Alexandros Makriyannis - US grants

The proposed study is directed towards understanding the molecular mechanism of action of cannabinoids. It will seek to identify, within the cannabinoid structures, those molecular features required to produce the membrane perturbations that result in alterations of the cellular functions. The project will focus on a carefully selected group of cannabinoid analogs closely related in structure but covering a wide range of potencies, and will include detailed studies on: (a) the conformations of the cannabinoids in solution using high resolution NMR techniques; (b) the interactions of cannabinoids with representative phospholipid model membranes using 2H, 13C and 31P solid state NMR techniques (these studies will require the synthesis of specifically 2H and 13C labeled phospholipids as well as the synthesis of specifically 2H labeled cannabinoids); (c) the interactions of cannabinoids with a natural membrane (synaptosomal plasma membrane) using 2H solid state NMR techniques. The above findings will be correlated with the effects of cannabinoids on the uptake of biogenic amines (norepinephrine, dopamine, serotonin) aby brain synaptosomes.

biomedical equipment resource; biomedical equipment purchase;

This is a competitive renewal for a project (DA-3801) aimed at understanding the molecular mechanism of action of cannabinoids. It will seek to identify the molecular properties required for cannabimimetic activity by studying experimentally the conformational properties of cannabimimetic agents and their interactions with membrane preparations and individual membrane components. During the past three years our studies have focused on the classical cannabinoids which include the naturally occurring analogs and others having closely related structures. We now propose to expand the scope of this work by including two additional groups of drugs shown to have cannabimetic activities, namely; (a) the non- classical cannabinoids synthesized by Pfizer (NCCs); and (b) some aminoalkylindole analogs (AAIs) synthesized by Sterling Research. Each group of drugs is chosen judiciously and includes analogs closely related in structure but having a wide range of potencies. The project includes detailed studies on (a) the conformational properties of the drug molecules in solution and in membrane environments using high resolution NMR methods; (b) the interactions of the drug molecules with membrane using 2H, 13C, and 31P solid state NMR and their orientations in membranes using 2H solid state NMR; (c) the topographical and geometrical features of the drug:membrane interactions using small angle x-ray and neutron diffraction; (d) the local environment of the drug in the membrane using high resolution NMR techniques for solids (MASS) and Fourier transform infrared (FTIR); (e) representation of the experimentally determined drug:membrane interactions using computer graphics. Our findings will be correlated with several biochemical effects of the cannabimimetic agents in brain synaptosomes. Direct information regarding the active site of the "cannabinoid receptor" will be obtained with the help of photoaffinity labels. Future plans include studies of drug:receptor interactions using purified cannabinoid receptor preparations as these become available. Such studies will be conducted by following the above-mentioned approaches with appropriate modification when necessary. Information on the sites of cannabimetic activity in the brain will be obtained using 19F NMR in whole animals.

biomedical equipment purchase;

The proposed study will seek to identify the molecular features involved in cannabimimetic activity through the design, synthesis and biological testing of novel compounds. It is motivated by the recent discovery of a cannabinoid receptor in mammals and man. Our design recognizes four molecular fragments contributing to cannabimimetic activity; (a) a phenolic hydroxyl, (b) a side chain attached to the phenolic ring, (c,d) two aliphatic hydroxyl groups in the northern and southern ends of the molecule. The proposal focuses on the side chain (SC) and the southern aliphatic hydroxyl (SAH) fragments whose stereochemical requirements are not well characterized. We shall study the stereoelectronic properties of the novel cannabinoids; (a) in solution and in the membrane using modern high resolution NMR techniques, (b) theoretically using molecular mechanics and ab initio calculations. The data from the above methodologies will then be integrated to give detailed information on the molecular properties of each of the cannabinoids. Analogs will be tested (a) on a variety of behavioral test systems and for analgesic activity in mice, (b) for THC discrimination in rats. Both agonistic and antagonistic activities will be considered. Special attention will also be given to identifying analogs which demonstrate a separation between analgesic and psychotropic properties. Analogs will also be tested for their affinities for the cannabinoid receptor sites in the brain. Our studies will seek to correlate the molecular properties of the novel cannabinoids with their pharmacological and biochemical (affinities for receptor) properties. These correlations should provide valuable information on specific structural requirements for cannabimimetic activity. The studies should also help us identify subtypes of cannabinoid receptors if such subtypes exist. The therapeutic targets of the proposal include the development of (a) non-opioid analgesics which are also devoid of the known psychotropic properties of cannabinoids, and the addictive properties of opioids (b) specific antagonists which can antagonize the ill effects of cannabinoids. Hopefully, this project will either result in the development of new therapeutic analogs or will provide useful information for the design of such drug molecules.

This is a request for ADAMHA RSCDA II. The proposed study is directed towards understanding the molecular mechanism of action of cannabinoids. It will seek to identify, within the cannabinoid structures, those molecular features required to produce the membrane perturbations that result in alterations of the cellular functions. The project will focus on a carefully selected group of cannabinoid analogs closely related in structure but covering a wide range of potencies and will include detailed studies on: (a) the conformational properties of the cannabinoids in solu- tion using high resolution NMR techniques; (b) the interactions of cannabinoids and their orientations in model and biological membranes (synaptosomal plasma membranes) using (2)H,(13)C and (31)P solid state NMR techniques; (c) the topographical and geometrical features of the drug:membrane interactions using x-ray and neutron diffraction; (d) the cannabinoid local environment in the membrane using high resolution NMR techniques for solids (MASS) and Fourier transform infrared; (e) representation of the cannabinoid:membrane interactions based on information from our experimental findings using computer graphics. Our findings will be correlated with several biochemical effects of cannabinoids on by brain synaptosomes. Future plans include a) studying cannabinoid effects on other relevant membrane systems (lipid and/or protein components); b) obtaining direct evidence on the site of cannabinoid action through the use of NMR techniques (e.g. (19)F NMR in membranes, cell cultures and whole animals) and by using affinity labeling. Our studies will require extensive synthesis of specifically (2)H,(13)C and (19)F labeled cannabinoid analogs and phospholipids including a small number of novel cannabinoid analogs. Although the bulk of the studies described in this proposal deal with the effects of cannabinoids on the lipid component of the membrane, the methods described here are equally applicable for studying the effects of cannabinoids on membrane-associated proteins if such relevant proteins are identified by ourselves or by other groups during the course of our investigations. The methodologies developed for the study of cannabinoids will be applicable to other drugs of abuse e.g. opioids and cocaine. Data obtained from such studies will be useful for the design of novel therapeutic agents.

