Integrated Approaches for Genome-wide Interrogation of the Druggable Non-olfactory G Protein-coupled Receptor Superfamily

G-protein-coupled receptors (GPCRs) are frequent and fruitful targets for drug discovery and development, as well as being off-targets for the side effects of a variety of medications. Much of the druggable non-olfactory human GPCR-ome remains under-interrogated, and we present here various approaches that we and others have used to shine light into these previously dark corners of the human genome

Discovery and Characterization of Novel GPR39 Agonists Allosterically Modulated by Zinc

In this study, we identified two previously described kinase inhibitors—3-(4-chloro-2-fluorobenzyl)-2-methyl-N-(3-methyl-1H-pyrazol-5-yl)-8-(morpholinomethyl)imidazo[1,2-b]pyridazin-6-amine (LY2784544) and 1H-benzimidazole-4-carboxylic acid, 2-methyl-1-[[2-methyl-3-(trifluoromethyl)phenyl]methyl]-6-(4-morpholinyl)- (GSK2636771)—as novel GPR39 agonists by unbiased small-molecule-based screening using a β-arrestin recruitment screening approach (PRESTO-Tango). We characterized the signaling of LY2784544 and GSK2636771 and compared their signaling patterns with a previously described “GPR39-selective” agonist N-[3-chloro-4-[[[2-(methylamino)-6-(2-pyridinyl)-4- pyrimidinyl]amino]methyl]phenyl]methanesulfonamide (GPR39-C3) at both canonical and noncanonical signaling pathways. Unexpectedly, all three compounds displayed probe-dependent and pathway-dependent allosteric modulation by concentrations of zinc reported to be physiologic. LY2784544 and GS2636771 at GPR39 in the presence of zinc were generally as potent or more potent than their reported activities against kinases in whole-cell assays. These findings reveal an unexpected role of zinc as an allosteric potentiator of small-molecule-induced activation of GPR39 and expand the list of potential kinase off-targets to include understudied G protein–coupled receptors

Tuning up the right signal: chemical and genetic approaches to study GPCR functions

The G protein-coupled receptor (GPCR) family is among the most druggable families in the human proteome. GPCRs are involved in most physiological processes, and our ability to modulate their activity is a hallmark of modern pharmacology. The means by which the activity of GPCRs can be modulated have been expanded by emerging data and concepts in pharmacology, which has created new strategies for their control. These new approaches will lead to the generation of more potent, selective, and efficient pharmaceutics, while reducing inappropriate actions and adverse effects. Herein, we review and comment on some recent advances in chemical and genetic approaches to the profiling of GPCR function, as well as the validation of orphan GPCRs as potential therapeutic targets using engineered receptors

Magic shotguns versus magic bullets: Selectively non-selective drugs for mood disorders and schizophrenia

Most common central nervous system disorders — such as depression, bipolar disorder and schizophrenia — seem to be polygenic in origin, and the most effective medications have exceedingly complex pharmacologies. Attempts to develop more effective treatments for diseases such as schizophrenia and depression by discovering drugs selective for single molecular targets (that is, 'magic bullets') have, not surprisingly, been largely unsuccessful. Here we propose that designing selectively non-selective drugs (that is, 'magic shotguns') that interact with several molecular targets will lead to new and more effective medications for a variety of central nervous system disorders

PRESTO-Tango as an open-source resource for interrogation of the druggable human GPCRome

G protein-coupled receptors (GPCRs) are essential mediators of cellular signaling and important targets of drug action. Of the approximately 350 non-olfactory human GPCRs, more than 100 are still considered “orphans” as their endogenous ligand(s) remain unknown. Here, we describe a unique open-source resource that provides the capacity to interrogate the druggable human GPCR-ome via a G protein-independent β-arrestin recruitment assay. We validate this unique platform at more than 120 non-orphan human GPCR targets, demonstrate its utility for discovering new ligands for orphan human GPCRs, and describe a method (PRESTO-TANGO; Parallel Receptor-ome Expression and Screening via Transcriptional Output - TANGO) for the simultaneous and parallel interrogation of the entire human GPCR-ome

Assessing serotonin receptor mRNA editing frequency by a novel ultra high-throughput sequencing method

RNA editing is a post-transcriptional modification of pre-mRNA that results in increased diversity in transcriptomes and proteomes. It occurs in a wide variety of eukaryotic organisms and in some viruses. One of the most common forms of pre-mRNA editing is A-to-I editing, in which adenosine is deaminated to inosine, which is read as guanosine during translation. This phenomenon has been observed in numerous transcripts, including the mammalian 5-HT2C receptor, which can be edited at five distinct sites. Methods used to date to quantify 5-HT2C receptor editing are labor-intensive, expensive and provide limited information regarding the relative abundance of 5-HT2C receptor editing variants. Here, we present a novel, ultra high-throughput method to quantify 5-HT2C receptor editing, compare it to a more conventional method, and use it to assess the effect of a range of genetic and pharmacologic manipulations on 5-HT2C editing. We conclude that this new method is powerful and economical, and we provide evidence that alterations in 5-HT2C editing appear to be a result of regional changes in brain activity, rather than a mechanism to normalize 5-HT2C signaling

HL-Histamine receptor affinity predicts short-term weight gain for typical and atypical antipsychotic drugs

