Dale G. Deutsch - US grants

The main thrust of this proposal is based upon the hypothesis that the cannabinoids interact with the cytochrome P-450s at the molecular genetic level. In support of this hypothesis, experimental data is presented showing that the cannabinoids do indeed modulate the mRNA levels of specific cytochrome P-450s. The specific aims are to: 1) Determine which of the cannabinoids and compounds with cannabinmimetic activity) modulate the level of the cytochrome P-450 mRNAs. 2) Study the mechanism by which the cannabinoids affect the levels of mRNA (activation or repression of gene transcription; stabilization or destabilization of mRNA) and 3) Relate the changes found at the level of a specific cytochrome P-450 genes to the activity of the corresponding enzymes. Cannabinoids are widely used in our society (20 million Americans smoke marijuana). In addition, THC is used clinically (Dronabinol) for nausea and new man made analogs of the cannabinoids, some of which are 100 times more potent than THC, have been synthesized by various groups. Some of these congeners are also candidates for therapeutic agents (as analgesics, in movement disorders, as antiasthmatics, as secondary antiepileptics, etc.). Knowledge of the cannabinoids' role in drug interactions at the molecular genetic level is important since this interaction will influence the metabolism of endogenous substances, such as steroids, as well as exogenous substances such as therapeutic drugs and potential carcinogens. Cloning techniques have resulted in the synthesis of DNA complementary (cDNA) to the cytochrome P-450 mRNAs allowing quantitation of constitutive and inducible forms of the messengers. Using these probes, the novel observation has been made that co-administered of a cannabinoid with phenobarbital results in the superinduction of certain cytochrome P-450 mRNAs. The 1st set of experiments are designed to determine which of the cannabinoids (and compounds with cannabinmimetic activity) modulate the level of the cytochrome P-450 mRNAs in the rat. In these experiments hepatic mRNA levels of the cytochromes will be determined by northern and dot-blot analysis after isolation of RNA from rat liver. The 2nd set of experiments are designed to study the mechanism by which the cannabinoids affect the levels of mRNA (activation or repression of gene transcription; stabilization or destabilization of mRNA). Nuclear run-off transcription, kinetic analysis of mRNA stability, and gel-retardation analysis will be employed. Cloned fragments of the P-450 genes, containing upstream regulatory regions, which are transcribed in freeze-thawed rat liver nuclei, will provide a method to study the mechanism by which the cannabinoids regulate gene expression. The 3rd set of experiments are designed to relate the changes found at the level of a specific cytochrome P-450 genes to the activity of the corresponding enzymes employing isozyme specific substrates.

Anandamide (arachidonyiethanolamide) is an endogenous compound that binds to one of the most ubiquitous receptors in the brain, the delta9- tetrahydrocannabinol (THC) receptor. Anandamide has been demonstrated to be a cannabinoid agonist and may serve as a genuine neurotransmitter. The long term goal of this project is to characterize the enzymes that are responsible for the metabolism of anandamide. From the properties of the enzymes, it will be possible to design a series of inhibitors to prevent the breakdown of anandamide. The development of drugs that block anandamide's breakdown will be important for therapeutics since THC receptors mediate behavior involving memory, appetite, movement, pain, and mood. Preliminary studies indicate that enzymatic activities in the brain, do indeed, mediate the formation of anandamide from arachidonic acid and ethanolamine (a synthase) and the breakdown of anandamide to arachidonic acid (an amidase). The specific goals of this proposal are the following: 1) to characterize these enzymes in terms of kinetics and substrate specificity. Kinetic experiments will be conducted to determine the effects of time, temperature, pH, ions, substrate and enzyme concentrations upon the rate of reaction. In order to determine enzyme specificity for the amidase and the synthase reactions, a series of: fatty acid ethanolamide analogs of arachidonylethanolamide and a series of fatty acids analogs of arachidonic acid, respectively, will be tested as competitive inhibitors and/or substrates analogs for these enzymatic reactions; 2) to synthesize a series of ketonic arachidonylethanolamide analog "transition" state inhibitors, such as trifluoromethyl ketones, alpha-keto ester derivatives, or alpha-keto amide derivatives. The ability of these compounds to inhibit the amidase in tissue preparations and cell culture will be determined. In addition, these inhibitors will be tested for their effect upon the synthase, their ability to bind to the THC receptor, and their ability to potentiate the effect of anandamide in the rat vas deferens twitch response assay. Preliminary studies indicate that one of these compounds (an alpha-keto ester) is a potent inhibitor of anandamide amidase with a Ki in the submicromolar range and that it does not bind tightly to the THC receptor. The inhibitors discovered in these studies could be radiolabelled and used as probes for the detection of the amidase in brain slices; 3) to determine the subcellular localization of these enzymatic activities in the nerve terminals and to purify the enzymes. The enzymes will be purified by a combination of classical and affinity chromatography procedures. The purified enzymes will be utilized for the enzymatic studies described above, and for partial amino acid sequence determination. The purified protein could be used for the production of antibodies for immunocytochemical localization of the enzymes involved in anandamide metabolism.

