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Selective allosteric modulators bear great potential to fine-tune neurotransmitter-induced brain receptor responses. Promising targets are metabotropic glutamate (mGlu) receptors, which are associated to different brain diseases. These multidomain class C GPCRs experience concerted structural rearrangements and rely on allosteric modulation of agonist action to be fully activated. Here we establish live cell compatible fluorescence labeling of mGlu2 by click chemistry through genetic code expansion. Using lanthanide resonance energy transfer, we establish multiple FRET sensors to monitor ligand effects on conformational changes in mGlu2 extracellular domain and subsequently dissect the underlying conformational states by smFRET. Using three distinct FRET sensors, we demonstrate that mGlu activation relies on a ligand-induced sampling of three conformational states. Orthosteric agonists act by promoting the closure of the mGlu2 ligand binding domains, leading to an equilibrium between an inactive intermediate and the active state. Allosteric modulator further push this equilibrium toward the active state, promoting and stabilizing the relative reorientation of the mGlu protomers. These results underline the complex and dynamic nature of such type of neuroreceptors, pointing out that ligands fine-tune activation by differentially acting on the equilibria between multiple states.

International audience; Cell surface receptors represent a vast majority of drug targets. Efforts have been conducted to develop biosensors reporting their conformational changes in live cells for pharmacological and functional studies. Although Förster resonance energy transfer (FRET) appears to be an ideal approach, its use is limited by the low signal-to-noise ratio. Here we report a toolbox composed of a combination of labeling technologies, specific fluorophores compatible with time-resolved FRET and a novel method to quantify signals. This approach enables the development of receptor biosensors with a large signal-to-noise ratio. We illustrate the usefulness of this toolbox through the development of biosensors for various G-protein-coupled receptors and receptor tyrosine kinases. These receptors include mGlu, GABAB, LH, PTH, EGF and insulin receptors among others. These biosensors can be used for high-throughput studies and also revealed new information on the activation process of these receptors in their cellular environment.

International audience; The main inhibitory neurotransmitter, γ-aminobutyric acid (GABA), modulates many synapses by activating the G protein-coupled receptor GABAB, which is a target for various therapeutic applications. It is an obligatory heterodimer made of GB1 and GB2 that can be regulated by positive allosteric modulators (PAMs). The molecular mechanism of activation of the GABAB receptor remains poorly understood. Here, we have developed FRET-based conformational GABAB sensors compatible with high-throughput screening. We identified conformational changes occurring within the extracellular and transmembrane domains upon receptor activation, which are smaller than those observed in the related metabotropic glutamate receptors. These sensors also allow discrimination between agonists of different efficacies and between PAMs that have different modes of action, which has not always been possible using conventional functional assays. Our study brings important new information on the activation mechanism of the GABAB receptor and should facilitate the screening and identification of new chemicals targeting this receptor.

The main inhibitory neurotransmitter, γ-aminobutyric acid (GABA), modulates many synapses by activating the G protein-coupled receptor GABA

The Na(+)/I(-) symporter (NIS) mediates iodide uptake in the thyroid gland as well as in other NIS-expressing cells. This transport is the basis for an emerging approach to selective cancer cell destruction by using radioiodide after targeted NIS gene transfer. Therapeutic efficacy requires that radioiodide retention be maximized in tumor cells. A first generation of forty imidazo[2,1-b]thiazole derivatives as iodide efflux inhibitors is described along with the evaluation of their biological properties. Structure-activity relationship studies by using radioiodide uptake in rat thyroid-derived cells (FRTL5) revealed that the 5,6-dihydroimidazo[2,1-b]thiazole heterocycle is required for activity. Introduction of electron-donor substituents on the 3-biphenyl moiety led to the discovery of novel potent compounds. A compound was identified with enhanced potency compared to reference 1. These molecules give the possibility to increase the cellular retention of radioiodide in NIS-expressing tumors, leading to higher absorbed doses and killing efficacy.

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.

The Na(+)/I(-) symporter (NIS) mediates iodide uptake into thyroid follicular cells. Although NIS has been cloned and thoroughly studied at the molecular level, the biochemical processes involved in post-translational regulation of NIS are still unknown. The purpose of this study was to identify and characterize inhibitors of NIS function. These small organic molecules represent a starting point in the identification of pharmacological tools for the characterization of NIS trafficking and activation mechanisms. The screening of a collection of 17,020 druglike compounds revealed new chemical inhibitors with potencies down to 40 nM. Fluorescence measurement of membrane potential indicates that these inhibitors do not act by disrupting the sodium gradient. They allow immediate and total iodide discharge from preloaded cells in accord with a specific modification of NIS activity, probably through distinct mechanisms.

A high-throughput screening method based on radioiodide uptake in human embryonic kidney 293 cells expressing the human sodium/iodide symporter was developed. Central to assay development was a homogeneous cell culture in the 96-well microplate coupled with the use of scintillation proximity technology. The assay is fast and highly reproducible with a Z' greater than 0.8. The automated procedure allows the screening of 4,000 compounds per day. Using this methodology, several known substrates of the sodium/iodide symporter were evaluated in a single day. Inhibition of iodide uptake was shown to follow the series PF(6)(-)ClO(4)(-)BF(4)(-)SCN(-)NO(3)(-)IO(4)(-)N(3)(-)Br(-), in accord with the literature. This method represents an initial approach to the search for inhibitors of iodide transport mediated by the sodium/iodide symporter.

