Mutagenesis Analysis Reveals Distinct Amino Acids of the Human Serotonin 5‑HT_2C Receptor Underlying the Pharmacology of Distinct Ligands

While exploring the structure–activity relationship of 4-phenyl-2-dimethylaminotetralins (PATs) at serotonin 5-HT_2C receptors, we discovered that relatively minor modification of PAT chemistry impacts function at 5-HT_2C receptors. In HEK293 cells expressing human 5-HT_2C‑INI receptors, for example, (−)-<i>trans</i>-3′-Br-PAT and (−)-<i>trans</i>-3′-Cl-PAT are agonists regarding Gα_q-inositol phosphate signaling, whereas (−)-<i>trans</i>-3′-CF₃-PAT is an inverse agonist. To investigate the ligand–receptor interactions that govern this change in function, we performed site-directed mutagenesis of 14 amino acids of the 5-HT_2C receptor based on molecular modeling and reported G protein-coupled receptor crystal structures, followed by molecular pharmacology studies. We found that S3.36, T3.37, and F5.47 in the orthosteric binding pocket are critical for affinity (<i>K</i>_i) of all PATs tested, we also found that F6.44, M6.47, C7.45, and S7.46 are primarily involved in regulating EC/IC₅₀ functional potencies of PATs. We discovered that when residue S5.43, N6.55, or both are mutated to alanine, (−)-<i>trans</i>-3′-CF₃-PAT switches from inverse agonist to agonist function, and when N6.55 is mutated to leucine, (−)-<i>trans</i>-3′-Br-PAT switches from agonist to inverse agonist function. Notably, most point-mutations that affected PAT pharmacology did not significantly alter affinity (<i>K</i>_D) of the antagonist radioligand [³H]­mesulergine, but every mutation tested negatively impacted serotonin binding. Also, amino acid mutations differentially affected the pharmacology of other commercially available 5-HT_2C ligands tested. Collectively, the data show that functional outcomes shared by different ligands are mediated by different amino acids and that some 5-HT_2C receptor residues important for pharmacology of one ligand are not necessarily important for another ligand

Sensitive and Label-Free Fluorescent Detection of Transcription Factors Based on DNA-Ag Nanoclusters Molecular Beacons and Exonuclease III-Assisted Signal Amplification

Transcription factors (TFs) regulate gene expression by binding to regulatory regions, and their dysregulation is involved in numerous diseases. Thus, they are therapeutic targets and potential diagnostic markers. However, widely used methods for TFs detection are either cumbersome or costly. Herein, we first applied DNA-Ag nanoclusters molecular beacons (AgMBs) in TFs analysis and designed an assay based on the switchable fluorescence of AgMBs. In the absence of TFs, a single-stranded DNA functioned as a reporter is released from a double-stranded DNA probe (referred as dsTFs probe) under exonuclease III (Exo III) digestion. Then, the reporter triggers downstream Exo III-assisted signal amplification by continuously consuming the guanine-rich enhancer sequences in AgMBs, resulting in significant fluorescent decrease eventually. Conversely, the presence of TFs protects the dsTFs probe from digestion and blocks the downstream reaction to keep a highly fluorescent state. To testify this rationale, we utilized nuclear factor-kappa B p50 (NF-κB p50) as a model TFs. Owing to the amplification strategy, this method exhibited high sensitivity toward NF-κB p50 with a limit of detection of 10 pM, and a broad linear range from 30 pM to 1.5 nM. Furthermore, this method could detect multiple TFs in human colon cancer DLD-1 cells and reflect the variation in their cellular levels after stimulation. Finally, by conducting an inhibition assay we revealed the potential of this method for screening TFs-targeted drugs and calculating the IC₅₀ of corresponding inhibitors

An Orally Active Phenylaminotetralin-Chemotype Serotonin 5‑HT₇ and 5‑HT_1A 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-HT_1A partial agonist with relevant affinity at 5-HT₇ receptors. Herein, we report the synthesis, <i>in vitro</i> molecular pharmacology, behavioral pharmacology, and pharmacokinetic parameters in mice after subcutaneous and oral administration of (+)-5-(2′-fluorophenyl)-<i>N</i>,<i>N</i>-dimethyl-1,2,3,4-tetrahydronaphthalen-2-amine ((+)-5-FPT), a new, dual partial agonist targeting both 5-HT₇ (<i>K</i>_i = 5.8 nM, EC₅₀ = 34 nM) and 5-HT_1A (<i>K</i>_i = 22 nM, EC₅₀ = 40 nM) receptors. Three unique, heterogeneous mouse models were used to assess the efficacy of (<i>+</i>)-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 (<i>+</i>)-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 (<i>+</i>)-5-FPT is a promising medication for treating stereotypy in psychiatric disorders

COVID-19 Host Genetics Initiative. A first update on mapping the human genetic architecture of COVID-19

The COVID-19 pandemic continues to pose a major public health threat, especially in countries with low vaccination rates. To better understand the biological underpinnings of SARS-CoV-2 infection and COVID-19 severity, we formed the COVID-19 Host Genetics Initiative1. Here we present a genome-wide association study meta-analysis of up to 125,584 cases and over 2.5 million control individuals across 60 studies from 25 countries, adding 11 genome-wide significant loci compared with those previously identified2. Genes at new loci, including SFTPD, MUC5B and ACE2, reveal compelling insights regarding disease susceptibility and severity.</p