John R. Traynor - US grants

DESCRIPTION (provided by applicant): The long-term objective of this proposal is to identify regulators of G protein signaling (RGS) proteins as therapeutic targets for medications to treat the misuse of mu-opioids and other drugs of abuse. RGS proteins are GAPS (G protein accelerating proteins) that negatively regulate signaling through G protein-coupled receptors, including receptors for mu-opioids and dopamine. For example, the rewarding effects of morphine and cocaine are increased in mice lacking the gene for one particular RGS protein (RGS-9). RGS proteins also serve to control the severity of the withdrawal response seen on removal of chronic morphine. On the other hand they may promote tolerance. These findings suggest the ability to stimulate RGS activity would decrease the rewarding effects of opioids and stimulants and decrease the severity of withdrawal symptoms. Conversely, an inhibitor of RGS protein action might be a useful adjunct to morphine analgesia, without promoting tolerance. The potential of RGS proteins as drug targets is exciting but does depend upon a better understanding of the specificity of RGS action since there are >30 proteins with RGS action. Our previous studies suggest that RGS proteins by acting as modulators also control which signaling pathways are activated: an effect that could lead to selectivity of pharmacological action. The present proposal has 3 specific aims (1) to study how RGS protein GAP activity can selectively regulate signaling, (2) to identify the RGS proteins that modulate signaling through the mu-opioid receptor and (3) to examine the consequences of this modulation on opioid efficacy and potency. The work will study mu-opioid receptor-G protein coupling and signaling to adenylyl cyclase, increases in intracellular calcium, inwardly rectifying K+ channels and the MAP kinase pathway. Roles for RGS proteins will be determined using Ga proteins that are insensitive to the GAP activity of all RGS proteins and techniques including RNAi to identify which specific RGS proteins are responsible for the modulation and control of mu-opioid signaling. An understanding of roles for RGS proteins in the actions of mu-opioids may lead to improvements in the treatment of pain as well as treatment of opioid and stimulant abuse.

Abstract The overall purpose of this training grant is to provide postdoctoral training to young basic scientists and physicians in the area of neurobiology of substance abuse, with a focus on the mode of action of psychostimulant and opiate drugs at the genetic, molecular, circuit and behavioral levels. Our objective is to provide an exciting and innovative environment with world class facilities and faculty to develop the next generation of scientists working in drug abuse. Training will take place in the multidisciplinary setting at the University of Michigan. The faculty members are all NIDA grantees (or co-Investigators) and have expertise in the neurobiology of substance abuse, with particular emphasis on the area of opioid and psychostimulant drugs. The focus of the proposal is the training of postdoctoral fellows in state-of-the-art approaches for studying mechanisms underlying abuse of psychoactive drugs. This includes studying the genetic, developmental and environmental factors that lead to vulnerability to substance abuse; the mode of action of drugs of abuse at the molecular, cellular, anatomical and behavioral levels; and the long-term consequences of psychoactive drugs on the brain, as mediated through mechanisms of neural plasticity; and the development of medications, and prevention strategies. The working assumption is that the functional and structural brain remodeling associated with chronic drug use lies at the basis of tolerance, sensitization, physical dependence, and psychological addiction to these drugs. The drug abuse research community at the University of Michigan is of high quality and has a long history in the field. Beyond their individual strengths, the training faculty members have long-standing collaborative scientific and training relationships with each other. These historical strengths have recently been further enhanced with a number of initiatives at the University of Michigan, designed to facilitate life science research in general, and neuroscience research in particular. They include state-of-the- art tools for mouse and rat genetics, genomics, proteomics, and informatics. Thus, our postdoctoral fellows will profit from a highly sophisticated, yet supportive, research and training environment.

