4 research outputs found
Mutagenesis Analysis Reveals Distinct Amino Acids of the Human Serotonin 5‑HT<sub>2C</sub> Receptor Underlying the Pharmacology of Distinct Ligands
While exploring the structure–activity
relationship of 4-phenyl-2-dimethylaminotetralins
(PATs) at serotonin 5-HT<sub>2C</sub> receptors, we discovered that
relatively minor modification of PAT chemistry impacts function at
5-HT<sub>2C</sub> receptors. In HEK293 cells expressing human 5-HT<sub>2C‑INI</sub> receptors, for example, (−)-<i>trans</i>-3′-Br-PAT and (−)-<i>trans</i>-3′-Cl-PAT
are agonists regarding Gα<sub>q</sub>-inositol phosphate signaling,
whereas (−)-<i>trans</i>-3′-CF<sub>3</sub>-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<sub>2C</sub> 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><sub>i</sub>) of all PATs tested,
we also found that F6.44, M6.47, C7.45, and S7.46 are primarily involved
in regulating EC/IC<sub>50</sub> functional potencies of PATs. We
discovered that when residue S5.43, N6.55, or both are mutated to
alanine, (−)-<i>trans</i>-3′-CF<sub>3</sub>-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><sub>D</sub>) of the antagonist radioligand [<sup>3</sup>H]Âmesulergine, but every mutation tested negatively impacted
serotonin binding. Also, amino acid mutations differentially affected
the pharmacology of other commercially available 5-HT<sub>2C</sub> ligands tested. Collectively, the data show that functional outcomes
shared by different ligands are mediated by different amino acids
and that some 5-HT<sub>2C</sub> 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 IC<sub>50</sub> of corresponding
inhibitors
An Orally Active Phenylaminotetralin-Chemotype Serotonin 5‑HT<sub>7</sub> and 5‑HT<sub>1A</sub> 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-HT<sub>7</sub> 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<sub>1A</sub> partial agonist with
relevant affinity at 5-HT<sub>7</sub> 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-HT<sub>7</sub> (<i>K</i><sub>i</sub> = 5.8 nM, EC<sub>50</sub> = 34 nM)
and 5-HT<sub>1A</sub> (<i>K</i><sub>i</sub> = 22 nM, EC<sub>50</sub> = 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-HT<sub>2</sub> 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