Discovery of 3-((4-Benzylpyridin-2-yl)amino)benzamides as Potent GPR52 G Protein-Biased Agonists.

Orphan GPR52 is emerging as a promising neurotherapeutic target. Optimization of previously reported lead 4a employing an iterative drug design strategy led to the identification of a series of unique GPR52 agonists, such as 10a (PW0677), 15b (PW0729), and 24f (PW0866), with improved potency and efficacy. Intriguingly, compounds 10a and 24f showed greater bias for G protein/cAMP signaling and induced significantly less in vitro desensitization than parent compound 4a, indicating that reducing GPR52 ß-arrestin activity with biased agonism results in sustained GPR52 activation. Further exploration of compounds 15b and 24f indicated improved potency and efficacy, and excellent target selectivity, but limited brain exposure warranting further optimization. These balanced and biased GPR52 agonists provide important pharmacological tools to study GPR52 activation, signaling bias, and therapeutic potential for neuropsychiatric and neurological diseases.

Functionally selective dopamine D1 receptor endocytosis and signaling by catechol and non-catechol agonists.

The dopamine D1 receptor (D1R) has fundamental roles in voluntary movement and memory and is a validated drug target for neurodegenerative and neuropsychiatric disorders. However, previously developed D1R selective agonists possess a catechol moiety which displays poor pharmacokinetic properties. The first selective non-catechol D1R agonists were recently discovered and unexpectedly many of these ligands showed G protein biased signaling. Here, we investigate both catechol and non-catechol D1R agonists to validate potential biased signaling and examine if this impacts agonist-induced D1R endocytosis. We determined that most, but not all, non-catechol agonists display G protein biased signaling at the D1R and have reduced or absent Beta-arrestin recruitment. A notable exception was compound (Cmpd) 19, a non-catechol agonist with full efficacy at both D1R-G protein or D1R Beta-arrestin pathways. In addition, the catechol ligand A-77636 was a highly potent, super agonist for D1R Beta-arrestin activity. When examined for agonist-induced D1R endocytosis, balanced agonists SKF-81297 and Cmpd 19 induced robust D1R endocytosis while the G protein biased agonists did not. The Beta-arrestin super agonist, A-77636, showed significantly increased D1R endocytosis. Moreover, Beta-arrestin recruitment efficacy of tested agonists strongly correlated with total D1R endocytosis. Taken together, these results indicate the degree of D1R signaling functional selectivity profoundly impacts D1R endocytosis regardless of pharmacophore. The range of functional selectivity of these D1R agonists will provide valuable tools to further investigate D1R signaling, trafficking and therapeutic potential.

Retraction of "Synthesis and Pharmacological Evaluation of Noncatechol G Protein Biased and Unbiased Dopamine D1 Receptor Agonists".

[This retracts the article DOI: 10.1021/acsmedchemlett.9b00050.].

Evaluation of lorcaserin as an anticonvulsant in juvenile Fmr1 knockout mice.

