Structure-based discovery of conformationally selective inhibitors of the serotonin transporter.

The serotonin transporter (SERT) removes synaptic serotonin and is the target of anti-depressant drugs. SERT adopts three conformations: outward-open, occluded, and inward-open. All known inhibitors target the outward-open state except ibogaine, which has unusual anti-depressant and substance-withdrawal effects, and stabilizes the inward-open conformation. Unfortunately, ibogaine's promiscuity and cardiotoxicity limit the understanding of inward-open state ligands. We docked over 200 million small molecules against the inward-open state of the SERT. Thirty-six top-ranking compounds were synthesized, and thirteen inhibited; further structure-based optimization led to the selection of two potent (low nanomolar) inhibitors. These stabilized an outward-closed state of the SERT with little activity against common off-targets. A cryo-EM structure of one of these bound to the SERT confirmed the predicted geometry. In mouse behavioral assays, both compounds had anxiolytic- and anti-depressant-like activity, with potencies up to 200-fold better than fluoxetine (Prozac), and one substantially reversed morphine withdrawal effects.

ß-Arrestin-Biased Allosteric Modulator of NTSR1 Selectively Attenuates Addictive Behaviors.

Small molecule neurotensin receptor 1 (NTSR1) agonists have been pursued for more than 40 years as potential therapeutics for psychiatric disorders, including drug addiction. Clinical development of NTSR1 agonists has, however, been precluded by their severe side effects. NTSR1, a G protein-coupled receptor (GPCR), signals through the canonical activation of G proteins and engages ß-arrestins to mediate distinct cellular signaling events. Here, we characterize the allosteric NTSR1 modulator SBI-553. This small molecule not only acts as a ß-arrestin-biased agonist but also extends profound ß-arrestin bias to the endogenous ligand by selectively antagonizing G protein signaling. SBI-553 shows efficacy in animal models of psychostimulant abuse, including cocaine self-administration, without the side effects characteristic of balanced NTSR1 agonism. These findings indicate that NTSR1 G protein and ß-arrestin activation produce discrete and separable physiological effects, thus providing a strategy to develop safer GPCR-targeting therapeutics with more directed pharmacological action.

Bespoke library docking for 5-HT2A receptor agonists with antidepressant activity.

There is considerable interest in screening ultralarge chemical libraries for ligand discovery, both empirically and computationally1-4. Efforts have focused on readily synthesizable molecules, inevitably leaving many chemotypes unexplored. Here we investigate structure-based docking of a bespoke virtual library of tetrahydropyridines-a scaffold that is poorly sampled by a general billion-molecule virtual library but is well suited to many aminergic G-protein-coupled receptors. Using three inputs, each with diverse available derivatives, a one pot C-H alkenylation, electrocyclization and reduction provides the tetrahydropyridine core with up to six sites of derivatization5-7. Docking a virtual library of 75 million tetrahydropyridines against a model of the serotonin 5-HT2A receptor (5-HT2AR) led to the synthesis and testing of 17 initial molecules. Four of these molecules had low-micromolar activities against either the 5-HT2A or the 5-HT2B receptors. Structure-based optimization led to the 5-HT2AR agonists (R)-69 and (R)-70, with half-maximal effective concentration values of 41 nM and 110 nM, respectively, and unusual signalling kinetics that differ from psychedelic 5-HT2AR agonists. Cryo-electron microscopy structural analysis confirmed the predicted binding mode to 5-HT2AR. The favourable physical properties of these new agonists conferred high brain permeability, enabling mouse behavioural assays. Notably, neither had psychedelic activity, in contrast to classic 5-HT2AR agonists, whereas both had potent antidepressant activity in mouse models and had the same efficacy as antidepressants such as fluoxetine at as low as 1/40th of the dose. Prospects for using bespoke virtual libraries to sample pharmacologically relevant chemical space will be considered.

Postsynaptic Mechanisms Render Syn I/II/III Mice Highly Responsive to Psychostimulants.

