Laura M. Bohn, Ph.D. - US grants

The determination of the mechanism of the neurotrophic actions of opioids is the broad goal of the ongoing research. The rat C6 glioma cell line will be used as an astrocytic model system. Recent evidence suggests that this cell line expresses at least two different opioid receptor types following treatment with desipramine (DMI). The receptors will be identified by northern blot analysis using radiolabeled intact mu, kappa and delta cDNAs. Addition of opioid agonist to DMI-treated C6 cells inhibits mitogen stimulated cell proliferation. Identification of the specific receptor(s) involved in the opioid inhibition of cell proliferation will be accomplished by using specific antagonists to each of the opioid receptors detected by northern blot analysis. Mitogen stimulation of C6 cells leads to an increase in the production of nitric oxide (NO) which has been positively correlated with an increase in cell proliferation. The ability of opioids to modulate NO production will be examined. The regulation of astrocyte proliferation in developing brain may prove to be a normal function of endogenous opioids in the control of cell numbers during ontogeny. Moreover, this effect of opioids on the fetus of drug-addicted mothers may contribute to the physiological and psychological delays observed in offspring. Finally, the role that opioids play in regulating gliogenesis may provide insight into mechanisms of neural regeneration after brain injury where astrocytic proliferation is prevalent.

Morphine has been used for centuries to alleviate pain and still remains the analgesic standard to which newly developed drugs are compared. However, prolonged use of morphine, as well as other opiates, can lead to the development of tolerance and ultimately, addiction. The neuronal signaling mechanisms by which options mediate antinociception through their G protein-coupled receptors is not clearly understood. Likewise, the process of tolerance development remains to be defined at the cellular level. There is some evidence to suggest that desensitization (discontinued signaling) and/or downregulation (loss of receptors) of opioid receptors may be involved in controlling these processes. Two major classes of proteins involved in GPCR regulation are GPCR kinases (GRKs) and arrestins. Although a number of recent studies have examined the contribution of GRKs and arrestins to opioid receptor regulation in cell cultures, there have been no conclusive in vivo studies of functional significance to this date. Our laboratory has a number of transgenic mouse lines that lack different GRKs or barrestins. This report proposes the following specific aims: AIM I: To determine differences in pain perception after morphine treatment between wild type and GRK or barrestin knockout mice. AIM II: To identify whether morphine induces different degrees of tolerance between wild type and GRK or barrestin knockout mice. AIM III: To evaluate m opioid receptor function in brain and in primary neural cultures derived from wildtype and knockout mice. AIM IV: To generate a transgenic mouse that overexpresses a GRK or barrestin. By this approach we hope to identify which GRKs/arrestins are essential in morphine's actions regarding analgesia and the development of tolerance. We envision that these observations may lead to greater understanding of the mechanisms behind morphine's potent actions in vivo. Advances in this field may not only lead to potential gains in pain control, but may also lead to better treatment and possible prevention of opiate addiction.

