A Genome-Wide RNAi Screen in Caenorhabditis elegans Identifies the Nicotinic Acetylcholine Receptor Subunit ACR-7 as an Antipsychotic Drug Target

We report a genome-wide RNA interference (RNAi) screen for Suppressors of Clozapine-induced Larval Arrest (scla genes) in Caenorhabditis elegans, the first genetic suppressor screen for antipsychotic drug (APD) targets in an animal. The screen identifies 40 suppressors, including the α-like nicotinic acetylcholine receptor (nAChR) homolog acr-7. We validate the requirement for acr-7 by showing that acr-7 knockout suppresses clozapine-induced larval arrest and that expression of a full-length translational GFP fusion construct rescues this phenotype. nAChR agonists phenocopy the developmental effects of clozapine, while nAChR antagonists partially block these effects. ACR-7 is strongly expressed in the pharynx, and clozapine inhibits pharyngeal pumping. acr-7 knockout and nAChR antagonists suppress clozapine-induced inhibition of pharyngeal pumping. These findings suggest that clozapine activates ACR-7 channels in pharyngeal muscle, leading to tetanus of pharyngeal muscle with consequent larval arrest. No APDs are known to activate nAChRs, but a number of studies indicate that α7-nAChR agonists may prove effective for the treatment of psychosis. α-like nAChR signaling is a mechanism through which clozapine may produce its therapeutic and/or toxic effects in humans, a hypothesis that could be tested following identification of the mammalian ortholog of C. elegans acr-7

BCL::EMAS — Enantioselective Molecular Asymmetry Descriptor for 3D-QSAR

Stereochemistry is an important determinant of a molecule’s biological activity. Stereoisomers can have different degrees of efficacy or even opposing effects when interacting with a target protein. Stereochemistry is a molecular property difficult to represent in 2D-QSAR as it is an inherently three-dimensional phenomenon. A major drawback of most proposed descriptors for 3D-QSAR that encode stereochemistry is that they require a heuristic for defining all stereocenters and rank-ordering its substituents. Here we propose a novel 3D-QSAR descriptor termed Enantioselective Molecular ASymmetry (EMAS) that is capable of distinguishing between enantiomers in the absence of such heuristics. The descriptor aims to measure the deviation from an overall symmetric shape of the molecule. A radial-distribution function (RDF) determines a signed volume of tetrahedrons of all triplets of atoms and the molecule center. The descriptor can be enriched with atom-centric properties such as partial charge. This descriptor showed good predictability when tested with a dataset of thirty-one steroids commonly used to benchmark stereochemistry descriptors (r2 = 0.89, q2 = 0.78). Additionally, EMAS improved enrichment of 4.38 versus 3.94 without EMAS in a simulated virtual high-throughput screening (vHTS) for inhibitors and substrates of cytochrome P450 (PUBCHEM AID891)

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BCL::EMAS — Enantioselective Molecular Asymmetry Descriptor for 3D-QSAR

Stereochemistry is an important determinant of a molecule’s biological activity. Stereoisomers can have different degrees of efficacy or even opposing effects when interacting with a target protein. Stereochemistry is a molecular property difficult to represent in 2D-QSAR as it is an inherently three-dimensional phenomenon. A major drawback of most proposed descriptors for 3D-QSAR that encode stereochemistry is that they require a heuristic for defining all stereocenters and rank-ordering its substituents. Here we propose a novel 3D-QSAR descriptor termed Enantioselective Molecular ASymmetry (EMAS) that is capable of distinguishing between enantiomers in the absence of such heuristics. The descriptor aims to measure the deviation from an overall symmetric shape of the molecule. A radial-distribution function (RDF) determines a signed volume of tetrahedrons of all triplets of atoms and the molecule center. The descriptor can be enriched with atom-centric properties such as partial charge. This descriptor showed good predictability when tested with a dataset of thirty-one steroids commonly used to benchmark stereochemistry descriptors (r<sup>2</sup> = 0.89, q<sup>2</sup> = 0.78). Additionally, EMAS improved enrichment of 4.38 versus 3.94 without EMAS in a simulated virtual high-throughput screening (vHTS) for inhibitors and substrates of cytochrome P450 (PUBCHEM AID891)

Discovery of Small-Molecule Modulators of the Human Y₄ Receptor

<div><p>The human neuropeptide Y₄ receptor (Y₄R) and its native ligand, pancreatic polypeptide, are critically involved in the regulation of human metabolism by signaling satiety and regulating food intake, as well as increasing energy expenditure. Thus, this receptor represents a putative target for treatment of obesity. With respect to new approaches to treat complex metabolic disorders, especially in multi-receptor systems, small molecule allosteric modulators have been in the focus of research in the last years. However, no positive allosteric modulators or agonists of the Y₄R have been described so far. In this study, small molecule compounds derived from the Niclosamide scaffold were identified by high-throughput screening to increase Y₄R activity. Compounds were characterized for their potency and their effects at the human Y₄R and as well as their selectivity towards Y₁R, Y₂R and Y₅R. These compounds provide a structure-activity relationship profile around this common scaffold and lay the groundwork for hit-to-lead optimization and characterization of positive allosteric modulators of the Y₄R.</p></div

YR subtype selectivity of Y₄R PAMs.

<p>Effect of 30 μM compound on the pEC₅₀ of Y-receptor agonists in COS-7 cells stable expressing a Y receptor subtype and the chimeric G-protein G_α6qi4myr. Receptors were stimulated with their native ligands (Y₁R, Y₂R, Y₅R: NPY; Y₄R: PP). For Y-axis values, positive modulation represents an increase in the apparent potency (pEC₅₀) of the native agonist and negative modulation represents a decrease in the apparent potency of the native agonist. Data represent the mean ± SEM of at least two independent experiments (for full concentration-response curves see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157146#pone.0157146.s002" target="_blank">S2 Fig</a>) (*p < .05, ***p < .001 Bonferroni).</p

Distinct positions of the Niclosamide scaffold were shown to be relevant for Y₄R PAM activity and YR selectivity.

<p>Substitutions in the benzoyl ring are important for Y₄R potency (green), and offer a potential modification site (grey). Modifications in the aniline ring engender selectivity towards Y₁R / Y₅R subtype (red).</p

Y₄R PAM activity of Niclosamide-like compounds.

<p>Potency of the Y₄R PAMs was investigated with an inositol phosphate accumulation assay through potentiation of a PP EC₂₀ response. Data have been normalized to the maximum IP accumulation caused by the Y₄R native ligand PP. Data represent the mean ± SEM of three independent experiments performed in duplicate.</p

Validation of Y₄R PAM activity and subtype selectivity of initial Ca²⁺-flux-based screen hit compounds in an inositol phosphate accumulation assay.

<p>(A) Compound structures. (B) Effect of 10 μM compound on submaximal YR activation by 1 nM ligand, which represents EC₂₀-EC₆₀ (Y_1,2,5R: NPY; Y₄R: PP). Data represent the mean ± SEM of two independent experiments each performed in quadruplicate (*** p ≤ .001 Bonferroni).</p