DESCRIPTION (Applicant's Abstract): The study of the naturally occurring "cannabinoid" system in our bodies will help us elucidate any harmful and beneficial effects of marijuana use. The long term goal of this research is to understand how the actions of the endogenous cannabinomimetic (anandamide) are enzymatically terminated (by an amidase) in the nervous system and peripheral tissues. In this proposal, we plan to build upon our progress and capitalize upon recent advances in the area by others (cloning of the enzyme) to answer questions of fundamental importance. The specific aims are to: 1) Identify the active-site and the transmembrane region (s) of anandamide amidase. The amino acids crucial for the active site of the enzyme (a putative serine) will be identified as well as those regions of the enzyme which may be responsible for insertion or association of the enzyme into the membrane. These studies will employ site-directed mutagenesis, radiolabeling of the active site with an irreversible inhibitor, and deletion mutagenesis. 2) Characterize the enzymatic properties of the amidase. We will answer the fundamental questions: (a) Does the enzyme catalyze the reverse reaction; i.e., the synthesis of anandamide from the condensation of ethanolamine and arachidonic acid; (b) What are the effects of some novel irreversible inhibitors upon the enzyme; and How many molecules of anandamide can the enzyme degrade per unit time (turnover number). These experiments will utilize purified amidase preparations from tissue or from the cloned amidase. 3) Expression of the amidase in tissue. These experiments are designed to determine the levels of the enzyme and its mRNA in organs where the cannabinoid receptors (CB1 and CB2) have been shown to occur. In addition, any variants of the mRNA that are detected will be analyzed by sequencing. In situ hybridization experiments will be undertaken to determine the cell specific expression of the amidase mRNA in some selected organs including uterus, kidney and brain.

DESCRIPTION (provided by applicant): FAAH, the fatty acid amide hydrolase, is responsible for the termination of the action of anandamide, an endocannabinoid. The overall goal of this proposal is to understand the mechanism by which FAAH regulates the levels of anandamide. The first Specific Aim is to characterize the mouse FAAH gene promoter elements responsible for FAAH expression in the brain and in other tissues. The activity of FAAH is regulated between cells, between organs, and by hormones and our research is designed to elucidate the mechanism by which this occurs. The second Specific Aim is to explain how FAAH, an intracellular enzyme, terminates anandamide's action by driving its cellular uptake. Anandamide is inactivated after being transported into cells and we postulate that FAAH contributes to anandamide uptake by creating and maintaining an inward concentration gradient. We will undertake FAAH inhibitor studies with analogs of methylarachidonyl fluorophosphonate (MAFP). We will employ various cell lines including primary cultured neuronal cells (striatal and cortical) from FAAH knockout and FAAH wild type mice. FAAH is a target for the design of therapeutic drugs since its inhibition (a chemical knockout) will raise the levels of extracellular anandamide at cannabinoid receptors. These studies have practical applications in the field of drug abuse where it would be clinically desirable to raise the levels of the endocannabinoids (e.g., during cannabinoid or opiate withdrawal). We will test the selectivity of a series MAFP analogs that inhibit FAAH towards five other enzymes. In the 3rd Specific Aim, the expression of FAAH and uptake of anandamide will be characterized in a model system (the mouse retina) where the cellular organization is very well characterized. We will determine if the transport of anandamide is greatest in retina cells that contain FAAH to validate the results of our cell culture experiments in Specific Aim 2. We will employ FAAH retina from knockout and FAAH wild type mice to undertake the first anandamide uptake studies in intact tissues where the individual cells can be identified. The long-term objective of the proposed research is to understand the role that FAAH plays in the inactivation of the endocannabinoids.