Morphine has been used for centuries to alleviate pain and still remains the analgesic standard to which newly developed drugs are compared. However, prolonged use of morphine, as well as other opiates,, can lead to the development of tolerance, dependence, and ultimately, addiction. Recently, we showed that a G protein-coupled receptor regulatory element, betaarrestin-I, is required for mu opioid receptor (muOR) desensitization in vivo. Mice lacking the Betaarrestin-2 molecule, but not Betaarrestin-I, experience enhanced and prolonged morphine analgesia. Moreover, muOR desensitization proved to be essential in the development of morphine antinociceptive tolerance yet the presence of a functional desensitization mechanism was not required to develop morphine dependence. The dissociation of morphine tolerance and dependence in these mice raises the question whether other manifestations of muOR activation could be differently effected by a loss of Betaarestin-2. Using the Betaarrestin-2-knockout (betaarr2-KO) mice, we have the opportunity to evaluate whether other physiological parameters are subject to tolerance or sensitization after chronic morphine in an animal which lacks tolerance to morphine antinociceptive effects. We anticipate that not all of the physiological functions effected by chronic morphine will experience a change in regulation in the absence of Betaarrestin-2 just as the Betaarr2-KO mice experienced the same degree of morphine dependence as their wild-type littermates. This proposal aims to examine the following questions: AIM I: To test whether morphine tolerance occurs in the suppression of respiration or gastrointestinal transit in mice that lack morphine antinociceptive tolerance. AIM II: To test the effect of chronic morphine on the dopamine system in mice that lack morphine on the dopamine system in mice that lack morphine antinociceptive tolerance. AIM III: To examine the rewarding properties of morphine in mice that lack morphine antinoci ceptive tolerance. My hope is that this research plan will shed light on the role of muOR regulation in mediating the diverse physiological effects that occur with chronic morphine use or abuse. This research proposal has also been designed to direct my scientific development, allowing for the acquisition of diverse experimental approaches to better address the problem of drug abuse in an animal model. By combining the knowledge of systems relating to whole animal physiology, complex animal behaviors, as well as highly intricate neurochemical assessments, I hope to broaden my scientific approach in the pursuit of several independent avenues for my continuing research in the field of drug abuse. I envision that the observation made in the Betaarrestin-2 knockout mice, may lead to greater understanding of the mechanisms behind the potential for developing morphine tolerance, dependence, and addiction following chronic use or abuse.

DESCRIPTION (provided by applicant): Morphine, the gold standard for pain relief, is clinically limited by several adverse properties including tolerance, dependence and the onset of respiratory suppression. Opiate analgesics, such as morphine, mediate their biological effects mainly via activation of the mu opioid receptor (muOR). The muOR, a G protein coupled receptor (GPCR), is regulated by GPCR kinase (GRK) phosphorylation and subsequent binding of betaarrestins in a process known as receptor desensitization. The genetic ablation of betaarrestin2 (betaarr2)in mice has seemingly paradoxical effects; it leads to enhanced and prolonged morphine analgesia and dramatically reduces morphine tolerance. Moreover, these mice display no change in physical dependence; in contrast, respiratory suppression is practically eliminated. The effects of morphine in this mouse model approach a hypothetical, optimal opiate analgesic for clinical use. The coupling of the muOR to G proteins is elevated in brain regions associated with morphine analgesia in the betaarr2knockout (betaarr2-KO) mice which may contribute to the enhanced analgesic responses. However, the role of betaarr2 in morphine-induced respiratory suppression is unclear. Furthermore, while morphine analgesia is enhanced in the betaarr2-KO mice, lresponses to other opiates such as fentany and methadone, are unaltered. We have hypothesized that GRKs and betaarrestins regulate muORs and the specificity of this regulation is determined by the opiate agonist onboard. These regulatory differences at the level of the muOR may thereby underlie the diverse pharmacological effects produced by a wide-range of clinically relevant opiates such as morphine, fentanyl, methadone and buprenorphine. We have the unique opportunity to evaluate the contributions of GRKs and betarr2 to opiate responses in vivo by studying opiatemediated behaviors and physiological responses in strains of mice lacking individual GRKs (GRK2, GRK3, GRK4, GRK5, and GRK6) or betarr2. Neurochemical alterations will be assessed in these same animals in parallel to the behavioral studies with a focus on receptor trafficking, receptor desensitization, and downstream neuroadaptive changes. HEK-293 cells wil be used as a model system to further elucidate the molecular mechanisms underlying the observed in vivo phenomena. The overall objective of this study is to gain a greater understanding of muOR regulation in determining the specificity of drug effects on physiological and pathological conditions. Toward this goal, these studies focus on examining the contribution of GRKs and parrestins to muOR regulation in the development of opiate tolerance (AIM I), dependence (AIM II), and the side effects induced by opiates (AIM III). Illuminating the intricacies of muOR regulation may point to fine-tuning receptor responsiveness to increase analgesic efficacy, limit abuse liabilityand eliminate adverse side effects in developing opiate pharmaceutical therapies.