This Core Facility will serve as a technical and scientific support unit for three projects (#1, #2, #3) in this Program Project. Its major goals involve 1) the production of radiolabeled and nonradiolabeled analogs in suitable quantities to fulfill the needs of the research laboratories; 2) the testing of all new analogs for their affinities for the cannabinoid receptor; 3) the testing of the "successful covalent ligands" for their abilities to irreversibly bind to the cannabinoid receptor; 4) the biochemical and pharmacological characterization of the ligands. The biochemical and pharmacological characterization of all successful ligands will involve the determination of their activities as agonists or antagonists (reversible and irreversible). Biochemically, the analogs will be tested a) for their effects on adenylate cyclase as a functional test; b) for their partitioning properties in the membrane. The pharmacological characterization will include peripheral in vitro tests for the cannabinoid receptor including their effects on the electrically- evoked contractions of the mouse vas deferens and/or guinea pig myenteric plexus preparation; b) in vivo bioassays in mice to measure locomotor activity, hypothermia, antinociception and ring immobility (Pertwee ring test).

This project serves as the "chemistry component" of a comprehensive collaborative effort, the ultimate goal of which is to develop useful novel drug analogs which produce their effects by interacting with the newly discovered cannabinoid receptor(s). The proposed project will seek to develop novel covalent and non-covalent high affinity ligands for the cannabinoid receptor. These novel analogs will be used to obtain detailed information on the molecular features of the cannabinoid receptor sites and the manner with which the different cannabimimetic ligands interact with these sites. This will be accomplished by covalently labeling the active sites of cannabinoid receptor preparations which will be produced using recombinant methods. Information obtained from such work could lead to the development of a novel non-opioid analgesic devoid of psychotropic properties or a cannabinoid antagonist to counter the ill effects of cannabinoids. Analogs from four structurally different classes of cannabimimetic agents including, classical and non-classical cannabinoids, aminoalkylindoles and anandamides will be synthesized. The ligands are designed either as photoactivatable or electrophilic, irreversible probes or as high affinity reversible probes. Their conformational properties will be studied using high resolution NMR and computational methods. The biochemical and pharmacological testing of analogs will determine their usefulness as probes and as drug prototypes (agonists, antagonists) and will be carried out under the auspices of Core 2. The most promising analogs will be radioiodinated (Core 2) and used in experiments with preparations containing the cloned receptor or receptor mutants (Project 2) in order to determine the amino acid residues involved in the binding of each group of analogs in the cannabinoid receptor site (Project 3). The most promising reversible radioiodinated ligands will be used to study cannabinoid receptor distribution and the possible presence of new receptor subtypes (Project 3 and also collaboratively).

This program project application represents a comprehensive collaborative effort the ultimate goal of which is to develop novel drug analogs which produce their therapeutic effects by acting on the newly discovered cannabinoid receptor(s). A central hypothesis of this program is that the recent availability of such receptor(s) offers the opportunity to rationally design analogs with a high degree of selectivity for inducing certain actions of cannabinoids including analgesia, inhibition of vomiting and reduction of intraocular pressure without their undesirable psychoactive effects. Similarly, there will be an opportunity for developing novel ligands which can successfully block the actions of cannabinoids. Such a process will require detailed knowledge of the molecular, biochemical and anatomical features of this receptor and its subtypes which are associated with cannabinoid activity. The receptor active site(s) could thus be used as template(s) for the successful design of these novel analogs. Obtaining intimate knowledge of the receptor's structure and function will require an interdisciplinary approach which could be accomplished through concerted collaborative efforts between several laboratories with a commitment for cannabinoid research and/or the high degree of technical expertise required for an effective approach to this problem. Strong collaborative interactions will be emphasized with the following major specific aims: (1) the development of high affinity ligands for the receptor(s) which will be used for obtaining molecular information on the cannabinoid site(s) of action. The group of ligands to be developed will encompass all four classes of molecules which are associated with cannabimimetic activity including classical cannabinoids (CCs), non- classical cannabinoids (NCCs), aminoalkylindoles (AAIs) and arachidonic acid amides (AAAs). (2) The expression isolation, purification and reconstitution of the cannabinoid receptor(s) and its mutants in viruses and bacteria. (3) Studying the conformational properties of the novel ligands in solution and in the membrane. (4) Obtaining direct information on the structure of the receptor active site(s) with the help of high affinity covalent receptor ligands and by determining the amino acid residues with which the ligands reacted. (5) The testing of the novel analogs on the cannabinoid receptor/G/i protein/adenylate cyclase system, in isolated tissue (mouse vas deferens, guinea pig ileum), and the intact animal (hypokinesia, antinociception, ring test, hypothermia).

The proposed research is directed towards understanding the molecular basis of cannabinoid activity. Cannabinoids and other cannabimimetic agents produce a complex pattern of pharmacological actions some of which are believed to be related to their effects on cellular membranes while others are thought to be produced through an interaction with cannabinoid receptors, two of which have already been identified (CB1,CB2). The work to be carried out under the auspices of this award in involves a multifaceted approach to gain information on the molecular features required for the different types of cannabinoid activity within the known cannabimimetic structures. With regard to the membrane related cannabinoid effects, we will focus on carefully selected groups of structurally related analogs covering a wide range of potencies from each of the known cannabimimetic classes and study: (a) their conformational properties in solution and in membrane-like environments using state of the art 2-dimensional multinuclear NMR in combination with computational methods; (b) the orientation of these molecules in model and biological membranes using 2/H-solid-state NMR; (c) their location and conformation in the membranes using small angle X-ray and neutron diffraction; (d) their local environment in the membranes using high resolution NMR techniques for solids. To probe the stereoelectronic requirements of the cannabinoid receptors we plan to synthesize conformationally and electronically defined analogs. To obtain information on the molecular features of the cannabinoid receptor sites and the manner with which the different ligands interact with these sites we plan to synthesize suitable covalent ligands. These will be used to label or radiolabel cannabinoid receptor clones or mutants in an effort to determine the amino acid residues with which the ligand interacts. The biochemical and pharmacological properties of all analogs will be studied to determine their usefulness as receptor probes or drug prototypes. The information obtained from our studies will be used for the design and synthesis of novel cannabimimetic agents possessing high potency and selectivity. Such agents could be therapeutically useful as non-opioid analgesics, new drugs against glaucoma or as immunomodulatory agents.

