Bryan L. Roth - US grants

DESCRIPTION (provided by applicant): This K02 Award will fund the salary support for all of Dr. Roth's research related activities. Three major goals are proposed for this funding cycle including: (1) Characterization of the structure and function of 5-HT2-family serotonin receptors; (2) Discovering the cellular and molecular mechanisms responsible for the regulation of 5-HT2A receptors and (3) Administering the National Institute of Mental Health Psychoactive Drug Screening Program. Characterizing the structure and function of 5-HT2A receptors is of great importance for mental health-related research because a number of psychoactive compounds including atypical antipsychotic drugs, antidepressants and some anxiolytic medications exert their actions via interacting with 5-HT2A receptors. Understanding how such drugs interact with 5-HT2-family receptors at the atomic level may lead to novel insights into drug design and development. Discovering how the 5-HT2A receptor is regulated is important for understanding the pharmacological mechanisms by which drugs may regulate neurotransmitter receptor levels. 5-HT2A receptors are regulated in a paradoxical manner by antagonists and insights into the cellular and molecular mechanisms by which these alterations occur could be of importance for understanding 6-protein receptor coupled regulation. Finally, the NIMH Psychoactive Drug Screening Program is responsible for characterizing the pharmacology of novel psychoactive compounds, some of which may represent new therapeutic agents. A full molecular pharmacologic characterization of these compounds is essential prior to their use in humans.

Alterations in the functional activity and/or number of serotonin2 (5-HT2) and/or serotonin1C (5-HT-1c) receptors have been implicated in the pathophysiology of a large number of mental illnesses including obsessive- compulsive disorder, schizophrenia, depression, anxiety, dysthymia, suicide, aggression and eating disorders. Additionally, a number of psycho- and neuropharmacologic agents including antidepressants (mianserin, amitriptyline, imipramine), antipsychotic drugs (loxapine, clozapine), hallucinogens (lysergic acid diethylamide) and anti-dysthymic agents (ritanserin) bind to 5-HT2 and 5-HT-1c receptors. Determining the precise roles the 5-HT2 and 5-HT-1c receptors have in mediating the effects of these agents is difficult because no truly subtype selective agents are currently available. Insights we gain in understanding how drugs bind to each of these receptors should enhance our ability to design novel receptor-specific agents which can be used to treat mental illnesses. This proposal will determine how agonists and antagonists bind to 5-HT2 and 5-HT-1c receptors. We will utilize the techniques of molecular biology and receptor pharmacology to identify specific amino acids essential for specifying the ligand binding domains of each receptor. We are utilizing four major techniques. (1) chimeric protein construction (2) site directed mutagenesis, (3) molecular modelling and (4) structural biochemical measurements. Our strategy is to test three-dimensional molecular models of 5- HT2 and 5- HT-1c receptor-ligand interactions using a combination of molecular biological and structural-biochemical techniques. We will also take advantage of unique mutations we have produced to probe the molecular details responsible for hallucinogen- induced alterations of serotonin receptors in vitro. These findings will be useful for the design and synthesis of new medications to treat psychiatric diseases.

The purpose of the NIMH Psychoactive Drug Screening Program is to provide pharmacological and functional screening of novel synthetic compounds and natural products for potential use as PET (positron emission tomography) ligands for functional brain imaging, research tools or probes for basic and clinical research, and therapeutic agents for mental disorders. The objectives of the contract are to receive and test coded samples (synthetic compounds, small molecules, gene products, and natural product extracts) in broad-based human and rodent CNS (central nervous system) receptor and enzyme binding assays, to test active samples in secondary functional assays, and to provide an electronic data file for each of the screened compounds. Approximately 60% of these samples are to be tested in human recombinant receptor assays. Samples for screening can include, but are not limited to, novel chemical entities, structural analogs of lead compounds, genes or gene products, small molecules, enzyme inhibitors, and natural products. It is anticipated that NIMH- approved and coded samples will be submitted from NIH- or federally- funded research programs and other academic research laboratories. It is expected that the screening information derived from this program will provide individual investigators with conceptual or structural leads for the design and/or development of new chemical entities, small molecules, gene products, tissue- or cell-specific drug delivery systems, therapeutic entities, or PET/SPECT ligands for human brain imaging.