DESCRIPTION (provided by applicant): We have previously described a novel opioid agonist, termed herkinorin, that activates mu opioid receptors without recruiting ?arrestin2. ?arrestins are involved in desensitizing and regulating opioid receptors and the removal ?arrestin2 in mice increases morphine's analgesic potency while attenuating morphine tolerance. Mice that lack ?arrestin2 also display significantly reduced morphine-associated side effects, such as constipation and respiratory suppression. Therefore, the development of a ligand that could activate the mu opioid receptor, but not invoke the ?arrestin2 interaction, may pharmacologically recapitulate the effects of morphine in ?arrestin2 knockout mice. Such compounds would ideally produce analgesia with very limited tolerance or side effects. Opioid tolerance has been associated with the desensitization of the mu opioid receptor. One would predict that in the absence of ?arrestins, the MOR would not be desensitized. However, chronic herkinorin treatment in cells leads to a desensitized MOR. This proposal has been developed to assess whether that desensitization is due to protein kinase C-dependent regulatory mechanisms as these kinases have been previously shown to regulate mu opioid receptor responsiveness. Further, we will directly test whether herkinorin will lead to analgesic tolerance in mice. Such studies are necessary to determine the feasibility of developing opioid agonists that do not recruit ?arrestins to promote analgesia in the absence of tolerance and side effects. The Public Health Relevance: Our studies are designed to explore the feasibility of designing new opioid analgesics that we predict will produce less analgesic tolerance and limited side effects such as constipation and respiratory suppression. We will base our studies on a lead compound that we have recently described;this compound has very promising biochemical properties that would lead us to predict it would possess these favorable characteristics.

DESCRIPTION (provided by applicant): As an abundant neurotransmitter in the brain and throughout the body, serotonin plays a significant role in determining many biological processes. Serotonin mediates its actions by binding to and activating G protein-coupled receptors (GPCR) on the surface of neurons and other cells. The activity of a particular GPCR, the serotonin 2A receptor, is associated with several neuropsychiatric disorders and many efforts spanning several decades have been made to modulate the activity of this receptor to improve psychiatric treatments. For example, the atypical antipsychotic drug clozapine was designed to block the activity of this receptor. Additional pharmaceutical therapeutics, such as selective serotonin uptake inhibitors (SSRIs) and related antidepressants, may indirectly affect activity at this receptor by altering brain serotonin levels. The serotonin 2A receptor is also a prominent target for drugs of abuse, particularly those classified as hallucinogens;lysergic acid diethylamide (LSD) mediates its hallucinogenic effects by directly activating this receptor. Other drugs of abuse that indirectly act at the 2A receptor are amphetamines, particularly MDMA or "ecstacy". We are proposing a new basis for drug development targeting the regulation of the serotonin 2A receptor. We have found that serotonin activates the 2A receptor and downstream signaling is mediated by an intracellular regulatory protein called ?arrestin. Regulation of the serotonin 2A receptor in vivo may set the tone for neurological sensitivity to endogenous levels of serotonin as well as the responsiveness to pharmacological agents. Our studies explore the biochemical, physiological and behavioral consequences of disrupting the serotonin 2A receptor-?arrestin interactions in vivo. We predict that drugs that disrupt the serotonin 2A receptor-?arrestin interaction might provide a means to alter the sensitivity of the receptor to the levels of serotonin present in the brain. These findings may inspire the development of drugs that could maintain a desired basal serotonergic tone while eliminating excessive receptor responsiveness to endogenous serotonin. Such strategies in drug design may prove to be clinically useful for treating neuropsychiatric disorders including those associated with drugs of abuse. PUBLIC HEALTH RELEVANCE: Our studies explore the biochemical, physiological and behavioral consequences of disrupting serotonin receptor regulation. We predict that drugs that disrupt this regulation might provide a means to alter the sensitivity of the receptor to the levels of serotonin present in the brain. These findings may inspire the development of drugs that could be clinically useful for treating neuropsychiatric disorders such as schizophrenia as well as those associated with drug addiction.

