Val J. Watts - US grants

DESCRIPTION (provided by applicant): Prolonged activation of D2 dopamine receptors enhances subsequent drug-stimulated cyclic AMP accumulation.This heterologous sensitization of adenylate cyclase occurs following persistent activation of several Gai/o-coupled receptors, however, the precise mechanisms involved remain unknown. Previous studies support a hypothesis that persistent activation of a Gai/o-Coupled receptor promotes the dissociation of Ga and By'subunits in a pertussis toxinsensitive fashion that induces sensitization through a Gots-dependent mechanism. Recent evidence suggests that the pertussis toxin-dependent signaling events that modulate protein kinases may act directly or indirectly to regulate individual adenylate cyclases and play a role in heterologous sensitization. We propose to refine further this hypothetical model using experiments that will manipulate the expression of recombinant Got subunits and adenylate cyclase isoforms in characterized cell lines expressing recombinant D2 dopamine receptors. The first objective is to define the relative contributions of individual Gai/o and By subunits for D2 receptor-induced sensitization. This objective will be accomplished by expressing individual constitutively active Got subunits or pertussis toxin-resistant Ga subunits containing mutations that alter GTP hydrolysis. Additional studies will use recombinant proteins that modulate G protein signaling (GBy sequestering proteins and AGS proteins). These studies will use representative recombinant adenylate cyclases that show short-term sensitization. The second objective is to determine the role of protein kinases in sensitization using wild-type and mutant isoforms of adenylate cyclase. These studies will use selective adenylate cyclase activators in combination with site-directed mutagenesis to examine the kinase involvement and to identify potential cellular mechanisms for agonist-induced sensitization. The third objective will determine the absolute requirement of Gas and role of Gas-adenylate cyclase interactions in heterologous sensitization. These studies will use a newly discovered cellular model deficient in Gas signaling. The ability of wild-type and mutant Gas to rescue sensitization in that model will be explored. The isoform specificity and the role of Gas-adenylate cyclase interactions in heterologous sensitization will also be examined using recombinant adenylate cyclases that are deficient in Gas binding and using dominant negative mutants of Gas. The goals of the present studies are to examine and elucidate the biochemical pathways and mechanism(s) responsible for D2 receptorinduced heterologous sensitization. The data that we obtain are likely to increase our understanding of the ] pathophysiology of central nervous system disorders and may also lead to improved treatment strategies.

DESCRIPTION (provided by applicant): D1-like dopamine receptors have been implicated in CNS function and psychiatric disorders including the therapy of Parkinson's disease, schizophrenia, and drug abuse. Current therapeutic agonists that target D1 receptors are very limited and have potentially serious side effects. We hypothesize that developing a strategy that mimics the natural pulsatile activation of D1 dopamine receptors (activity- dependent) would offer a significant advantage for treating disorders associated with hypodopaminergic function of D1 receptors. To test this hypothesis it is necessary to develop positive allosteric modulators of D1 dopamine receptors that could be used to enhance the agonist-stimulated receptor signal. Identifying scaffold molecules to initiate this process was the primary goal for a recently completed high throughput screening (HTS) assay that was funded through the NIH Molecular Libraries and Imaging Roadmap Initiative. The specific aims described below will take five of the identified lead compounds as scaffolds for the synthesis and molecular characterization of putative allosteric modulators of D1 dopamine receptors. Specific Aim 1 will use the five previously identified lead probes to guide the synthesis of novel structural analogs. Specific Aim 2 will initiate the pharmacologic characterization of novel compounds in cells expressing D1 dopamine receptors. Each of the newly synthesized compounds will be tested for allosteric activity in a potentiator assay and a fold-shift analysis. Subsequent studies will also explore the potential signaling mechanisms of the test compounds. Specific Aim 3 will complete secondary assays to identify the potential sites of action and the mechanisms for the newly identified allosteric modulators of D1 dopamine receptors. These studies will include a series of radioreceptor binding studies, a receptor-specificity analysis, and mechanistic signaling studies. The studies in each of the Specific Aims will be iterative and the results from Specific Aims 2 and 3 will be used to drive the synthetic work proposed in Specific Aim 1. In summary, we propose to identify and characterize a novel class of chemical probes that can be used to modulate D1 dopamine receptor signaling. PUBLIC HEALTH RELEVANCE: Dopamine receptors are critical neuroreceptors that are involved in motor function, cognition, and addictive behaviors. The D1 dopamine receptor has been implicated as a target in the treatment of Parkinson's disease, schizophrenia, and drug abuse. Because current therapeutic agonists that target D1 dopamine receptors are very limited, we propose to identify a novel class of chemical probes that can be used to safely and effectively modulate D1 dopamine receptors to improve human health.