[unreadable] DESCRIPTION (provided by applicant): The overall hypothesis of this proposal is that anandamide (an endogenous ligand that binds to cannabinoid and vanilloid receptors) is taken up into cells by a mechanism of simple diffusion rather than, as currently believed, by an anandamide transporter. Examining approximately a dozen immortalized and primary cell lines reported in the literature to have an anandamide transporter, the specific aims of the proposal are: 1) to determine if anandamide uptake occurs by simple diffusion (non-saturable) or facilitated diffusion (saturable). These experiments will differ from other published reports in that uptake of anandamide will be studied at initial times (25 seconds). These initial rates will distinguish actual uptake from downstream events such as metabolism or intracellular sequestration, which may give the appearance of saturation and an anandamide carrier. 2) to investigate the role of the "anandamide transport inhibitors" described in the literature. These "anandamide transport inhibitors" will be tested as uptake competitors using initial uptake rates as opposed to the prevailing studies in the literature that employed times greater than one minute that measures net uptake. If our hypothesis is correct, none of these will inhibit transport but will, in fact, be shown to inhibitor FAAH. Therefore the approach being used by other investigators in the field to synthesize "anandamide transport inhibitors" may be flawed from a theoretical viewpoint. 3) to elucidate the cellular distribution of FAAH with particular emphasis on its intracellular localization. If immunofluorescence indicates that FAAH staining resides mainly on intracellular membranes of neuroblastoma cells, it suggests that anandamide hydrolysis will appear as a downstream event that will be confirmed by the observed kinetics of anandamide hydrolysis in Specific Aims 1 and 2. In contrast to prevailing studies, this proposal will unequivocally determine if anandamide uptake is a process of simple diffusion. The clarification of the mechanism of anandamide inactivation is important for the development of therapeutics in areas such as drug addiction, mood, pain, fertility, and appetite regulation. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The endocannabinoids, anandamide (AEA) and 2-arachidonoylglycerol (2-AG), are the major signaling molecules for the cannabinoid receptors CB1 and CB2. All neurotransmitters require an efficient mechanism to terminate their action at the receptor. Water-soluble neurotransmitters are removed from the synapse by specific plasma transmembane transporters and they then diffuse unassisted within the cell to an enzyme for breakdown. The hypothesis of this proposal is that, owing to their neutral lipid structure the inactivation of AEA and 2-AG occurs by a unique pathway. It is proposed that this inactivation occurs by two steps: 1) Passive diffusion through the plasma membrane, independently of a transmembrane transporter, and 2) intracellular trafficking with chaperone proteins to their inactivating enzymes. To examine whether AEA and 2-AG can passively diffuse across the membrane, synthetic lipid vesicles (liposomes) will be employed with internalized FAAH (fatty acid amide hydrolase) or MAGL (monoacylglycerol lipase). These inactivating enzymes maintain the outward/inward gradient that drives the uptake. This model system will also be valuable in determining if simple diffusion demonstrates saturation kinetics, owing to an unstirred water layer, and if membrane composition regulates uptake. The latter will be addressed by comparing uptake in liposomes with and without lipid rafts. Of the possible mechanisms for the movement of AEA and 2-AG from the plasma membrane to their inactivating enzymes in the cell: unassisted simple diffusion, endocytosis, or trafficking by a chaperone protein, the latter appears most plausible. Accordingly, these proposed studies will examine endogenous levels of endocannabinoids trafficking proteins in cells and will elucidate the role of these proteins by comparing endocannabinoid metabolism rates and cellular uptake when the proteins are up-regulated by transfection and down-regulated by RNA interference or specific inhibitors. From a health related viewpoint, understanding the mechanism of endocannabinoid inactivation may lead to drug targets for addiction, mood disorders, pain and inflammation, cognition, and appetite regulation. PUBLIC HEALTH RELEVANCE: Maintenance of the levels of the two endocannabinoid neurotransmitters, anandamide and 2-AG, is required for normal physiological functioning in humans. The steps by which these compounds are inactivated in the body are the focus of this proposal and will lead to new targets for pharmaceutical drugs. Since anandamide and 2-AG signaling molecules are involved in such diverse functions as reinforcement, mood, memory, appetite, pain and movement;drugs that prevent their inactivation may be good for treating drug addiction, depression, compulsive behaviors, neuropathic pain and movement disorders.