In many streptococci, competence for natural DNA transformation is regulated by the Rgg-type regulator ComR and the pheromone ComS, which is sensed intracellularly. We compared the ComRS systems of four model streptococcal species using in vitro and in silico approaches, to determine the mechanism of the ComRS-dependent regulation of competence. In all systems investigated, ComR was shown to be the proximal transcriptional activator of the expression of key competence genes. Efficient binding of ComR to DNA is strictly dependent on the presence of the pheromone (C-terminal ComS octapeptide), in contrast with other streptococcal Rgg-type regulators. The 20 bp palindromic ComR-box is the minimal genetic requirement for binding of ComR, and its sequence directly determines the expression level of genes under its control. Despite the apparent species-specific specialization of the ComR-ComS interaction, mutagenesis of ComS residues from Streptococcus thermophilus highlighted an unexpected permissiveness with respect to its biological activity. In agreement, heterologous ComS, and even primary sequence-unrelated, casein-derived octapeptides, were able to induce competence development in S. thermophilus. The lack of stringency of ComS sequence suggests that competence of a specific Streptococcus species may be modulated by other streptococci or by non-specific nutritive oligopeptides present in its environment.

In this paper, we describe the screening of a 14640-compound library using a novel whole mycobacteria phenotypic assay to discover inhibitors of EthR, a transcriptional repressor implicated in the innate resistance of Mycobacterium tuberculosis to the second-line antituberculosis drug ethionamide. From this screening a new chemical family of EthR inhibitors bearing an N-phenylphenoxyacetamide motif was identified. The X-ray structure of the most potent compound crystallized with EthR inspired the synthesis of a 960-member focused library. These compounds were tested in vitro using a rapid thermal shift assay on EthR to accelerate the optimization. The best compounds were synthesized on a larger scale and confirmed as potent ethionamide boosters on M. tuberculosis-infected macrophages. Finally, the cocrystallization of the best optimized analogue with EthR revealed an unexpected reorientation of the ligand in the binding pocket. © 2012 American Chemical Society.

Mycobacterial transcriptional repressor EthR controls the expression of EthA, the bacterial monooxygenase activating ethionamide, and is thus largely responsible for the low sensitivity of the human pathogen Mycobacterium tuberculosis to this antibiotic. We recently reported structure-activity relationships of a series of 1,2,4-oxadiazole EthR inhibitors leading to the discovery of potent ethionamide boosters. Despite high metabolic stability, pharmacokinetic evaluation revealed poor mice exposure; therefore, a second phase of optimization was required. Herein a structure-property relationship study is reported according to the replacement of the two aromatic heterocycles: 2-thienyl and 1,2,4-oxadiazolyl moieties. This work was done using a combination of structure-based drug design and in vitro/ex vivo evaluations of ethionamide boosters on the targeted protein EthR and on the human pathogen Mycobacterium tuberculosis. Thanks to this process, we identified compound 42 (BDM41906), which displays improved efficacy in addition to high exposure to mice after oral administration.

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., JOURNAL ARTICLE, SCOPUS: ar.j, info:eu-repo/semantics/published

We report in this article an extensive structure-activity relationships (SAR) study with 58 thiophen-2-yl-1,2,4-oxadiazoles as inhibitors of EthR, a transcriptional regulator controling ethionamide bioactivation in Mycobacterium tuberculosis. We explored the replacement of two key fragments of the starting lead BDM31343. We investigated the potency of all analogues to boost subactive doses of ethionamide on a phenotypic assay involving M. tuberculosis infected macrophages and then ascertained the mode of action of the most active compounds using a functional target-based surface plasmon resonance assay. This process revealed that introduction of 4,4,4-trifluorobutyryl chain instead of cyanoacetyl group was crucial for intracellular activity. Replacement of 1,4-piperidyl by (R)-1,3-pyrrolidyl scaffold did not enhance activity but led to improved pharmacokinetic properties. Furthermore, the crystal structures of ligand-EthR complexes were consistent with the observed SAR. In conclusion, we identified EthR inhibitors that boost antibacterial activity of ethionamide with nanomolar potency while improving solubility and metabolic stability.

Four photoreactive analogues of 3-biphenyl-4'-yl-5,6-dihydroimidazo[2,1-b]thiazole were prepared and evaluated as iodide sequestering agents in sodium iodide symporter-expressing cells. One of these new photoactivatable compounds retained biological activity and was further radiolabeled with tritium. This compound may provide a useful tool for labeling, purification, and identification of target protein responsible for iodide efflux in thyrocytes.

The ability of the sodium/iodide symporter (NIS) to take up iodide has long provided the basis for cytoreductive gene therapy and cancer treatment with radioiodide. One of the major limitations of this approach is that radioiodide retention in NIS-expressing cells is not sufficient for their destruction. We identified and characterized a small organic molecule capable of increasing iodide retention in HEK293 cells permanently transfected with human NIS cDNA (hNIS-HEK293) and in the rat thyroid-derived cell line FRTL-5. In the presence of 3-biphenyl-4′-yl-5,6-dihydroimidazo[2,1-b]thiazole (ISA1), the transmembrane iodide concentration gradient was increased up to 4.5-fold. Our experiments indicate that the imidazothiazole derivative acts either by inhibiting anion efflux mechanisms, or by promoting the relocation of iodide into subcellular compartments. This new compound is not only an attractive chemical tool to investigate the mechanisms of iodide flux at the cellular level, but also opens promising perspectives in the treatment of cancer after NIS gene transfer.