DREADDs for Neuroscientists

To understand brain function, it is essential that we discover how cellular signaling specifies normal and pathological brain function. In this regard, chemogenetic technologies represent valuable platforms for manipulating neuronal and non-neuronal signal transduction in a cell-type-specific fashion in freely moving animals. Designer Receptors Exclusively Activated by Designer Drugs (DREADD)-based chemogenetic tools are now commonly used by neuroscientists to identify the circuitry and cellular signals that specify behavior, perceptions, emotions, innate drives, and motor functions in species ranging from flies to nonhuman primates. Here I provide a primer on DREADDs highlighting key technical and conceptual considerations and identify challenges for chemogenetics going forward

Engineered G-protein Coupled Receptors are Powerful Tools to Investigate Biological Processes and Behaviors

Understanding how discreet tissues and neuronal circuits function in relation to the whole organism to regulate physiological processes and behaviors is a fundamental goal of modern biological science. Powerful and important new tools in this discovery process are modified G-protein coupled receptors (GPCRs) known as ‘Receptors Activated Solely by Synthetic Ligands (RASSLs),’ and ‘Designer Receptors Exclusively Activated by a Designer Drug (DREADDs).’ Collectively, these are GPCRs modified either through rational design or directed molecular evolution, that do not respond to native ligand, but functionally respond only to synthetic ligands. Importantly, the utility of these receptors is not limited to examination of the role of GPCR-coupled effector signal transduction pathways. Due to the near ubiquitous expression of GPCRs throughout an organism, this technology, combined with whole animal transgenics to selectively target expression, has the ability to regulate activity of discreet tissues and neuronal circuits through effector pathway modulation to study function and behavior throughout the organism. Advantages over other systems currently used to modify in vivo function include the ability to rapidly, selectively and reversibly manipulate defined signal transduction pathways both in short term and long term studies, and no need for specialized equipment due to convenient systemic treatment with activating ligand

DREADD: A Chemogenetic GPCR Signaling Platform

Recently, we created a family of engineered G protein-coupled receptors (GPCRs) called DREADD (designer receptors exclusively activated by designer drugs) which can precisely control three major GPCR signaling pathways (Gq, Gi, and Gs). DREADD technology has been successfully applied in a variety of in vivo studies to control GPCR signaling, and here we describe recent advances of DREADD technology and discuss its potential application in drug discovery, gene therapy, and tissue engineering

β-Fluorofentanyls Are pH-Sensitive Mu Opioid Receptor Agonists

The concept recently postulated by Stein and co-workers (Science2017, 355, 966) that mu opioid receptor (MOR) agonists possessing amines with attenuated basicity show pH-dependent activity and can selectively act at damaged, low pH tissues has been additionally supported by in vitro studies reported here. We synthesized and tested analogs of fentanyl possessing one or two fluorine atoms at the beta position of the phenethylamine side chain, with additional fluorines optionally added to the benzene ring of the side chain. These compounds were synthesized in 1 to 3 steps from commercial building blocks. The novel bis-fluorinated analog RR-49 showed superior pH sensitivity, with full efficacy relative to DAMGO, but with 19-fold higher potency (IC50) in a MOR cAMP assay at pH 6.5 versus 7.4. Such compounds hold significant promise as analgesics for inflammatory pain with reduced abuse potential

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

Structure and function of serotonin G protein-coupled receptors

Serotonin receptors are prevalent throughout the nervous system and the periphery, and remain one of the most lucrative and promising drug discovery targets for disorders ranging from migraine headaches to neuropsychiatric disorders such as schizophrenia and depression. There are 14 distinct serotonin receptors, of which 13 are G protein coupled receptors (GPCRs), which are targets for approximately 40% of the approved medicines. Recent crystallographic and biochemical evidence has provided a converging understanding of the basic structure and functional mechanics of GPCR activation. Currently, two GPCR crystal structures exist for the serotonin family, the 5-HT1B and 5-HT2B receptor, with the antimigraine and valvulopathic drug ergotamine bound. The first serotonin crystal structures not only provide the first evidence of serotonin receptor topography but also provide mechanistic explanations into functional selectivity or biased agonism. This review will detail the findings of these crystal structures from a molecular and mutagenesis perspective for driving rational drug design for novel therapeutics incorporating biased signaling

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