Raymond G. Booth - US grants

This application is focused on the study of a novel neuroreceptor we have termed to sigma3 receptor. We have designed a series of phenylamino- substituted tetrahydro-naphthalene (phenylaminotetralin, PAT) ligands and radioligands that have very high affinity (e.g., 50 pM) and selectivity for activation of sigma3 receptors that lead to changes in brain catecholamine nerve terminal function. We propose to elucidate recognition and functional characteristics of the sigma3 receptor that should lead to a better understanding of its role in the central nervous system and neurodegeneration. Critical tools for this work are the selective, high affinity PAT-type sigma3 ligands and radioligands we have developed. [3H]PATs will be used in radioreceptor studies with rat and monkey brain homogenates to unequivocally establish that the sigma3 site is not any known receptor. The affinity of a wide range of CNS receptor- active ligands in competition for [3H]PAT-labeled sigma3 sites will be determined to characterize the pharmacological profile of the sigma3 receptor and screen for candidate endogenous sigma3 ligands. The PAT binding pharmacophore at sigma3 receptors will be established by characterizing the affinity, selectivity, and structure-activity requirement (SAR) of PATs and other tetrahydronaphthalenes for [3H]PAT- labeled sigma3 sites. Localization of [3H]PAT-labeled sigma3 receptors in mammalian brain will be determined using quantitative receptor autoradiography in rat, guinea pig, and monkey brain slices. Preliminary data suggest that trans-PATs can potently stimulate striatal dopamine synthesis in vitro and after in vivo administration in rats, while cis- PATs can block this effect. Additional studies will be undertaken using rat brain to establish the PAT agonist and antagonist functional pharmacophore at sigma3 receptors that modulate catecholamine synthesis, and perhaps release, in caudate and other catecholaminergic terminal fields. Selected PAT sigma3 ligands will be screened in vitro for functional effects on catecholamine synthesis by measuring tyrosine hydroxylase activity in rat brain minces. In vivo sigma3 receptor- mediated effects on synthesis and release of 3,4-dihydroxyphenylalanine (DOPA) and catecholamines will be measured in specific brain regions by HPLC-EC, after administering PATs by IP and ICV injection and by intracerebral microdialysis in rats. Post-mortem accumulation of neurotoxic quinone metabolites will be documented in specified brain regions by UV/Vis spectrophotometry. Nerve terminal integrity will be assessed by quantitative autoradiographic assessment of catecholamine neurotransporter density. Additional PAT derivatives to be synthesized will examine: 1) cis vs. trans stereochemical requirement, 2) requirements of the N-binding domain; 3) conformational orientation of the phenethylamine and 1-phenyl moieties; and 4) substitution on the tetrahydronaphthalene and 1-phenyl rings. Enantiomeric resolution of highly active PATs will be undertaken to further examine stereochemical requirements of sigma3 receptor activation. A preliminary sigma3 receptor model has been established that aligns low energy conformations of PATs and non-PATs. Neuropharmacological studies will provide quantitative data for molecular mechanics-based development of a PAT sigma3 receptor model that will be useful in understanding the role of sigma3 receptors to affect brain catecholaminergic nerve terminal function and in neurodegeneration.

DESCRIPTION (provided by applicant): This application is to design and synthesize novel phenylaminotetralin (PAT) compounds as biochemical probes and drugs that target histamine H1-type G protein-coupled receptors (GPCRs) to modulate brain catecholamine neurotransmitter synthesis in neuropsychiatric disorders. Preliminary results suggest PATs are functionally selective ligands that can distinguish and selectively activate brain H1 receptors that couple to the inositol phosphates (IP) vs. cAMP intracellular signaling pathways to modulate tyrosine hydroxylase (TH) activity and catecholamine synthesis. Such ligand-directed functional heterogeneity is proposed for other GPCRs, however, there is a paucity of selective medicinal chemical probes to map the molecular determinants of ligand-receptor interactions that form the molecular basis of differential signaling. Likewise, brain H1 receptors are unexploited as psycho- and neuro-therapeutic targets due to lack of potent, functionally selective agonist ligands that can enter into brain. We developed the stereoselective H1 probe, [3H]-(-)-trans-PAT, that putatively distinguishes the subset of H1 receptors that activate cAMP signaling. Syntheses of 34 PATs is proposed to delineate the PAT-H1 pharmacophore - the specific PAT steric, lipophilic, and electronic chemical determinants for H1 receptor differential binding and signaling. Molecular interaction of PATs with the H1 active site is examined in radioreceptor studies using recombinant human H1 receptors with point mutatations of 13 amino acids hypothesized to be molecular determinants for PAT-H1 binding. We test a hypothesis of ligand-directed functional heterogeneity, i.e., stereochemical and other structural parameters of PATs determines H1 functionally selective binding and activation of IP vs. cAMP signaling to stimulate TH and catecholamine synthesis in mammalian brain. Structure-activity data is correlated using CoMFA 3DQSAR and pharmacophore mapping to develop an H1 receptor model for PAT ligand docking studies. Results will indicate amino acids involved in PAT binding and inferences of H1 receptor 3D structure. QSAR models are iteratively refined to predict PAT chemistry for H1 functionally selective binding and signaling. As most untoward H1-mediated effects proceed via IP signaling, PATs that selectively enhance H1 cAMP signaling will provide a mechanistic basis for exploiting brain H1 receptors as drug targets in neuropsychiatric diseases.

