Martin Kaczocha, Ph.D. - US grants

DESCRIPTION (provided by applicant): Nuclear receptors regulate a diverse set of physiological processes and represent attractive targets for therapeutic applications. Peroxisome proliferator-activated receptor alpha (PPAR1) are nuclear receptors that are activated by members of the N-acylethanolamine (NAE) family of lipids, which includes oleoylethanolamide (OEA) and palmitoylethanolamide (PEA), and the endocannabinoid anandamide. PPAR1 receptors mediate the anti-inflammatory and anorexigenic effects of OEA and PEA and may therefore represent attractive therapeutic targets for the treatment of inflammation and pain. Due to their hydrophobicity, NAEs are unable to traverse the aqueous cytosol unassisted. It is currently not known how OEA or PEA navigate the cellular cytoplasm to reach nuclear PPAR1 receptors. Recently, we identified fatty acid binding proteins (FABPs) as intracellular carriers for the endocannabinoid anandamide. FABPs are cytosolic fatty acid trafficking proteins whose binding sites accommodate a broad range of lipophilic ligands and may likewise bind OEA and PEA. Their small size and ability to enter the nucleus renders FABPs as likely carriers for NAEs to PPAR1 receptors. We hypothesize that FABPs act as OEA and PEA transporters, and by affecting ligand availability, may regulate PPAR1 activity. The first aim of the current application is to determine whether FABPs transport NAEs to nuclear PPAR1 receptors. We will employ chemical and genetic approaches to inhibit FABP function and delineate the contribution of FABPs towards PPAR1 activity. This study will identify the first intranuclear NAE carriers and will shed insights into the regulation of NAE signaling. The other major goal of this application involves identifying the molecular target(s) of endocannabinoid/anandamide transport inhibitors. Owing to their lipophilic nature, NAEs have been proposed to passively diffuse through cellular membranes. However, carrier-mediated uptake of NAEs via a putative endocannabinoid membrane transporter has also been proposed. Despite lacking molecular evidence to substantiate its existence, hundreds of inhibitors targeting this putative transporter have been synthesized and continue to be actively used in the endocannabinoid research community. The lack of a bona fide cellular target for transport inhibitors raises questions about the specificity and validity of these compounds as research tools. We have recently shown that FABP inhibitors reduce the intracellular transport of anandamide, effects that are mimicked by transport inhibitors. The goal of this aim is to provide evidence that cytosolic FABPs are targets of endocannabinoid transport inhibitors. We will employ overexpression, knockdown, and direct binding approaches to demonstrate that these compounds interact with FABPs. Because transport inhibitors possess analgesic properties, identifying cellular targets for these compounds may unmask novel therapeutic targets and will clarify ambiguities associated with their use in drug abuse research. The overall outcome of this study will greatly enhance our understanding of NAE signaling and will ascribe novel functions to FABPs in endocannabinoid biology. PUBLIC HEALTH RELEVANCE: Modulation of N-acylethanolamine signaling offers promising therapeutic avenues for the treatment of various disorders. This proposal focuses upon characterizing proteins that regulate N-acylethanolamine signaling within the cell. The findings of this study may lead to future therapies for the treatment of pain and inflammation.