Recent preclinical and clinical studies suggest that lorcaserin, a preferential serotonin 2C receptor (5-HT2CR) agonist that was approved for the treatment of obesity, possesses antiepileptic properties. Here, we tested whether lorcaserin (1, 3, 5.6, 10 mg/kg) is prophylactic against audiogenic seizures (AGSs) in juvenile Fmr1 knockout mice, a mouse model of fragile X syndrome (FXS). MPEP (30 mg/kg), a non-competitive mGluR5 receptor antagonist, was used as a positive control. As lorcaserin likely engages 5-HT2ARs at therapeutic doses, we pretreated one group of mice with the selective 5-HT2AR antagonist/inverse agonist, M100907 (0.03 mg/kg), alone or before administering lorcaserin (5.6 mg/kg), to discern putative contributions of 5-HT2ARs to AGSs. We also assessed lorcaserin's in vitro pharmacology at human (h) and mouse (m) 5-HT2CRs and 5-HT2ARs and its in vivo interactions at m5-HT2CRs and m5-HT2ARs. MPEP significantly decreased AGS prevalence (P = 0.011) and lethality (P = 0.038). Lorcaserin, 3 mg/kg, attenuated AGS prevalence and lethality by 14 % and 32 %, respectively, however, results were not statistically significant (P = 0.5 and P = 0.06); other doses and M100907 alone or with lorcaserin also did not significantly affect AGSs. Lorcaserin exhibited full efficacy agonist activity at h5-HT2CRs and m5-HT2CRs, and near full efficacy agonist activity at h5-HT2ARs and m5-HT2ARs; selectivity for activation of 5-HT2CRs over 5-HT2ARs was greater for human (38-fold) compared to mouse (13-fold) receptors. Lorcaserin displayed relatively low affinities at antagonist-labeled 5-HT2CRs and 5-HT2ARs, regardless of species. Lorcaserin (3 and 5.6 mg/kg) increased the 5-HT2AR-dependent head-twitch response (HTR) elicited by (±)-2,5-dimethoxy-4-iodoamphetamine (DOI) in mice (P = 0.03 and P = 0.02). At 3 mg/kg, lorcaserin alone did not elicit an HTR. If mice were treated with the selective 5-HT2CR antagonist SB 242084 (0.5 or 1 mg/kg) plus lorcaserin (3 mg/kg), a significantly increased HTR was observed, relative to vehicle (P = 0.01 and P = 0.03), however, the HTR was much lower than what was elicited by DOI or DOI plus lorcaserin. Lorcaserin, 3 mg/kg, significantly reduced locomotor activity on its own, an effect reversed by SB 242084, and lorcaserin also dose-dependently reduced locomotor activity when administered prior to DOI (Ps<0.002). These data suggest that lorcaserin may engage 5-HT2CRs as well as 5-HT2ARs in mice at doses as low as 3 mg/kg. The similar activity at m5-HT2CRs and m5-HT2ARs suggests careful dosing of lorcaserin is necessary to selectively engage 5-HT2CRs in vivo. In conclusion, lorcaserin was ineffective at preventing AGSs in Fmr1 knockout mice. Lorcaserin may not be a suitable pharmacotherapy for seizures in FXS.

Maternal Opioid Exposure Culminates in Perturbed Murine Neurodevelopment and Hyperactive Phenotype in Adolescence.

Opioid use by women during pregnancy has risen dramatically since 2004, accompanied by a striking increase in the prevalence of neonatal opioid withdrawal syndrome (NOWS) and other long-term neurological deficits. However, the mechanisms underlying the impact of prenatal opioid exposure on fetal neurodevelopment are largely unknown. To translate from the clinical presentation, we developed a novel mouse model to study the neurodevelopmental consequences of maternal opioid use and management. Female mice were treated with oxycodone (OXY) before mating to mimic opioid use disorder (OUD) in humans. Following pregnancy confirmation, dams were switched to buprenorphine (BUP) via oral administration, simulating medication management of OUD (MOUD) in pregnant women. Here, we document critical changes in fetal brain development including reduced cortical thickness, altered corticogenesis, and ventriculomegaly in embryos from dams that were treated with opioids before and throughout pregnancy. Maternal care giving behavior was slightly altered without affecting gross growth of offspring. However, adolescent offspring exposed to maternal opioid use during pregnancy exhibited hyperactivity in late adolescence. Remarkably, we also show increased generation of dopaminergic neurons within the ventral tegmental area (VTA) of mice exposed to prenatal opioids. These data provide critical evidence of teratogenic effects of opioid use during pregnancy and suggest a causal relationship between maternal opioid use and neurodevelopmental/behavioral anomalies in adolescence.

Discovery of Potent and Brain-Penetrant GPR52 Agonist that Suppresses Psychostimulant Behavior.