BACKGROUND: Synapsins are encoded by SYN I, SYN II, and SYN III, and they regulate neurotransmitter release by maintaining a reserve pool of synaptic vesicles. METHODS: Presynaptic dopamine responses to cocaine were examined by microdialysis, and postsynaptic responses were evaluated to various dopamine receptor agonists in the open field with SynI/SynII/SynIII triple knockout mice. RESULTS: Triple knockout mice showed enhanced spontaneous locomotion in a novel environment and were hyper-responsive to indirect and direct D1 and D2 dopamine agonists. Triple knockout animals appeared sensitized to cocaine upon first open field exposure; sensitization developed across days in wild-type controls. When mutants were preexposed to a novel environment before injection, cocaine-stimulated locomotion was reduced and behavioral sensitization retarded. Baseline dopamine turnover was enhanced in mutants and novel open field exposure increased their striatal dopamine synthesis rates. As KCl-depolarization stimulated comparable dopamine release in both genotypes, their readily releasable pools appeared indistinguishable. Similarly, cocaine-induced hyperlocomotion was indifferent to blockade of newly synthesized dopamine and depletion of releasable dopamine pools. Extracellular dopamine release was similar in wild-type and triple knockout mice preexposed to the open field and given cocaine or placed immediately into the arena following injection. Since motor effects to novelty and psychostimulants depend upon frontocortical-striatal inputs, we inhibited triple knockout medial frontal cortex with GABA agonists. Locomotion was transiently increased in cocaine-injected mutants, while their supersensitive cocaine response to novelty was lost. CONCLUSIONS: These results reveal presynaptic dopamine release is not indicative of agonist-induced triple knockout hyperlocomotion. Instead, their novelty response occurs primarily through postsynaptic mechanisms and network effects.

The G protein biased serotonin 5-HT2A receptor agonist lisuride exerts anti-depressant drug-like activities in mice.

There is now evidence from multiple Phase II clinical trials that psychedelic drugs can exert long-lasting anxiolytic, anti-depressant, and anti-drug abuse (nicotine and ethanol) effects in patients. Despite these benefits, the hallucinogenic actions of these drugs at the serotonin 2A receptor (5-HT2AR) limit their clinical use in diverse settings. Activation of the 5-HT2AR can stimulate both G protein and ß-arrestin (ßArr) -mediated signaling. Lisuride is a G protein biased agonist at the 5-HT2AR and, unlike the structurally-related lysergic acid diethylamide (LSD), the drug does not typically produce hallucinations in normal subjects at routine doses. Here, we examined behavioral responses to lisuride, in wild-type (WT), ßArr1-knockout (KO), and ßArr2-KO mice. In the open field, lisuride reduced locomotor and rearing activities, but produced a U-shaped function for stereotypies in both ßArr lines of mice. Locomotion was decreased overall in ßArr1-KOs and ßArr2-KOs relative to wild-type controls. Incidences of head twitches and retrograde walking to lisuride were low in all genotypes. Grooming was decreased in ßArr1 mice, but was increased then decreased in ßArr2 animals with lisuride. Serotonin syndrome-associated responses were present at all lisuride doses in WTs, but they were reduced especially in ßArr2-KO mice. Prepulse inhibition (PPI) was unaffected in ßArr2 mice, whereas 0.5 mg/kg lisuride disrupted PPI in ßArr1 animals. The 5-HT2AR antagonist MDL100907 failed to restore PPI in ßArr1 mice, whereas the dopamine D2/D3 antagonist raclopride normalized PPI in WTs but not in ßArr1-KOs. Clozapine, SCH23390, and GR127935 restored PPI in both ßArr1 genotypes. Using vesicular monoamine transporter 2 mice, lisuride reduced immobility times in tail suspension and promoted a preference for sucrose that lasted up to 2 days. Together, it appears ßArr1 and ßArr2 play minor roles in lisuride's actions on many behaviors, while this drug exerts anti-depressant drug-like responses without hallucinogenic-like activities.

The G protein biased serotonin 5-HT 2A receptor agonist lisuride exerts anti-depressant drug-like activities in mice.