DESCRIPTION (provided by applicant): All neurotransmitters require an efficient mechanism to terminate their action. Water-soluble neurotransmitters diffuse unassisted in the cytosol to their respective catabolic enzyme for breakdown, after being transported into cells. The endocannabinoids, anandamide (AEA) and 2-arachidonoylglycerol (2-AG), are the major signaling molecules for the cannabinoid receptors CB1 and CB2. Unlike most other neurotransmitters, the endocannabinoids are neutral hydrophobic molecules. The hypothesis of this proposal is that, owing to their neutral lipid structure and their insolubility in cytosol, the inactivation of AEA and 2-AG occurs by a unique pathway. It is proposed that they bind to fatty acid binding proteins (FABPs) for intracellular trafficking to their inactivating enzymes. Accordingly, these studies will examine endogenous levels of FABPs in different cells. Furthermore, these studies will elucidate the role of FABPs upon the rate of endocannabinoid metabolism by assaying the cellular uptake and hydrolysis of AEA and 2-AG. The proof that FABPs are intracellular transporters of endocannabinoids will be demonstrated by measuring commensurate changes in metabolism after up-regulation of FABPs by transfection, down-regulation of FABPs by RNA interference or inactivation of FABPs by specific inhibitors. Herein we describe, for the first time, proteins that bind endocannabinoids and function as intracellular transporters. To further strengthen our confidence in the hypothesis that FABPs are intracellular transporters of endocannabinoids, experiments will also be conducted to show that two alternative mechanisms (simple diffusion or endocytosis) are less likely. From a health related viewpoint, understanding the mechanism of endocannabinoid inactivation and identifying FABPs as a new drug target drug target may lead to treatment for addiction, mood disorders, pain and inflammation, cognition, and appetite regulation. For example, competitive inhibitors of AEA and 2-AG binding to specific FABPs will decrease the hydrolysis of AEA and 2-AG as well as their cellular uptake, raising their levels at the synapse and producing a cannabinoid tone. PUBLIC HEALTH RELEVANCE: The two endocannabinoid neurotransmitters, anandamide and 2-AG, are required for normal physiological functioning in humans. Understanding how these compounds are inactivated in the body are the focus of this proposal and will lead to new targets for pharmaceutical drugs. Since anandamide and 2-AG signaling molecules are involved in such diverse functions as reinforcement, mood, memory, appetite, pain and movement;drugs that prevent their inactivation may be good for treating drug addiction, depression, compulsive behaviors, neuropathic pain and movement disorders.

DESCRIPTION (provided by applicant): Anandamide (AEA) is a naturally occurring ligand that binds to cannabinoid receptors and is widely distributed in the body, particularly the nervous system. Although AEA was described in 1992, the pathway for its synthesis has not been definitively determined. Three pathways have been published that all start from a single precursor called N-arachidonoyl-phosphatidylethanolamine (NArPE). The first pathway utilizes one enzyme called NAPE-PLD to form AEA. A second pathway has three steps that utilize two enzymes called ??? hydrolase 4 (Abhd4) and a phosphodiesterase called GDE1. The third pathway has two enzymes (phospholipase C and a phosphatase) and has phospho-AEA (pAEA) as an intermediate. In all three pathways intermediates have been identified and the pathways have been shown to be valid when exogenous substrates are added to tissue homogenates or to cells in culture. A conventional mouse knockout (called NAPE-PLD-/-) has been characterized and indicated that the second pathway was dominant in AEA production in brain. However, this conventional NAPE-PLD knockout may introduce compensatory changes in anandamide's biosynthetic pathway(s) during embryogenesis and development and these changes may drive the synthesis of anandamide by one of the parallel pathways. To circumvent the major limitation of a conventional NAPE-PLD-/-, we propose to develop an adult conditional NAPE-PLD knockout by breeding mice in which the exon encoding the enzyme's catalytic residues is floxed. These mice will also be transgenic for a Cre recombinase gene that is under the control of a tetracycline operon that can be turned on in the absence of doxycycline. The tetracycline transactivator will be under the control of a neuron-specific enolase promoter so that -/- NAPE-PLD will be restricted to the nervous system. In this model the ablation of NAPE-PLD will occur during the adult stage in the nervous system, so that compensatory pathways cannot take effect. The brains from the control and experimental animals will be analyzed for AEA and NArPE, their respective congeners as well as pAEA. The NAPE-PLD pathway is the only one that shows calcium dependence, a property one would expect for a postsynaptic enzyme involved in AEA synthesis. The trigger for AEA synthesis would be an increase in postsynaptic calcium resulting from presynaptic neurotransmitter release. Understanding the synthesis of anandamide is essential owing to its effects upon movement, memory, nociception, endocrine regulation, thermoregulation, sensory perception, cognitive functions, mood, and addictive behavior for opiates, alcohol and nicotine. PUBLIC HEALTH RELEVANCE: Maintenance of the levels of anandamide, an endocannabinoid neurotransmitter, is required for normal physiological functioning in humans. The steps by which anandamide is synthesized in the body is the focus of this proposal and may lead to new targets for pharmaceutical drugs. Since anandamide is involved in such diverse functions as reinforcement, mood, memory, appetite, pain and movement, understanding how it is synthesized may lead to treatment for drug addiction, depression, compulsive behaviors, neuropathic pain and movement disorders.

