bluuebunny/crossref_metadata_2025_split huggingface dataset:
Analogs of the designer drug, 3,4‐methylenedioxymethcathinone (methylone), differentially affect monoamine transporters in rat brain
Authors: Baumann Michael Howard , Bukhari Mohammad Omar , Lehner Kurt Richard , Partilla John S 
Published: November 2023

The abuse of synthetic cathinone compounds, often sold as “bath salts” or “research chemicals”, is an emerging public health concern. 3,4‐Methylenedioxymethcathinone (methylone) is a popular synthetic cathinone that is now illegal, but new analogs are being marketed as replacements. The purpose of the present investigation was to examine the interaction of new methylone analogs with transporters for dopamine (DAT) and 5‐HT (SERT). β‐Keto‐N‐methylbenzodioxolylbutanamine (butylone) and β‐keto‐N‐methylbenzodioxolylpentanamine (pentylone) were the analogs studied. In vitro assays were carried out in rat brain synaptosomes to assess drug‐induced effects on transporter‐mediated uptake and release. In vivo microdialysis was carried out in the nucleus accumbens of conscious rats to assess drug‐induced changes in extracellular dopamine and 5‐HT. Methylone, butylone and pentylone were fully efficacious uptake blockers at DAT and SERT, but pentylone was more DAT‐selective than the other drugs. Methylone was a substrate‐type releaser at both transporters, while butylone evoked release at SERT but not at DAT. Pentylone failed to evoke release at either transporter. Administration of butylone (1–3 mg/kg, i.v.) increased extracellular concentrations of 5‐HT more than dopamine, while pentylone (1–3 mg/kg. i.v.) increased both neurotransmitters to the same extent. All drugs produced significant locomotor activation, but the effects of butylone were blunted compared to pentylone. Our data show that each of the compounds examined displays a unique profile of in vitro transporter activity. Butylone has “hybrid” transporter effects, acting as a substrate at SERT but a blocker at DAT. Pentylone is a DAT‐preferring uptake blocker which lacks substrate activity. Overall, the data show that increasing the α‐carbon chain length of methylone creates compounds with reduced releasing activity, converting them to DAT blockers. All of the compounds elevate extracellular dopamine and stimulate motor activity to some degree, suggesting the drugs possess a significant risk for abuse.Support or Funding InformationThis research was generously supported by the Intramural Research Program of NIDA, NIH, DHHS.
The Effects of Chronic Cigarette Smoke Extract Exposure on Somatic Withdrawal
Authors: Reynaga Daisy , Leslie Frances , Belluzzi James 
Published: November 2023

Tobacco dependence is extremely difficult to treat and the vast majority of those who try to quit will relapse within the first year. Tobacco withdrawal is a major cause of relapse during the first few days of the quitting attempt. Adult rodent models of nicotine exposure show similar symptoms of withdrawal as humans. However, although human adolescents have an increased sensitivity to the effects of smoking cessation, rodent adolescents experience little to no somatic withdrawal symptoms after chronic nicotine exposure (DiFranza et al., 2007; O'Dell, 2009; Torres et al., 2013). This discrepancy may be due to the lack of other non‐nicotine smoke constituents in current animal models. We have shown that rats exposed to aqueous cigarette smoke extract (CSE), a solution containing nicotine and other cigarette smoke constituents, are more sensitive to stress‐induced reinstatement of self‐administration (Costello et al., 2014). The goal of this study is to determine whether adult and adolescents rats show enhanced spontaneous somatic withdrawal symptoms after chronic exposure to CSE, and to investigate the involvement of nicotinic acetylcholine receptors (nAChRs) in CSE cessation‐induced withdrawal using mecamylamine, a nonselective and noncompetitive nAChR antagonist, to precipitate withdrawal. To compare withdrawal effects, adult and adolescent rats were exposed to nicotine or CSE (1.5 mg nicotine/kg/day; i.v) for 10 days and analyzed for spontaneous or precipitated somatic withdrawal. We found that adult rats that received chronic CSE exposure showed enhanced spontaneous somatic withdrawal that emerged sooner than in animals that received chronic nicotine exposure. Adolescent rats showed significant spontaneous somatic withdrawal after chronic CSE exposure but not after nicotine. Mecamylamine (1 mg/kg; s.c.) administration precipitated withdrawal after chronic CSE exposure in both adult and adolescent rats. This provides evidence that non‐nicotine constituents in CSE mediate withdrawal processes, possibly via sensitization of nAChRs.Support or Funding InformationTobacco Related Disease Research Program (TRDRP): 21RT‐0136National Institute on Drug Abuse (NIDA): 1RO1DA040440‐01
A Novel Structural Landscape for Ligand Binding to the α7 Nicotinic Acetylcholine Receptor
Authors: Camacho‐Bustamante Gisela Andrea , Kaczanowska Kasia , Harel Michal , Cornejo‐Bravo Jose , Taylor Palmer 
Published: November 2023