The G protein-coupled receptor 52 (GPR52) is an orphan receptor that is selectively expressed in the striatum and regulates various brain functions through activation of cAMP-dependent pathways. GPR52 has been identified as a promising therapeutic target for central nervous system disorders including schizophrenia and substance use disorders. Here, a series of novel GPR52 agonists were designed, synthesized, and evaluated based on compound 4. Several potent and efficacious GPR52 agonists (12c, 23a, 23d, 23e, 23f, and 23h) were identified with nanomolar range potency based on a systematic structure-activity relationship exploration. Further studies of 12c indicate enhanced efficacy, excellent target selectivity, and pharmacokinetic properties including good brain permeability. In vivo proof-of-concept investigations revealed that 12c displayed antipsychotic-like activity by significantly inhibiting amphetamine-induced hyperlocomotor behavior in mice. Collectively, our findings have resulted in an efficacious, brain-penetrant GPR52 agonist as a valuable pharmacological tool for investigating the physiological and therapeutic potential of GPR52 activation.

Synthesis of novel 5-substituted-2-aminotetralin analogs: 5-HT1A and 5-HT7 G protein-coupled receptor affinity, 3D-QSAR and molecular modeling.

The serotonin 5-HT7 G protein-coupled receptor (GPCR) is a proposed pharmacotherapeutic target for a variety of central and peripheral indications, albeit, there are no approved drugs selective for binding 5-HT7. We previously reported that a lead analog based on the 5-substituted-N,N-disubstituted-1,2,3,4-tetrahydronaphthalen-2-amine (5-substituted-2-aminotetralin, 5-SAT) scaffold binds with high affinity at the 5-HT7 GPCR, and can treat symptoms of autism in mouse models; subsequently, the lead was found to have high affinity at the 5-HT1A GPCR. Herein, we report the synthesis of novel 5-SAT analogs to develop a 3-dimensional quantitative structure-affinity relationship (3D-QSAR) at the human 5-HT7 receptor for comparison with similar studies at the highly homologous 5-HT1A receptor. We report 35 new 5-SAT ligands, some with very high affinity (Ki ≤ 1 nM) and stereoselectivity at 5-HT7 + or 5-HT1A receptors, several with modest selectivity (up to 12-fold) for binding at 5-HT7, and, several ligands with high selectivity (up to 40-fold) at the 5-HT1A receptor. 3D-QSAR results indicate that steric extensions at the C(5)-position improve selectivity for the 5-HT7 over 5-HT1A receptor, while steric and hydrophobic extensions at the chiral C(2)-amino position impart 5-HT1A selectivity. In silico receptor homology modeling studies, supplemented with molecular dynamics simulations and binding free energy calculations, were used to rationalize experimentally-determined receptor selectivity and stereoselective affinity results. The data from these studies indicate that the 5-SAT chemotype, previously shown to be safe and efficacious in rodent paradigms of neurodevelopmental and neuropsychiatric disorders, is amenable to structural modification to optimize affinity at serotonin 5-HT7 vs. 5-HT1A GPCRs, as may be required for successful clinical translation.

Serotonin 5-HT2C Receptor Cys23Ser Single Nucleotide Polymorphism Associates with Receptor Function and Localization In Vitro.

A non-synonymous single nucleotide polymorphism of the human serotonin 5-HT2C receptor (5-HT2CR) gene that converts a cysteine to a serine at amino acid codon 23 (Cys23Ser) appears to impact 5-HT2CR pharmacology at a cellular and systems level. We hypothesized that the Cys23Ser alters 5-HT2CR intracellular signaling via changes in subcellular localization in vitro. Using cell lines stably expressing the wild-type Cys23 or the Ser23 variant, we show that 5-HT evokes intracellular calcium release with decreased potency and peak response in the Ser23 versus the Cys23 cell lines. Biochemical analyses demonstrated lower Ser23 5-HT2CR plasma membrane localization versus the Cys23 5-HT2CR. Subcellular localization studies demonstrated O-linked glycosylation of the Ser23 variant, but not the wild-type Cys23, may be a post-translational mechanism which alters its localization within the Golgi apparatus. Further, both the Cys23 and Ser23 5-HT2CR are present in the recycling pathway with the Ser23 variant having decreased colocalization with the early endosome versus the Cys23 allele. Agonism of the 5-HT2CR causes the Ser23 variant to exit the recycling pathway with no effect on the Cys23 allele. Taken together, the Ser23 variant exhibits a distinct pharmacological and subcellular localization profile versus the wild-type Cys23 allele, which could impact aspects of receptor pharmacology in individuals expressing the Cys23Ser SNP.

