Physiological and Molecular Function of the Sodium/Hydrogen Exchanger NHA2 (SLC9B2)

NHA2, also known as SLC9B2, is an orphan intracellular Na+/H+ exchanger (NHE) that has been associated with arterial hypertension and diabetes mellitus in humans. The objective of this NCCR TransCure project was to define the physiological and molecular function of NHA2, to develop a high resolution kinetic transport assay for NHA2 and to identify specific and potent compounds targeting NHA2. In this review, we summarize the results of this highly interdisciplinary and interfaculty effort, led by the groups of Proffs. Jean-Louis Reymond, Christoph von Ballmoos and Daniel Fuster

Discovery of novel drug-like antitubercular hits targeting the MEP pathway enzyme DXPS by strategic application of ligand-based virtual screening.

In the present manuscript, we describe how we successfully used ligand-based virtual screening (LBVS) to identify two small-molecule, drug-like hit classes with excellent ADMET profiles against the difficult to address microbial enzyme 1-deoxy-d-xylulose-5-phosphate synthase (DXPS). In the fight against antimicrobial resistance (AMR), it has become increasingly important to address novel targets such as DXPS, the first enzyme of the 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway, which affords the universal isoprenoid precursors. This pathway is absent in humans but essential for pathogens such as Mycobacterium tuberculosis, making it a rich source of drug targets for the development of novel anti-infectives. Standard computer-aided drug-design tools, frequently applied in other areas of drug development, often fail for targets with large, hydrophilic binding sites such as DXPS. Therefore, we introduce the concept of pseudo-inhibitors, combining the benefits of pseudo-ligands (defining a pharmacophore) and pseudo-receptors (defining anchor points in the binding site), for providing the basis to perform a LBVS against M. tuberculosis DXPS. Starting from a diverse set of reference ligands showing weak inhibition of the orthologue from Deinococcus radiodurans DXPS, we identified three structurally unrelated classes with promising in vitro (against M. tuberculosis DXPS) and whole-cell activity including extensively drug-resistant strains of M. tuberculosis. The hits were validated to be specific inhibitors of DXPS and to have a unique mechanism of inhibition. Furthermore, two of the hits have a balanced profile in terms of metabolic and plasma stability and display a low frequency of resistance development, making them ideal starting points for hit-to-lead optimization of antibiotics with an unprecedented mode of action

Discovery of novel drug-like antitubercular hits targeting the MEP pathway enzyme DXPS by strategic application of ligand-based virtual screening

In the present manuscript, we describe how we successfully used ligand-based virtual screening (LBVS) to identify two small-molecule, drug-like hit classes with excellent ADMET profiles against the difficult to address microbial enzyme 1-deoxy-D-xylulose-5-phosphate synthase (DXPS). In the fight against antimicrobial resistance (AMR), it has become increasingly important to address novel targets such as DXPS, the first enzyme of the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway, which affords the universal isoprenoid precursors. This pathway is absent in humans but essential for pathogens such as Mycobacterium tuberculosis, making it a rich source of drug targets for the development of novel anti-infectives. Standard computer-aided drug-design tools, frequently applied in other areas of drug development, often fail for targets with large, hydrophilic binding sites such as DXPS. Therefore, we introduce the concept of pseudo-inhibitors, combining the benefits of pseudo-ligands (defining a pharmacophore) and pseudo-receptors (defining anchor points in the binding site), for providing the basis to perform a LBVS against M. tuberculosis DXPS. Starting from a diverse set of reference ligands showing weak inhibition of the orthologue from Deinococcus radiodurans DXPS, we identified three structurally unrelated classes with promising in vitro (against M. tuberculosis DXPS) and whole-cell activity including extensively drug-resistant strains of M. tuberculosis. The hits were validated to be specific inhibitors of DXPS and to have a unique mechanism of inhibition. Furthermore, two of the hits have a balanced profile in terms of metabolic and plasma stability and display a low frequency of resistance development, making them ideal starting points for hit-to-lead optimization of antibiotics with an unprecedented mode of action

Devenir à long terme d'une série d'adolescents souffrant d'anorexie mentale hospitalisés dans un service de pédiatrie

AIX-MARSEILLE2-BU Méd/Odontol. (130552103) / SudocPARIS-BIUM (751062103) / SudocSudocFranceF

Discovery and characterization of a novel non-competitive inhibitor of the divalent metal transporter DMT1/SLC11A2

Divalent metal transporter-1 (SLC11A2/DMT1) uses the H+ electrochemical gradient as the driving force to transport divalent metal ions such as Fe2+, Mn2+ and others metals into mammalian cells. DMT1 is ubiquitously expressed, most notably in proximal duodenum, immature erythroid cells, brain and kidney. This transporter mediates H+-coupled transport of ferrous iron across the apical membrane of enterocytes. In addition, in cells such as to erythroid precursors, following transferrin receptor (TfR) mediated endocytosis; it mediates H+-coupled exit of ferrous iron from endocytic vesicles into the cytosol. Dysfunction of human DMT1 is associated with several pathologies such as iron deficiency anemia hemochromatosis, Parkinson's disease and Alzheimer's disease, as well as colorectal cancer and esophageal adenocarcinoma, making DMT1 an attractive target for drug discovery. In the present study, we performed a ligand-based virtual screening of the Princeton database (700,000 commercially available compounds) to search for pharmacophore shape analogs of recently reported DMT1 inhibitors. We discovered a new compound, named pyrimidinone 8, which mediates a reversible linear non-competitive inhibition of human DMT1 (hDMT1) transport activity with a Ki of ∼20 μM. This compound does not affect hDMT1 cell surface expression and shows no dependence on extracellular pH. To our knowledge, this is the first experimental evidence that hDMT1 can be allosterically modulated by pharmacological agents. Pyrimidinone 8 represents a novel versatile tool compound and it may serve as a lead structure for the development of therapeutic compounds for pre-clinical assessment