In our previous study we identified a series of 4,6‐disubstituted 2‐aminopyrimidines interacting with Acetylcholine Binding Proteins (AChBP) in a cooperative fashion [Kaczanowska et al., Proc. Natl. Acad. Sci. USA 111, 10749 (2014)]. Based on X‐ray crystal structures of AChBP in complex with the cooperative ligands, we designed a new subset of these molecules that target the α7‐nicotinic acetylcholine receptor (nAChR). Seventeen compounds corresponding to this design were synthesized and characterized in cell based functional assays and radioligand binding assay using HEK cell lines overexpressing transfected cDNAs of human α7‐nAChRs, α4β2‐nAChRs and mouse 5HT3A receptors and Lymnaea (Ls) AChBP, respectively. We found a subset of structures that show Kd values in radioligand binding for AChBP between 51–68 nM and elicited responses for α7‐nAChRs in cell based assays (EC50's) as low as 60–70 nM, reflecting high affinity for α7‐nAChRs. Differential selectivity was characterized using α4β2‐nAChRs and 5HT3A receptors as references. Selected Ls‐AChBP and α7‐nAChRs ligands (AC‐171A, AC‐171A2, AC‐171C, AC‐171D) were then crystallized with the Ls‐AChBP revealing a unique binding pose at the five subunit interfaces. These 4,6‐disubsituted 2‐aminopyrimidines may hold potential in treatment of various CNS developmental disorders. For example, in schizophrenia nicotine addiction and resistance to abstinence prevails and may be modulated by these α7‐nAChRs ligands.Support or Funding InformationSupported by NIH grant GM18360 and the Tobacco‐related Disease Research Program (TRDRP).
Pharmacology of a novel TRPM2 antagonist, JNJ‐28583113
Authors: Fourgeaud Lawrence , Faouzi Malika , Starkus John , Wang Qi , Coates Heather , He Yingbo , Lovenberg Timothy , Penner Reinhold , Dvorak Curt , Bhattacharya Anindya 
Published: November 2023

TRPM2 is a calcium‐permeable non‐selective cation channel that is activated, in a synergistic manner, by both intracellular adenosine diphosphate ribose (ADPR) and calcium. It is also activated indirectly by reactive oxygen nitrogen species (ROS/NOS) due to increased ADPR production during oxidative/nitrosative stress. TRPM2 is primarily expressed in the immune cells and central nervous system (CNS) where it plays important roles in pathologies involving neuroimmune interactions, particularly in the context of oxidative stress. Within the CNS, TRPM2 is expressed mainly in neurons and microglia. Here, we present the pharmacological properties of JNJ‐28583113, a newly discovered compound representing the most potent TRPM2 antagonist described to date. Our data show that this compound has a potency range of 100–200 nM in blocking H2O2‐induced calcium flux in several recombinant systems over‐expressing human, chimpanzee and rat TRPM2. In whole‐cell patch clamp recordings, JNJ‐285831113 blocked intracellular ADPR‐induced human TRPM2 currents with a potency of 13 ± 3 nM. Similar pharmacology was observed for native TRPM2 in a human macrophage cell line (U937) and in a rat pancreatic cell line (INS‐1). In line with TRPM2 microglial expression, JNJ‐28583113 also modulated lipopolysaccharide (LPS)‐induced changes gliotransmitter secretion in mouse microglia in vitro, indicating a possible role of this channel in neuroinflammation. Using multi‐electrode arrays to record hippocampal field potentials in acute mouse brain slices, we observed that JNJ‐285831113 prevented hypoxia‐induced synaptic depression at CA3 to CA1 synapses, suggesting a role for TRPM2 in synaptic changes occurring during oxidative stress. Finally, JNJ‐28583113 inhibited H2O2‐induced dephosphorylation of glycogen synthase kinase‐3 (GSK3) alpha and beta in recombinant cells over‐expressing human TRPM2 channels, confirming the importance of TRPM2 in modulating GSK3 signaling. Together our results demonstrate that JNJ‐28583113 is a very potent TRPM2 antagonist that can be used to further elucidate TRPM2 physiological functions as well as to probe the role of TRPM2 in disease biology under conditions of oxidative stress.Support or Funding InformationJanssen
The extent of vascular remodeling is dependent on the balance between ERα and GPER(GPR30)
Authors: Gros Robert , Ding Qingming , Hussain Yasin , Chorazyczewski Jozef , Pickering Geoffery , Feldman Ross D 
Published: November 2023