DESCRIPTION (provided by applicant): Transporters (carriers, chaperones) for the endocannabinoid, anandamide (AEA) have recently been identified. One class of these transporters are the fatty acid binding proteins (FABPs). The FABPs are a family of small soluble carrier proteins for lipophilic substances. These FABPs have recently been shown to solubilize anandamide so it can move inside the cell for degradation by enzymes at the endoplasmic reticulum. The major goal of this proposal is to develop drugs that bind to these anandamide carriers. We hypothesize that targeting these FABPs with inhibitors will prevent anandamide being transported for breakdown inside the cell and subsequently raise the extracellular levels of anandamide, resulting in anti-nociceptive and anti-inflammatory effects. We will test this hypothesis by identifying and designing inhibitors of the anandamide transporters. This approach will involve a combination of interrelated techniques including in silico screening of commercial drug libraries using DOCK followed by re- ranking with a novel footprint-based scoring function. The most promising compounds will then be tested employing a fluorescent displacement assay for their ability to bind mainly to FABP5 and FABP7 that occur in the nervous system. From these binding data, chemical synthesis of lead compounds will be undertaken to authenticate their structure. The most promising compounds will be tested in cell culture uptake assays, engineered to contain specific FABPs. The best transport inhibitors will be tested for efficacy in mice using models of pain and inflammation. Finally, X-ray crystallographic structures of a select number of the aforementioned FABP-inhibitor complexes will be determined. This X-ray data will be employed for more accurate in silico screening and for the chemical synthesis of more potent and specific FABP inhibitors. Anandamide transport inhibitors found in this fashion may lead to novel approaches for treatment of pain and inflammation and eventually to medications for drug abuse and addiction. Our preliminary studies have identified a class of compounds, the truxilloids, that are anti-nociceptive and anti inflammatory in mice.

DESCRIPTION (provided by applicant): Transporters (carriers, chaperones) for the endocannabinoid, anandamide (AEA) have recently been identified. One class of these transporters are the fatty acid binding proteins (FABPs). The FABPs are a family of small soluble carrier proteins for lipophilic substances. These FABPs have recently been shown to solubilize anandamide so it can move inside the cell for degradation by enzymes at the endoplasmic reticulum. The major goal of this proposal is to develop drugs that bind to these anandamide carriers. We hypothesize that targeting these FABPs with inhibitors will prevent anandamide being transported for breakdown inside the cell and subsequently raise the extracellular levels of anandamide, resulting in anti-nociceptive and anti-inflammatory effects. We will test this hypothesis by identifying and designing inhibitors of the anandamide transporters. This approach will involve a combination of interrelated techniques including in silico screening of commercial drug libraries using DOCK followed by re- ranking with a novel footprint-based scoring function. The most promising compounds will then be tested employing a fluorescent displacement assay for their ability to bind mainly to FABP5 and FABP7 that occur in the nervous system. From these binding data, chemical synthesis of lead compounds will be undertaken to authenticate their structure. The most promising compounds will be tested in cell culture uptake assays, engineered to contain specific FABPs. The best transport inhibitors will be tested for efficacy in mice using models of pain and inflammation. Finally, X-ray crystallographic structures of a select number of the aforementioned FABP-inhibitor complexes will be determined. This X-ray data will be employed for more accurate in silico screening and for the chemical synthesis of more potent and specific FABP inhibitors. Anandamide transport inhibitors found in this fashion may lead to novel approaches for treatment of pain and inflammation and eventually to medications for drug abuse and addiction. Our preliminary studies have identified a class of compounds, the truxilloids, that are anti-nociceptive and anti inflammatory in mice.