Advances in Dopamine D1 Receptor Ligands for Neurotherapeutics.

The dopamine D1 receptor (D1R) is essential for neurotransmission in various brain pathways where it modulates key functions including voluntary movement, memory, attention and reward. Not surprisingly, the D1R has been validated as a promising drug target for over 40 years and selective activation of this receptor may provide novel neurotherapeutics for neurodegenerative and neuropsychiatric disorders. Several pharmacokinetic challenges with previously identified small molecule D1R agonists have been recently overcome with the discovery and advancement of new ligands, including drug-like non-catechol D1R agonists and positive allosteric modulators. From this, several novel molecules and mechanisms have recently entered clinical studies. Here we review the major classes of D1R selective ligands including antagonists, orthosteric agonists, non-catechol biased agonists and positive allosteric modulators, highlighting their structure-activity relationships and medicinal chemistry. Recent chemistry breakthroughs and innovative approaches to selectively target and activate the D1R also hold promise for creating pharmacotherapy for several neurological diseases.

Synthesis and Pharmacological Evaluation of Noncatechol G Protein Biased and Unbiased Dopamine D1 Receptor Agonists.

Noncatechol heterocycles have recently been discovered as potent and selective G protein biased dopamine 1 receptor (D1R) agonists with superior pharmacokinetic properties. To determine the structure-activity relationships centered on G protein or ß-arrestin signaling bias, systematic medicinal chemistry was employed around three aromatic pharmacophores of the lead compound 5 (PF2334), generating a series of new molecules that were evaluated at both D1R Gs-dependent cAMP signaling and ß-arrestin recruitment in HEK293 cells. Here, we report the chemical synthesis, pharmacological evaluation, and molecular docking studies leading to the identification of two novel noncatechol D1R agonists that are a subnanomolar potent unbiased ligand 19 (PW0441) and a nanomolar potent complete G protein biased ligand 24 (PW0464), respectively. These novel D1R agonists provide important tools to study D1R activation and signaling bias in both health and disease.

Ligand-directed serotonin 5-HT2C receptor desensitization and sensitization.

Exposure of G protein-coupled receptors (GPCRs) to agonists can desensitize receptor signaling and lead to drug tolerance, whereas inverse agonists can sensitize signaling. For example, activation of serotonin 5-HT2C GPCRs is pharmacotherapeutic for obesity, but there is tolerance to the anorectic effect of the only approved 5-HT2C agonist, lorcaserin. We tested the hypothesis that different agonists or inverse agonists differentially desensitize or sensitize, respectively, canonical 5-HT2C-mediated activation of phospholipase C (PLC) signaling in vitro. Lorcaserin, which displays potency and efficacy equal to 5-HT, desensitized the 5-HT2C receptor significantly more than 5-HT (p<0.05). Agonist chemotypes such as 2-aminotetralins, with similar potency but lower efficacy than 5-HT, produced little 5-HT2C desensitization. The piperazine agonist 1-(3-chlorophenyl)piperazine (mCPP), with lower potency but similar efficacy as 5-HT, elicited desensitization indistinguishable from 5-HT, while the piperazine agonist aripiprazole, with lower potency and efficacy, did not desensitize 5-HT2C-PLC signaling. Several 5-HT2C agonists also were assessed for ß-arrestin recruitment-lorcaserin was a 'super-agonist', but a 2-aminotetralin and aripiprazole had nil activity, suggesting they are biased towards 5-HT2C-PLC signaling. We observed robust positive correlations between the magnitude of 5-HT2C desensitization and agonist efficacy to stimulate PLC or to recruit ß-arrestin. In contrast, different inverse agonists caused different magnitudes of 5-HT2C sensitization that did not correlate with efficacy (or potency) to inhibit constitutive 5-HT2C-PLC signaling.  Assessment of the 5-HT2C-S407A point-mutated receptor indicated this residue's involvement in ligand-dependent desensitization, but we did not observe a role for protein kinase C.These data show that ligand structure uniquely impacts 5-HT2C desensitization and sensitization processes..