Estrogens are important physiological and pathophysiological regulators of cardiovascular function. The traditional view of the mechanism for these cardiovascular effects has focused on the activation of “classical” steroid receptors (i.e., estrogen receptors‐ERs). However, recent studies have elucidated the mechanism of estrogen's cardiovascular effects mediated by an alternate GPCR‐based mechanism viz., via GPER (aka GPR30), formerly characterized as an “orphan receptor”. We are beginning to appreciate that to understand the effect of estradiol in cardiovascular regulation one must understand the balance between GPER‐ and ER‐mediated effects. In vascular smooth muscle cells, estrogen‐mediated regulation of apoptotic cell death and proliferation is divergently regulated by activation of ER vs. GPER. However, the significance of this divergence in the in vivo regulation of vascular growth processes was unknown. To determine the role of GPER in regulating vascular remodeling we studied the effects of altering GPER expression vs. altering the expression of ERα in a rat model of carotid ligation. Under baseline conditions, 1 week following carotid ligation and endothelial disruption with distilled water installation there was a 51±7.6% (n=3) increase in medial thickness, paralleling neointimal proliferation in female rats. This medial hypertrophy paralleled down‐regulation of GPER protein content (26±5% of control; n=3) and mRNA expression (29±13% of control; n=3). Re‐introduction of GPER, via abluminal adenoviral delivery, significantly attenuated the extent of medial hypertrophy (adeno‐GPER: 28±2.2% increase; n=10 vs. adeno‐GFP/control: 48±3.3% increase; n=16, p<0.05). Similarly, inhibition of ERα expression using an adeno‐shERαRNA construct resulted in an attenuation of injury‐mediated medial hypertrophy. In these studies we show that in vivo the balance between GPER and ERα is a significant regulator in the extent of vascular remodeling. Receptor‐specific modulation of estrogen's growth regulatory effects may be an important new approach in modifying the extent of vascular remodeling in both acute settings like vascular injury and perhaps in longer term models of regulation like in hypertension.Support or Funding InformationThese studies were supported by grants from the Heart and Stroke Foundation of Canada (RG and RDF).
G‐protein Coupled Receptor Kinase 4 gamma (GRK4γ) Mediated Activation of NF‐κB
Authors: Kanassatega Rhye‐Samuel , Andresen Bradley T 
Published: November 2023