[unreadable] DESCRIPTION (provided by applicant): There is currently no pharmacotherapy for cocaine abuse. This public health crisis is addressed in this application that is directly responsive to RFA-DA-07-006. This application is for development of novel serotonin 5HT2C receptor agonist drugs with 5HT2A/5HT2B receptor antagonist activity to attenuate the behavioral and neurochemical effects of cocaine use and dependence. Preclinical data indicate activation of brain 5HT2C receptors attenuates the reinforcing effects of cocaine, whereas discriminative stimulus and reinstating effects of cocaine are sensitive to attenuation by both 5HT2C activation and 5HT2A blockade. Meanwhile, activation of brain 5HT2A receptors produces psychotomimetic effects and activation of peripheral 5HT2B receptors produces cardiac valvulopathy and pulmonary hypertension. Currently, there is no 5HT2C receptor agonist reported that does not also activate 5HT2A and/or 5HT2B receptors. Preliminary Data reported here, however, demonstrate that a molecule synthesized in our lab, (1R,3S)-(-)-trans-1-phenyl-3-dimethylamino-1,2,3,4-tetrahydronaphthalene (PAT), is a full-efficacy agonist at human 5HT2C receptors, plus, an antagonist at 5HT2A and 5HT2B receptors. This dual activity (activation/blockade) at multiple serotonin receptors is unique to (-)-trans-PAT and is hypothesized to provide pharmacological treatment for cocaine addiction without cardiotoxicity. Innovative approaches include targeted syntheses of novel PAT-type stereoprobes to map 3D molecular determinants for selective activation of 5HT2C receptors and sophisticated self-administration procedures to identify cocaine's abuse-related effects that are sensitive to modulation by PAT analogs. Microdialysis with capillary electrophoresis/ laser-induced fluorescence will allow 15-sec temporal resolution of neurochemical changes in cocaine self-administering rats to identify neurochemical mechanisms of PAT therapeutic effects. The Specific Aims are: (1) PAT analog syntheses and quantitative structure-activity relationship modeling, (2) in vitro characterization of PAT 5HT2 affinity and functional activity, (3) in vivo behavioral pharmacology studies to evaluate PAT modulation of the abuse-related effects of cocaine, and (4) in vivo analysis of the PAT-cocaine neurochemical interactions. This research is undertaken by a multidisciplinary team of researchers for preclinical development of novel compounds that likely will translate to an innovative pharmacological intervention for cocaine abuse. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): There is compelling evidence that activation of brain serotonin 5HT2C G protein-coupled receptors (GPCRs) produces anti-obesity effects in humans, attenuation of psychomimetic activity, and other neuropsychiatric effects. Meanwhile, activation of brain 5HT2A GPCRs produces psychomimetic effects and activation of peripheral 5HT2B GPCRs produces cardiac valvulopathy and pulmonary hypertension. Currently, there is no 5HT2C receptor agonist reported that does not also activate 5HT2A and/or 5HT2B receptors. This research proposes to exploit a compound synthesized in our lab, (1R,3S)-(-)-trans-1-phenyl-3-dimethylamino-1,2,3,4- tetrahydronaphthalene (PAT), that is a full-efficacy agonist at human 5HT2C receptors, plus, it is an antagonist at 5HT2A and 5HT2B receptors. As a small (MW=250) lipophilic molecule, (-)-trans-PAT readily penetrates mouse brain after peripheral (IP) administration to inhibit food consumption, produce weight loss, and inhibit amphetamine-induced locomotion, neurobehavioral effects consistent with 5HT2C agonism and 5HT2A antagonism. This research proposes preclinical evaluation of (-)-trans-PAT as pharmacotherapy for obesity and neuropsychiatric disorders, and, synthesis of other PATs with potent and efficacious 5HT2C agonist activity;PATs with potent 5HT2A/5HT2B antagonism may be lead drugs for psychiatric or cardiovascular diseases. We also have identified a potent PAT-type 5HT2C inverse agonist useful especially to characterize molecular determinants involved in ligand-directed 5HT2C