DESCRIPTION (provided by applicant): D2 dopamine receptors have been implicated in neuropsychiatric and neurologic disorders including schizophrenia, drug abuse, and Parkinson's disease. Acute activation of D2 dopamine receptors inhibits cyclic AMP accumulation; however, persistent activation of D2 dopamine receptors enhances subsequent drug-stimulated cyclic AMP accumulation. This heterologous sensitization of adenylyl cyclase (AC) signaling occurs following persistent activation of several G?i/o-coupled receptors in vitro and in vivo. The overall objective of this research proposal is to elucidate the molecular mechanisms involved in heterologous sensitization of AC following persistent activation of D2-like dopamine receptors. Previous studies support a hypothesis that heterologous sensitization requires the activation of G?i/o subunits to induce sensitization through a G??-dependent mechanism. We hypothesize that G?? subunits lead to heterologous sensitization of individual AC isoforms through both direct and indirect mechanisms. The indirect mechanisms may involve protein-protein interactions as well as G?s. The general approach for these studies will be to express heterologously D2L dopamine receptors together with well characterized wild-type or mutant ACs (e.g. AC1, AC2, and AC5) for intact cell experiments in unique cellular backgrounds (i.e., G protein subunit deficient). This strategy takes advantage of recently discovered molecular and cellular tools to study G protein signaling as well as novel fluorescent technologies. The first specific aim will test the hypothesis that heterologous sensitization of select isoforms of AC involves G??-AC interactions and requires G?? subunit signaling. These studies will use a series of AC mutants, unique cellular models, small molecule inhibitors of G?? subunit signaling, and striatal neurons. The second specific aim will determine the roles and requirements for G protein subunits in modulating receptor-AC and AC-AC interactions. These experiments will use bimolecular fluorescence complementation (BiFC) to probe the specific role of G?? and G?s subunits in modulating basal and drug-induced protein-protein interactions in living cells. The third specific aim will identify and characterize the AC sensitization interactome using BiFC in a neuronal cell model. These studies will use BiFC to perform cDNA library screening to identify sensitization-induced interacting proteins of AC in living cells. Completion of the proposed studies will deliver mechanistic information regarding specific G protein subunits and new protein targets that could ultimately be used to prevent the development and expression of heterologous sensitization in vivo.

DESCRIPTION (provided by applicant): Persistent activation of G?i/o-coupled receptors leads to enhanced adenylyl cyclase (AC) signaling that has been described using many different names, including cAMP overshoot and heterologous sensitization of AC. This adaptive response has been implicated in several psychiatric and neurological conditions. Previous studies support a hypothesis that persistent activation of G?i/o-coupled receptors promotes the dissociation/rearrangement of G? and G?? subunits in a pertussis toxin-sensitive manner that induces sensitization through a yet unknown mechanism. We hypothesize that drug-induced protein interactions with AC are responsible for the enhanced AC response. Previous studies have examined closely-related proteins or established AC interacting partners preventing the discovery of truly novel mechanisms. Thus, an unprecedented approach will be used to identify the sensitization interactome of adenylyl cyclase type 5 (AC5) in a neuronal cell model. These studies will use Bimolecular Fluorescence Complementation (BiFC) to perform cDNA library screening to identify sensitization-induced interacting proteins of AC5 in living cells. Specific am 1 will construct and characterize a neuronal cellular model for drug-induced BiFC of AC5. These studies will use CAD cells stably expressing an engineered AC5 fusion construct capable of fluorescence complementation with appropriate binding partners. Specific aim 2 shall construct the retrovirus-based BiFC cDNA library of potential AC5 binding partners for FACS screening. Specific aim 3 shall execute both primary and secondary screening for drug-induced BiFC. These studies will infect the neuronal cell model with the retroviral cDNA library followed by treatment with a G?i/o receptor agonist to induce heterologous sensitization of AC activity. Cells revealing drug-induced BiFC will be identified using FACS and isolated for cDNA amplification. Specific aim 4 will initiate a series of biochemical and functional studies to characterize those interacting proteins that represent the AC5 sensitization interactome. The anticipated outcome of these aims is the identification and characterization of novel AC5 interacting proteins relevant to heterologous sensitization. The impact of these scientific outcomes is substantial and will address a long-standing scientific question, develop a novel methodological approach, and have the potential to provide drug targets for in vivo studies.

