Potent prearranged positive allosteric modulators of the glucagon-like peptide-1 receptor

Drugs that allosterically modulate G protein-coupled receptor (GPCR) activity display higher specificity and may improve disease treatment. However, the rational design of compounds that target the allosteric site is difficult, as conformations required for receptor activation are poorly understood. Guided by photopharmacology, a set of prearranged positive allosteric modulators (PAMs) with restricted degrees of freedom was designed and tested against the glucagon-like peptide-1 receptor (GLP-1R), a GPCR involved in glucose homeostasis. Compounds incorporating a trans-stilbene comprehensively outperformed those with a cis-stilbene, as well as the benchmark BETP, as GLP-1R PAMs. We also identified major effects of ligand conformation on GLP-1R binding kinetics and signal bias. Thus, we describe a photopharmacology-directed approach for rational drug design, and introduce a new class of stilbene-containing PAM for the specific regulation of GPCR activity

Structural activation of the transcriptional repressor EthR from Mycobacterium tuberculosis by single amino acid change mimicking natural and synthetic ligands

Ethionamide is an antituberculous drug for the treatment of multidrug-resistant Mycobacterium tuberculosis. This antibiotic requires activation by the monooxygenase EthA to exert its activity. Production of EthA is controlled by the transcriptional repressor EthR, a member of the TetR family. The sensitivity of M. tuberculosis to ethionamide can be artificially enhanced using synthetic ligands of EthR that allosterically inactivate its DNA-binding activity. Comparison of several structures of EthR co-crystallized with various ligands suggested that the structural reorganization of EthR resulting in its inactivation is controlled by a limited portion of the ligand-binding-pocket. In silico simulation predicted that mutation G106W may mimic ligands. X-ray crystallography of variant G106W indeed revealed a protein structurally similar to ligand-bound EthR. Surface plasmon resonance experiments established that this variant is unable to bind DNA, while thermal shift studies demonstrated that mutation G106W stabilizes EthR as strongly as ligands. Proton NMR of the methyl regions showed a lesser contribution of exchange broadening upon ligand binding, and the same quenched dynamics was observed in apo-variant G106W. Altogether, we here show that the area surrounding Gly106 constitutes the molecular switch involved in the conformational reorganization of EthR. These results also shed light on the mechanistic of ligand-induced allosterism controlling the DNA binding properties of TetR family repressors

Identification d inhibiteurs du symporteur sodium-iode par criblage à haut débit

Ce travail de thèse a été consacré à la recherche d'inhibiteurs du Symporteur Sodium-Iode ou NIS. Le NIS est une protéine membranaire localisée sur la membrane basolatérale des thyrocytes. Il permet le transport de ions iodures du plasma dans le thyrocyte, ce qui constitue la première étape de la biosynthèse des hormones iodées T 3 et T 4 produites par la thyroïde. La campagne de criblage à haut débit, mise au point sur les cellules HEK-293 sur exprimant la protéine NIS, sur une chimiothèque de 16 720 composés a permis de sélectionner 413 composés bloquant l'entrée d'iodures dans les cellules. Des tests de validation ainsi que la caractérisation de ces composés ont finalement permis d'identifier 10 inhibiteurs puissants et spécifiques du NIS humain, du rat et de la souris possédant des ECSO proches du micro-molaire. Un inhibiteur supplémentaire a été identifié en criblant une chimiothèque interne de 320 peptidomimétiques issus de chimie combinatoire. Parallèlement, un criblage orienté d'une chimiothèque d'organotrifluoroborates a permis de caractériser 3 autres inhibiteurs spécifiques du NIS. Un autre composé a été identifié comme activateur d'entrée d'iodures dans les thyrocytes de rats en culture. L'analyse de son mode d'action a permis de suggérer un mécanisme de maintien des ions iodures dans le thyrocyte. La synthèse d'analogues de ce composé a permis d'identifier un composé encore plus actif et aussi de proposer des relations de structure-activité de cette famille de molécules. Enfin, la synthèse de sondes photoactivables a été initiée afin d'identifier le mode d'action de ce composé sur les thyrocytes.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Caracterización de inhibidores de molécula pequeña del simportador de yoduro de sodio

The sodium/iodide symporter (NIS) mediates the active transport of iodide from the bloodstream into thyrocytes. NIS function is strategic for the diagnosis and treatment of various thyroid diseases. In addition, a promising anti-cancer strategy based on targeted NIS gene transfer in non-thyroidal cells is currently developed. However, only little information is available concerning the molecular mechanism of NIS-mediated iodide translocation. Ten small molecules have recently been identified using a high-throughput screening method for their inhibitory effect on iodide uptake of NIS-expressing mammalian cells. In the present study, we analyzed these compounds for their rapid and reversible effects on the iodide-induced current in NIS-expressing Xenopus oocytes. Four molecules almost completely inhibited the iodide-induced current; for three of them the effect was irreversible, for one compound the initial current could be fully re-established after washout. Three molecules showed a rapid inhibitory effect of about 75%, half of which was reversible. Another three compounds inhibited the iodide-induced current from 10 to 50%. Some molecules altered the membrane conductance by themselves, i.e. in the absence of iodide. For one of these molecules the observed effect was also found in water-injected oocytes whereas for some others the iodide-independent effect was associated with NIS expression. The tested molecules show a surprisingly high variability in their possible mode of action, and thus are promising tools for further functional characterization of NIS on a molecular level, and they could be useful for medical applications

Caracterización de inhibidores de molécula pequeña del simportador de yoduro de sodio

The sodium/iodide symporter (NIS) mediates the active transport of iodide from the bloodstream into thyrocytes. NIS function is strategic for the diagnosis and treatment of various thyroid diseases. In addition, a promising anti-cancer strategy based on targeted NIS gene transfer in non-thyroidal cells is currently developed. However, only little information is available concerning the molecular mechanism of NIS-mediated iodide translocation. Ten small molecules have recently been identified using a high-throughput screening method for their inhibitory effect on iodide uptake of NIS-expressing mammalian cells. In the present study, we analyzed these compounds for their rapid and reversible effects on the iodide-induced current in NIS-expressing Xenopus oocytes. Four molecules almost completely inhibited the iodide-induced current; for three of them the effect was irreversible, for one compound the initial current could be fully re-established after washout. Three molecules showed a rapid inhibitory effect of about 75%, half of which was reversible. Another three compounds inhibited the iodide-induced current from 10 to 50%. Some molecules altered the membrane conductance by themselves, i.e. in the absence of iodide. For one of these molecules the observed effect was also found in water-injected oocytes whereas for some others the iodide-independent effect was associated with NIS expression. The tested molecules show a surprisingly high variability in their possible mode of action, and thus are promising tools for further functional characterization of NIS on a molecular level, and they could be useful for medical applications