As a result of superior efficacy and overall tolerability, atypical antipsychotic drugs have become the treatment of choice for schizophrenia and related disorders, despite their side effects. Weight gain is a common and potentially serious complication of some antipsychotic drug therapy, and may be accompanied by hyperlipidemia, hypertension and hyperglycemia and, in some extreme cases, diabetic ketoacidosis. The molecular mechanism(s) responsible for antipsychotic drug-induced weight gain are unknown, but have been hypothesized to be because of interactions of antipsychotic drugs with several neurotransmitter receptors, including 5-HT2A and 5-HT2Cserotonin receptors, H1-histamine receptors, α1- and α2-adrenergic receptors, and m3-muscarinic receptors. To determine the receptor(s) likely to be responsible for antipsychotic-drug-induced weight gain, we screened 17 typical and atypical antipsychotic drugs for binding to 12 neurotransmitter receptors. H1-histamine receptor affinities for this group of typical and atypical antipsychotic drugs were significantly correlated with weight gain (Spearman ρ=−0.72; p less than 0.01), as were affinities for α1A adrenergic (ρ=−0.54; p less than 0.05), 5-HT2C (ρ=−0.49; p less than 0.05) and 5-HT6 receptors (ρ=−0.54; p less than 0.05), whereas eight other receptors' affinities were not. A principal components analysis showed that affinities at the H1, α2A, α2B, 5-HT2A, 5-HT2C, and 5-HT6 receptors were most highly correlated with the first principal component, and affinities for the D2, 5-HT1A, and 5-HT7 receptors were most highly correlated with the second principal component. A discriminant functions analysis showed that affinities for the H1 and α1A receptors were most highly correlated with the discriminant function axis. The discriminant function analysis, as well as the affinity for the H1-histamine receptor alone, correctly classified 15 of the 17 drugs into two groups; those that induce weight gain and those that do not. Because centrally acting H1-histamine receptor antagonists are known to induce weight gain with chronic use, and because H1-histamine receptor affinities are positively correlated with weight gain among typical and atypical antipsychotic drugs, it is recommended that the next generation of atypical antipsychotic drugs be screened to avoid H1-histamine receptors

The human polyomavirus, JCV, uses serotonin receptors to infect cells

The human polyomavirus, JCV, causes the fatal demyelinating disease progressive multifocal leukoencephalopathy in immunocompromised patients. We found that the serotonergic receptor 5HT2AR could act as the cellular receptor for JCV on human glial cells. The 5HT2Areceptor antagonists inhibited JCV infection, and monoclonal antibodies directed at 5HT2Areceptors blocked infection of glial cells by JCV, but not by SV40. Transfection of 5HT2Areceptor–negative HeLa cells with a 5HT2A receptor rescued virus infection, and this infection was blocked by antibody to the 5HT2A receptor. A tagged 5HT2A receptor colocalized with labeled JCV in an endosomal compartment following internalization. Serotonin receptor antagonists may thus be useful in the treatment of progressive multifocal leukoencephalopathy

Single Amino Acid Variation Underlies Species-Specific Sensitivity to Amphibian Skin-Derived Opioid-like Peptides

It has been suggested that the evolution of vertebrate opioid receptors (ORs) follow a vector of increased functionality. Here we test this idea comparing human and frog ORs. Interestingly, some of the most potent opioid peptides known have been isolated from amphibian skin secretions. Here we show that such peptides (dermorphin and deltorphin) are highly potent in the human receptors and inactive in frog ORs. The molecular basis for the insensitivity of the frog ORs to these peptides was studied using chimeras and molecular modeling. Interestingly, the insensitivity of the delta opioid receptor (DOR) to deltorphin was due to variation of a single amino acid– Trp7.35—which is a leucine in mammalian DORs. Notably, Trp7.35 is completely conserved in all known DOR sequences from lamprey, fish and amphibians. The deltorphin-insensitive phenotype was verified in fish. Our results provide a molecular explanation for the species selectivity of skin-derived opioid peptides

Quantitative Analysis of Focused A-To-I RNA Editing Sites by Ultra-High-Throughput Sequencing in Psychiatric Disorders

A-to-I RNA editing is a post-transcriptional modification of single nucleotides in RNA by adenosine deamination, which thereby diversifies the gene products encoded in the genome. Thousands of potential RNA editing sites have been identified by recent studies (e.g. see Li et al, Science 2009); however, only a handful of these sites have been independently confirmed. Here, we systematically and quantitatively examined 109 putative coding region A-to-I RNA editing sites in three sets of normal human brain samples by ultra-high-throughput sequencing (uHTS). Forty of 109 putative sites, including 25 previously confirmed sites, were validated as truly edited in our brain samples, suggesting an overestimation of A-to-I RNA editing in these putative sites by Li et al (2009). To evaluate RNA editing in human disease, we analyzed 29 of the confirmed sites in subjects with major depressive disorder and schizophrenia using uHTS. In striking contrast to many prior studies, we did not find significant alterations in the frequency of RNA editing at any of the editing sites in samples from these patients, including within the 5HT2C serotonin receptor (HTR2C). Our results indicate that uHTS is a fast, quantitative and high-throughput method to assess RNA editing in human physiology and disease and that many prior studies of RNA editing may overestimate both the extent and disease-related variability of RNA editing at the sites we examined in the human brain