Selectivity Challenges in Docking Screens for GPCR Targets and Antitargets

To investigate large library docking’s ability to find molecules with joint activity against on-targets and selectivity versus antitargets, the dopamine D₂ and serotonin 5-HT_2A receptors were targeted, seeking selectivity against the histamine H₁ receptor. In a second campaign, κ-opioid receptor ligands were sought with selectivity versus the μ-opioid receptor. While hit rates ranged from 40% to 63% against the on-targets, they were just as good against the antitargets, even though the molecules were selected for their putative lack of binding to the off-targets. Affinities, too, were often as good or better for the off-targets. Even though it was occasionally possible to find selective molecules, such as a mid-nanomolar D₂/5-HT_2A ligand with 21-fold selectivity versus the H₁ receptor, this was the exception. Whereas false-negatives are tolerable in docking screens against on-targets, they are intolerable against antitargets; addressing this problem may demand new strategies in the field

Selectivity Challenges in Docking Screens for GPCR Targets and Antitargets

To investigate large library docking’s ability to find molecules with joint activity against on-targets and selectivity versus antitargets, the dopamine D₂ and serotonin 5-HT_2A receptors were targeted, seeking selectivity against the histamine H₁ receptor. In a second campaign, κ-opioid receptor ligands were sought with selectivity versus the μ-opioid receptor. While hit rates ranged from 40% to 63% against the on-targets, they were just as good against the antitargets, even though the molecules were selected for their putative lack of binding to the off-targets. Affinities, too, were often as good or better for the off-targets. Even though it was occasionally possible to find selective molecules, such as a mid-nanomolar D₂/5-HT_2A ligand with 21-fold selectivity versus the H₁ receptor, this was the exception. Whereas false-negatives are tolerable in docking screens against on-targets, they are intolerable against antitargets; addressing this problem may demand new strategies in the field

Selectivity Challenges in Docking Screens for GPCR Targets and Antitargets

To investigate large library docking’s ability to find molecules with joint activity against on-targets and selectivity versus antitargets, the dopamine D₂ and serotonin 5-HT_2A receptors were targeted, seeking selectivity against the histamine H₁ receptor. In a second campaign, κ-opioid receptor ligands were sought with selectivity versus the μ-opioid receptor. While hit rates ranged from 40% to 63% against the on-targets, they were just as good against the antitargets, even though the molecules were selected for their putative lack of binding to the off-targets. Affinities, too, were often as good or better for the off-targets. Even though it was occasionally possible to find selective molecules, such as a mid-nanomolar D₂/5-HT_2A ligand with 21-fold selectivity versus the H₁ receptor, this was the exception. Whereas false-negatives are tolerable in docking screens against on-targets, they are intolerable against antitargets; addressing this problem may demand new strategies in the field

Selectivity Challenges in Docking Screens for GPCR Targets and Antitargets

To investigate large library docking’s ability to find molecules with joint activity against on-targets and selectivity versus antitargets, the dopamine D₂ and serotonin 5-HT_2A receptors were targeted, seeking selectivity against the histamine H₁ receptor. In a second campaign, κ-opioid receptor ligands were sought with selectivity versus the μ-opioid receptor. While hit rates ranged from 40% to 63% against the on-targets, they were just as good against the antitargets, even though the molecules were selected for their putative lack of binding to the off-targets. Affinities, too, were often as good or better for the off-targets. Even though it was occasionally possible to find selective molecules, such as a mid-nanomolar D₂/5-HT_2A ligand with 21-fold selectivity versus the H₁ receptor, this was the exception. Whereas false-negatives are tolerable in docking screens against on-targets, they are intolerable against antitargets; addressing this problem may demand new strategies in the field

<i>In Silico</i> Molecular Comparisons of <i>C. elegans</i> and Mammalian Pharmacology Identify Distinct Targets That Regulate Feeding