? DESCRIPTION (provided by applicant): We propose to develop new kappa opioid receptor (KOR) modulators for early stage development towards treating addictive and mood disorders. Dynorphins are stress peptides and act at the KOR. Therefore, to suppress dynorphin-mediated effects, negative regulators of KOR are sought. There is considerable evidence that KOR signals through ?arrestin2 to mediate certain side effects (sedation and dysphoria) and through G proteins to mediate its analgesic and antipruritic effects. Therefore, we propose to develop compounds that antagonize the ?arrestin2-interacting receptor. Specifically we aim to deliver: 1. Competitive antagonists that are potent and efficacious in suppressing ?arrestin2 recruitment; 2. Partial agonists that are potently competitive at blocking dynorphin-stimulated ?arrestin2 recruitment while preserving full agonism in G protein signaling; 3. Negative allosteric modulators that will decrease KOR responsiveness to dynorphins. In this proposal, we present an update on the extensive progress we have made in introducing the first small molecule, G protein biased KOR agonists to the field. We also provide substantial preliminary data supporting a successful campaign to develop the aforementioned antagonists, biased partial agonists and negative allosteric modulators. In particular, the negative allosteric modulators will be first in class for this receptor. This proposal seeks 5 years of support to provide the initia preclinical characterizations and chemical optimizations of these compounds into drug candidates. In line with this goal, we will fully characterize the pharmacological properties of th compounds across functionally diverse cell-based assays with of a goal of identifying compounds capable of fine-tuning KOR responsiveness. Cell-based responses will be validated in mouse models assessing locomotor responses, antinociceptive activity and suppressing pruritis (itch response) to determine that compound maintains the pharmacological profiles in vivo. Drug metabolism and pharmacokinetics of the compounds will be performed to provide information for continued medicinal chemistry optimization rounds and to advance compounds to clinical development. Our enthusiastic team consists of established medicinal and synthetic chemists; an opioid neuropharmacologist (with both molecular and behavioral pharmacology expertise); and an expert in pharmacokinetics and drug metabolism. The development of pharmacological tools across diverse pharmacophores and correlating their properties with in vivo response profiles will provide guiding evidence of the optimal chemical and pharmacological properties required to produce the desired physiological responses.

Opiate analgesics act at the mu opioid receptor (MOR) in humans to alleviate pain but also to produce unwanted effects such as constipation, respiratory suppression/overdose and addiction. The overall potency and efficacy of an agonist at the receptor may be determined not only by how well the drug binds the receptor but also by how well the receptor engages with intracellular signaling proteins, such as barrestin2. Our studies over the last decade have led us to hypothesize that if a drug could activate the MOR yet not induce barrestin2 interactions with the receptor, then such a drug might be an efficacious analgesic with limited side effects, producing less tolerance, constipation and overdose potential. In the last 5 years, we have generated more than 50 new MOR agonists that activate G protein signaling pathways in a highly biased manner, such that they do not recruit barrestins. Using these compounds, we have tested our hypothesis and have found that this is approach will allow for the separation of analgesic efficacy in vivo from respiratory suppression. We have identified lead candidates and have filed for patent protection of this series of compounds and are currently pursuing clinical development. While certain physiological side effects have been limited, there is no indication thus far that the compounds will not produce reward or be subject to abuse. In this current proposal, we are seeking to test whether they promote drug preference and to further refine candidate compounds and also to introduce affinity at an additional receptor target as a means to introduce abuse deterrence into the compounds. In our initial screens for target selectivity, we noted some affinity for D3 dopamine receptors in a series that was not further pursued (as we focused on MOR selectivity). However, given that D3 dopamine receptors play an important role in maintaining dopamine homeostasis, can greatly impact drug reward thresholds, and have been identified as a drug abuse deterrence target by NIDA, we will focus on optimizing D3 antagonism while maintaining biased MOR agonism in this compound series. In this multidisciplinary study, the Bohn pharmacology laboratory will work in a highly collaborative manner with the Bannister medicinal chemistry laboratory to generate and optimize multiple derivatives on the compound series (Aim 1). We will use several cell-based assays to characterize the signaling parameters induced by these compounds with the goal of finding opiates that maintain G protein over barrestin signaling bias at MOR yet also display D3 DAR antagonism (Aim 2). These compounds will be tested in mouse models to determine if their signaling properties correlate with their ability to produce analgesia with less respiratory suppression and also if dopaminergic behaviors, such as locomotor activity and conditioned place preference are avoided (Aim 3). Finally, in collaboration with Dr. Michael Cameron of Scripps Florida, we will evaluate the DMPK properties of our best candidate compounds. The information garnered from this proposal will prove useful in the clinical development of pain relievers with limited side effects.