DESCRIPTION (provided by applicant): Adaptive signaling of adenylyl cyclase (AC) has been implicated in a variety of neuropsychiatric and neurologic disorders including schizophrenia, drug abuse, and Parkinson's disease. Heterologous sensitization of AC is thought to play a primary role in this process. Acute activation of G?i/o-linked receptors inhibits AC activity, whereas persistent activation of these receptors results in sensitization of AC signaling and increased levels of intracellular cAMP. Previous studies have demonstrated that this enhancement of AC responsiveness is observed both in vitro and in vivo following the chronic activation of several types of receptors including D2 dopamine and ? opioid receptors. Although heterologous sensitization of AC was first reported four decades ago, the mechanism(s) that underlie this phenomenon remain largely unknown. Therefore, the overall objective of this research proposal is to use a non-biased approach and advances in target discovery to identify the molecular pathways involved in heterologous sensitization of three neuronal AC isoforms (i.e., AC1, AC2, and AC6). Each of the ACs show robust expression in the brain; however, their regulatory mechanisms (including sensitization) are unique. Despite their regulatory differences, previous studies have implicated phosphorylation, G?? subunits, and signalosome assembly as potential overlapping components in the molecular mechanisms of heterologous sensitization of AC. To identify both unique and overlapping genes associated with sensitization of AC, we propose to develop and validate a series of three scalable cAMP sensitization assays for genome-wide siRNA library screening. Cell lines co-expressing individual AC isoforms and the D2L dopamine receptor will be used for this effort. However, an important first step will be the development of robust cell-based assays that can be used for high throughput screening (HTS) in 384 well format. Each HTS assay will also be validated for reverse transfection with siRNA with appropriate positive and negative controls. The genome-wide siRNA library screening will be completed in collaboration with the Center for Chemical Genomics (CCG) at the University of Michigan. The newly identified genes will be then validated and further characterized using several cellular assays to assess specificity for AC isoforms, cellular background, and receptor type. We anticipate that the proposed studies will identify new protein targets that could ultimately be used to prevent the heterologous sensitization of AC/cAMP signaling that occurs in vivo. The results from the proposed experiments are likely to increase our understanding of adaptive changes that occur in central nervous system disorders, and the findings may also lead to improved treatment strategies.

? DESCRIPTION (provided by applicant): Transformative solutions are required to control and eliminate diseases transmitted by mosquitoes and protect human health and biosecurity. Mosquito control relies heavily on insecticides that are neurotoxic to many organisms, including humans, and ineffective against insecticide-resistant mosquitoes. The long-term goal of our research is the development of new, safer interventions to control mosquito vectors. We lead a mature research effort to develop a new class of insecticides that target G protein-coupled receptors (GPCRs) in mosquitoes. Our chemistries are selective for mosquito dopamine receptors (DARs) and operate via modes that are different to existing products. This work is significant because new mode-of-action insecticides would provide continued yet safer mosquito and disease control. Our goal on this two-year developmental project is to advance novel, complementary allosteric modulator technology to enhance the safety profile and insecticidal properties of GPCR-directed insecticides. Small molecule allosteric modulators are widely used in human medicine to improve the specificity of GPCR acting drugs and minimize adverse side-effects. Allosteric drugs can stimulate or inhibit receptor activity. These chemistries offer selectivity through binding at unique sites on the receptor and/or by causing the receptor to engage a specific cell-signaling pathway (signaling bias). Allosteric modulators of GPCRs can be detected using in vitro pharmacological assays. On this project, we will explore the potential of allosteric modulator technology at the DAR AaDOP2 from the Aedes aegypti mosquito vector of dengue and yellow fever. Our project goal is to identify and evaluate the pharmacology of mosquito DAR modulators. This objective will be accomplished via work under two Specific Aims: Specific Aim 1: Discover inhibitors that modulate activity at AaDOP2. Allosteric modulation of receptor activity can present as changes to the potency, efficacy and affinity of a chemistries at the receptor. We will develop cellular assays to identify negative allosteric modulators (NAMs) that reduce the potency, efficacy and affinity of chemistries at AaDOP2. Specific Aim 2: Discover negative allosteric modulators that selectively inhibit AaDOP2 signaling pathways. Modulation can present as the biased engagement of a receptor-mediated cell signaling pathway. Cellular assays that measure common signaling endpoints will be used to screen for biased NAMs and explore the phenomenon of signaling bias at AaDOP2. At the successful completion of the proposed exploratory studies, we will have identified and assessed the pharmacological properties of one or more NAMs active at the Ae. aegypti DAR. This will enable expanded efforts to develop unique small molecule technologies against multiple mosquito vectors of disease.