[unreadable] DESCRIPTION (provided by applicant): Opioid dependence plays a major role in abuse of these drugs and is a concern in the clinic. It is important to understand mechanisms underlying dependence if we are to improve the treatment of pain and to treat and/or prevent opioid abuse. Acutely, mu- opioids activate Gai/o proteins to inhibit adenylyl cyclase, but following chronic treatment dependence can be demonstrated at the cellular level by the compensatory increase in adenylyl cyclase activation, referred to as sensitization. The objective of this project is to better understand the mechanisms of the development of dependence at the cellular level, particularly how the compensatory changes in adenylyl cyclase are modulated by co-localization with other signaling proteins in plasma membrane microdomains referred to as lipid rafts. Mu-opioid receptors are found in lipid rafts and the adenylyl cyclase isoforms which are sensitized tend to be localized in lipid rafts, while some signaling proteins which have a role in sensitization are able to relocalize into or out of lipid rafts. To determine the role of lipid rafts in the development and expression of mu-opioid mediated adenylyl cyclase sensitization, we will: 1) Examine the distribution of mu-opioid signaling components in lipid rafts and non-raft fractions and develop assays to measure their function. 2) Prepare lipid-raft enriched and non-raft plasma membrane fractions from cells treated with mu-opioid agonist to compare sensitization of adenylyl cyclase and determine if changes in sensitization are related to relocalization of signaling proteins into or out of lipid rafts. 3) Determine if lipid rafts play a necessary role in adenylyl cyclase sensitization across different receptors and in different cell types. Changes occur in signaling proteins, such as adenylyl cyclase, in membranes of neuronal cells following long term exposure to opioid drugs, such as morphine or heroin. These compensatory changes result in withdrawal on removal of the opioid. To better treat and prevent opioid dependence, the goal is to understand the mechanisms causing these changes, particularly in relation to the location of modulatory signaling proteins in or out of cell membrane microdomains referred to as lipid rafts. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Depression is an illness that is poorly understood and treatments are only effective in a certain percentage of the population. Antidepressant medications, including selective serotonin reuptake inhibitors (SSRIs), are the most prescribed antidepressant medications and are effective in animal models of depression. SSRI's act indirectly via 5HT receptors including receptors that couple to heterotrimeric G proteins. Regulators of G protein signaling (RGS) are a family of intracellular proteins that negatively modulate receptor-mediated G protein signaling. Using mutant mice expressing an RGS-insensitive G?i2 protein we have identified a pathway downstream of 5HT1A receptors that mediates antidepressant and anti-anxiety behaviors. This led us to consider that RGS protein modulation of 5HT1A receptor signaling may play an important role in depression and so present a novel target for the treatment of depression. However, there are >20 mammalian RGS proteins and a general inhibitor will likely have a broad range of effects. In this proposal we seek to understand the function of G?i2-coupled 5HT1A receptors in animal models of depression by identifying the location of receptors responsible for the observed phenotype, the biochemical mechanism underlying the antidepressant-like effect, and the particular RGS proteins involved. The proposed work will use a combination of behavioral, biochemical and molecular biological approaches in genetically modified mice with knock-in of RGS-insensitive G?i2 protein, including conditional knock-in animals that avoid problems associated with compensatory changes and allow for site-specific expression of the G?i2 mutation. These studies will provide knowledge of RGS proteins in serotonin signaling in animal models of depression and identify novel targets for antidepressant therapy. In particular, RGS proteins may be direct drug targets and/or act to promote the antidepressant actions of currently available SSRI's without enhancing their unwanted effects. PUBLIC HEALTH RELEVANCE: Depression is a poorly understood illness that affects approximately 10 million Americans. Moreover, treatments for depression are ineffective in a majority of patients. We have recently identified a transgenic mouse model that shows antidepressant-like behavior and responds very well to selective serotonin reuptake inhibitors (SSRIs). These mice carry a mutation that renders specific intracellular signaling proteins (G proteins) unresponsive to their endogenous regulators (RGS proteins). This mutation also increases the effectiveness of drugs targeting the serotonin 5HT1A receptor, a receptor that is linked to several mood disorders. In this proposal we seek to increase our understanding of 5HT1A receptors using behavioral and biochemical measures and identify the particular RGS protein(s) involved in their regulation. Our aim is to identify novel targets for the development of agents to treat depressive illness.

DESCRIPTION (provided by applicant): Depression is an illness that is poorly understood and treatments are only effective in a certain percentage of the population. Selective serotonin reuptake inhibitors (SSRIs) are the most prescribed antidepressant medications. SSRIs act indirectly via 5HT receptors, including the 5HT1A receptor that couples to heterotrimeric G proteins. Regulators of G protein signaling (RGS) are a family of intracellular proteins that negatively modulate receptor-mediated G protein signaling. Using mutant mice we have identified a subset of 5HT1A receptors coupled to G?i2 and negatively regulated by RGS proteins that appear to mediate the beneficial actions of SSRIs. This led us to consider that RGS protein modulation of 5HT1A receptor signaling may play an important role in depression and so present a novel target for the treatment of depression. However, there are >20 mammalian RGS proteins and a general inhibitor will likely have a broad range of effects. In this proposal we seek to identify the specific RGS protein that modulates 5HT1A receptors and therefore controls behavioral responses to 5HT1A receptor activation related to mood disorders. The proposed work will use a combination of behavioral, biochemical and molecular biological approaches to provide knowledge of RGS proteins in serotonin signaling in animal models of depression and implicate RGS proteins as drug targets to promote the antidepressant actions of currently available SSRI's without enhancing their unwanted effects. PUBLIC HEALTH RELEVANCE: Depression is a poorly understood illness that affects approximately 10 million Americans. Moreover, treatments for depression are ineffective in a majority of patients. We have recently identified a family of intracellular proteins (RGS proteins) that appear to enhance the beneficial actions of selective serotonin reuptake inhibitors (SSRIs) which are the major drugs used for the treatment of depression. In this proposal we seek to identify the particular RGS protein(s) involved and so identify a novel target for treatment of depressive illness.