There is now evidence from multiple Phase II clinical trials that psychedelic drugs can exert longlasting anxiolytic, anti-depressant, and anti-drug abuse (nicotine and ethanol) effects in patients. Despite these benefits, the hallucinogenic actions of these drugs at the serotonin 2A receptor (5-HT2AR) limit their clinical use in diverse settings. Activation of the 5-HT2AR can stimulate both G protein and ß-arrestin (ßArr) -mediated signaling. Lisuride is a G protein biased agonist at the 5-HT2AR and, unlike the structurally-related LSD, the drug does not typically produce hallucinations in normal subjects at routine doses. Here, we examined behavioral responses to lisuride, in wild-type (WT), ßArr1-KO, and ßArr2-KO mice. In the open field, lisuride reduced locomotor and rearing activities, but produced a U-shaped function for stereotypies in both ßArr lines of mice. Locomotion was decreased overall in ßArr1-KOs and ßArr2-KOs, relative to WT controls. Incidences of head twitches and retrograde walking to lisuride were low in all genotypes. Grooming was depressed in ßArr1 mice, but was increased then decreased in ßArr2 animals with lisuride. Prepulse inhibition (PPI) was unaffected in ßArr2 mice, whereas 0.5 mg/kg lisuride disrupted PPI in ßArr1 animals. The 5-HT2AR antagonist MDL100907 failed to restore PPI in ßArr1 mice, whereas the dopamine D2/D3 antagonist raclopride normalized PPI in WTs but not in ßArr1-KOs. Using vesicular monoamine transporter 2 mice, lisuride reduced immobility times in tail suspension and promoted a preference for sucrose that lasted up to 2 days. Together, it appears ßArr1 and ßArr2 play minor roles in lisuride's actions on many behaviors, while this drug exerts anti-depressant drug-like responses without hallucinogenic-like activities.

Dopamine-Depleted Dopamine Transporter Knockout (DDD) Mice: Dyskinesia with L-DOPA and Dopamine D1 Agonists.

L-DOPA is the mainstay of treatment for Parkinson's disease (PD). However, over time this drug can produce dyskinesia. A useful acute PD model for screening novel compounds for anti-parkinsonian and L-DOPA-induced dyskinesia (LID) are dopamine-depleted dopamine-transporter KO (DDD) mice. Treatment with α-methyl-para-tyrosine rapidly depletes their brain stores of DA and renders them akinetic. During sensitization in the open field (OF), their locomotion declines as vertical activities increase and upon encountering a wall they stand on one leg or tail and engage in climbing behavior termed "three-paw dyskinesia". We have hypothesized that L-DOPA induces a stereotypic activation of locomotion in DDD mice, where they are unable to alter the course of their locomotion, and upon encountering walls engage in "three-paw dyskinesia" as reflected in vertical counts or beam-breaks. The purpose of our studies was to identify a valid index of LID in DDD mice that met three criteria: (a) sensitization with repeated L-DOPA administration, (b) insensitivity to a change in the test context, and (c) stimulatory or inhibitory responses to dopamine D1 receptor agonists (5 mg/kg SKF81297; 5 and 10 mg/kg MLM55-38, a novel compound) and amantadine (45 mg/kg), respectively. Responses were compared between the OF and a circular maze (CM) that did not hinder locomotion. We found vertical counts and climbing were specific for testing in the OF, while oral stereotypies were sensitized to L-DOPA in both the OF and CM and responded to D1R agonists and amantadine. Hence, in DDD mice oral stereotypies should be used as an index of LID in screening compounds for PD.

Identification and Characterization of ML321: A Novel and Highly Selective D2 Dopamine Receptor Antagonist with Efficacy in Animal Models That Predict Atypical Antipsychotic Activity.

We have developed and characterized a novel D2R antagonist with exceptional GPCR selectivity - ML321. In functional profiling screens of 168 different GPCRs, ML321 showed little activity beyond potent inhibition of the D2R and to a lesser extent the D3R, demonstrating excellent receptor selectivity. The D2R selectivity of ML321 may be related to the fact that, unlike other monoaminergic ligands, ML321 lacks a positively charged amine group and adopts a unique binding pose within the orthosteric binding site of the D2R. PET imaging studies in non-human primates demonstrated that ML321 penetrates the CNS and occupies the D2R in a dose-dependent manner. Behavioral paradigms in rats demonstrate that ML321 can selectively antagonize a D2R-mediated response (hypothermia) while not affecting a D3R-mediated response (yawning) using the same dose of drug, thus indicating exceptional in vivo selectivity. We also investigated the effects of ML321 in animal models that are predictive of antipsychotic efficacy in humans. We found that ML321 attenuates both amphetamine- and phencyclidine-induced locomotor activity and restored pre-pulse inhibition (PPI) of acoustic startle in a dose-dependent manner. Surprisingly, using doses that were maximally effective in both the locomotor and PPI studies, ML321 was relatively ineffective in promoting catalepsy. Kinetic studies revealed that ML321 exhibits slow-on and fast-off receptor binding rates, similar to those observed with atypical antipsychotics with reduced extrapyramidal side effects. Taken together, these observations suggest that ML321, or a derivative thereof, may exhibit ″atypical″ antipsychotic activity in humans with significantly fewer side effects than observed with the currently FDA-approved D2R antagonists.

Establishment of multi-stage intravenous self-administration paradigms in mice.

Genetically tractable animal models provide needed strategies to resolve the biological basis of drug addiction. Intravenous self-administration (IVSA) is the gold standard for modeling psychostimulant and opioid addiction in animals, but technical limitations have precluded the widespread use of IVSA in mice. Here, we describe IVSA paradigms for mice that capture the multi-stage nature of the disorder and permit predictive modeling. In these paradigms, C57BL/6J mice with long-standing indwelling jugular catheters engaged in cocaine- or remifentanil-associated lever responding that was fixed ratio-dependent, dose-dependent, extinguished by withholding the drug, and reinstated by the presentation of drug-paired cues. The application of multivariate analysis suggested that drug taking in both paradigms was a function of two latent variables we termed incentive motivation and discriminative control. Machine learning revealed that vulnerability to drug seeking and relapse were predicted by a mouse's a priori response to novelty, sensitivity to drug-induced locomotion, and drug-taking behavior. The application of these behavioral and statistical-analysis approaches to genetically-engineered mice will facilitate the identification of neural circuits driving addiction susceptibility and relapse and focused therapeutic development.

Working memory deficits in neuronal nitric oxide synthase knockout mice: potential impairments in prefrontal cortex mediated cognitive function.

Neuronal nitric oxide synthase (nNOS) forms nitric oxide (NO), which functions as a signaling molecule via S-nitrosylation of various proteins and regulation of soluble guanylate cyclase (cGC)/cyclic guanosine monophosphate (cGMP) pathway in the central nervous system. nNOS signaling regulates diverse cellular processes during brain development and molecular mechanisms required for higher brain function. Human genetics have identified nNOS and several downstream effectors of nNOS as risk genes for schizophrenia. Besides the disease itself, nNOS has also been associated with prefrontal cortical functioning, including cognition, of which disturbances are a core feature of schizophrenia. Although mice with genetic deletion of nNOS display various behavioral deficits, no studies have investigated prefrontal cortex-associated behaviors. Here, we report that nNOS knockout (KO) mice exhibit hyperactivity and impairments in contextual fear conditioning, results consistent with previous reports. nNOS KO mice also display mild impairments in object recognition memory. Most importantly, we report for the first time working memory deficits, potential impairments in prefrontal cortex mediated cognitive function in nNOS KO mice. Furthermore, we demonstrate Disrupted-in-Schizophrenia 1 (DISC1), another genetic risk factor for schizophrenia that plays roles for cortical development and prefrontal cortex functioning, including working memory, is a novel protein binding partner of nNOS in the developing cerebral cortex. Of note, genetic deletion of nNOS appears to increase the binding of DISC1 to NDEL1, regulating neurite outgrowth as previously reported. These results suggest that nNOS KO mice are useful tools in studying the role of nNOS signaling in cortical development and prefrontal cortical functioning.

LSD-stimulated behaviors in mice require ß-arrestin 2 but not ß-arrestin 1.

Recent evidence suggests that psychedelic drugs can exert beneficial effects on anxiety, depression, and ethanol and nicotine abuse in humans. However, their hallucinogenic side-effects often preclude their clinical use. Lysergic acid diethylamide (LSD) is a prototypical hallucinogen and its psychedelic actions are exerted through the 5-HT2A serotonin receptor (5-HT2AR). 5-HT2AR activation stimulates Gq- and ß-arrestin- (ßArr) mediated signaling. To separate these signaling modalities, we have used ßArr1 and ßArr2 mice. We find that LSD stimulates motor activities to similar extents in WT and ßArr1-KO mice, without effects in ßArr2-KOs. LSD robustly stimulates many surrogates of psychedelic drug actions including head twitches, grooming, retrograde walking, and nose-poking in WT and ßArr1-KO animals. By contrast, in ßArr2-KO mice head twitch responses are low with LSD and this psychedelic is without effects on other surrogates. The 5-HT2AR antagonist MDL100907 (MDL) blocks the LSD effects. LSD also disrupts prepulse inhibition (PPI) in WT and ßArr1-KOs, but not in ßArr2-KOs. MDL restores LSD-mediated disruption of PPI in WT mice; haloperidol is required for normalization of PPI in ßArr1-KOs. Collectively, these results reveal that LSD's psychedelic drug-like actions appear to require ßArr2.

D2 Dopamine Receptor G Protein-Biased Partial Agonists Based on Cariprazine.

Functionally selective G protein-coupled receptor ligands are valuable tools for deciphering the roles of downstream signaling pathways that potentially contribute to therapeutic effects versus side effects. Recently, we discovered both Gi/o-biased and ß-arrestin2-biased D2 receptor agonists based on the Food and Drug Administration (FDA)-approved drug aripiprazole. In this work, based on another FDA-approved drug, cariprazine, we conducted a structure-functional selectivity relationship study and discovered compound 38 (MS1768) as a potent partial agonist that selectively activates the Gi/o pathway over ß-arrestin2. Unlike the dual D2R/D3R partial agonist cariprazine, compound 38 showed selective agonist activity for D2R over D3R. In fact, compound 38 exhibited potent antagonism of dopamine-stimulated ß-arrestin2 recruitment. In our docking studies, compound 38 directly interacts with S1935.42 on TM5 but has no interactions with extracellular loop 2, which appears to be in contrast to the binding poses of D2R ß-arrestin2-biased ligands. In in vivo studies, compound 38 showed high D2R receptor occupancy in mice and effectively inhibited phencyclidine-induced hyperlocomotion.

Designing Functionally Selective Noncatechol Dopamine D1 Receptor Agonists with Potent In Vivo Antiparkinsonian Activity.

Dopamine receptors are important G protein-coupled receptors (GPCRs) with therapeutic opportunities for treating Parkinson's Disease (PD) motor and cognitive deficits. Biased D1 dopamine ligands that differentially activate G protein over ß-arrestin recruitment pathways are valuable chemical tools for dissecting positive versus negative effects in drugs for PD. Here, we reveal an iterative approach toward modification of a D1-selective noncatechol scaffold critical for G protein-biased agonism. This approach provided enhanced understanding of the structural components critical for activity and signaling bias and led to the discovery of several novel compounds with useful pharmacological properties, including three highly GS-biased partial agonists. Administration of a potent, balanced, and brain-penetrant lead compound from this series results in robust antiparkinsonian effects in a rodent model of PD. This study suggests that the noncatechol ligands developed through this approach are valuable tools for probing D1 receptor signaling biology and biased agonism in models of neurologic disease.

Parvalbumin Interneurons of the Mouse Nucleus Accumbens are Required For Amphetamine-Induced Locomotor Sensitization and Conditioned Place Preference.

To determine the requirement for parvalbumin (PV) expressing GABAergic interneurons of the nucleus accumbens (NAc) in the behavioral adaptations induced by amphetamine (AMPH), we blocked synaptic vesicle release from these neurons using Cre-inducible viral expression of the tetanus toxin light chain in male and female PV-Cre mice. Silencing PV+ interneurons of the NAc selectively inhibited the expression of locomotor sensitization following repeated injections of AMPH and blocked AMPH-induced conditioned place preference (CPP). AMPH induced significantly more expression of the activity-dependent gene Fos in both D1 and D2 dopamine receptor-expressing medium spiny neurons (MSNs) of the NAc of PV+ interneuron silenced mice, suggesting a function for PV+ interneuron-mediated MSN inhibition in the expression of AMPH-induced locomotor sensitization and CPP. These data show a requirement for PV+ interneurons of the NAc in behavioral responses to AMPH, and they raise the possibility that modulation of PV+ interneuron function may alter the development or expression of psychostimulant-induced behavioral adaptations.

Use of a platform in an automated open-field to enhance assessment of anxiety-like behaviors in mice.

The present report describes a setup for simultaneously measuring anxiety-like behaviors and locomotor activity in mice. Animals are placed in a brightly lit, standard automated open-field (OF) in which a rectangular ceramic platform 8 cm high covers one quadrant of the floor. Mice preferred to stay under the platform, avoiding the area with bright illumination. Activities under and outside the platform were measured for 5 min. Chlordiazepoxide and buspirone dose-dependently increased time spent outside the platform (L-Time) and the light distance to total OF distance ratio (L:T-TD) in both genders without changing total OF distance. By contrast, amphetamine decreased L-Time and L:T-TD in males, thus displaying an anxiogenic effect. Imipramine was without selective effect on L-Time or L:T-TD, but decreased total OF distance at the highest dose indicative of a sedative effect. Drug effects were also evaluated in the OF without platform using conventional anxiety measures. Introduction of the platform into the OF apparatus strongly enhanced the sensitivity to anxiolytics. Comparison of strains differing in activity or anxiety levels showed that L-Time and L:T-TD can be used as measures of anxiety-like behavior independent of locomotor activity. Changes in motor activity are reflected in the total distance traveled under and outside the platform. Therefore, the platform test is fully automated, sensitive to both anxiolytic and anxiogenic effects of drugs and genetic phenotypes with little evidence of gender-specific responses, and can be easily utilized by most laboratories measuring behavior.

Distinct neuronal populations in the basolateral and central amygdala are activated with acute pain, conditioned fear, and fear-conditioned analgesia.

Fear-conditioned analgesia (FCA) is modulated by brain areas involved in the descending inhibitory pain pathway such as the basolateral (BLA) and central amygdala (CEA). The BLA contains Ca2+/calmodulin-dependent protein kinase II (CaMKII) and parvalbumin (PV) neurons. CEA neurons are primarily inhibitory (GABAergic) that comprise enkephalin (ENK) interneurons and corticotropin-releasing factor (CRF) - neurons that project to the periaqueductal grey. The purpose of our experiment was to determine the pattern of activation of CaMKII/PV and ENK/CRF neurons following the expression of acute pain, conditioned fear, and FCA. A significant reduction was observed in nociceptive behaviors in mice re-exposed to a contextually-aversive environment. Using NeuN and cFos as markers for activated neurons, CaMKII, PV, ENK, or CRF were used to identify neuronal subtypes. We find that mice expressing conditioned fear displayed an increase in c-Fos/CaMKII co-localization in the lateral amygdala and BLA compared to controls. Additionally a significant increase in cFos/CRF co-localization was observed in mice expressing FCA. These results show that amygdala processing of conditioned contextual aversive, nociceptive, and FCA behaviors involve different neuronal phenotypes and neural circuits between, within, and from various amygdala nuclei. This information will be important in developing novel therapies for treating pain and emotive disorders in humans.

5-HT2C Agonists Modulate Schizophrenia-Like Behaviors in Mice.

