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

DINC 2.0: A New Protein–Peptide Docking Webserver Using an Incremental Approach

Molecular docking is a standard computational approach to predict binding modes of protein–ligand complexes by exploring alternative orientations and conformations of the ligand (i.e., by exploring ligand flexibility). Docking tools are largely used for virtual screening of small drug-like molecules, but their accuracy and efficiency greatly decays for ligands with more than 10 flexible bonds. This prevents a broader use of these tools to dock larger ligands, such as peptides, which are molecules of growing interest in cancer research. To overcome this limitation, our group has previously proposed a meta-docking strategy, called DINC, to predict binding modes of large ligands. By incrementally docking overlapping fragments of a ligand, DINC allowed predicting binding modes of peptide-based inhibitors of transcription factors involved in cancer. Here, we describe DINC 2.0, a revamped version of the DINC webserver with enhanced capabilities and a more user-friendly interface. DINC 2.0 allows docking ligands that were previously too challenging for DINC, such as peptides with more than 25 flexible bonds. The webserver is freely accessible at http://dinc.kavrakilab.org, together with additional documentation and video tutorials. Our team will provide continuous support for this tool and is working on extending its applicability to other challenging fields, such as personalized immunotherapy against cancer

Genetic and pharmacological characterization of clozapine-induced larval arrest.

<p>(A) Clozapine induced developmental delay in wild-type <i>C. elegans</i> in a concentration-dependent manner. <i>acr-7(tm863)</i> partially suppressed this developmental delay, and the suppression was rescued by expression of ACR-7 in the mutant background. Expression was driven by a putative <i>acr-7</i> promoter in the case of <i>acr-7(tm863) mchEx40</i> or by the pharyngeal muscle-specific <i>myo-2</i> promoter in the case of <i>acr-7(tm863) mchEx207</i>. Growth was measured as the percentage of different development stages 72 hours after loading synchronized L1 animals into the drug plates. Different concentrations (80, 160, 320 µM) of clozapine were dissolved in DMSO with the maximum concentration of DMSO being 0.1%. 0.1% DMSO alone was used for control wells. (B) nAChR agonists nicotine (Nico) and levamisole (Leva) induced developmental delay in a concentration-dependent manner, mimicking the effect of clozapine. (C) Nicotine-induced developmental delay is suppressed by <i>acr-7(tm863)</i>, and suppression was partially rescued by expression of full-length ACR-7 in the mutant background. (D) Nicotine receptor antagonists d-TC (100 and 500 µM) and DHβE (100 and 500 µM) suppressed clozapine (Cloz)-induced developmental delay. d-TC or DHβE alone did not affect the growth of the animals at 100 and 500 µM.</p

A genome-wide RNAi screen for <i>scla</i> genes yielded the nAChR gene <i>acr-7</i>.

<p>(A) Experimental design of the feeding RNAi screen in liquid culture. On day 1, <i>rrf-3</i> animals were synchronized, and bacteria was cultured from the Ahringer RNAi feeding library. Synchronized L1 animals were added to induced cultures on day 2 and were allowed to grow to adulthood for 3 days. Clozapine was added on day 5, and progeny were allowed to develop for 3 days. Progeny were scored for the Scla phenotype on day 8. (B1) N2 animals grew to adulthood within 3 days in the presence of 0.1% DMSO alone. (B2) 320 µM clozapine caused larval arrest in N2 animals exposed to feeding RNAi bacteria with empty vector alone. (B3) <i>acr-7(RNAi)</i> animals in 0.1% DMSO alone displayed normal development. (B4) <i>acr-7(RNAi)</i> suppressed developmental delay caused by 320 µM clozapine. (B5) The <i>acr-7(tm863)</i> knockout suppressed developmental delay caused by 320 µM clozapine. Note that experiments depicted in (B) were performed on NGM plates, not in liquid culture, to allow clear photographs.</p

ACR-7 protein sequence alignment and <i>acr-7</i> gene structure.

<p>(A) The <i>C. elegans</i> ACR-7 protein and human α7-nAChR subunit CHRNA7 were aligned using ClustalW. The four putative transmembrane domains, as predicted for the human CHRNA7 protein, were boxed. nAChRs are members of the cys-loop family of ionotropic neurotransmitter receptors, and this conserved di-cysteine loop is dotted line-boxed. α-nAChR subunits contain a pair of vicinal cysteines within the Ach binding site, and these residues are marked with asterisks. The <i>C. elegans</i> and human proteins share 36% identity overall and 41% identity within the pore-lining transmembrane domain II (M2). (B) The <i>acr-7</i> gene contains 13 exons, and the region deleted in the <i>tm863</i> allele is marked with a line. <i>tm863</i> is an out-of-frame deletion which removes exons 6–7 and most of exons 5 and 8. (C) The <i>tm863</i> knockout is predicted to lack all four transmembrane domains and to be a null allele.</p

Functional diversity of <i>scla</i> genes.

<p>Genes were classified by biological process based on Gene Ontology terms from Wormbase. The percentages for each functional class of genes required for suppression of clozapine-induced larval arrest are shown.</p

Ach release was not the mechanism of clozapine-induced larval arrest.

<p>Mutations in genes required for Ach release failed to suppress clozapine-induced developmental delay. Strong loss-of-function of the choline acetyltransferase gene <i>cha-1</i> or the synaptic vesicle Ach transporter gene <i>unc-17</i> alone produced developmental delay. Therefore, both weak (<i>n2411</i> or <i>e113</i>) and strong (<i>p1152</i> or <i>e245</i>) loss-of-function alleles were tested.</p

Suppression by <i>acr-7(lf)</i> is specific.

<p>A series of 22 nAChR mutants were tested for suppression of clozapine-induced larval arrest, and only <i>acr-7(lf)</i> produced robust suppression.</p

ACR-7 acted in the pharyngeal muscle.

<p>(A) ACR-7 was highly expressed in the pharynx as indicated by the arrows. A, anterior; P, posterior; D, dorsal; V, ventral. Scattered expression was also observed in the tail and vulval regions. (B) Clozapine inhibited pharyngeal pumping in wild-type animals in a concentration-dependent manner. Suppression of clozapine-induced inhibition of pharyngeal pumping was seen in <i>acr-7(tm863)</i> animals, and rescue was observed with expression of ACR-7 in the mutant background. A putative <i>acr-7</i> promoter was utilized to drive expression for the <i>acr-7(tm863) mchEx40</i> strain, whereas the pharyngeal muscle-specific <i>myo-2</i> promoter was utilized for the <i>acr-7(tm863) mchEx207</i> strain. (C) Both nicotine and clozapine inhibited the pumping rate in N2 animals. d-TC partially blocked these effects. Importantly, d-TC alone had no effect on pumping. (D) d-TC failed to block clozapine- or nicotine-induced inhibition of pumping in <i>acr-7(lf)</i> animals. * <i>P</i><0.05; ** <i>P</i><0.01; *** <i>P</i><0.0001.</p