Small-Molecule Neuromedin U Receptor 2 Agonists Suppress Food Intake and Decrease Visceral Fat in Animal Models.

Obesity is a growing public health concern, with 37.5% of the adult population in need of therapeutics that are more efficacious with a better side effect profile. An innovative target in this regard is neuromedin U, a neuropeptide shown to suppress food intake and attenuate weight gain in animal models. These effects of neuromedin U on feeding behavior are thought to be related to agonism at the centrally expressed neuromedin U receptor 2 (NMUR2). As peptides present unique challenges that limit their therapeutic potential, the discovery of small-molecule NMUR2 agonists is needed to validate the targets therapeutic value, but to date, none have been evaluated in any animal model of disease. We therefore assessed two small-molecule NMUR2 agonists for their in vitro signaling and their in vivo efficacy. The NMUR2 agonists were synthesized and both NMUR2 agonists, NY0116 and NY0128, decreased cAMP while stimulating calcium signaling in stably expressing NMUR2 HEK293 cells. When small-molecule NMUR2 agonists were tested in vivo, acute administration significantly decreased high-fat diet consumption. Repeated administration of the compounds decreased body weight and more specifically, decreased the percentage of visceral adipose tissue (VAT) in obese mice. These results have confirmed small-molecule NMUR2 agonists are efficacious in animal models to decrease fat content, food intake, and body weight, suggesting NMUR2 is a promising therapeutic target for metabolic disorders.

Biophysical validation of serotonin 5-HT2A and 5-HT2C receptor interaction.

The serotonin (5-HT) 5-HT2A receptor (5-HT2AR) and 5-HT2C receptor (5-HT2CR) in the central nervous system are implicated in a range of normal behaviors (e.g., appetite, sleep) and physiological functions (e.g., endocrine secretion) while dysfunctional 5-HT2AR and/or 5-HT2CR are implicated in neuropsychiatric disorders (e.g., addiction, obesity, schizophrenia). Preclinical studies suggest that the 5-HT2AR and 5-HT2CR may act in concert to regulate the neural bases for behavior. Here, we utilize three distinct biophysical and immunocytochemistry-based approaches to identify and study this receptor complex in cultured cells. Employing a split luciferase complementation assay (LCA), we demonstrated that formation of the 5-HT2AR:5-HT2CR complex exists within 50 nm, increases proportionally to the 5-HT2CR:5-HT2AR protein expression ratio, and is specific to the receptor interaction and not due to random complementation of the luciferase fragments. Using a proximity ligation assay (PLA), we found that cells stably expressing both the 5-HT2AR and 5-HT2CR exhibit 5-HT2AR:5-HT2CR heteroreceptor complexes within 40 nm of each other. Lastly, bioluminescence resonance energy transfer (BRET) analyses indicates the formation of a specific and saturable 5-HT2AR:5-HT2CR interaction, suggesting that the 5-HT2AR and 5-HT2CR form a close interaction within 10 nm of each other in intact live cells. The bioengineered receptors generated for the LCA and the BRET exhibit 5-HT-mediated intracellular calcium signaling as seen for the native receptors. Taken together, this study validates a very close 5-HT2AR:5-HT2CR interaction in cultured cells.

Homology Modeling Inspired Synthesis of 5-HT2A Receptor Inhibitors: A Diazepine Analogue of the Atypical Antipsychotic JL13.

BACKGROUND: The benzoxazepine JL13 is an analogue of the clozapine family of antipsychotic agents which target the 5-HT2A receptor, and has showed promise as an atypical antipsychotic agent. Based on the dearth of clinically effective anti-psychotic agents available, we sought to design and chemically synthesize additional analogues. METHODS: Structure function analysis was conducted using state of art computational methods, which were designed to highlight new candidates for chemical synthesis. Efficient syntheses were then conducted and the products screened for affinity to the receptor. RESULTS: Among many new analogues prepared, an aza analogue demonstrated seventeen times greater affinity for the receptor than JL13. CONCLUSION: An efficient synthetic route to an aza-analogue of JL13 was developed and will allow rapid modifications of the core and synthesis of related libraries.