Alterations in the functional activity and/or number of serotonin2 (5-HT2) and/or serotonin1C (5-HT-1c) receptors have been implicated in the pathophysiology of a large number of mental illnesses including obsessive- compulsive disorder, schizophrenia, depression, anxiety, dysthymia, suicide, aggression and eating disorders. Additionally, a number of psycho- and neuropharmacologic agents including antidepressants (mianserin, amitriptyline, imipramine), antipsychotic drugs (loxapine, clozapine), hallucinogens (lysergic acid diethylamide) and anti-dysthymic agents (ritanserin) bind to 5-HT2 and 5-HT-1c receptors. Determining the precise roles the 5-HT2 and 5-HT-1c receptors have in mediating the effects of these agents is difficult because no truly subtype selective agents are currently available. Insights we gain in understanding how drugs bind to each of these receptors should enhance our ability to design novel receptor-specific agents which can be used to treat mental illnesses. This proposal will determine how agonists and antagonists bind to 5-HT2 and 5-HT-1c receptors. We will utilize the techniques of molecular biology and receptor pharmacology to identify specific amino acids essential for specifying the ligand binding domains of each receptor. We are utilizing four major techniques. (1) chimeric protein construction (2) site directed mutagenesis, (3) molecular modelling and (4) structural biochemical measurements. Our strategy is to test three-dimensional molecular models of 5- HT2 and 5- HT-1c receptor-ligand interactions using a combination of molecular biological and structural-biochemical techniques. We will also take advantage of unique mutations we have produced to probe the molecular details responsible for hallucinogen- induced alterations of serotonin receptors in vitro. These findings will be useful for the design and synthesis of new medications to treat psychiatric diseases.

DESCRIPTION (Verbatim from the Applicant's Abstract): 5HT2A serotonin receptors represent a major site of action for atypical antipsychotic drugs, antidepressants and hallucinogens. Long-term treatment with a variety of 5HT2A receptor agonists and antagonists leads to down-regulation and altered trafficking of 5HT2A receptors in vitro and in vivo. The goal of this grant is to understand the molecular and cellular mechanisms by which 5HT2A receptors are regulated by alterations in their targeting and trafficking. To accomplish this goal, five specific aims are proposed. The first specific aim proposes to identify proteins which interact with the 5HT2A receptor using the yeast-two-hybrid method. The second specific aim proposes to characterize the binding of accessory proteins such as MAP-1A and arrestin to intracellular regions of the 5HT2A receptor. The third specific aim proposes to determine how binding of these accessory proteins modulates the trafficking and targeting of 5HT2A receptors in cortical neurons in vitro. The fourth specific aim proposes to determine the mechanisms responsible for agonist- and antagonist-induced internalization of 5HT2A receptors while the fifth specific aim proposes to determine if arrestins are involved in both agonist- and antagonist-induced endocytosis of 5HT2A receptors. Because a large number of drugs used in treating schizophrenia and depression mediate their actions by inducing a down-regulation of 5HT2A receptor number, these studies could clarify the mechanism of action of these psychiatrically important drugs. It is also possible that novel treatment strategies for disorders such as hallucinogen-induced psychosis may come from the basic studies outlined in this application. Finally, novel insights into the mechanism by which receptors are regulated at the cellular and molecular levels will be obtained with the proposed studies.

DESCRIPTION (provided by applicant): This K02 Award will fund the salary support for all of Dr. Roth's research related activities. Three major goals are proposed for this funding cycle including: (1) Characterization of the structure and function of 5-HT2-family serotonin receptors; (2) Discovering the cellular and molecular mechanisms responsible for the regulation of 5-HT2A receptors and (3) Administering the National Institute of Mental Health Psychoactive Drug Screening Program. Characterizing the structure and function of 5-HT2A receptors is of great importance for mental health-related research because a number of psychoactive compounds including atypical antipsychotic drugs, antidepressants and some anxiolytic medications exert their actions via interacting with 5-HT2A receptors. Understanding how such drugs interact with 5-HT2-family receptors at the atomic level may lead to novel insights into drug design and development. Discovering how the 5-HT2A receptor is regulated is important for understanding the pharmacological mechanisms by which drugs may regulate neurotransmitter receptor levels. 5-HT2A receptors are regulated in a paradoxical manner by antagonists and insights into the cellular and molecular mechanisms by which these alterations occur could be of importance for understanding 6-protein receptor coupled regulation. Finally, the NIMH Psychoactive Drug Screening Program is responsible for characterizing the pharmacology of novel psychoactive compounds, some of which may represent new therapeutic agents. A full molecular pharmacologic characterization of these compounds is essential prior to their use in humans.

