Fragile X syndrome (FXS) is a neurodevelopmental disorder characterized by intellectual disabilities and a plethora of neuropsychiatric symptoms. FXS is the leading monogenic cause of autism spectrum disorder (ASD), which is defined clinically by repetitive and/or restrictive patterns of behavior and social communication deficits. Epilepsy and anxiety are also common in FXS and ASD. Serotonergic neurons directly innervate and modulate the activity of neurobiological circuits altered in both disorders, providing a rationale for investigating serotonin receptors (5-HTRs) as targets for FXS and ASD drug discovery. Previously we unveiled an orally active aminotetralin, (S)-5-(2'-fluorophenyl)-N,N-dimethyl-1,2,3,4-tetrahydronaphthalen-2-amine (FPT), that exhibits partial agonist activity at 5-HT1ARs, 5-HT2CRs, and 5-HT7Rs and that reduces repetitive behaviors and increases social approach behavior in wild-type mice. Here we report that in an Fmr1 knockout mouse model of FXS and ASD, FPT is prophylactic for audiogenic seizures. No FPT-treated mice displayed audiogenic seizures, compared to 73% of vehicle-treated mice. FPT also exhibits anxiolytic-like effects in several assays and increases social interactions in both Fmr1 knockout and wild-type mice. Furthermore, FPT increases c-Fos expression in the basolateral amygdala, which is a preclinical effect produced by anxiolytic medications. Receptor pharmacology assays show that FPT binds competitively and possesses rapid association and dissociation kinetics at 5-HT1ARs and 5-HT7Rs, yet has slow association and rapid dissociation kinetics at 5-HT2CRs. Finally, we reassessed and report FPT’s affinity and function at 5-HT1ARs, 5-HT2CRs, and 5-HT7Rs. Collectively, these observations provide mounting support for further development of FPT as a pharmacotherapy for common neuropsychiatric symptoms in FXS and ASD.

Background and Purpose The 5‐HT receptor subtypes 5‐HT2A and 5‐HT2C are important neurotherapeutic targets, though, obtaining selectivity over 5‐HT2B and H1 receptors is challenging. Here, we delineated molecular determinants of selective binding to 5‐HT2A and 5‐HT2C receptors for novel 4‐phenyl‐2‐dimethylaminotetralins (4‐PATs). Experimental Approach We synthesized 42 novel 4‐PATs with halogen or aryl moieties at the C(4)‐phenyl meta‐position. Affinity, function, molecular modeling and 5‐HT2A receptor mutagenesis studies were performed to understand structure–activity relationships at 5‐HT2‐type and H1 receptors. Lead 4‐PAT‐type 5‐HT2A/5‐HT2C receptor inverse agonists were compared with pimavanserin, a selective 5‐HT2A/5‐HT2C receptor inverse agonist approved to treat Parkinson's disease‐related psychosis, in the mouse head twitch response and locomotor activity assays, models relevant to antipsychotic drug development. Key Results Most 4‐PAT diastereomers in the (2S,4R)‐configuration bound non‐selectively to 5‐HT2A, 5‐HT2C and H1 receptors, with >100‐fold selectivity over 5‐HT2B receptors, whereas diastereomers in the (2R,4R)‐configuration bound preferentially to 5‐HT2A over 5‐HT2C receptors and had >100‐fold selectivity over 5‐HT2B and H1 receptors. Results suggest that G2385.42 and V2355.39 in 5‐HT2A receptors (conserved in 5‐HT2C receptors) are important for high affinity binding, whereas interactions with T1945.42 and W1584.56 determine H1 receptor affinity. The 4‐PAT analog (2S,4R)‐4‐(4'‐(dimethylamino)‐[1,1'‐biphenyl]‐3‐yl)‐N,N‐dimethyl‐1,2,3,4‐tetrahydronaphthalen‐2‐amine, (2S,4R)‐2k, a potent and selective 5‐HT2A/5‐HT2C receptor inverse agonist, had activity like pimavanserin in the mouse head twitch response assay but was distinct in not suppressing locomotor activity. Conclusions and Implications The novel 4‐PAT chemotype can yield selective 5‐HT2A/5‐HT2C receptor inverse agonists for antipsychotic drug development by optimizing ligand–receptor interactions in transmembrane domain 5. Chirality can be exploited to attain selectivity over H1 receptors, which may circumvent sedative effects.