An Orally Active Phenylaminotetralin-Chemotype Serotonin 5-HT7 and 5-HT1A Receptor Partial Agonist that Corrects Motor Stereotypy in Mouse Models.

Stereotypy (e.g., repetitive hand waving) is a key phenotype of autism spectrum disorder, Fragile X and Rett syndromes, and other neuropsychiatric disorders, and its severity correlates with cognitive and attention deficits. There are no effective treatments, however, for stereotypy. Perturbation of serotonin (5-HT) neurotransmission contributes to stereotypy, suggesting that distinct 5-HT receptors may be pharmacotherapeutic targets to treat stereotypy and related neuropsychiatric symptoms. For example, preclinical studies indicate that 5-HT7 receptor activation corrects deficits in mouse models of Fragile X and Rett syndromes, and clinical trials for autism are underway with buspirone, a 5-HT1A partial agonist with relevant affinity at 5-HT7 receptors. Herein, we report the synthesis, in vitro molecular pharmacology, behavioral pharmacology, and pharmacokinetic parameters in mice after subcutaneous and oral administration of (+)-5-(2'-fluorophenyl)-N,N-dimethyl-1,2,3,4-tetrahydronaphthalen-2-amine ((+)-5-FPT), a new, dual partial agonist targeting both 5-HT7 (Ki = 5.8 nM, EC50 = 34 nM) and 5-HT1A (Ki = 22 nM, EC50 = 40 nM) receptors. Three unique, heterogeneous mouse models were used to assess the efficacy of (+)-5-FPT to reduce stereotypy: idiopathic jumping in C58/J mice, repetitive body rotations in C57BL/6J mice treated with the NMDA antagonist, MK-801, and repetitive head twitching in C57BL/6J mice treated with the 5-HT2 agonist, DOI. Systemic (+)-5-FPT potently and efficaciously reduced or eliminated stereotypy in each of the mouse models without altering locomotor behavior on its own, and additional tests showed that (+)-5-FPT, at the highest behaviorally active dose tested, enhanced social interaction and did not cause behaviors indicative of serotonin syndrome. These data suggest that (+)-5-FPT is a promising medication for treating stereotypy in psychiatric disorders.

A novel aminotetralin-type serotonin (5-HT) 2C receptor-specific agonist and 5-HT2A competitive antagonist/5-HT2B inverse agonist with preclinical efficacy for psychoses.

Development of 5-HT2C agonists for treatment of neuropsychiatric disorders, including psychoses, substance abuse, and obesity, has been fraught with difficulties, because the vast majority of reported 5-HT2C selective agonists also activate 5-HT2A and/or 5-HT2B receptors, potentially causing hallucinations and/or cardiac valvulopathy. Herein is described a novel, potent, and efficacious human 5-HT2C receptor agonist, (-)-trans-(2S,4R)-4-(3'[meta]-bromophenyl)-N,N-dimethyl-1,2,3,4-tetrahydronaphthalen-2-amine (-)-MBP), that is a competitive antagonist and inverse agonist at human 5-HT2A and 5-HT2B receptors, respectively. (-)-MBP has efficacy comparable to the prototypical second-generation antipsychotic drug clozapine in three C57Bl/6 mouse models of drug-induced psychoses: the head-twitch response elicited by [2,5]-dimethoxy-4-iodoamphetamine; hyperlocomotion induced by MK-801 [(5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (dizocilpine maleate)]; and hyperlocomotion induced by amphetamine. (-)-MBP, however, does not alter locomotion when administered alone, distinguishing it from clozapine, which suppresses locomotion. Finally, consumption of highly palatable food by mice was not increased by (-)-MBP at a dose that produced at least 50% maximal efficacy in the psychoses models. Compared with (-)-MBP, the enantiomer (+)-MBP was much less active across in vitro affinity and functional assays using mouse and human receptors and also translated in vivo with comparably lower potency and efficacy. Results indicate a 5-HT2C receptor-specific agonist, such as (-)-MBP, may be pharmacotherapeutic for psychoses, without liability for obesity, hallucinations, heart disease, sedation, or motoric disorders.