DESCRIPTION (provided by applicant): Regulators of G protein Signaling (RGS) proteins are a family of G protein-coupled receptor (GPCR) accessory proteins that are essential for the temporal and spatial control of cell signaling, including signaling downstream of the mu-opioid receptor (MOR). RGS proteins are GTPase accelerating proteins (GAPs) that accelerate the hydrolysis of Galpha bound GTP and promote the formation of inactive Galpha GDP to switch off signaling by GPCRs. We have shown that RGS proteins serve to terminate signaling of MOR to adenylate cyclase and the MAP kinase pathway. On the other hand, efficient signaling of MOR to release intracellular calcium requires RGS protein GAP activity. Thus, RGS activity controls the balance of signaling pathways within a single cell. We recently used a novel tool to explore regulation of GPCR signaling by RGS proteins: a transgenic knock-in mouse that expresses Galpha proteins that are insensitive to the GAP activity of RGS proteins. In these mice antinociception is dependent on the opioid agonist and pain assay employed. For example, in the hot-plate assay loss of RGS regulation potentiates morphine, without affecting methadone, antinociception. In contrast, in the tail-withdrawal assay removal of RGS activity decreases both morphine and methadone antinociception. This suggests the different neuronal pathways involved in these two behaviors show differential sensitivity to RGS protein action. We propose to continue our exploration of these mice to tackle a series of fundamental questions concerning MOR signaling and its relationship to antinociception. For example: Are the differences between antinociceptive tests due to site-specific variation in RGS action? What is the basis of the observed agonist differences? Can the differences be explained by effects at the level of cell signaling? What is the role of other neurotransmitter systems that also use Galpha proteins? Are more clinically-related pain models also regulated by RGS proteins? Our overall conceptual framework is as follows: 1) RGS activity controls the balance of GPCR signaling to multiple pathways and this balance may be disrupted in pain and 2) RGS-induced changes in MOR-mediated-antinociception may reflect an alteration in the balance between neurotransmitter systems, particularly the nociceptin (NOP) system. The proposed studies will advance our understanding of opioid signaling pathways and their regulation by RGS proteins and explain these actions in the context of altered behaviors. Results from this study may be exploited to develop better analgesic drugs.

? DESCRIPTION (provided by applicant): The current opioid prescription misuse epidemic has renewed interest in obtaining strong analgesic agents with decreased tolerance and addiction liability. Moreover, the high incidence of other side-effects with currently used opioid analgesics such as respiratory depression and constipation reduces their effectiveness in the pain clinic. Hence, there is a need either to develop new analgesics or to improve the clinical profile of existing medications. Morphine and related opioids exert their effects by acting at the orthosteric site on the mu-opioid receptor (MOR), i.e. the site where the endogenous opioid peptides bind. Recent advances in our knowledge of the structure of G-protein coupled receptors (GPCRs) has highlighted the possibility that GPCR function may be controlled by compounds binding at a separate, allosteric, site on the receptors. In this regard we have recently identified positive allosteric modulators (PAMs) that act at MOR (MOR-PAMs). These compounds increase the binding affinity of MOR agonists and the potency and/or maximal response to MOR agonists in vitro. Therefore, MOR-PAMs could possibly to be used as adjuncts to morphine and so reduce the level of opiate required to afford analgesia; thus producing the same functional analgesic response as a higher dose of morphine, but with reduced likelihood of the appearance of side-effects, including of tolerance and dependence. Perhaps more importantly, MOR-PAMs have the potential to be used alone to enhance the activity of endogenous opioid peptides. This would preserve the temporal and spatial characteristics of neuronal signaling and so avoid receptor down-regulation and other compensatory mechanisms that are induced by chronic MOR activation. In this application we seek to establish the value of allosteric modulation of MOR in vitro using an approach that involves investigation of the properties of allosteric modulation of MOR, structural biology to identify an allosteric binding site on MOR and development of improved allosteric probes. Detailed understanding of the actions of allosteric modulators of MOR will provide new information on mechanisms by which MOR function may be controlled and, together with the identification of high affinity probes, will pave the way for future drug development efforts of MOR modulators as analgesic adjunct drugs and/or novel analgesics.