PROJECT SUMMARY Of the twenty thousand proteins encoded in the human genome, three thousand of them are considered as part of the `druggable genome' by their estimated capability to bind drug-like molecules. Ion channels, kinases and G protein-coupled receptors (GCPRs) are three important components of the `druggable genome'. GPCRs are the most successful class of druggable targets. Currently, GPCRs are the target of over ~26% of the Food and Drug Administration (FDA) approved drugs. GPCRs are a family of seven-transmembrane (7TM) receptors that regulate important physiological functions through a diverse array of the endogenous ligands, which include light, odor, neurotransmitter, ion, hormone, peptide, lipid, metabolite, etc. Yet, despite of their functional importance and excellent druggability, a large number of non-olfactory GPCRs are still understudied, which is largely due to the unknown nature of the endogenous ligands and physiological functions. Therefore, there is an urgent need to characterize the understudies GPCRs by providing new research tools and characterize the physiological function of these receptors. Metabolic diseases, including diabetes and obesity, have become a major health problem worldwide. Sedentary lifestyles and the abundance of palatable and calorie-dense foods in modern societies have undoubtedly contributed to the increasing prevalence of obesity, which is associated with the incidence of multiple co-morbidities including diabetes and cardiovascular diseases. The brain is a key regulator for energy balance, owing to its ability to sense nutrients, control reward/motivation behavior, and orchestrate peripheral responses. The overarching goal of our research program is to understand the molecular mechanisms of neurohormonal pathways critical for feeding and glucose metabolism. Specifically, we aim to focus on the understudied GPCRs in the neuroendocrine system and study their roles in the pathophysiology of obesity and diabetes. Our preliminary study showed that GPR162 expression in the hypothalamus is regulated by feeding conditions and correlated with metabolic derangements. Our data, together with data from the public domain, strongly suggest that more investigations are needed to understand the upstream signals and downstream activities of this understudied GPCR and its relevance to metabolic disease pathophysiology. By characterizing the tissue / cell expression and the signaling properties of this GPCR, we will establish key background knowledge that is necessary to develop developing screening assays and performing pilot screening to obtain agonist and antagonist for GPR162. Moreover, the results from this work will have the potential to translate to humans and to the development of novel therapeutic reagents for metabolic diseases. This complementary expertise of the investigators in GPCR biology and metabolism and the on-going collaboration support the feasibility and increase the likelihood of success. Successful completion of this study will serve as preliminary data for the subsequent NIDDK R01 applications and/or drug discovery projects.

Chronic pain costs the US more than $635 billion per year, however, patients fail to receive adequate relief from the available drugs and often become drug-dependent. These observations highlight the importance for identifying new agents acting on unique targets to treat chronic pain. Genetic, neurobiological, and preclinical studies have suggested that adenylyl cyclase type 1 (AC1) may provide that new drug target. AC1 knock out mice (AC1-/-) show reduced or absent inflammatory and neuropathic pain when compared to littermate controls. Preclinical studies with a small molecule inhibitor of AC1, NB001 revealed that NB001 reduced chronic pain responses (i.e. inflammatory and neuropathic) in both mice and rats. Similarly, we have recently shown that a novel AC1 inhibitor, ST034307 also reduced inflammatory pain in a mouse model. These studies are consistent with the premise that AC1 is a new target for inhibitors of chronic pain. Unfortunately, both NB001 and ST034307 have significant issues and liabilities preventing further development. To that end, we have recently screened a chemical library collection that allowed us to identify a pyrimidinone scaffold for the development of novel AC1 inhibitors. This scaffold was prioritized for hit-to-lead optimization based on several promising criteria. Preliminary structure-activity relationship (SAR) studies have revealed for the first time compounds with sub-micromolar potency at AC1, as well as selectivity versus the closely-related AC8. Further, initial in vivo studies with a lead compound reveal activity in an animal model of chronic pain. Despite these promising observations, the lead compounds suffer from extremely low aqueous solubility. We propose medicinal chemistry optimization of this scaffold to develop potent and selective inhibitors of AC1 activity as novel probes under the following Specific Aims: Specific aim 1 will use medicinal chemistry optimization of the pyrimidinone scaffold to develop potent drug-like AC1-selective molecular probes. Specific aim 2 will establish the pharmacological specificity of the probe molecules using a set of in vitro model assays and explore the mechanisms for probe activity. Additionally, we will execute in vivo preclinical pharmacokinetic testing with iterative medicinal chemistry and pharmacology. Specific aim 3 will then use the best molecules to explore the in vivo pharmacological activity of the AC1 inhibitors in a mouse model of inflammatory pain, conditioned place preference, and opioid withdrawal. At the end of this study, we shall provide the research community with chemical probes with < 100 nM AC1 potency, > 30-fold selectivity vs other ACs and related CNS targets, and in vivo efficacy. These new probes will provide essential tools to validate AC1 as a new and safe drug target in the treatment of chronic pain.