OTHER PROJECT INFORMATION - SUMMARY/ABSTRACT The endocannabinoid system plays a major role in modulating pain perception and mood and therefore, ligands that act at the cannabinoid receptors may prove to have wide-ranging therapeutic utility. Prior drug development efforts at the CB1 receptor have involved using one-dimensional signaling outputs to determine overall efficacy and potency. However, recent advances in understanding receptor pharmacology indicate that receptors are capable of engaging in multiple signaling cascades and that the chemical nature of the ligand can direct these downstream signaling pathways. Furthermore, there is increasing evidence that diverse signaling pathways can give rise to distinct physiological responses produced by a drug. This paradigm yields a novel manner by which to fine-tune receptor signaling in order to enhance desirable biological effects (such as pain relief) while simultaneously eliminating unwanted side effects (such as sedation or negative effects on mood). The project described herein is focused on the characterization of functionally selective ligands at the CB1 cannabinoid receptor. We hypothesize that ligands that bind to certain regions of the receptor will lead to different signaling profiles than those that bind to other regions. Moreover, our goal is to use probe compounds characterized for such functional selectivity to determine if they induce certain behavioral responses while sparing other physiological responses. To this end we will work closely with Project 1 to evaluate compounds that are shown to bind to particular residues in the CB1R in multiple signaling assays, validate signaling in neurons (AIM 1) and then test behaviors in mice (AIM 2, Project 3). The development of such important tools will not only serve to allow for the testing the hypothesis that functional selectivity can fine tune drug efficacies in vivo, but may also serve as the building blocks for the development of future therapeutics for pain, depression and addiction.

DESCRIPTION (provided by applicant): Prescription opioid narcotics, such as morphine, oxycodone, and fentanyl, produce analgesia and side effects through activation of the mu opioid receptor (MOR), a G protein coupled receptor (GPCR). Our long-standing goal is to understand how MOR signals to produce distinct biological effects and to ultimately inform the development of therapeutics that will take advantage of good receptor signaling (pain relief) and avoid bad receptor signaling (tolerance, dependence, constipation and other side effects). It has become increasingly evident that different drug structures can elicit different receptor signaling cascade at a single receptor, likely by changing the affinities for association with intracellular binding partners. Further, the intracellular binding partner profile differs between neuronal populations. Therefore, the nature of a drug response can be determined not only by the chemical properties of the drug, but also by the complement of signaling proteins found in residence with the receptor; making it critical to study receptor signaling in physiologically relevant systems. One particular intracellular protein that influences MOR function is ?arrestin2. betaArrestin2 is a scaffolding protein that can act as desensitizing element or as a signal transduction facilitator. Our studies have shown that morphine-induced analgesia is enhanced while tolerance is attenuated in mice lacking betaarrestin2, which implicates betaarrestin2 as a desensitizing factor in pain regulating brain regions. Our collective body of work shows that the severity of certain side effects, including physical dependence and constipation, are significantly reduced in mice lacking betaarrestin2 suggesting that in some organ systems and brain regions, betaarrestin2 facilitates MOR signaling. Since receptor responsiveness to a drug in vivo is ultimately dependent upon the cellular environment that encompasses the receptor, we hypothesize that betaarrestin2 dampens morphine responsiveness in analgesia pathways while it mediates morphine-associated side effects such as physical dependence and constipation. To this end, we propose to elucidate the mechanisms by which betaarrestins regulate MOR in brain regions and tissues that mediate morphine-induced antinociception and tolerance (brainstem), physical dependence (striatum) and constipation (colon). We will utilize new MOR agonists that are functionally selective for activating G protein signaling pathways (we hypothesize this will promote antinociception) and against recruiting betaarrestin2 (we hypothesize that recruiting ?arrestin2 leads to tolerance, dependence and constipation). Published and preliminary evidence suggests that the G protein biased agonists promote antinociception with fewer side effects. We will use these tools to gain a greater understanding of MOR regulation in the endogenous setting as it pertains to in vivo physiologies. These studies should provide guidance for developing therapeutics that preferentially enhance desired effects such as improving pain therapy while preventing adverse reactions.