DESCRIPTION (provided by applicant): Salvinorin A - the active ingredient of the hallucinogenic sage Salvia divinorum - along with Salvia divinorum represent emerging drugs of abuse in the US and elsewhere. Salvinorin A and extracts of Salvia divinorum induce an intense and short-lived hallucinatory experience in humans unlike that of the classical hallucinogens LSD, psilocybin and mescaline. In 2002, my lab discovered that salvinorin A potently and specifically targets ?-opioid receptors (KOR) (Roth et al., PNAS 2002);these findings have been widely replicated. Despite intensive interest in the actions of salvinorin A, we still do not fully understand the molecular and atomic mechanisms responsible for its exquisite potency and selectivity at KOR. The goal of these studies is to discover how salvinorin A binds to and activates KOR. To accomplish these goals we will conduct two specific aims: (1) elucidate the structural features of ?-opioid receptors essential for salvinorin A's actions using high-throughput molecular biology and (2) elucidate the structural features of ?-opioid receptors responsible for salvinorin A's actions via direct biochemical approaches. These studies will clarify how this drug of abuse exerts its actions at the atomic level. These studies are significant as follows: Conceptual: Salvinorin A is an emerging drug of abuse which has profound effects on human perception. Salvinorin A exerts these actions via potent and selective activation of ?-opioid receptors. Understanding how salvinorin A achieves its remarkable selectivity and potency for KORs is an essential first step toward elucidating the mechanism of action of salvinorin A. Technical: We will utilize novel and extraordinarily robust yeast-based technologies we have invented (Armbruster et al., 2007a) to identify the molecular and atomic mechanisms by which salvinorin A binds to and activates KORs. We will also employ newly synthesized pM-affinity and irreversible salvinorin A analogues to identify residues on KOR responsible for binding. To our knowledge, these will be the first studies in the opioid receptor field to biochemically identify residues in the binding pocket. PUBLIC HEALTH RELEVANCE: Salvia divinorum and its active ingredient salvinorin A represent emerging drugs of abuse in the US. These studies could lead to novel treatments for drug abuse.

DESCRIPTION (provided by applicant): Serotonin (5-hydroxytryptamine; 5-HT) is essential for a number of central nervous system processes including the modulation of mood, perception and anxiety, the regulation of feeding behavior and the control of sleep and wakefulness. To mediate this large number of processes, multiple (>14) 5-HT receptors divided into 7 main families have evolved. This grant focuses on the 5-HT2-family of receptors, which is involved in regulating perception, appetite, anxiety, mood and cardiac development. These studies are of clinical significance because activation of 5-HT2A receptors is responsible for the actions of hallucinogens (e.g. LSD, psilocybin, mescaline) while 5-HT2B activation is involved in the cardiac side-effects of fenfluramine and related compounds (e.g. Redux, Phen/Fen) and 5-HT2C activation is important for the anorectic actions of fenfluramine. By contrast, many atypical antipsychotic and antidepressant drugs are high affinity antagonists for 5-HT2A and 5-HT2C receptors Understanding the molecular and atomic mechanisms responsible for these unique actions could lead to novel pharamacotherapies for a number of diseases including schizophrenia, depression, anxiety and feeding disorders (anorexia, bulimia) and cardiac valvulopathies. In the first specific aim, we will clarify the atomic and molecular mechanisms responsible for subtype-selective activation of 5-HT2-family receptors. The second and third specific aims will determine the relevance of conserved features of 5-HT2-family receptors for receptor-G protein activation. The fourth specific aim will determine the molecular features responsible for 5-HT2A/Gq interactions while the fifth specific aim will uncover the molecular features of Gq responsible for interacting with the 5-HT2A receptors. Novel techniques of protein biochemistry (hydroxyl-mediated 1H/2H exchange), cell biology (yeast 2-hybrid screening) and spectroscopy (FRET/BRET) will be used to arrive at testable models for 5-HT2A-Gq interactions.