Characterisation of the spermosphere of the common bean seed (Phaseolus vulgaris)

International audienceSeed plants have co-evoluted with microorganisms by forming beneficial relationships in order to facilitate their adaptation to terrestrial lifestyle. Indeed, more and more studies are demonstrating the inter-relationships of the microbiome and their hosts and the resulting impacts of such relationships in terms of the host’s fitness, environmental response, adaptation and evolution. These studies have given rise to a relatively new concept of the holobiont which views the host organism and its associated microbiota as a single functional unit, where evolutionary selection occurs between both the host and its microbiota, as well as among different microbial species within the microbiota. While plant microbiome in the phyllosphere and rhizosphere has been well studied, seed spermosphere and microbiome have remain relatively unnoticed. Yet, seeds have an important distinctive function in the inheritance process by connecting plant generations and serving as a critical point in the continuity of the plant microbiome. However, the fate of seed-borne microorganisms after germination is relatively unexplored. Seed germination, a transient process giving rise to a microenvironment known as the spermosphere, is a critical point for the establishment of the future plant microbiome. Indeed, seed exudation after imbibition is hypothesized to be the driving force for microbial selection, growth and interaction in the spermosphere. The aim of this study is to characterize the germinating bean spermosphere with multi-omics technologies to understand the complex interactions between seed bornemicroorganisms and seed exudate

Optimization of TRPV6 Calcium Channel Inhibitors Using a 3D Ligand-Based Virtual Screening Method

Herein, we report the discovery of the first potent and selective inhibitor of TRPV6, a calcium channel overexpressed in breast and prostate cancer, and its use to test the effect of blocking TRPV6-mediated Ca2+-influx on cell growth. The inhibitor was discovered through a computational method, xLOS, a 3D-shape and pharmacophore similarity algorithm, a type of ligand-based virtual screening (LBVS) method described briefly here. Starting with a single weakly active seed molecule, two successive rounds of LBVS followed by optimization by chemical synthesis led to a selective molecule with 0.3 μM inhibition of TRPV6. The ability of xLOS to identify different scaffolds early in LBVS was essential to success. The xLOS method may be generally useful to develop tool compounds for poorly characterized targets

Recombinant Human Melatonin Receptor MT1 Isolated in Mixed Detergents Shows Pharmacology Similar to That in Mammalian Cell Membranes

<div><p>The human melatonin MT1 receptor—belonging to the large family of G protein-coupled receptors (GPCRs)—plays a key role in circadian rhythm regulation and is notably involved in sleep disorders and depression. Structural and functional information at the molecular level are highly desired for fine characterization of this receptor; however, adequate techniques for isolating soluble MT1 material suitable for biochemical and biophysical studies remain lacking. Here we describe the evaluation of a panel of constructs and host systems for the production of recombinant human MT1 receptors, and the screening of different conditions for their solubilization and purification. Our findings resulted in the establishment of an original strategy using a mixture of Fos14 and CHAPS detergents to extract and purify a recombinant human MT1 from <i>Pichia pastoris</i> membranes. This procedure enabled the recovery of relatively pure, monomeric and ligand-binding active MT1 receptor in the near-milligram range. A comparative study based on extensive ligand-binding characterization highlighted a very close correlation between the pharmacological profiles of MT1 purified from yeast and the same receptor present in mammalian cell membranes. The high quality of the purified MT1 was further confirmed by its ability to activate its cognate Gαi protein partner when reconstituted in lipid discs, thus opening novel paths to investigate this receptor by biochemical and biophysical approaches.</p></div

Gi coupling of MT1 reconstituted in lipid nanodiscs.

<p>Kinetics of GTPγS binding to Gi in the Gαiβ1γ2 trimer triggered by MT1R-containing nanodiscs in the absence of ligand (open circles) or in the presence of 10 µM of the melatonin agonist (closed circles). The kinetics measured in the presence of empty nanodiscs is given as open squares. These data were treated as described in the Materials and Methods section to extract the basal exchange rates.</p

Correlation plots between binding affinities of MT1 receptors in CHO membranes, in <i>P. pastoris</i> membranes, and purified.

<p><b><i>A</i></b>, pKi correlation of MT1 in <i>P. pastoris</i> membranes <i>vs.</i> MT1 in CHO membranes. <b><i>B</i></b>, pKi correlation of MT1 purified in Fos14/CHAPS <i>vs.</i> MT1 in CHO membranes. <i>C</i>, pKi correlation of MT1 in <i>P. pastoris</i> membranes <i>vs.</i> MT1 purified in Fos14/CHAPS. Processed data are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100616#pone-0100616-t001" target="_blank">Table 1</a>. Pearson's correlation analyses revealed r coefficients of 0.908 (<i>p</i><0.0001, n = 4), 0.840 (<i>p</i><0.0001, n = 4), and 0.840 (<i>p</i><0.0001, n = 4) for <i>A</i>, <i>B</i>, and <i>C</i>, respectively.</p