DESCRIPTION (provided by applicant): Chronic pain accounts for billions of dollars of lost productivity and medical expenses annually. Current treatment strategies suffer from partial efficacy across the population, resulting in inadequate pain relief. Furthermore, many chronically administered analgesics (e.g., morphine or oxycontin), while actually effective, lead to tolerance and addiction. Consequently, it is imperative to identify novel drug targets for the development of non-addictive analgesics. Bioactive lipids such as endocannabinoids and N-acylethanolamines (NAEs) regulate nociception throughout the nervous system. Preclinical studies suggest that modulation of endocannabinoid and NAE catabolism represents an attractive strategy for the treatment of pain that is also devoid of psychotropic effects. Recently we identified fatty acid binding proteins (FABPs) as the first intracellular carriers that regulate endocannabinoid and NAE transport and inactivation in vitro. To date, it is not known whether FABPs regulate the endocannabinoid and NAE tone in vivo. The central goals of this project are to determine whether FABPs regulate endocannabinoid and NAE signaling and inactivation in vivo and to ascertain whether inhibition of FABPs produces endocannabinoid- and NAE-mediated antinociception. We will accomplish this by first determining whether ablation of FABPs reduces nociception in models of inflammatory pain. We will then identify the FABP subtypes that modulate pain and inflammation through a combination of complementary approaches: pharmacological manipulation and transgenic FABP knockout mice lacking specific subsets of FABPs. In the second aim of this proposal, we will employ mass spectrometry-based lipidomics to determine whether FABPs regulate endocannabinoid and NAE levels in vivo at relevant anatomical sites and consequently whether FABP inhibition produces endocannabinoid- and NAE-mediated analgesia. Finally, in the last aim, we will examine changes in peripheral cytokine and prostaglandin levels that accompany FABP inhibition and determine whether FABP inhibition alters the sensitization of nociceptive neurons. In summary, this study will identify FABPs as novel proteins that regulate nociception and inflammation and will evaluate the roles for individual FABPs in endocannabinoid and NAE inactivation in vivo. By ascribing novel roles to FABPs in nociception, this work will provide a foundation for the development of future FABP targeting therapeutics that may lead to improved analgesics.

Project Summary Despite advances in anti-androgen and taxane-based therapies, prostate cancer (PC) often becomes castration-resistant, metastatic, and incurable. Consequently, there is an urgent need to develop novel interventions to treat metastatic PC. Lipid signaling and metabolism are major drivers of PC metastasis and present ideal targets for therapeutic intervention. However, therapeutic exploitation of lipid signaling systems is hampered by the existence of multiple lipid metabolizing enzymes and nuclear receptors, which would necessitate targeting these systems in parallel. Fatty acid binding protein 5 (FABP5) is an intracellular carrier that shuttles bioactive lipids to nuclear receptors, thereby activating gene transcription programs that enhance tumor growth and metastasis. FABP5 is not expressed in the normal prostate but becomes highly upregulated in advanced metastatic PC. Our group has obtained preliminary data demonstrating that FABP5 is indispensable for the delivery of pro-tumorigenic lipids produced by multiple cytosolic to nuclear receptors to promote PC metastasis. This positions FABP5 as an essential node in a PC lipid signaling network and an attractive target for the development of therapeutics to treat metastatic PC. Despite the considerable promise of FABP5 inhibitors as potential PC therapeutics, potent and selective inhibitors have yet to emerge. The major goal of this proposal is to develop and optimize novel potent and selective FABP5 inhibitors. The proposed multidisciplinary project will be carried out by a highly qualified team with expertise in computer-aided drug design, medicinal chemistry, and PC biology. Aim 1 will leverage structure-based drug design and iterative chemical synthesis approaches to identify and optimize FABP5 inhibitors for potency and selectivity. Aim 2 will employ a robust in vitro inhibitor testing platform including assessments of inhibitor potency, efficacy, selectivity, stability, and cytotoxicity in PC cell-lines and non-transformed cells. Aim 3 will assess the efficacy of candidate inhibitors in mouse models of PC, including a novel genetically engineered mouse model of androgen-dependent and castration-resistant PC. We will also assess the efficacy of FABP5 inhibitors when used as monotherapies and in combination with FDA approved therapeutics. Successful completion of the proposed studies will lead to the development of optimized FABP5 inhibitor scaffolds that can be advanced to late stage IND-enabling studies and eventual clinical deployment.