<div><p>Phenotypic screens can identify molecules that are at once penetrant and active on the integrated circuitry of a whole cell or organism. These advantages are offset by the need to identify the targets underlying the phenotypes. Additionally, logistical considerations limit screening for certain physiological and behavioral phenotypes to organisms such as zebrafish and <i>C. elegans</i>. This further raises the challenge of elucidating whether compound-target relationships found in model organisms are preserved in humans. To address these challenges we searched for compounds that affect feeding behavior in <i>C. elegans</i> and sought to identify their molecular mechanisms of action. Here, we applied predictive chemoinformatics to small molecules previously identified in a <i>C. elegans</i> phenotypic screen likely to be enriched for feeding regulatory compounds. Based on the predictions, 16 of these compounds were tested <i>in vitro</i> against 20 mammalian targets. Of these, nine were active, with affinities ranging from 9 nM to 10 µM. Four of these nine compounds were found to alter feeding. We then verified the <i>in vitro</i> findings <i>in vivo</i> through genetic knockdowns, the use of previously characterized compounds with high affinity for the four targets, and chemical genetic epistasis, which is the effect of combined chemical and genetic perturbations on a phenotype relative to that of each perturbation in isolation. Our findings reveal four previously unrecognized pathways that regulate feeding in <i>C. elegans</i> with strong parallels in mammals. Together, our study addresses three inherent challenges in phenotypic screening: the identification of the molecular targets from a phenotypic screen, the confirmation of the <i>in vivo</i> relevance of these targets, and the evolutionary conservation and relevance of these targets to their human orthologs.</p></div

SEA predictions that were confirmed by <i>in vitro</i> testing.

<p>SEA predictions that were confirmed by <i>in vitro</i> testing.</p

Structure–Functional Selectivity Relationship Studies of β‑Arrestin-Biased Dopamine D₂ Receptor Agonists

Functionally selective G protein-coupled receptor (GPCR) ligands, which differentially modulate canonical and noncanonical signaling, are extremely useful for elucidating key signal transduction pathways essential for both the therapeutic actions and side effects of drugs. However, few such ligands have been created, and very little purposeful attention has been devoted to studying what we term: “structure–functional selectivity relationships” (SFSR). We recently disclosed the first β-arrestin-biased dopamine D₂ receptor (D₂R) agonists UNC9975 (<b>44</b>) and UNC9994 (<b>36</b>), which have robust in vivo antipsychotic drug-like activities. Here we report the first comprehensive SFSR studies focused on exploring four regions of the aripiprazole scaffold, which resulted in the discovery of these β-arrestin-biased D₂R agonists. These studies provide a successful proof-of-concept for how functionally selective ligands can be discovered

Interaction matrix of all binary combinations of compounds and gene knockdowns that individually increase pharyngeal pumping.

<p>(A) The differences in the pumping rates of compound-treated versus vehicle (0.1% DMSO) treatment on each genetic background for all pair-wise combinations of compounds and mutants were evaluated. Compound concentrations used were 10 µM for B16, D20, K9, F15, and H6; 200 nM for L-371257 and SB 222200; and 2 µM for MMPIP, 5-flurox. The predicted compound–target interactions are outlined in yellow. Red- and blue-labeled interactions indicate pumping rates significantly different (ANOVA, <i>p</i><0.05 Dunnett's multiple comparison test) from the corresponding vehicle control-treated mutant. (B) The implied genetic interactions on pharyngeal pumping of <i>mgl-2</i>, <i>ver-2</i>, <i>ver-3</i>, and <i>gnrr-1</i> mutants assayed by mutant–RNAi combinations. Twelve animals per condition were analyzed. Error bars represent 1 standard deviation. * <i>p</i><0.001 one-way ANOVA using Bonferroni's multiple comparison test.</p

Several compounds with predicted and confirmed human targets increase pharyngeal pumping.

<p>(A) Wild-type <i>C. elegans</i> were cultured on media supplemented with either 0.1% DMSO (vehicle control) or 10 µM of each compound. (B) The effects of the compounds on the pharyngeal pumping rate when exposed for differing developmental periods was evaluated for <i>C. elegans</i> exposed to each 10 uM of each compound during different times: L1 to L4 (2 d at 20°C), L1 to gravid adult (3 d at 20°C, and naïve day 1 gravid adults exposed to B16, F15, and D20 for 1 h, H6 for 16 h). The pharyngeal pumping rate of 10–13 animals per condition was quantified. Error bars represent the standard deviation. *<i>p</i><0.01: ANOVA, Dunnett's multiple comparisons test. In (B) gravid adults exposed to H6 for 16 h was compared to DMSO 16 h (<i>t</i> test: two tailed *<i>p</i><0.01).</p

Overview of the ligand-target predictions for <i>C. elegans</i> screen actives.

<p>(A) Distribution of ligand predictions per compound expressed as a histogram. (B) Target classes more frequently (positive %) or less frequently (negative %) predicted for <i>C. elegans</i> screen actives, using predictions on ChEMBL's ligands as a baseline. Data are calculated based on ligand–target interactions at a minimum significance threshold of <i>E</i><0.00001.</p