordingly, our results demonstrate that perinuclear actin dynamics are orchestrated by the NE itself. By binding of Vpr to the NPC, the virus could possibly exploit this pool of polymerizing actin as a new strategy to overcome the nuclear membrane.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF