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.

There are no approved medicines for fragile X syndrome (FXS), a monogenic, neurodevelopmental disorder. Electroencephalogram (EEG) studies show alterations in restingstate cortical EEG spectra, such as increased gamma-band power, in patients with FXS that are also observed in Fmr1 knockout models of FXS, offering putative biomarkers for drug discovery. Genes encoding serotonin receptors (5-HTRs), including 5-HT 1A , 5-HT 1B , and 5-HT 1D Rs, are differentially expressed in FXS, providing a rationale for investigating them as pharmacotherapeutic targets. Previously we reported pharmacological activity and preclinical neurotherapeutic effects in Fmr1 knockout mice of an orally active 2-aminotetralin, (S)-5-(2′-fluorophenyl)-N,Ndimethyl-1,2,3,4-tetrahydronaphthalen-2-amine (FPT). FPT is a potent (low nM), high-efficacy partial agonist at 5-HT 1A Rs and a potent, low-efficacy partial agonist at 5-HT 7 Rs. Here we report new observations that FPT also has potent and efficacious agonist activity at human 5-HT 1B and 5-HT 1D Rs. FPT's K i values at 5-HT 1B and 5-HT 1D Rs were <5 nM, but it had nil activity (>10 μM K i ) at 5-HT 1F Rs. We tested the effects of FPT (5.6 mg/kg, subcutaneous) on EEG recorded above the somatosensory and auditory cortices in freely moving, adult Fmr1 knockout and control mice. Consistent with previous reports, we observed significantly increased relative gamma power in untreated or vehicle-treated male and female Fmr1 knockout mice from recordings above the left somatosensory cortex (LSSC). In addition, we observed sex effects on EEG power. FPT did not eliminate the genotype difference in relative gamma power from the LSSC. FPT, however, robustly decreased relative alpha power in the LSSC and auditory cortex, with more pronounced effects in Fmr1 KO mice. Similarly, FPT decreased relative alpha power in the right SSC but only in Fmr1 knockout mice. FPT also increased relative delta power, with more pronounced effects in Fmr1 KO mice and caused small but significant increases in relative beta power. Distinct impacts of FPT on cortical EEG were like effects caused by certain FDA-approved psychotropic medications (including baclofen, allopregnanolone, and clozapine). These results advance the understanding of FPT's pharmacological and neurophysiological effects.

Pharmacological magnetic resonance imaging (phMRI) is a noninvasive method used to evaluate neural circuitry involved in the behavioral effects of drugs like ketamine, independent of their specific biochemical mechanism. The study was designed to evaluate the immediate effect of esketamine, the S‐isomer of (±) ketamine on brain activity in awake mice using blood oxygenation level dependent (BOLD) imaging. It was hypothesized the prefrontal cortex, hippocampus, and brain areas associated with reward and motivation would show a dose‐dependent increase in brain activity. Mice were given vehicle, 1.0, 3.3, or 10 mg/kg esketamine I.P. and imaged for 10 min post‐treatment. Data for each treatment were registered to a 3D MRI mouse brain atlas providing site‐specific information on 134 different brain areas. There was a global change in brain activity for both positive and negative BOLD signal affecting over 50 brain areas. Many areas showed a dose‐dependent decrease in positive BOLD signal, for example, cortex, hippocampus, and thalamus. The most common profile when comparing the three doses was a U‐shape with the 3.3 dose having the lowest change in signal. At 1.0 mg/kg there was a significant increase in positive BOLD in forebrain areas and hippocampus. The anticipated dose‐dependent increase in BOLD was not realized; instead, the lowest dose of 1.0 mg/kg had the greatest effect on brain activity. The prefrontal cortex and hippocampus were significantly activated corroborating previous imaging studies in humans and animals. The unexpected sensitivity to the 1.0 mg/kg dose of esketamine could be explained by imaging in fully awake mice without the confound of anesthesia and/or its greater affinity for the N‐methyl‐d‐aspartate receptor (NMDAR) receptor than (±) ketamine.

Fragile X syndrome (FXS)—caused by FMR1 gene silencing—is a severe neurodevelopmental disorder characterized by intellectual disabilities that are often comorbid with seizures, sensory hypersensitivities, anxiety, social deficits, and repetitive behaviors. Neuronal hyperexcitability is an overarching neurophysiological characteristic of FXS that may underlie FXS symptoms. About 33% of Fmr1 KO mice from our colony exhibit spontaneous seizures, a newly observed phenotype that more closely parallels seizures in FXS, compared to the audiogenic seizure phenotype in Fmr1 KO mice. In addition, we and others show that Fmr1 KO mice, like individuals with FXS, have cortical EEG gamma‐band power alterations, and at the single‐cell level, have hyperexcitable pyramidal neurons in multiple brain regions. We are using a combinatorial approach—from behavioral to EEG to single‐cell experiments—to advance FXS drug discovery. Based on our observations of altered brain expression and in vivo function of serotonin 1A receptors (5‐HT1ARs) in Fmr1 KO mice and correction of the audiogenic seizure phenotype by our novel 2‐aminotetralin‐type 5‐HT1R modulator, FPT, we are testing the hypothesis that selectively activating 5‐HT1ARs prevents seizures and corrects neurophysiological abnormalities. We evaluated the efficacy of FPT (5.6 mg/kg), a potent and efficacious 5‐HT1AR agonist, to correct EEG abnormalities in Fmr1 KO mice. We also tested the antiepileptic effects of the selective 5‐HT1AR agonist, NLX‐112 (0.25‐2.5 mg/kg), and are currently testing the effects of FPT and NLX‐112 on CA1 pyramidal neuron hyperexcitability in Fmr1 KO mice. In parallel experiments, we are evaluating the pharmacology of FPT and NLX‐112 at each of the 5‐HT G protein‐coupled receptors. Recordings from above the left somatosensory cortex showed a significantly elevated high gamma (65‐100 Hz) power ratio in Fmr1 KO mice relative to control mice at baseline (n=16, P=0.0357) and after vehicle injection (n=16, P=0.0066), a genotype difference that FPT eliminated (n=15‐16, P=0.6279). Comparisons between baseline and first injection conditions also revealed an increased delta power in Fmr1 KO mice relative to controls. Separately, NLX‐112 prevented audiogenic seizures in Fmr1 KO mice (n=10‐12, P≤0.0002), and preliminary data suggest NLX‐112 and FPT modulate CA1 pyramidal neuron activity. For example, FPT (10 µM) showed a reversible reduction of firing frequency of hippocampal CA1 neurons in Fmr1 KO mice (n=8, P<0.05). Forthcoming experiments will include evaluating the effects of NLX‐112 on cortical EEG activity in Fmr1 KO and control mice and the effects of chronic NLX‐112 and FPT on spontaneous seizures in Fmr1 KO mice. Tests of the selective 5‐HT1AR antagonist, WAY100635, will be conducted to examine a 5‐HT1AR mechanism underlying positive outcomes of NLX‐112 and FPT. At present, our convergent data suggest that 5‐HT1AR activation may ameliorate neuronal hyperexcitability, at multiple levels of analysis, in Fmr1 KO mice. Potent and selective 5‐HT1AR agonists might ...