DESCRIPTION (provided by applicant): 5-HT2A serotonin receptors represent the principal neuropharmacological target for LSD-like hallucinogens and an important molecular target for many psychiatric medications (e.g. selected atypical antipsychotics, antidepressants and atypical anxiolytics). Not surprisingly, 5-HT2A receptors continue to be actively targeted for psychiatric drug discovery in many therapeutic areas including schizophrenia, sleep disorders and cognition enhancement. Thus, study of 5-HT2A receptors is likely to lead to novel insights into psychiatric drug actions as well as insights into the molecular mechanisms responsible for psychosis and related disorders. We have recently discovered that 5-HT2A receptors functionally interact with RSK-2 and that knocking-out RSK-2 augments 5-HT2A mediated signaling (see Preliminary studies). These results imply that the psychosis and impaired cognition, which occur in Coffin-Lowry Syndrome, are due, in part, to over-active 5-HT2A signaling. In favor of this hypothesis are findings that 5-HT2A receptor agonists induce psychosis and cognitive impairment reminiscent of schizophrenia in humans and that 5-HT2A antagonists improve cognition and reduce psychosis in schizophrenia. For more than 50 years it has been suggested that the psychosis, which occurs in schizophrenia is due, in part, to dysregulation of serotonergic signaling-without any direct evidence. These findings represent the first direct evidence in favor of the hypothesis that psychosis is associated with augmented 5-HT2A receptor signaling. The ultimate goal of these studies is to elucidate the molecular actions by which 5-HT2A receptors are regulated and targeted to neuronal subdomains and how this system is dysregulated in various psychotic states: Specific Aim 1: To determine the relevance of 5-HT2A receptor interactions with PDZ-domain containing proteins on the functional activity and sorting of 5-HT2A receptors in vitro and, ultimately, in vivo. Specific Aim 2: To determine the relevance of 5-HT2A receptor interactions with caveolins-1 and flotillin, which target 5-HT2A receptors. Specific Aim 3: To determine whether the interaction between ribosomal S6-kinase-2 (RSK2) and 5-HT2A receptors has functional significance.

behavioral /social science research tag; drug discovery /isolation; laboratory mouse; psychosis

DESCRIPTION (provided by applicant): In classical pharmacology, an agonist activates a[unreadable] single linear signal transduction pathway, whereas an antagonist blocks the action of the agonist and[unreadable] possesses no intrinsic activity. A rapidly evolving idea is that a given receptor, through various ligand induced[unreadable] functional conformations, can engage multiple modalities through interaction with different[unreadable] signaling partners. Hence, a given ligand can bind a receptor and act as an antagonist for one signaling[unreadable] pathway while serving as an agonist at another or vice versa. This property is established for several G[unreadable] protein-coupled receptors ? the most important targets for therapeutic intervention. Importantly, none[unreadable] of the drugs in clinical use have been developed with these multiple signaling considerations in mind.[unreadable] Additionally, agonists and antagonists are rarely completely selective and, for a given receptor, may[unreadable] alter signaling by influencing various receptor-mediated processes such as interaction with G proteins,[unreadable] desensitization, internalization, down-regulation, and receptor-mediated scaffolding of non-G protein[unreadable] signaling components. The physiological relevance of these properties is not fully appreciated. Thus,[unreadable] identifying the FUNCTIONAL SELECTIVITY of compounds may help reveal not only distinct biological[unreadable] processes, but also specific functional outcomes. Currently, the relevance of functional selectivity to[unreadable] psychiatry is unknown. This is particularly important for atypical antipsychotics, where dopamine (DA)[unreadable] D2 and 5-HT2A serotonin receptor antagonism is essentially a prerequisite for all these drugs;[unreadable] however, their other intrinsic activities are obscure. The overall goal of the proposed research is to[unreadable] elucidate signal transduction mechanisms that are essential for antipsychotic efficacy in preclinical[unreadable] genetic and pharmacological mouse models of schizophrenia-like behaviors. The behavioral core will[unreadable] analyze effects of anti psychotics on locomotion, prepulse inhibition, latent inhibition, and social[unreadable] behavior in DA transporter knockout, NMDA NR1-subunit knockdown, and C57BL/6 mice treated with[unreadable] amphetamine or phencyclidine to reproduce schizophrenia-like states. Molecular fingerprinting studies[unreadable] will be performed to analyze effects of antipsychotic drugs on various signal transduction modalities[unreadable] that include the PKA and DARPP-32, AktiGSK, PLC, and ERK pathways. Understanding the relevance[unreadable] of functional selectivity of anti psychotics may provide novel targets with fewer side-effects, greater[unreadable] therapeutic selectivity, and enhanced efficacy for treating individuals with schizophrenia. [unreadable] [unreadable]

Administrative Core for Collaborative Drug Discovery Center Grant 'Functional selectivity: a novel approach for CNS drug discovery' The goal of the Administrative Core is to oversee the organizational, budgeting and reporting aspects and provide the leadership for scientific and programmatic activities of the CDDG. The budget for the administrative core is relatively modest as is its scope.

DESCRIPTION (provided by applicant): Despite the introduction of dozens of antipsychotic drugs during the past 50 years, there is evidence that the overall community functioning of individuals with schizophrenia has demonstrated relatively little improvement. This has led to the suggestion that the model of drug development for serious mentally illnesses is not working and should be replaced by new paradigm in which discoveries in basic neuroscience are translated into new and more innovative drugs. The overall goal of this R13 grant will be to facilitate this translation through a process that brings basic neuroscientists, neuropsychologists, and clinical investigators together to accomplish the following: (1) Identify novel molecular targets for improving treatments for domains of psychopathology in schizophrenia that contribute to the profound disability associated with this disorder (e.g., cognitive impairment, negative symptoms, refractory positive symptoms);(2) Identify biomarkers that will accelerate the process of drug discovery and development: (3) Use accumulated new information from recent NIMH initiatives and clinical trials to improve the design of clinical studies. This will be accomplished through a series of 3 meetings that will include experts in basic and clinical neuroscience from industry, academia, and government as well as representatives from the US Food and Drug Administration and consumer groups. The findings from each meeting will be rapidly disseminated to the field.

DESCRIPTION (provided by applicant): The successful sequencing of the human genome has given rise to the next scientific opportunity of the twenty-first century: functional annotation of the proteome. About one-fifth of the genome encodes secretory proteins, a small number of which represent signaling molecules for G-protein coupled receptors (GPCRs). The major challenge that we will explore in this proposal is the comprehensive 'de-orphanization' (and thereby annotation) of the universe of peptides involved in neuronal GPCR signaling. There are currently about 100 non-olfactory orphan GPCRs, many of which are expected to use peptide ligands; however, fewer than a dozen novel peptides have been identified within the last eight years- and none at all within the last few years. Bioinformatics analyses indicate that the genome contains about 150 untested secretory proteins which possess biochemical similarities to known peptide precursors. We postulate that these proteins contain the missing peptide ligands for orphan GPCRs. However, in order to make these new orphan receptor- peptide matches, fresh approaches to peptide ligand identification are urgently needed. To identify novel peptide neurotransmitters we propose to take an innovative approach integrating expertise in bioactive peptide synthesis (Lindberg laboratory) with expertise in GPCR screening (Roth laboratory). We will experimentally confirm the presence of prohormone convertase-cleavable sites in a bioinformatically-derived list of putative precursors. Bioactive peptides will be generated from all validated precursors through large-scale in vitro posttranslational modification reactions using physiological enzymes (Lindberg laboratory). We will then discover cognate receptors to these peptides via functional screening against the entire genomic complement of known and orphan peptide receptors using facile screening technologies (Roth laboratory). Our results will enable us to match orphan receptors with novel peptide ligands, thus providing new neuropeptide-receptor signaling pairs. Since neuropeptide signaling pathways are critical to brain function and include pathways involved in mood and cognition, mental disorders, and drug reward, our results will significantly advance our understanding of mental health and disease, and may also generate new drug targets. While we will focus on obtaining and testing neuronally-expressed precursors/ligands and receptors, our research is also likely to uncover other ligand-receptor matches; thus a major impact on the many other physiological processes controlled by peptide- GPCR receptor signaling pathways is also anticipated.

Screening Core: which will provide all of the in vitro screening and profiling required to advance compounds. This core will be assisted by the National Institute of Mental Health Psychoactive Drug Screening Program at the University of North Carolina Chapel Hill Medical School.