? DESCRIPTION (provided by applicant): The aims of the proposal are in accord with the mission of the Addiction Treatment Discovery Program (ATDP): To discover potential pharmacological treatments for substance abuse in humans, with an emphasis on relapse prevention, through preclinical testing and evaluation of compounds. The principal goal of the project is to provide potential treatment agents for polydrug dependence by targeting single chemical entities that mimic the profile produced by a buprenorphine/naltrexone combination. There is evidence from both clinical and preclinical research that this combination can help prevent relapse to drug taking behaviour, including both opioids and cocaine. The target compounds lack of, or very limited, mu opioid receptor efficacy will render the compounds safe and ethically acceptable for use in both opioid using and non-opioid using addicts. Efficacy as treatment agents will also come from the compounds kappa opioid receptor antagonist activity, coupled with agonism at NOP (nociceptin) receptors and antagonism at delta opioid receptors. Lead compounds have been identified during the current funding period from within the orvinol and naltrexone series. These series have already produced several compounds of clinical or veterinary utility, including buprenorphine and naltrexone. Key, recent medicinal chemistry discoveries by our group including, 1) the role of a methyl group at C7 in the orvinol series in reducing efficacy at kappa receptors and increasing NOP activity and 2) the role of the C14 side chain in the naltrexone series in allowing substantial NOP activity to be introduced to this series of opioid antagonists, has allowed compounds with the desired profile to be developed. These discoveries will be extended; in particular, ligands from the orvinol series have been identified for further evaluation with one currently undergoing extensive in vivo testing and evaluation of its ADME-tox profile. Follow-on/back-up compounds are also being developed. The significance of this work is that it will tell us whether single compounds can be developed that can block relapse to both opioid and cocaine use, what types of relapse are blocked (drug-, stress- or cue-primed) and will allow us to move our identified candidate therapeutics closer to the clinic.

DESCRIPTION (provided by applicant): Regulators of G protein Signaling (RGS) proteins are a family of G protein-coupled receptor (GPCR) accessory proteins that are essential for the temporal and spatial control of cell signaling, including signaling downstream of the mu-opioid receptor (MOR). RGS proteins are GTPase accelerating proteins (GAPs) that accelerate the hydrolysis of Galpha bound GTP and promote the formation of inactive Galpha GDP to switch off signaling by GPCRs. We have shown that RGS proteins serve to terminate signaling of MOR to adenylate cyclase and the MAP kinase pathway. On the other hand, efficient signaling of MOR to release intracellular calcium requires RGS protein GAP activity. Thus, RGS activity controls the balance of signaling pathways within a single cell. We recently used a novel tool to explore regulation of GPCR signaling by RGS proteins: a transgenic knock-in mouse that expresses Galpha proteins that are insensitive to the GAP activity of RGS proteins. In these mice antinociception is dependent on the opioid agonist and pain assay employed. For example, in the hot-plate assay loss of RGS regulation potentiates morphine, without affecting methadone, antinociception. In contrast, in the tail-withdrawal assay removal of RGS activity decreases both morphine and methadone antinociception. This suggests the different neuronal pathways involved in these two behaviors show differential sensitivity to RGS protein action. We propose to continue our exploration of these mice to tackle a series of fundamental questions concerning MOR signaling and its relationship to antinociception. For example: Are the differences between antinociceptive tests due to site-specific variation in RGS action? What is the basis of the observed agonist differences? Can the differences be explained by effects at the level of cell signaling? What is the role of other neurotransmitter systems that also use Galpha proteins? Are more clinically-related pain models also regulated by RGS proteins? Our overall conceptual framework is as follows: 1) RGS activity controls the balance of GPCR signaling to multiple pathways and this balance may be disrupted in pain and 2) RGS-induced changes in MOR-mediated-antinociception may reflect an alteration in the balance between neurotransmitter systems, particularly the nociceptin (NOP) system. The proposed studies will advance our understanding of opioid signaling pathways and their regulation by RGS proteins and explain these actions in the context of altered behaviors. Results from this study may be exploited to develop better analgesic drugs.