Polymorphisms within GRK4γ strongly associate with hypertension, and recently A142V GRK4γ was implicated in NF‐κB‐mediated expression of the angiotensin type 1 receptor (AT1R). Luciferase assays driven by the 2x κB pGL3 vector, nuclear fractionation, and pull‐down assays were utilized to investigate the mechanism via which wild‐type (wt) GRK4γ and single nucleotide polymorphisms (SNPs) R65L, A142V, and A486V activate NF‐κB. Luciferase assays were conducted in growth media and serum‐free media. Only in growth media did the SNPs increase NF‐κB activity; wtGRK4γ decreased NF‐κB activity compared to the vector control. Utilizing kinase‐dead mutants of GRK4γ demonstrated that only A142V GRK4γ‐mediated NF‐κB activity is dependent on the kinase activity. The luciferase assays demonstrate that GRK4γ SNPs act as an amplifier, but do not constitutively activate NF‐κB. Nuclear fraction confirmed the luciferase data and demonstrated that RelB is the NF‐κB isoform activated through the GRK4γ pathway. Pull‐down affinity assays with S‐tagged wtGRK4γ and GRK4γ SNPs showed significantly increased interaction between SNP GRK4γ and NEMO (IKKγ). No interaction between the other IKK isoforms (α, β, ɛ) and wtGRK4γ or GRK4γ SNPs was detected. Currently, the IκBs are being examined to complete the NF‐κB‐mediated signaling pathway that is activated by SNPs of GRK4γ. Collectively, the data indicates that polymorphic GRK4γ amplifies serum‐mediated activation of the canonical NF‐κB pathway through a kinase‐mediated (A142V) and protein‐protein interaction‐mediated (R65L & A486V) mechanism. Further elucidation of these mechanisms may allow for the development of drugs that block polymorphic GRK4γ‐mediated NF‐κB activity and consequently reduce AT1R expression and thus blood pressure.Support or Funding InformationThis work was funded from Internal funds from Western University of Health Sciences and American Indian Graduate Center STEM Student Fellowship.
Tankyrases Are Druggable Targets for the Treatment of Metabolic Disorders
Authors: Obianom Obinna , Yang Wei , Xue Fengtian , Shu Yan 
Published: November 2023

PurposeTankyrases (TNKS) catalyze the poly‐ADP‐ribosylation of axin, targeting it for ubiquitination and proteasomal degradation. Along with adenomatous polyposis coli (APC), glycogen synthase kinase 3 (GSK3) and caseine kinase 1α (CK1α), axin makes up the β‐catenin destruction complex that promotes the degradation of β‐catenin, a critical effector in the canonical WNT signaling pathway that has been recently recognized to play a role in glucose homeostasis and the pathogenesis of metabolic disorders. This study sought to understand the relevance of TNKS1/2 inhibition and subsequent axin stabilization for the treatment of metabolic disorders.MethodsWe investigated the effect of a TNKS1/2 inhibitor, XAV939, on WNT signaling and gluconeogenesis pathway. TNKS1/2 inhibition leading to axin stabilization was measured by repression of WNT signaling pathway using dual luciferase assay. Western blots and quantitative PCR analysis were employed for the assessment of gene expression. Glucose production assay was used to measure direct effect on gluconeogenesis pathway.ResultsCell based dual‐luciferase assay in HEK293 cells transiently transfected with a TCF/LEF‐responsive reporter showed potent inhibition (IC50 = 34 nM) of Wnt signaling as a result of TNKS1/2 inhibition by XAV939. Western blot analysis with lysates from XAV939 treated hepG2 cells confirmed increase in phospho‐β‐catenin and axin1 protein levels. More so, mRNA and protein expression of the Wnt signaling targets c‐Myc and cyclin D1 were repressed. In the gluconeogenesis pathway, gene expression of glucose 6‐phosphatase was significantly decreased by XAV939, the anti‐diabetic metformin, and a hybrid drug containing both metformin and XAV939 (YW1083). Glucose production assays using hepG2 and mouse primary hepatocytes showed that XAV939 was able to decrease glucose production even more potently than metformin.ConclusionThis study adds to the meager scientific information that stabilization of axin via inhibition of tankyrase1/2 is valuable in combating metabolic disorders such as type 2 diabetes.Support or Funding Informationthe National Institute of General Medical Sciences of the National Institutes of Health (NIH) under Award R01GM099742
The Effect of PHLPP2 Removal on Cardiac Hypertrophy
Authors: Purcell Nicole , Yeh Szu‐Tsen , Koch Walter J , Zambrano Cristina M 
Published: November 2023