DESCRIPTION (provided by applicant): Our ultimate goal is to synthesize and characterize novel 5-HT2c serotonin agonists that may be useful for treating schizophrenia and related disorders. Our preliminary findings, as well as those of others, indicate that 5-HT2C agonists are effective in animal models predictive of efficacy against the positive and cognitive symptoms of schizophrenia. Because 5-HT2C agonists are not associated with the metabolic and motoric side- effects characteristic of current typical and atypical antipsychotic drugs, 5-HT2C agonists would afford novel treatment strategies for schizophrenia and related disorders. To achieve this overall goal we have three specific aims. Aim 1: To further expand and improve upon the potent 5-HT2c ligands that we have already identified using rational drug design principles and chemical intuition. Structural alterations will be made to enhance 5-HT2c subtype selectivity and to avoid any 5-HT2B valvuopathic-associated activity, while also improving upon compound solubility and ADMET parameters as needed to achieve the desired efficacy in preclinical animal studies. Aim 2: Characterize binding affinities and functional activities of putative 5-HT2c agonists for the human and mouse 5-HT2c-INI, 5-HT2c-NVN and 5-HT2c-VSV receptor-isoforms. We will also evaluate specificity of putative 5-HT2c agonists by assessing 5-HT2A and 5-HT2B receptor activities by radioligand binding and functional assays. The best compounds emerging from these studies will be subjected to a large battery of assays for identification of off-target activity. Aim 3: To evaluate the best 5-HT2c ligands identified in Specific Aims 1 and 2 in a battery of schizophrenia- related behavioral assays to test for antipsychotic efficacy and possible pro-cognitive effects. The behavioral studies will be conducted with pharmacological and genetic models of schizophrenia-like behaviors; 5-HT2C- knockout mice will serve as controls. The strength of this proposal lies in: (1) Targeting a receptor for which there are no currently approved medications (i.e. a novel molecular target); (2) 5-HT2C agonists are likely to have clinical indications beyond schizophrenia including bipolar disorder, depression, obesity, and drug abuse (i.e. many potential clinical indications); (3) With respect to schizophrenia, two different 5-HT2C agonists have already shown efficacy in animal models predictive of efficacy for positive- and cognitive-like schizophrenia symptoms. Since cognitive symptoms are very difficult to treat in schizophrenia, the 5-HT2C compounds may represent a novel treatment strategy; (4) Finally, it is likely that 5-HT2C agonists will not only be devoid of the metabolic ad motoric side- effects associated with current medications but may also be beneficial from a metabolic perspective.

Production of large quantities of hiighly purified receptors is an underlying requirement for all the projects in ttie Program Project -Structure-Function of Opioid Receptors for Drug Discovery. The production of these samples will be carried out using well established protocols that have now been optimized for use with GPCRs as part of GPCR Network's GPCR Structure Determination Pipeline. These production protocols now include the use of newly developed GPCR fusion partner toolchest for stabilization and crystallization that has increased the quantity and quality of structures that we are able to determine. Most notably, with the use of a new fusion partner, apo-cytochrome b562 (RIL) mutant, BRIL, we were able to determine the structure of the A2A adenosine receptor- ZM241385 to 1.8 A resolution, the structure of NOP, and more recently 2 agonist bound serotonin receptors in an activated state. Structural studies will demand the use of large volumes of highly purified protein for crystallization studies while others doing functional studies will need much less. Studies that led to the structure solution of the K-opioid receptor and the nociception/oprhanin FQ peptide receptor required a large number of constructs and an average of 25 mgs of highly purified protein or about 100 liters of biomass. For receptor-ligand complex structural studies we estimate that 5-10 mgs will be required per structure.

Substances addressing the opioid system are widely used pharmaceuticals. Agonists (narcotic analgesics) are used for the treatment of chronic pain, whereas antagonists are generally effective in the treatment of addictive disorders, including substance abuse (opiate, alcohol, amphetamine) and non-substance, i.e. behavioral addictions such as gaming, gambling and over-eating. The proposed program project will use structural, biophysical, biochemical, and pharmacological approaches to understand ligand binding properties of all opioid receptors (p-, 6, K (MOR, DOR, KOR), and the nociceptin/orphanin FQ peptide receptor (NOP)) and to define the structural basis for molecular recognition and activation. The three overall aims are (1): Understand opioid receptor molecular recognition and functional selectivity using X-ray crystallography to determine structures of ligand-receptor complexes and NMR to understand dynamic behavior. (2) Understand the molecular mechanisms for functional and pharmacological selectivity for the four opioid receptors using computational approaches, and (3) Understand ligand-directed signaling (G- protein-, arrestin-, JNK-dependent) and its relevance in mediating opioid receptor action. Management and administration of the project will be carried out by the Management and Administration Core. Its main responsibilities include (1) Overall management of all project to ensure progress, (2) Prioritization of studies, (3) Assembly of a Scientific Advisory Board, (4) Preparation of required progress reports, (5) Establishment and facilitation of communication routes within the POI as well as with external stakeholders (e.g. SAB, NIH), and (6) Assurance ofthe high quality of studies and results.

DESCRIPTION: G-Protein Coupled Receptors (GPCRs) represent both the largest class of signaling receptors in the human genome and the family most targeted by therapeutic drugs. Responding to ligands that vary from protons to bioamines to lipids to chemokines, their attractiveness for drug discovery reflects the importance of the signals they transduce and, as has become apparent with the determination of their structures, the intrinsic ligand- ability of their binding sites. Despite intense interest, most GPCRs remain sparsely annotated by chemical matter. In this grant we will take a two-pronged approach to overcome these difficulties. In Specific Aim 1 we will develop and validate scalable assays in yeast and mammalian cells with which to screen a library of 5321 drugs and reagents. In Specific Aim 2 we will develop and validate scalable computational screens against modeled structures of the orphan GPCRs, leveraging the empirical hits. By the end of the project period we anticipate validating and executing physical screens against 30 orphan GPCRs and producing computationally optimized lead-like compounds for 20.

? DESCRIPTION (provided by applicant): The Brain Research through Advancing Innovative Neurotechnologies (BRAIN) Initiative has the ambitious goal of elucidating how neuronal ensembles interactively encode higher brain processes. To accomplish this goal, new and improved methods for both recording and manipulating neuronal activity will be needed. In this application, we focus on technologies for manipulating neuronal activity. The major significance of this application is that we will provide an enhanced chemogenetic toolbox that allows non-invasive, multiplexed spatiotemporal control of neuronal activity in domains ranging from single synapses to ensembles of neurons. To achieve this, we will provide: Chemical actuators with improved pharmacokinetics and pharmacodyamics suited for use with current DREADDs in eukaryotes ranging from Drosophila to primates (Specific Aim #1) Photo-caged CNO and other chemical actuators to provide millisecond-scale control (Specific Aim #1) Novel DREADDs and 'split-DREADDs' targeted to distinct neuronal pathways to enable multiplexed interrogation of neuronal circuits (Specific Aims #2 and 3) Chemogenetic platforms with minimized desensitization and down-regulation (Specific Aim #3)

We propose to develop a deeper understanding of the structural basis for the behavior/function of the opioid receptor subfamily of G protein-coupled receptors (GPCR). Structures of all three opioid receptors: ? -opioid receptor (MOR), ? -opioid receptor (KOR), ?-opioid receptor (DOR), and the opioid-like nociceptin/orphanin FQ opioid-like receptor (NOP), have recently been solved. This breakthrough created a unique opportunity to pursue a comprehensive understanding of ligand recognition and selectivity, as well as structural mechanisms of opioid receptor activation and biased signaling. Attaining this goal will require solving a large number of structures of ligand-receptor complexes similar to what is routinely done in structure based drug design targeting soluble proteins. This is now possible since our GPCR Structure Determination Pipeline (GSDP) and our newly developed GPCR Protein Fusion Toolchest has enabled large-scale structure determination of ligand-receptor complexes. The GSDP has been used by us in the successful structure determination of 14 different human GPCRs and their complexes, including two new receptors in activated states with bound agonist. In this proposed study, we will conduct similar efforts toward the crystallization and structural determination of ligand-receptor complexes of all of the receptors in the opioid family using agonists, antagonists, inverse agonists, and allosteric modulators. Criteria for opioid receptor and signaling pathway selectivity will be approached by attempting structure determination of several complexes of functional near-wild type constructs and rationally designed mutants. Within three specific aims we will solve several ligand-receptor structures on (1) KOR, (2) NOP, and (3) MOR and DOR.

DESCRIPTION (provided by applicant): Substances addressing the opioid system are widely used pharmaceuticals; agonists (narcotic analgesics) for the treatment of chronic pain; antagonists generally for the treatment of addictive disorders, including substance abuse (opiate, alcohol, amphetamine) and non-substance, i.e. behavioral addictions. Given the difficulties associated with opioid agonist therapy, high risk of death due to overdose (more victims were reported in 2008, than overdose of heroin and cocaine combined) and the development of tolerance and addiction, there is a need for safer narcotic analgesics. The proposed research program uses structural, computational, biophysical, biochemical, and pharmacological approaches: (1) to develop a new level of understanding of the action the opioid receptors, | j , 6, K, and the nociceptin receptor, defining the structural basis for molecuar recognition and activation, and (2) to design new binding ligands. Aims are to (1) understand opioid receptor molecular recognition and functional selectivity using X-ray crystallography and NMR; (2) understand the molecular mechanisms for functional and pharmacological selectivity for the four receptors using computational approaches; and (3) understand ligand-directed signaling (G-protein-, arrestin-, JNK-dependent) and its relevance in mediating opioid receptor action. Three projects supported by four core groups via inter-project collaborations will address important but difficult questions at a scale not imagined prior to this year. Project 1 will carry ut structural determination on ligand bound complexes. Project 2 will carry out computational studies to define the binding pockets of opioid receptors and perform computer-assisted SBDD to design a new series of optimized compounds including allosteric, functionally selective and bitopic ligands. Project 3, will carry out in vitro and in vivo studies to characterize pharmacological properties of ligands and generate new receptor biased mutants to test hypotheses related to functional selectivity and opioid receptor actions. Core groups support the aims of all 3 projects and will be responsible for (1) large-scale production of purified opioid receptors; (2) synthesis of opiate ligands including newly designed tool compounds for structural and functional studies; (3) molecular profiling and screening of new small molecules, and (4) management and administration of the overall program. Facility resources will be provided by the NIGMS GPCR Networks' Structural Determination Pipeline for large-scale protein production and structure solution and the NIMH Psychoactive Drug Screening Program resources for selectivity profiling of novel small molecules.