DESCRIPTION: (Applicant's Abstract) This is a competitive renewal request for a project seeking to identify the molecular features involved in cannabimimetic activity through the design and synthesis of novel compounds. In the current funding period on this project, the initial goals of the project were to focus on the requirements for activity at the cannabinoid receptor within two classes of compounds, the classical and the non-classical cannabinoids. These goals have been expanded, however, in order to take advantage of three developments in the field: first, the identification of a cannabinoid receptor subtype, CB2; second, the identification of a putative cannabinoid receptor ligand, anandamide; third, the characterization of an amidase enzyme that inactivates anandamides. The expanded scope of the project now includes the design and synthesis of anandamide analogs and the addition of the CB2 and the amidase enzyme sites as targets for structure-activity correlations. Our drug design encompasses two approaches: (a) the synthesis of novel conformationally restricted classical and non-classical cannabinoids to elaborate the first generation analogs from the current funding period, focusing on the side-chain and southern aliphatic hydroxyl pharmacophores; (b) the synthesis of novel anandamides in which the highly flexible arachidonyl fragment is conformationally restricted (this group also includes some ligands that can be considered as hybrids between the classical or non-classical cannabinoids and anandamide). The stereoelectronic properties of the novel cannabinoids will be studied, both in solution and in the membrane using high resolution NMR as well as theoretically using molecular mechanics and molecular dynamics calculations. All analogs will be tested for their affinities and functional properties on CB1 and CB2 receptors in membrane and tissue preparations, as well as in whole animals. The nature of the ligand-receptor interactions will also be explored on 3D computer models of the receptors. The anandamide analogs will also be tested for their effectiveness as anandamide amidase substrates. The data will be integrated to give detailed information on the molecular properties of the new compounds. The long-term therapeutic targets of the proposal include the development of non-opioid analgesics that are also devoid of psychotropic and immunosuppressive properties of cannabinoids; immunoregulatory agents devoid of the central effects of cannabinoids; and specific CB1 and/or CB2 receptor antagonists. It is hoped that the project will result in the development of new therapeutic analogs or will provide useful information for the design of such drug analogs.

The program project represents a comprehensive collaborative effort the ultimate goal of which is to develop novel drug analogs which produce their therapeutic effects by acting on the cannabinoid receptors. A central hypothesis of this program is that the recent availability of such receptor(s) offers the opportunity to rationally design analogs with a high degree of selectivity for inducing certain actions of cannabinoids including analgesia, inhibition of vomiting and reduction of intraocular pressure and immuno-modulation without their undesirable psychoactive effects. Similarly, there will be an opportunity for developing novel ligands which can successfully block the actions of cannabinoids. Such a process will require detailed knowledge of the molecular, biochemical and anatomical features of this receptor and its subtypes which are associated with cannabinoid activity. The receptor active sites could thus be used as template(s) for the successful design of these n ovel analogs. Obtaining intimate knowledge of the receptors' structure and function will require an interdisciplinary approach which will be accomplished through concerted collaborative efforts between several laboratories with a commitment for cannabinoid research and/or the high degree of technical expertise required for an effective approach to this problem. Strong collaborative interactions will be emphasized with the following major specific aims (1) the development of high affinity ligands for the receptor(s) which will be used for obtaining molecular information on the cannabinoid site(s) of action. The group of ligands to be developed will encompass all four classes of molecules which are associated with cannabimimetic activity including classical cannabinoids (CCs), non-classical cannabinoids (NCCs), aminoalkylindoles (AAIs) and arachidonic acid amides (AAAs). (2) The expression isolation, purification and reconstitution of the cannabinoid receptor(s) and its mutants in virus es and bacteria. (3) Obtaining receptor active sites(s) with the help of high affinity covalent receptor ligands and by determining the amino acid residues with which the ligands reacted (using photoaffinity labeling). Determination of the specific sites on the receptor (length = 472 amino acids) that are photoaffinity labeled will be accomplished using high performance liquid chromatography and mass spectrometry. Several different mass spectrometric techniques are being used I. MALDI-TOF mass spectrometric mapping of proteolytic digests of the receptor prior to and after reaction. This measurement will provide definition of the binding site to within a particular proteolytic peptide. II LC-ESI mass spectrometry of proteolytic digests of the receptor. This measurement will provide similar information to that obtained in I above. However, since we will split the flow, fractions of interest will be collected for further study. MALDI-ITMS and ESI-triple quadrupole tandem mass spectrometry of peptides that have been identified to be modified by the ligands of interest (either through observed mass shifts in I & II above or by radioactive labeling). In this context, we have recently defined the binding site of E. coli RecA to single stranded DNA during the process of homologous recombination. In this case, photocrosslinking was used and the binding site determined by MALDI-ITMS/MS (at the single amino acid level) was independently confirmed by Edman sequencing.

X ray crystallography; blood brain barrier; inhibitor /antagonist

The program project represents a comprehensive collaborative effort the ultimate goal of which is to develop novel drug analogs which produce their therapeutic effects by acting on the cannabinoid receptors. A central hypothesis of this program is that the recent availability of such receptor(s) offers the opportunity to rationally design analogs with a high degree of selectivity for inducing certain actions of cannabinoids including analgesia, inhibition of vomiting and reduction of intraocular pressure and immuno-modulation without their undesirable psychoactive effects. Similarly, there will be an opportunity for developing novel ligands which can successfully block the actions of cannabinoids. Such a process will require detailed knowledge of the molecular, biochemical and anatomical features of this receptor and its subtypes which are associated with cannabinoid activity. The receptor active sites could thus be used as template(s) for the successful design of these novel analogs. Obtaining intimate knowledge of the receptors' structure and function will require an interdisciplinary approach which will be accomplished through concerted collaborative efforts between several laboratories with a commitment for cannabinoid research and/or the high degree of technical expertise required for an effective approach to this problem. Strong collaborative interactions will be emphasized with the following major specific aims (1) the development of high affinity ligands; for the receptor(s) which will be used for obtaining molecular information on the cannabinoid site(s) of action. The group of ligands to be developed will encompass all four classes of molecules which are associated with cannabimimetic activity including classical cannabinoids; (CCs), non-classical cannabinoids (NCCs), aminoalkylindoles (AAls) and arachidonic acid amides (AAAs). (2) The expression isolation, purification and reconstitution of the cannabinoid receptor(s) and its mutants in viruses and bacteria. (3) Obtaining receptor active sites(s) with the help of high affinity covalent receptor ligands and by determining the amino acid residues with which the ligands reacted (using photoaffinity labeling). Determination of the specific sites on the receptor (length = 472 amino acids) that are photoaffinity labeled will be accomplished using high performance liquid chromatographyand mass spectrometry. Several different mass spectrometric techniques are being used. MALDI-TOF mass spectrometric mapping of proteolytic digests of the receptor prior to and after reaction. This measurement will provide definition of the binding site to within a particular proteolytic peptide. 11 LC-ESI mass spectrometry of proteolytic digests of the receptor. This measurement will provide similar information to that obtained in I above. However, since we will split the flow, fractions of interest will be collected for further study. 111. MALDI-ITMS and ESI-triple quadrupole tandem mass spectrometry of peptides that have been identified to be modified by the ligands of interest (either through observed mass shifts in I & 11 above or by radioactive labeling). In this context, we have recently defined the binding site of E. coli RecA to single stranded DNA during the process of homologous recombination. In this case, photocrosslinking was used and the binding site determined by MALDI-ITMS/MS (at the single amino acid level) was independently confirmed by Edman sequencing.

The long-term goal of this project is to develop novel ligands for the dopamine transporter (DAT) which will be useful medications in the treatment of cocaine abuse. The successful new compounds are expected to act: a) either as cocaine antagonists by blocking its effects at the DAT but showing no intrinsic activity on their own or b) as cocaine substitutes by acting as DAT inhibitors without, however, many of the deleterious effects of cocaine. A two-pronged approach will be followed. The first of these involves conformationally restricted analogs of GBR12909 a dopamine uptake inhibitor that appears particular promising in preclinical studies in experimental animals and man. The goal will be uncover novel conformation prototypes possessing enhanced properties as anti-cocaine medications. The second approach is based on evidence indicating that the known DAT inhibitors, cocaine, benztropine, GBR 12909 and mazindol interact with the DAT but have non-identical binding domains. This approach involves the development of two sets of chimeric molecules: a) GBR 12909/benztropine; b) GBR 12909/mazindol hybrids. The three dimensional structures of a judiciously chosen group of key new molecules will be studied using NMR spectroscopy and molecular modeling and correlated with their biochemical/pharmacological properties. The new compounds will be tested for their abilities to compete with different radio-ligands at the hDAT, hSERT, hNET, and their ability to inhibit the uptake of dopamine, serotonin, and norepinephrine as well as their abilities to antagonize the effects of cocaine on the DAT. The compounds will be further evaluated in studies that include examination of both unconditioned and conditioned behavior in rats. Initially the effects of drugs in a locomotor activity assay will be used to obtain necessary information on time course, dose range and behavioral and physiological toxicity. Subsequently, the effects of newly synthesized drugs will be examine din well-validated drug discrimination and drug self-administration procedures to evaluate their ability to mimic or to attenuate the discriminative stimulus and reinforcing effects of cocaine. These procedures will also provide important safety information regarding the abuse potential of candidate medications. This integral approach should facilitate rapid identification of effective medications for advanced preclinical and eventually clinical testing.

DESCRIPTION: (Applicant's Abstract) This is a request for a K05 Senior Scientist Award, which will serve as a continuation of the K02 Independent Scientist Award I currently hold. During the past decade some important developments in the field of cannabinoid research have placed it in the center of biomedical research. The availability of the K02 Award during the past nine years has allowed me to expand my scientific activities in this field in order to meet this exciting challenge. The K05 Award will make it possible for me to continue this highly effective effort. It will also provide me with the opportunity to introduce novel exciting approaches and technologies in my research including a) the use of mass spectrometry to determine the structural features of cannabimimetic protein binding sites; b) the use of molecular biological and genomics approaches in search for novel cannabimimetic targets; c) the use of state of the art multidimensional NIVIR techniques to study the interaction of cannabimimetic agents with their sites of action; d) the development of novel in vivo imaging approaches. The proposed research is directed towards understanding the molecular basis of cannabinoid activity. Cannabinoids and other cannabimimetic agents produce a complex pattern of pharmacological actions many of which are believed to be elicited through an "endogenous cannabinoid biochemical system." This involves two families of endogenous ligands (endocannabinoids) represented by anandamide and 2-arachidonylglycerol (2-AG), both of which induce their physiological responses by interacting with at least two cannabinoid receptors (CB1 and C132). The endocannabinoid system is also modulated by the anandamide amidase which catalyzes the enzymatic deactivation of the endocannabinoid ligands and the recently discovered transporter system (AT) which is involved in the reuptake of the endocannabinoids. Cannabinoids and cannabimimetic drugs induce their receptor-based effects by modulating the functions of one or more of the above four known "cannabimimetic targets." The work to be carried out under the auspices of this award involves a multifaceted approach involving ligand design and synthesis, biophysical and computational chemistry as well as biochemical methods. It will seek to gain information on the interactions of cannabimimetic ligands with each of the four cannabimimetic targets. The results should reveal the molecular properties required for cannabimimetic activity (pharmacophoric requirements) within in the known cannabimimetic classes and serve as the basis for the design of more effective ligands and therapeutic drugs.

cannabinoid receptor; receptor binding; drug design /synthesis /production;

DESCRIPTION (provided by applicant): This is a competing renewal for a project aimed at identifying the structural requirements for cannabinoid activity through the synthesis of novel ligands for the known cannabinergic sites. The goals of the current funding period included the CB1 and CB2 receptors and anandamide amidase as targets for our structure activity correlations and focused on three classes of cannabimimetic ligands, the classical and nonclassical cannabinoids (CCs, NCCs) and anandamide (AN). However, this intervening period has witnessed the discovery of the anandamide transporter (AT) and a second family of endogenous ligands represented by 2-arachidonyl glycerol (2AG) as well as the development of diarylpyrazole (PY) analogs as a new class of CB1 and CB2 antagonists. These developments have motivated us to widen the scope of this project by including the design and synthesis of 2AG and PY analogs and adding the AT as a target for our structure activity correlations. Our drug design encompasses three structural targets; a) the synthesis of novel later generation CCs and NCCs incorporating conformationally restricted pharmacophoric features and additional heteroatoms; b) novel conformationally more defined AN and 2-AG analogs; c) novel pyrazole analogs as later generation CB1 antagonists with improved pharmacological properties. We shall study the stereoelectronic properties of the most successful novel cannabinergic agents both in solution and in the membrane using high resolution NMR as well as theoretically using molecular mechanics and molecular dynamics. Furthermore, the nature of the ligand-receptor interaction will also be explored on three dimensional computer models of CB1 and CB2 receptors. All analogs will be tested for their affinities and functional properties (as agonists, antagonists, and inverse agonists) with regard to CB1 and CB2 in membrane preparations and in whole animals. The AEA and 2AG analogs will also be tested for their effectiveness in inhibiting anandamide amidase and the anandamide transporter. The long term therapeutic targets of the project include the development of (a) therapeutic agents for nicotine, opioid, alcohol or cocaine addiction; (b) non-opioid analgesics devoid of the known psychotropic properties of cannabinoids.

[unreadable] DESCRIPTION (provided by applicant): Marijuana is the most widespread illegal drug of abuse in Western societies. Its main active ingredient, delta-9-tetrahydrocannabinol, acts by binding to specific membrane receptors called cannabinoid receptors. Activation of these receptors exerts intense effects in humans, suggesting that endogenous cannabinoid (endocannabinoid) substances may contribute in important ways to brain functions such as cognition, mood and pain sensation. Several endocannabinoid substances have been identified, including anandamide and 2-arachidonylglycerol (2-AG). Anandamide and 2-AG are released from neuronal and non-neuronal cells and activate cannabinoid receptors with high affinity. After release, anandamide and 2-AG undergo a rapid inactivation process, which may play an important role in terminating their biological actions. They may be taken up by cells via high-affinity transport system(s) and then broken down by distinct enzymatic activities: anandamide by fatty acid amide hydrolase (FAAH) and 2-AG by monoacylglycerol lipase (MGL). In initial studies, we have molecularly cloned a cDNA encoding for rat brain MGL and provided evidence that this enzyme may serve an important function in 2-AG inactivation. Based on these results, we propose to develop novel chemical probes that act as substrates or inhibitors for brain MGL. The first aim of the proposed research is to define the structural requirements involved in the recognition and hydrolysis of 2-AG by MGL, and develop a pharmacophore profile for MGL inhibition. In initial experiments we have molecularly cloned a cDNA encoding for rat brain MGL and developed an adenovirus-mediated MGL over-expression system in mammalian HeLa cells. Using this system, we will explore the structure-activity relationships of 2-AG hydrolysis by rat brain MGL. Specifically, we will design and synthesize novel 2-AG analogs and test them for their ability to serve as substrates or inhibitors for MGL and enhance 2-AG signaling in intact cells. The second aim of the proposed research is to develop chemically reactive 2-AG analogs, which may serve as covalent MGL ligands. Covalent, radioactively labeled ligands for MGL may be useful tools in molecular studies of this enzyme. We will design and synthesize potential covalent ligands for MGL based on the structure of 2-AG, and test them for their ability to interact irreversibly with MGL. These studies will set the stage for the elaboration of potent and selective inhibitors that target either 2-AG or anandamide deactivation and may open novel therapeutic avenues for the treatment of neuropsychiatric and substance abuse disorders. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): During the first cycle, this T32 Program has functioned under the auspices of the Center for Drug Discovery (CDD) initially at the University of Connecticut (UConn) and more recently at Northeastern University (NU). All slots have been filled, and by summer of 2006 seven students will have received Ph.D. degrees. Of the postdoctoral fellows who have participated in the program, four have progressed to careers involving drug abuse research. In this proposed renewal we will continue to produce scientists trained in the multidisciplinary effort required to develop medications for drug abuse. This request for renewal of the Program at the same level of activity as our current grant includes 5 postdoctoral positions and 6 predoctoral student trainee slots, which will spend 2-3y and 4-5y, respectively, in the Program. Our primary objective is to familiarize trainees with the chemical, physiological, pharmacological, and clinical aspects of drug discovery and development in the field of drugs of abuse. Currently drug discovery is experiencing a major transformation initiated by the introduction of powerful new technologies. This training program is committed to cover this new terrain by providing trainees with novel coursework and the opportunity to train in leading academic laboratories. Their training is broadened by the participation of premier scientists and laboratories from the pharmaceutical and biotechnology industries. We have sought to include in our training faculty scientists with a strong record of accomplishment representing some of the novel technologies that have become an integral part of drug discovery. These include high throughput chemistry and screening, the use of mass spectrometry to study genomics, proteomics and metabolomics, NMR and x-ray crystallography to extend the study of target-based discovery as well as in vivo imaging. Predoctoral students will register at NU with affiliation to the CDD and will earn a Ph.D. in either Pharmaceutical Sciences or in Chemistry and Chemical Biology. Post-doctoral fellows training in areas including medicinal chemistry, structural and molecular biology, biochemical pharmacology, behavioral science, separations technologies (LC-MS), and in vivo imaging may chose mentored academic projects at any of the program sites. Relevance: Societal and medical problems involving substance abuse continue to plague the United States. Few medications are yet available to help health care professionals treat dependent and addicted individuals, and this area is badly underrepresented within the pharmaceutical industry. By training junior scientists, this Program seeks to address this deficiency.

PAS-06-Q66 identifies a need for novel directions in the development of medications against drug addictions. To answer this need, we plan to synthesize and pharmacologically evaluate novel cannabinergic-1 (CB1) partial agonists and antagonists for the management of methamphetamine addiction. Metharnphetamine addiction has become a world-wide epidemic, and is recognized as the most serious drug abuse problem in this country today. Addiction to methamphetamine, like other abused drugs, can be characterized as a recurring disease involving abuse, addiction, transition to drug-free status, and relapse. Different medications may be effective at different points in the cycle. We propose that CB1 partial agonists, through actions that overlap those of methamphetamine, may-assist in the transition to drug-free status and that CB1 antagonists, by dampening or masking the effects of stimuli that trigger drug-seeking behavior, may help prevent relapse. Our approach is based upon an emerging literature and our own laboratory observations ndicating that a) CB1 agonists and methamphetamine have convergent neurochemical and behavioral actions; and b) CB1 antagonists may lessen the impact of drugs or conditions that instigate drug-seeking behavior. In our proposed research, we will use chemical templates that we and others have developed for cannabinergic ligands to design and construct novel lead compounds. Our goal is to generate analogs that vary in duration of action and pharmacological efficacy and will include both partial agonists and antagonists with neutral or inverse agonist activity. Our strategy will be to conduct side-by-side comparisons with the partial agonist A9THC and the inverse agonist SR141716A whenever possible. Novel CB1 ligands that meet efficacy criteria in biochemical studies will then be studied in rats to confirm their biological activity in hypothermia and drug discrimination studies. Next, we will use drug discrimination procedures to evaluate overlap in the 'subjective' effects of methamphetamine and novel CB1 partial agonists and, also, the ability of B1 antagonists to mute methamphetamine's stimulus effects. Finally, the most promising CB1 ligands will be further evaluated in our i.v. self-administration food/drug 'choice' procedures. We expect CB1 partial agonists to reduce methamphetamine's reinforcing strength, i.e., the proportion of behavior that it commands. We also expect CB1 antagonists to dampen the ability of low doses of noncontingently delivered methamphetamine to engender drug-seeking behavior, i.e., re-allocation of behavior towardi.v. njections and away from non-drug reinforcement. Overall, our proposed studies will permit highly significant progress toward the identification of cannabinergic candidates for drug development and, eventually, novel cannabinergic medications to combat different phases of methamphetamine addiction. This will be a significant contribution to the improvement of public health.

DESCRIPTION (provided by applicant): This is a competing renewal for a project (DA007215-14) whose goal is to identify the structural requirements for cannabinergic activity through the synthesis of novel ligands aimed at modulating the function(s) of key cannabinergic proteins. These include the two cannabinoid receptors (CB1, CB2), the endocannabinoid deactivating enzymes (FAAH, MGL), and the transporter system (AT). This project has led to the development of key cannabinergic ligands currently in wide use by the research community, as well as novel drug leads. Recent developments in the field have motivated us to widen the project's scope to include monoacylglycerol lipase (MGL) as a novel cannabinergic target as well as azetidine analogs a promising new class of CB1 antagonists, allowing for the development of novel ligands and drugs with improved pharmacological profiles and broader therapeutic utility. We are also intensifying our efforts to develop improved 2AG analogs and probes, a goal that has not received due attention in the field. Our drug design and synthesis efforts encompass three approaches: 1) Novel cannabinoid analogs with improved water solubility and peripheral action. This will be accomplished through the introduction of heteroatoms in the cannabinoid side chain and tricyclic ring structures. 2) Novel 2-arachidonoyl glycerol (2AG) and arachidonoylethanolamine (AEA) analogs, with well-defined conformations. 3) Azetidine analogs representing a novel class of CB1 antagonists with improved pharmacological profiles. We shall study the stereoelectronic and physiochemical properties of the most successful novel compounds theoretically and by high-resolution NMR in solution and in membranes. Additionally, the nature of the ligand- receptor interaction will be explored through CB1 and CB2 receptor models. All new compounds will be tested for their affinities and functional properties (as agonists, antagonists or inverse agonists) for CB1 and CB2, while the endocannabinoid analogs will also be evaluated as substrates or inhibitors of FAAH, MGL and AT. The ability of the most successful compounds to cross the blood-brain barrier will be determined in vivo. The in vivo evaluation of key compounds will be carried out collaboratively at no cost to this grant. PUBLIC HEALTH RELEVANCE The development of medications to combat substance abuse and its myriad collateral ill-effects is of primary importance to NIDA and to national public health. Furthermore, the use of opioids as analgesic agents introduces serious abuse potential and invites development of analgesic medications with low or no abuse potential. The long-term goals of this application are to develop: (a) novel therapeutic medications for the treatment of cannabis, nicotine and stimulant abuse and (b) novel non-opioid analgesics devoid of undesirable side effects.

DESCRIPTION (provided by applicant): This application, designed in response to RFA-DA-09-001, addresses the need for novel medications to manage cannabis dependence and addiction, recognized public health problems that are directly relevant to NIDA's mission. (-)-delta-9-Tetrahydrocannabinol (delta-9-THC), the principal active ingredient in illicitly smoked marijuana, is legally available in oral preparations (dronabinol) for the relief of pain and nausea associated with the occurrence or management of cancer or HIV/AIDS. Recently, it also has been shown to have positive effects in countering withdrawal symptoms that likely contribute to marijuana addiction and relapse. However, oral formulations of delta-9-THC suffer from a number of clinically unfavorable properties including poor bioavailability, erratic biodisposition, and unpredictable onsets and offsets of action. We propose to synthesize and efficiently identify novel cannabinoid agonists that will improve upon the pharmacological characteristics of delta-9-THC and that, consequently, may serve as viable medications for the management of cannabis dependence. In our chemistry program, we will modify delta-9-THC and nabilone to produce unique molecules that have improved 'druggability', i.e., increased polarity, water solubility, and designed for inactivation through enzymatic detoxification. In this 'soft drug' approach, the drug, after exercising its biological actions, is enzymatically inactivated to yield products with no or much lower activities. In our pharmacology program, we will use in vitro measures (receptor binding, functional efficacy, plasma and microsomal stability) and in vivo endpoints (brain penetrability, hypothermia, analgesia) to identify novel cannabinergic analogs that reliably enter the brain and have predictable time courses of action. Compounds with the most favorable preclinical characteristics will be evaluated in drug discrimination studies to gauge the presence and time course of THC-like 'subjective-like' effects. Finally, the most promising compounds will be given in repeated dosing regimens to evaluate their ability to counter withdrawal as well as their propensity to produce cannabinoid tolerance or dependence.

RESEARCH & RELATED - OTHER PROJECT INFORMATION - PROJECT SUMMARY/ABSTRACT The Administrative Core (Core A) is a centralized facility designed to manage all operations related to this PPG. It facilitates the interactions between the collaborating laboratories and is located within the Center for Drug Discovery (Northeastern University). During this cycle, the Administrative Core has been functioning very effectively. Suitable modifications were made to accommodate new directions and personnel changes. The major tasks of this Core component will continue to be: a) communicate the central scientific theme to which all component projects will direct their research efforts and maintain a focused approach while fulfilling the PPG's overall specific aims; b) manage the fiscal aspects of the PPG, maintain financial records and other administrative functions; and c) promptly and properly distribute or coordinate the distribution of materials (novel ligands; other ligands developed in the PI's laboratory; receptor mutants) produced under the auspices of Core B and the other projects. The Administrative Core also takes responsibility for synchronizing the operations and communications between the individual laboratories involved in the PPG and other laboratories which interact collaboratively at no cost to the project. This effort includes recording and distributing materials produced to the participant laboratories and to the collaborating laboratories, together with maintaining and updating the data base related to this collaborative effort. Additional specific coordinating efforts include maintaining regular telephone conferences, the yearly group meeting of participants and collaborators, as well as other communications and the quarterly meetings with the Internal Advisory Committee, as well as the yearly formal meeting with the External Scientific Advisory Committee. The Administrative Core will manage the shared resources that are required for the performance of the interrelated tasks for the individual research projects. General administrative support related to this Program Project, including financial management, correspondence and preparation of reports and proposals, will also be provided by this Core Facility. The administrative portion of this grant seeks to keep all three projects and Core B focused on the overall research goals and to enhance communication between each branch of the grant. It will enhance the ability of collaborating laboratories to focus on the central PPG's overall goal which is the development of therapies for addiction and pain.

OTHER PROJECT INFORMATION - SUMMARY/ABSTRACT This project serves as the ligand/drug development component of a comprehensive collaborative effort to study the structural and functional properties of CB1 and CB2, the two known cannabinoid receptors. During the current funding period we have developed high-affinity covalent and non-covalent CB1/CB2 ligands that have allowed us to probe the manner in which structurally diverse classes of ligands interact and activate or deactivate each of the two receptors. To study the foot-printing of individual ligands on each receptor, we developed a Ligand Assisted Protein Structure (LAPS) approach which includes the use of covalent ligands capable of irreversibly attaching to the receptor active site(s) in conjunction with judiciously designed receptor mutants and detailed LC/MS/MS methods. To date, our results provide initial evidence that different classes of cannabinergic ligands interact and activate/deactivate the CB1 and CB2 receptors through distinct binding motifs. We have hypothesized that the individual binding motifs we have identified may be associated with distinct identifiable signaling pathway(s) leading to different ligand-dependent pharmacological profiles (functional selectivity/biased agonism). The project will provide the basis for the development of novel functionally selective CB1 and CB2 ligands and includes two complementary components. This project encompasses two components: 1) The first is a ligand development aspect which proposes the design and synthesis of novel high-affinity photoactivatable and electrophilic covalent ligands for CB1 and CB2 in which the reactive groups are strategically introduced in one (monofunctional) or two (bifunctional) sites within the parent ligand. Additionally, this component includes the design and synthesis of high-affinity reversible functionally distinct ligands and a lead optimization aspect for the design and synthesis of functionally selective CB2 agonists; 2) In the second component strategically designed single and multiple site mutants of CB1 and CB2 (human and rodent) expressed in HEK293 cells will be used to test individual successful ligands for covalent attachment and for their functional properties using the cAMP assay as an initial screen (under the auspices of Core 2). Binding site characterization will be based on the complementary use of a) receptor mutants; and b) LC/MS/MS characterization of the specific amino acid residue(s) to which the ligand is attached using our LAPS methodology. The results and ligands produced under the auspices of this project will serve as a basis for the treatment of addiction and pain using novel medications with improved pharmacological profiles. This Project 1 will design and synthesize novel ligands to probe their interactions with the CB1 and CB2 cannabinoid receptors. The results can be used to develop improved medications for pain and addictive disorders.

? DESCRIPTION (provided by applicant): Substance-use disorders (SUDs) are pervasive threats to global public health and constitute one of the top 25 top risk factors in the worldwide burden of disease. The nature of SUDs as major unsolved medical problems reflects to some degree the gaps in our current knowledge about SUD disease mechanisms and treatment. A decisive contributor to the unremitting global-health burden of drug abuse is the limited number of safe, effective pharmacotherapeutics able to combat SUDs and help attain the usual primary SUD treatment goal, durable abstinence. Strong, interdisciplinary communication among researchers is critical to improving current understanding of the neurobiological basis of addiction, stimulating interest in abuse-related pharmaceutical R&D, and generating better, more effective anti-abuse therapies. A key aspect of achieving these goals is the integration of medicinal chemistry and pharmacology to inform the design, synthesis, optimization, and (pre)clinical profiling of new agents for their pharmacological effects and their therapeutic potential for treating SUDs. Significant advances are required in SUD-related medicinal chemistry to produce anti-abuse drug candidates and expand our pharmacological knowledge as to how SUD therapies alter the relationships between brain (dys)function and the complex addiction-cycle phenotypes to elicit a salutary response. Advances in medicinal chemistry as applied to SUDs should also empower evaluation of preclinical SUD models with improved utility and translational reliability, especially regarding experimental systems that would help expand implementation-oriented research focused on brain function related to addiction etiology and pathology. New medicinal chemistry approaches that would generate both candidate SUD therapies and molecular imaging tools could also provide insight into SUD etiology. We aim to organize and conduct an annual, two-day Chemistry and Pharmacology of Drugs of Abuse symposium on campus of Northeastern University in Boston, MA. The proposed meeting is intended to serve as an interdisciplinary exposition of the prior year's most important, research-related advances in drug-abuse research. The topical agendas will emphasize laboratory findings in medicinal chemistry that inform the pharmacology and pathological mechanisms underlying addiction and the search for SUD pharmacotherapies. This focused meeting should garner substantial interest from and active participation by diverse constituencies, from basic researchers to healthcare providers and public-health officials, given worldwide recognition of the Boston area as a premiere biotechnology hub and leading biomedical research center and the involvement of major local, research-intensive clinical centers (e.g., McLean Hospital/Harvard Medical School) in SUD research and treatment.

? DESCRIPTION (provided by applicant): Structure-function characterization of a key protein component of the endocannabinoid system, the human cannabinoid receptor 1 (CB1), is the central focus of this research proposal. It aims to develop a fundamental understanding of the structural basis of CB1 function, with the ultimate translational goal of establishing a robust structure-based drug design (SBDD) program based on experimentally determined 3-dimensional structures. The endocannabinoid system is a complex network of lipid ligands, receptors, and metabolic enzymes involved in a wide range of important physiological processes, including nociception, inflammation, sleep, and drug addiction. As with other G protein coupled receptors, CB1 can exhibit preferential signaling events in response to different ligands. This functional selectivity offers the opportunity to discover new medications with improved pharmacological profiles, enhanced therapeutic properties and reduced side effects. The study will provide the structural basis for the design and development of functionally distinct CB1 selective compounds as useful pharmacological tools and/or leads for the future development of therapeutics. 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. The study has three specific aims: (1) Design and synthesize covalent ligands representing key classes of cannabinergic ligands that have been shown to have distinct functional profiles, (2) Develop a better understanding of the CB1 orthosteric binding site by solving the 3D structure of several receptor-ligand complexes, and (3) Develop a better understanding of the CB1 active state by solving the structure of the CB1 signaling complex.

Abstract Many patients suffering from HIV will develop distal symmetrical neuropathy that is frequently accompanied by pain. In some patients, HIV antiretroviral treatments can elicit and exacerbate neuropathic pain. The mechanisms underlying HIV neuropathic pain (HIVNP) are not well understood. HIVNP is relatively resistant to treatments commonly used for other types of neuropathic pain (e.g., reuptake blockers, gabapentinoids). Clinically significant effects of smoked cannabis have been reported in HIVNP, suggesting that cannabinoid activity may provide significant analgesic benefit in these patients. First, we shall develop novel CB2 agonists with no or very low functional efficacy for CB1, with improved pharmacological profiles based on the AM1710 chemotype. These novel compounds will be potent CV2 agonists and neutral CB1 antagonists. The second approach will involve the design and synthesis of novel CB1/CB2 agonists that are peripherally acting. We shall seek to obtain novel compounds in which the CB2/CB1 contributions to their respective effects on HIVNP is optimized. We have shown that AM1710, a selective CB2 agonist developed in our laboratory, elicits analgesic actions in a model of HIVNP. We propose that CB2 agonists could prove to be an effective strategy for non-addictive treatment of HIVNP and possibly for other chronic pain conditions. We propose two approaches. Both approaches will develop novel ligands that encompass design controlled cannabinoid deactivation, a concept recently developed in our laboratory for cannabinergic compounds. This involves the introduction within each ligand of a serum esterase susceptible moiety, which when subjected to the enzyme?s hydrolytic action, transforms the molecule into inactive metabolites. The duration of action in vivo of the new compounds is controlled by the chemical nature and environment of the sessile group, as well as by the degree to which the novel compound is subject to a depot effect, a property largely controlled by the compound?s hydrophobic character. The project will involve 1) the design and synthesis of the novel ligands and 2) in vitro characterization evaluation for their CB2 and CB1 affinities and functional potencies, as well as their biochemical stabilities, or bioavailabilities, and abilities (or lack of) to cross the blood brain barrier. The successful compounds will be screened (Biochemical Core) for their effects in models of inflammatory pain. 3) A select number of compounds will be tested for their efficacy in a HIV neuropathic pain model for their side effects, tolerance, and potential abuse liability.

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.