function. Targeted medicinal chemical syntheses will provide PAT type stereo-probes as test drugs for preclincial evaluation and to map molecular determinants for 5HT2C binding/activation for inferences of receptor 3D structure. Forty PATs already are available and 32 new analogs will help delineate the PAT- 5HT2C pharmacophore - the optimal PAT steric, lipophilic, and electronic chemical molecular features. In vitro binding/functional studies will continue to be conducted using clonal cells expressing recombinant human 5HT2A, 5HT2B, or 5HT2C receptors - preliminary results and PAT-5HT2C 3D QSAR and receptor homology models lead us to propose construction and expression of D3.32A, S3.36A, A5.46N, A5.46S, F5.47A, F5.48A, W6.48A, F6.51A, F6.52A, Y7.43A, &Y7.53A point-mutated 5HT2C receptors to validate hypothesized PAT- 5HT2C binding/function interactions. In an iterative fashion, pharmacological results and molecular models generate additional hypotheses to test involving additional PAT syntheses and 5HT2C point-mutations for receptor characterization and development of PAT-type 5HT2C agonist drug structures. Preclinical studies to evaluate PATs as pharmacotherapy for obesity, eating and other neuropsychiatric disorders, as well as, to determine in vivo molecular mechanisms of action, are conducted using wild-type vs. genetically modified mice with global disruption ("knock-out") of 5HT2C and 5HT2A receptor signaling. PUBLIC HEALTH RELEVANCE: About 65% of adults and 16% of children aged 6-19 years in the U.S. currently (2005) are overweight or obese. Obesity is associated with increased risk for cardiovascular disease;diabetes;certain forms of cancer, depression, and various other physical, psychological, and social morbidities. Current pharmacotherapy available for obesity is unsatisfactory. This research seeks to develop new drugs to treat obesity, as well as, certain neuropsychiatric disorders.

ABSTRACT: This research is to advance novel internationally-patented phenylaminotetralin (PAT) analogs as functionally-selective serotonin 5HT2 receptor-based drug development candidates to treat amphetamine and methamphetamine addiction and drug-induced psychotic disorders. Activation of brain 5HT2C receptors or antagonism/inverse agonism of 5HT2A receptors attenuates psychostimulant effects in rodents. With the exception of PATs, all compounds reported in the literature that activate 5HT2C receptors also activate 5HT2A and/or 5HT2B receptors-unfortunately, activation of brain 5HT2A receptors produces psychotomimetic effects and activation of peripheral 5HT2B receptors produces cardiac valvulopathy and pulmonary hypertension. Conversely, co-antagonism of 5HTC and 5HT2A receptors contributes to weight-gain associated with antipsychotic drugs. PATs uniquely demonstrate 5HT2C receptor agonism simultaneously with 5HT2A and 5HT2B inverse agonism-this 5HT2 functional selectivity translates in vivo to therapeutic activity in behavioral models of amphetamine/methamphetamine addiction and induced psychoses, with no overt adverse effects (including, weight-gain) after peripheral administration in rodents. This novel 5HT2 functionally-selective single molecule approach is an advance over approaches targeting dopamine neuronal proteins that have proven to be sub-optimal for drug addiction pharmacotherapy, while multiple or bi-valent serotonin 5HT2 compounds pose numerous ADMET limitations. Based on a preliminary in vivo structure-activity relationship, we will optimize the PAT molecular scaffold for activity to attenuate amphetamine/methamphetamine addiction by synthesis of at least 50 novel single-enantiomer PATs with modifications to the (C2) amine, (C4) pendant phenyl, and tetrahydronaphthyl moieties. In vitro pharmacology studies include determination of PAT 5HT2A, 2B, and 2C affinity (Ki) and function (EC50/IC50). PAT docking studies using 5HT2 receptor molecular models will characterize PAT-5HT2 molecular interactions for design of additional target molecules. Modeling of 5HT2 GPCRs is based on homology to the structure of the human adrenergic ¿2 GPCR, an innovative advance over previous models