PROJECT SUMMARY: Scientific summary: The 5-HT2-serotonin and D2-dopamine families of receptors represent essential targets for most atypical antipsychotics as well as drugs useful in treating obesity, sleep disorders, psychosis and autistic-spectrum disorders. It is currently unknown how these drugs interact with their target receptors. Here we aim to obtain high resolution structures of 5-HT2A- and 5-HT2B- serotonin and D2- and D4-dopamine receptors with an array of small molecules. These studies are both innovative technically and conceptually by providing an unprecedented understanding of the molecular and atomic details responsible for psychoactive drug actions. Technical achievements will include the structure of LSD in complex with its key serotonin receptor targets and typical and atypical antipsychotic drugs in complex with their serotonin and dopamine receptor targets. Additionally, models which attempt to explain ligand-specific properties of biased signaling from the perspective of ligand-receptor interactions will be tested by a combination of direct structural studies, molecular modeling, docking, site-directed mutagenesis and functional studies. We anticipate that our studies will reveal key ligand-receptor interactions essential for biased signaling for serotonin and dopamine receptors. Elucidating the molecular details for such interactions provides a template for the structure-guided design of novel therapeutics.

Project Summary G-protein coupled receptors (GPCRs) represent the single largest class of druggable targets in the human genome. Of the 390 or so druggable and non-olfactory human GPCRs there exist many which are orphan and/or understudied; we refer to these as ?oGPCRs?. Here we will illuminate the pharmacology, signaling pathways, chemical biology, distribution and function of the 143 oGPCRs listed in the RFA. This project seeks to discover and develop specific, community accessible tools? chemical probe molecules and engineered animals?that enable investigators to interrogate the biological functions of oGPCRs. Given the central importance of GPCRs for all areas of biomedical research, illuminating the pharmacology, function, signaling and chemical biology of these oGPCRs will have far-reaching impact for both therapeutics and basic biomedical science.

Mechanistic insights into LSD actions at 5-HT2A-serotonin receptors LSD (lysergic acid diethylamide) -- the prototypical hallucinogen -- continues to be a frequently abused psychotomimetic agent with a life-time prevalence as high as 10.9% among all individual surveyed and as high as 3% among high school students. LSD has a complex pharmacology with significant interactions with dozens of G-protein coupled receptors (GPCRs) and it exerts primary actions at serotonin 2A (5-HT2A) receptors. Various experiments have shown that ligand binding to G protein-coupled receptors (GPCRs) can activate, inhibit, or exert no effects on the G protein-dependent signaling pathway while having similar or diverse actions on a G protein-independent pathway through ?-arrestin (?ARR). In brain, these actions can be mediated through ?ARR 1 and/or 2. In recent papers in Science and Cell, the Roth lab has reported that LSD is a potent ?ARR-biased 5-HT2A receptor agonist. In preliminary experiments with wild-type mice, we find that LSD produces hyperactivity in the open field, it disrupts prepulse inhibition, and stimulates repetitive and stereotyped responses (head-twitch, nose-pokes, retrograde walking, unsupported rearing, and grooming). In contrast, the hyperactivity is blunted and the other behaviors are abrogated in the global ?ARR2 knockout mice. Nevertheless, additional physiological and behavioral responses have been ascribed to LSD that may also involve G proteins or ?ARR1. The Overall Goal of the proposed research is to clarify the role of ?ARR in mediating the actions of LSD at 5-HT2A receptors in vitro and in vivo. Our Central Hypothesis is that ?ARR-mediated signaling through 5-HT2A receptors will play a major role in many responses to LSD. Relevance: We have already reported on the antipsychotic effects ?ARR-biased dopamine 2 receptor compounds exert on behavior. We have evidence that some of behavioral effects of LSD are mediated at least through ?ARR2. With various strains of mice we will define a role for ?ARR1 and ?ARR2 signaling through 5-HT2A receptors and, thereby, reveal how activation of this receptor may underlie hallucinations in humans.