All FDA-approved antipsychotic drugs (APDs) target primarily dopamine D2 or serotonin (5-HT2A) receptors, or both; however, these medications are not universally effective, they may produce undesirable side effects, and provide only partial amelioration of negative and cognitive symptoms. The heterogeneity of pharmacological responses in schizophrenic patients suggests that additional drug targets may be effective in improving aspects of this syndrome. Recent evidence suggests that 5-HT2C receptors may be a promising target for schizophrenia since their activation reduces mesolimbic nigrostriatal dopamine release (which conveys antipsychotic action), they are expressed almost exclusively in CNS, and have weight-loss-promoting capabilities. A difficulty in developing 5-HT2C agonists is that most ligands also possess 5-HT2B and/or 5-HT2A activities. We have developed selective 5-HT2C ligands and herein describe their preclinical effectiveness for treating schizophrenia-like behaviors. JJ-3-45, JJ-3-42, and JJ-5-34 reduced amphetamine-stimulated hyperlocomotion, restored amphetamine-disrupted prepulse inhibition, improved social behavior, and novel object recognition memory in NMDA receptor hypofunctioning NR1-knockdown mice, and were essentially devoid of catalepsy. However, they decreased motivation in a breakpoint assay and did not promote reversal learning in MK-801-treated mice. Somewhat similar effects were observed with lorcaserin, a 5-HT2C agonist with potent 5-HT2B and 5-HT2A agonist activities, which is approved for treating obesity. Microdialysis studies revealed that both JJ-3-42 and lorcaserin reduced dopamine efflux in the infralimbic cortex, while only JJ-3-42 decreased it in striatum. Collectively, these results provide additional evidence that 5-HT2C receptors are suitable drug targets with fewer side effects, greater therapeutic selectivity, and enhanced efficacy for treating schizophrenia and related disorders than current APDs.

Further Advances in Optimizing (2-Phenylcyclopropyl)methylamines as Novel Serotonin 2C Agonists: Effects on Hyperlocomotion, Prepulse Inhibition, and Cognition Models.

A series of novel compounds with two halogen substituents have been designed and synthesized to further optimize the 2-phenylcyclopropylmethylamine scaffold in the quest for drug-like 5-HT2C agonists. Compound (+)-22a was identified as a potent 5-HT2C receptor agonist, with good selectivity against the 5-HT2B and the 5-HT2A receptors. ADMET assays showed that compound (+)-22a possessed desirable properties in terms of its microsomal stability, and CYP and hERG inhibition, along with an excellent brain penetration profile. Evaluation of (+)-22a in animal models of schizophrenia-related behaviors revealed that it had a desirable activity profile, as it reduced d-amphetamine-stimulated hyperlocomotion in the open field test, it restored d-amphetamine-disrupted prepulse inhibition, it induced cognitive improvements in the novel object recognition memory test in NR1-KD animals, and it produced very little catalepsy relative to haloperidol. These data support the further development of (+)-22a as a drug candidate for the treatment of schizophrenia.

Novelty seeking and stereotypic activation of behavior in mice with disruption of the Dat1 gene.

Disruption of the dopamine (DA) transporter (Dat1) gene in mice leads to a 50% reduction or complete elimination of Dat1 expression in striatum of respective heterozygous (HZ) and knockout (KO) mice. Compared to wild-type (WT) controls, extracellular DA is increased approximately two- and five-fold in the mutants. Although open field (OF) activity is similar for WT and HZ animals, it is enhanced for KO mice. The purpose of the present investigations was to study spontaneously emitted behaviors and to determine the behavioral and neurochemical mechanisms that may contribute to the hyperactivity of KO animals. Heterozygotes are less anxious than other genotypes and they engage in novelty-seeking behaviors that include increased time spent in the center of the OF, enhanced investigation of objects, and augmented free exploration of a novel environment. By comparison, KO mice display neophobia when initially exposed to novel conditions. Over time the anxiety-like response habituates and behaviors become activated and stereotyped; these responses are unrelated to exploration or novelty seeking. No alterations in extracellular DA levels or tissue contents from several brain regions are detected at the time of stereotypic activation of KO mice. By contrast, this behavior is accompanied by changes in serotonin metabolism in basal ganglia. This feature may contribute to the behavioral inflexibility of KO mice in different experimental contexts. Collectively, these findings suggest that disruption of the Dat1 gene in mice leads to two different phenotypes; one related to anxiety-reducing and novelty seeking, while the other has some homology to disorders with a stereotypical-perseverative spectrum.