PROJECT SUMMARY The central focus of this Multi-PI R01 research proposal is the structure-function characterization of the human cannabinoid receptor 2 (CB2), a key protein component of the endocannabinoid system. We aim to develop a fundamental understanding of the structural basis of CB2 function, with the ultimate translational goal of establishing a robust structure-based drug design (SBDD) program. The ECS is a complex network of lipid ligands, receptors, and metabolic enzymes involved in a wide range of important physiological processes. There have been important implications that targeting CB2 may be useful as a means for treating inflammation, pain, neurological disorders and addiction. As with other G protein-coupled receptors (GPCRs), CB2 can exhibit preferential signaling events in response to different ligands. This functional selectivity offers the opportunity to refine therapeutic approaches, to improve beneficial properties, and reduce side effect liability. The study will provide the structural basis for the design and development of pharmacologically distinct CB2-selective compounds as useful biological probes and/or leads for the future development of therapeutics. To enhance our effort in obtaining high quality crystal structures, we shall use carefully designed ligands with high affinities and selectivities for CB2, and which are also capable of tight attachment at or near the receptor?s binding domain(s) coupled with their abilities to form crystallizable ligand-receptor complexes. The study has three specific aims: (1) Design and synthesize novel irreversible ligands representing key classes of CB2 selective compounds with distinct functional profiles. (2) Extensive characterization of the newly synthesized ligands in order to identify compounds with pharmacologically diverse profiles, including the partial agonists, inverse agonists, neutral antagonists and allosteric modulators. The crystallization candidates and their chemical derivatives will also be characterized for their reversible binding nature using functional assays. (3) Develop a clear understanding of CB2 ligand binding sites by determining the 3-D structures of the several receptor-ligand complexes. Towards these goals, several crystal structures will be solved to better understand molecular recognition, signaling, and to assist in the design of novel compounds that could then serve as prototypes for later generation leads and drug candidates.

Summary: The 2018 Molecular Pharmacology Gordon Research Conference (GRC) and Gordon Research Seminar (GRS) will highlight the latest advances in understanding G protein-coupled receptors (GPCRs) and how they mediate regulation of physiological processes. Broadly, our goals are to disucss: 1) How ligands and modulators interact with receptors; 2) How receptors respond to these interactions; 3) How context, both spatial and temporal, affects the signaling output; and importantly 4) How we can harness these events to treat disease. The GRS is the trainee organized component of the conference and will kick-off the meeting on the first 2 days. GPCRs are well established effective therapeutic targets and presentations will focus on how we can further improve therapeutic development across diverse disease states, including cancer, metabolic disorders, cardiovascular disease, and pain. There will be focused session on developing non-opioid pain therapeutics and also on means to attenuate opioid use disorders. Funding is requested to support trainee participation in the GRS and the GRC. The GRS consists of 12 student or post-doc podium presentations; the GRC has ~70 speakers and discussion leaders. The GRS also includes a Mentorship workshop on how to transition to an independent career in academia or industry.