that used homology to the bovine rhodopsin GPCR. Potent and efficacious PAT 5HT2C agonists with 5HT2A/2B inverse agonism are promoted to preclinical studies to evaluate pharmacotherapeutic efficacy in vivo to attenuate amphetamine and methamphetamine addiction and drug-induced psychotic disorders. A systematic behavioral analysis of PATs is undertaken to provide information on pharmacokinetic variables, functional activity in the 5HT2 system regarding 5HT2A inverse agonism vs. 5HT2C agonism, and attenuation of drug-induced psychotic disorders. Studies include using amphetamine and methamphetamine drug discrimination and self-administration procedures to validate 5HT2 receptor systems as targets for stimulant pharmacotherapies and assess attenuation of the reinforcing effects of these stimulants in a variety of behavioral procedures designed to mimic critical aspects of an addiction-like behavioral phenotype.

This application responds to PA-18-058 to address the epidemic levels of prescription opioid abuse and addiction that have resulted in an appalling number of overdose deaths. Unequivocally, there is an urgent need for effective pharmacologic treatment options for opioid use disorder (OUD) that are safe, non-addictive, and without substantial diversion liability to address the national public health emergency. Compelling evidence suggests that serotonin (5HT) 2A and 2C G protein-coupled receptor (GPCR) subtypes may provide a fruitful strategy to achieve this goal. In this regard, 5HT2C agonists can reduce self-administration and block relapse-related drug- seeking behavior in rodents and monkeys. This has prompted development of 5HT2C-specific agonists that are devoid of potentially deleterious effects mediated through activation of the 5HT2A (i.e., hallucinogenic) and 5HT2B (i.e., cardiotoxic) subtypes. Furthermore, 5HT2C-specific agonists are required to delineate the roles of 5HT2 receptor subtypes in substance use disorders. To this end, our medicinal chemistry program has produced 4- phenyl-2-aminotetralin (PAT) analogs with unique multifunctional pharmacology at 5HT2 GPCRs, i.e., activation of 5HT2C signaling with inactivation of 5HT2A and 5HT2B signaling in the same monovalent, orally bioavailable, small molecule. Our preliminary results indicate the unique PAT-type 5HT2 pharmacology translates effectively in models of OUD in rhesus monkeys, including, attenuation of heroin-primed reinstatement, suggesting efficacy for addressing relapse. PATs do not have stimulant or sedative effects and are without liability for addiction, encouraging us to pursue translational studies in nonhuman primates (NHP) to guide development of PAT-type 5HT2 modulators as pharmacologic intervention for prescription and illicit opioid abuse, as well as, delineate 5HT2 roles in OUD. The overarching hypothesis tested is that an optimal balance of PAT function at 5HT2A receptors (inverse agonism/antagonism, partial agonism) relative to agonist function at 5HT2C receptors (implicit is no activation of 5HT2B) translates to beneficial effects in primate models of OUD. In Aim 1A medicinal chemistry and Aim 1B molecular pharmacology studies we will develop PAT analogs with a range of functional activities, potencies, and efficacies at 5HT2 receptors, including 5HT2C-specific agonists with 5HT2A inverse agonism. In Aim 2A we will assess the PATs for in vivo potency and efficacy at 5HT2A and 5HT2C receptors to establish dosing parameters for NHP studies. Aim 2B studies will use drug discrimination studies to evaluate in vivo 5HT2 activity in squirrel monkeys. Aim 3 will assess PATs for safety and efficacy to attenuate oxycodone and fentanyl self-administration as well as drug- and cue-primed reinstatement in squirrel monkeys. Results will establish the role of serotonergic 5HT2 receptors in the pathophysiology and pharmacotherapy of OUD. PATs that selectively block responding for drug self-administration and have appropriate safety and efficacy parameters including with regard to relapse will be considered for development in collateral future studies in collaboration with an identified interested pharma.