Targeting Inflammasome with stable endocannabinoid ligand AMG315: CRISPR/Cas9 and nanotechnology study in the context of HIV and cannabinoid. PROJECT SUMMARY (ABSTRACT): Although the use of cannabis for medical purposes has shown great promise for the treatment of certain medical conditions, cannabinoid abuse exerts significant impairments in neurocognitive and behavioral functions and these effects are exacerbated in patients with HIV infection. Studies suggest that even after HIV-1 suppressing combined antiretroviral therapy (cART), HIV-1 Tat is being produced in the brain from proviral DNA and implicated as a causative agent for latent infection and development of inflammasome mediated neuroinflammation in HIV infected patients. CRISPR/Cas9 gene-editing technology has been shown by us and others to be effective for excising the HIV genome integrated into the host genome. Our preliminary studies using a recently discovered and potent metabolically stable endocannabinoid analog AMG315 demonstrate that this synthetic cannabinoid exerts anti-inflammatory properties by suppressing NLRP3 inflammasome and HIV infection. Accordingly, we hypothesize that elimination of the HIV-1 Tat gene in CNS cells using Tat specific CRISPR/Cas9 and suppression of inflammasome with CB1-specific stable endocannabinoid analog AMG315 can eliminate active HIV infection/induce permanent latency and prevent neurodegeneration, respectively. However, AMG-315 and CRISPR are impenetrable to the brain in sufficient quantities necessary to prevent HIV-infection, inflammasome activation, and subsequent neurodegeneration. To overcome this, we will use our patented magneto-electric nanoparticles (MENP) technology and liposomes to deliver CRISPR/Cas9 and AMG315, and for on-demand controlled-release. For the sustained release and to protect CRISPR from lysosomal degradation, MENP-bound CRISPR and AMG315 will be encapsulated in liposomes. Accordingly, in Specific Aim # 1, we will develop, characterize, and evaluate the delivery of Tat-specific CRISPR Cas9/gRNA and AMG315 across the in vitro BBB using MENP-based drug delivery approach to excise HIV-1 Tat gene, and attenuate cannabinoid and Tat-induced inflammasome, respectively. In Specific Aim # 2, we will study the in vivo therapeutic efficacy of MENP nanoformulation containing CRISPR, and AMG315 using doxycycline-inducible HIV-1 Tat transgenic mice (iTat) as an HIV/neuroAIDS and cannabinoid administration animal model. In Specific Aim-3, we will validate the effects of these nanoformulations on neuronal plasticity and neurocognitive functions in vivo. Successful completion of this grant will have a translational significance in preventing HIV-1 Tat and cannabinoid-mediated neurodegeneration in HIV infected cannabinoid-abusing patients. This multidisciplinary new break-through concept targeting inflammasome in the brain with stable endocannabinoid ligand AMG315 and Tat specific CRISPR/Cas9 using MENP-based technology is in response to RFA-DA-20-026 and will be useful for the suppression of HIV replication, NLRP3 inflammasome activity and to treat cannabinoid-induced neuronal impairments in HIV infected cannabinoid abusers.

PROJECT SUMMARY of the administrative supplement The major objectives of the parent R01 grant are 1) to develop, characterize, and evaluate the delivery of HIV Tat-specific CRISPR Cas9/gRNA and AMG315 across the in vitro BBB using the MENP-based drug delivery approach to excise HIV-1 Tat gene, and attenuate cannabinoid and Tat-induced inflammasome, respectively. 2) to study the in vivo therapeutic efficacy of MENP nanoformulation containing CRISPR, and AMG315 using doxycycline-inducible HIV-1 Tat transgenic mice (iTat) as an HIV/neuroAIDS and cannabinoid administration animal model. 3) to validate the effects of these nanoformulations on neuronal plasticity and neurocognitive functions in vivo. Thus the three major focuses in the parent R01 are HIV, magneto-electric nanoparticle (MENP)-based drug delivery, and the cannabinoid pathway, in particular the use of AMG315. Here we want to broaden the scope of these three major focuses by also evaluating the potential of using AMG315 to mitigate NLRP3 inflammasome-mediated markers of neuroinflammation, amyloid, and neurofibrillary tangle pathology in a comorbidity model of HIV and Alzheimer?s disease (AD). As more people living with HIV (PLHIV) are reaching geriatric ages when the risk of developing AD reaches exponential proportion, PLHIV has an additional burden of secreting toxic proteins such as HIV-Tat that might explain the prevalent HIV-associated neurocognitive disorder (HAND). More cases of HIV-infected individuals diagnosed with AD are being reported. This is not surprising given that similar to A?, HIV-Tat protein can activate inflammasome, increase A? levels by inhibiting neprilysin and enhancing BACE activity thereby predisposing HIV and AD comorbid patients for synergistic effects. Unfortunately, there has been no therapeutic success so far either for HIV-HAND or memory issues in AD, including clinical trials using cannabinoid compounds, although the endocannabinoid system (ECS) is known to play a fundamental role in memory. The failure of ECS ligands is mainly due to poor pharmacokinetics especially the short-life of compounds. This greatest challenge of stability was successfully addressed by our Co-I, Dr. Alexandros Makriyannis by discovering AMG315, a potent and more stable CB1 receptor ligand. Therefore in Specific Aim 1, similar to parent R01, using MENP nanoformulation (NF) we will administer AMG315, methanandamide (for comparison), or vehicle by i.v. route and assess whether AMG315 can mitigate inflammasome-mediated neuroinflammation in a bigenic mouse model of HIV and AD comorbidity, 3xTg/iTat. In Specific Aim 2, we will verify whether AMG315 can reduce amyloid plaque and neurofibrillary tangle burden and correlate with improved memory. Since we plan to test the AMG315 directly in a mouse model of HIV (iTAT) and AD (3xTg) and address three key questions in HIV/AD research, i.e., HIV and AD comorbidity, cannabinoid multitarget pathway, and efficient brain penetration, this administrative supplement request is directly related to Alzheimer?s disease and related dementias (ADRD). If AMG315 has beneficial effects in the comorbidity model in this study, it may be further developed for clinical use in future studies.

ABSTRACT In response to RFA-AA-20-007, which calls for the development of medications to treat Alcohol Use Disorders (AUD), this U01 application proposes research to advance the CB1 neutral antagonist AM6527 towards IND- enabling studies for treating AUD. The current therapies for AUD are either behavioral or is limited to drugs such as disulfiram, acamprosate and naltrexone, which are restricted to specific patient populations in terms of their therapeutic effects. Given these epidemiological, and economic issues related with AUD, there is an urgent need for novel pharmacological interventions that are more acceptable and selective towards treating AUD. Rimonabant, a CB1 antagonist, has proven to have unacceptable adverse effects, possibly resulting from its CB1 inverse agonist actions. In contrast, AM6527 is a novel, selective, CB1 cannabinoid-receptor neutral antagonist that is devoid of inverse agonist activity. The preclinical pharmacology of AM6527 in rodents and other prototypes within this class of compounds in nonhuman primates strongly supports their potential utility as therapy for AUD. AM6527 has a favorable safety profile, without evidence of adverse side effects (e.g., nausea, depression) that have been reported with CB1-receptor inverse agonists such as rimonabant and taranabant in preclinical and clinical studies. Based upon these positive preclinical data indicating its effectiveness as a CB1 antagonist, we plan to move AM6527 toward IND-enabling studies in preparation for first-in-man studies to treat AUD. Specifically, our work in this U01 proposal is designed to meet the following aims: Chemistry, manufacturing, and controls of drug substance, non-GLP preclinical safety studies including hERG, genotoxicity, ADME and dose escalation studies in two species. The subsequent steps will comprise: Chemistry, manufacturing, and controls of drug product including formulation development, cGMP scale-up, stability, and analytical methods; toxicokinetics with single and repeat 28-day dosing safety pharmacology, followed by advisement on product development planning, IND preparation and preclinical regulatory strategy.