Many important physiological processes are mediated by alpha2A-and alpha2C-adrenergic receptors (α2Rs), a subtype of class A G protein-coupled receptors (GPCRs). However, α2R signaling is poorly understood, and there are few approved medications targeting these receptors. Drug discovery aimed at α2Rs is complicated by the high degree of binding pocket homology between α2AR and α2CR, which confounds ligandmediated selective activation or inactivation of signaling associated with a particular subtype. Meanwhile, α2R signaling is complex and it is reported that activating α2AR is beneficial in many clinical contexts, while activating α2CR signaling may be detrimental to these positive effects. Here, we report on a novel 5-substituted-2aminotetralin (5-SAT) chemotype that, depending on substitution, has diverse pharmacological activities at α2Rs. Certain lead 5-SAT analogues act as partial agonists at α2ARs, while functioning as inverse agonists at α2CRs, a novel pharmacological profile. Leads demonstrate high potency (e.g., EC 50 < 2 nM) at the α2AR and α2CRs regarding Gα i -mediated inhibition of adenylyl cyclase and production of cyclic adenosine monophosphate (cAMP). To help understand the molecular basis of 5-SAT α2R multifaceted functional activity, α2AR and α2CR molecular models were built from the crystal structures and 1 μs molecular dynamics (MD) simulations and molecular docking experiments were performed for a lead 5-SAT with α2AR agonist and α2CR inverse agonist activity, i.e., (2S)-5-(2′-fluorophenyl)-N,N-dimethyl-1,2,3,4-tetrahydronaphthalen-2-amine (FPT), in comparison to the FDA-approved (for opioid withdrawal symptoms) α2AR/α2CR agonist lofexidine. Results reveal several interactions between FPT and α2AR and α2CR amino acids that may impact the functional activity. The computational data in conjunction with experimental in vitro affinity and function results provide information to understand ligand stabilization of functionally distinct GPCR conformations regarding α2AR and α2CRs.

Fragile X Syndrome (FXS) is the leading monogenetic cause of intellectual disabilities and autism. Seizures, severe anxiety, and sensory hypersensitivity are also common in FXS. The central serotonin system can modulate these symptoms, and certain serotonin receptors are functionally linked with proteins known to be altered in FXS, suggesting targeting them may be therapeutic for FXS. We are developing (S)‐5‐(2′‐Fluorophenyl)‐N, N‐dimethyl‐1,2,3,4‐tetrahydronaphthalen‐2‐amine (FPT), a partial agonist at both 5‐HT7 and 5‐HT1A receptors, as a treatment for FXS and autism. We previously reported that FPT corrects repetitive behaviors and enhances social interactions—core symptom domains in FXS and autism—in wild‐type (WT) mouse models. Herein, we report the results of tests assessing the efficacy of FPT to improve behavioral phenotypes in Fmr1 knockout (KO) mice, an etiologically valid genetic model of FXS. Fmr1 KO mice have seizures when exposed to a 120 dB alarm (audiogenic seizures, AGS). 73% of vehicle‐treated Fmr1 KO mice we tested had AGS (males and females, age P23–25, N=22), however, 0% of the Fmr1 KO mice pretreated with FPT (5.6 mg/kg) had AGS (N=20). This effect was very significant (P<0.0001). In addition, FPT significantly reduced marble burying (vehicle, N=11, FPT, N=10, P<0.001) and reduced rearing in Fmr1 KO mice (vehicle, N=12, FPT, N=12, P<0.05). These data corroborate our earlier studies that FPT corrects repetitive behaviors and may have anxiolytic properties. We next tested FPT's efficacy to promote social behavior. Mice were matched for genotype and sex and paired together for a 10 minute observation period. One mouse was given vehicle and the other was given FPT. Social behavior, scored by an observer blind to treatment, was defined as the number of approaches from one mouse to the other and was recorded for each mouse within the pair. FPT increased social behavior in both WT (P<0.005) and Fmr1 KO (P<0.05) mice. We did not observe overt behavioral toxicity (for example, hypolocomotion, flat body posture, tremor, or head weaving) in mice treated with FPT. Our data provide further evidence that FPT has activity in neurobehavioral assays suggesting anxiolytic and prosocial effects that extend from WT to Fmr1 KO mice.Support or Funding InformationDOD W81XWH‐17‐1‐0329, FRAXA Research FoundationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.