Project Summary Failure to adequately treat pain accounts for hundreds of billions of dollars of lost productivity and medical expenses annually. According to the Centers for Disease Control, each day in the United States over forty people die from an overdose of prescription pain killers (e.g. Vicodin and OxyContin). Consequently, there is an urgent need to develop new, safe, and potent non-opioid analgesics for the treatment of acute and chronic pain. Many surgical procedures induce significant acute pain that is difficult to treat. Patients who undergo such major surgical procedures are also at an increased risk of developing a subsequent opioid addiction. Therefore, improving acute pain control will not only enhance patient outcomes but may also lead to reduced prevalence of subsequent opioid abuse. The endocannabinoid 2-arachidonoylglycerol (2-AG) produces analgesia by activating cannabinoid receptors. However, 2-AG can also be hydrolyzed by the enzyme monoacylglycerol lipase (MAGL) to generate arachidonic acid, the precursor to downstream eicosanoids that can promote pain. In a recent publication, our group demonstrated that 2-AG levels were elevated in patients who developed greater acute postoperative pain, suggesting that 2-AG/eicosanoid crosstalk may directly modulate acute pain in humans. However, the contribution of 2-AG metabolism toward acute pain is poorly defined and its role in eicosanoid biosynthesis and pain in humans is lacking, highlighting a major gap in our understanding of endocannabinoid metabolism and pain. The current proposal leverages rodent surgical models and patient derived samples to test the major hypothesis that MAGL activity is essential for the biosynthesis of cyclooxygenase and 5-lipoxygenase (5-LOX) derived eicosanoids, which we hypothesize operate in parallel to promote acute pain. In Aim 1, we will employ complementary pharmacological and genetic approaches to test the hypothesis that MAGL inhibition suppresses acute pain by depriving cyclooxygenase and 5-LOX enzymes of arachidonic acid for eicosanoid biosynthesis within the incision site. This aim will also employ selective inhibitors and 5-LOX KO mice to test the hypothesis that 5-LOX inhibition attenuates acute pain. Aim 2 will leverage novel conditional MAGL knockout mice to identify peripheral cell populations wherein MAGL activity contributes to postoperative eicosanoid biosynthesis and pain. Aim 3 will characterize 2-AG/eicosanoid crosstalk in perioperative human tissue and will assess the contribution of 2-AG and eicosanoid levels toward acute pain in humans. The outcome of this study will provide fundamental insights into endocannabinoid/eicosanoid crosstalk and may identify MAGL as a novel target for the treatment of acute pain, thereby providing the foundation for the rapid translation of MAGL inhibitors to patients suffering from inadequately controlled pain.

Project Summary The endocannabinoid (eCB) system plays a key role in regulating synaptic function in the brain. Dysfunction of eCB signaling contributes to numerous psychiatric and neurological disorders including anxiety, depression, and autism. Consequently, the development of treatments for disorders involving eCB dysfunction requires a thorough understanding of the mechanisms regulating eCB signaling in the brain. It is well-established that physiological and/or pathological activation of postsynaptic neurons leads to the biosynthesis and release of the eCB 2-arachidonoylglycerol (2-AG). Once released, 2-AG traverses the synaptic cleft and activates cannabinoid receptors located on presynaptic axon terminals, which mediate its behavioral and physiological effects. Although considerable progress has been made in elucidating how 2-AG signaling controls synaptic function and behavioral outputs, the mechanism(s) governing synaptic 2-AG transport remains unknown, highlighting a major gap in our fundamental understanding of 2-AG signaling in the brain. The lipophilic nature of 2-AG limits its diffusion across the synapse, suggesting the existence of a carrier(s) that facilitates 2-AG transport to permit cannabinoid receptor activation. Identification of a synaptic 2-AG carrier would not only greatly enhance our basic understanding of 2-AG signaling but could also lead to the discovery of a new therapeutic target(s) to treat disorders involving eCB dysfunction. To that end, our group has recently identified fatty acid binding proteins (FABPs) as intracellular carriers for eCBs. In this application, we will build upon this progress and test the novel hypothesis that FABP5, secreted by astrocytes, functions as a synaptic carrier that is essential for 2-AG signaling in multiple brain areas. In Aim 1, we will employ complementary pharmacological and genetic approaches to test the hypothesis that FABP5 mediates retrograde 2-AG transport at inhibitory and excitatory synapses in the hippocampus and ventral tegmental area, brain areas involved in cognitive and emotional regulation. In Aim 2, we will employ our novel FABP5Flox/Flox mice to delineate the roles of astrocytic and neuronal FABP5 in controlling synaptic 2-AG transport. Aim 3 will characterize the contributions of intracellular and secreted FABP5 in mediating 2-AG transport at hippocampal and ventral tegmental area synapses. Successful completion of this proposal will position FABP5 as a synaptic carrier for 2-AG at central synapses, which will greatly enhance our basic understanding of eCB signaling and may facilitate the development of future therapeutics targeting disorders involving eCB dysfunction.