Crucial cellular events such as death, growth, metabolism, proliferation, and hypertrophy are regulated by phosphorylation and dephosphorylation of proteins. PH domain leucine‐rich repeat protein phosphatase (PHLPP) is a serine/threonine phosphatase that has been shown to directly dephosphorylate several members of the AGC family of kinases. Preliminary data suggests that removal of PHLPP2 in neonatal rat ventricular myocytes (NRVMs) activates fetal gene re‐expression at baseline and potentiates phenylephrine (PE) induced gene expression 2 fold over siControl. Since we have previously published that removal of PHLPP2 in NRVMs does not affect Akt phosphorylation as previously reported in other cells, we hypothesize that PHLPP2 must have the ability to repress cellular hypertrophy through the dephosphorylation of other unknown targets. G‐protein coupled receptor kinase 5 (GRK5), an AGC kinase, has been shown to regulate cardiac hypertrophy through de‐repression of gene transcription and by directly binding DNA. Upon stimulation of NRVMs with the hypertrophic agonist phenylephrine (PE), GRK5 translocates to the nucleus to regulate hypertrophic gene expression. We wanted to determine whether PHLPP2 directly affects GRK5 phosphorylation and localization to augment cardiomyocyte hypertrophy. We found that removal of PHLPP2 increased GRK5 translocation to the nucleus at baseline and following PE treatment. Conversely, overexpression of PHLPP2 blocked nuclear translocation of GRK5 following PE treatment. Immunoprecipitation experiments confirm an interaction between PHLPP2 and GRK5 and removal of PHLPP2 increased phosphorylation of GRK5 at baseline and following PE treatment. Ongoing studies using GRK5 phosphorylation mutants will determine whether the phosphatase activity of PHLPP2 is necessary for the inhibition of GRK5 translocation. Our preliminary data is the first to uncover GRK5 as a novel PHLPP2 target in cardiomyocytes. Since little is known about the non‐canonical regulation of GRK5, understanding whether phosphorylation and localization is regulated within the cardiomyocyte by PHLPP has potential for new therapeutic targets in the treatment of cardiac hypertrophy and failure.Support or Funding InformationThis work is supported by the National Heart, Lung and Blood Institute grant HL114949
Functions of Human RGS2 Missense Mutations in Hypertension
Authors: Phan Hoa , SjÖgren Benita , Neubig Richard 
Published: November 2023

RGS2 attenuates Gq/11 a signaling which mediates the action of a majority of vasoconstrictors. The association between RGS2 deficiency and hypertension has been demonstrated by genetic knock‐out mouse models and RGS2 mutations identified in Japanese hypertensive patients. Though biochemical functions of RGS2 are well characterized, the actual role of RGS2 in human hypertension is not known. 1.6% of individuals from the NHLBI exome sequencing project have missense mutations in RGS2, however, the functional significance of these mutations is unknown. The objective of this study is to examine the effect of human RGS2 missense mutations on protein expression levels, subcellular localization and how they differ from wild‐type RGS2 in their ability to regulate GPCR‐mediated signaling. Wild‐type and 14 RGS2 mutants were investigated by transient transfection in HEK293T and CHO cells. RGS2A4V showed reduced protein expression (30% of WT, n=10, p<0.01) and impaired inhibition of AT1R‐mediated calcium release (57% of WT vs 23% of A4V, n=5, p<0.01). In contrast, RGS2Q2L showed reduced expression (40% of WT, n=10, p<0.05) but did not exhibit a significant loss of function (57% of WT vs 51% of Q2L, n=5). In this functional model, RGS2R44H did not exhibit loss of function as previously reported. Preliminary data on other mutants suggesting functional deficits are currently being assessed as are potential differences in actions of RGS2 mutants on different GPCRs. Together, these studies will provide a molecular understanding of a number of human RGS2 mutant alleles in vitro. This will identify key candidate alleles for further study as contributors to human hypertension.Support or Funding InformationSupported by AHA predoctoral fellowship to H.P. and NIH R01GM110195 to R.R.N.
Using NMR‐Based Fragment Screening to Probe the Druggability of Regulator of G Protein Signaling 17
Authors: Hayes Michael Patrick , Fowler C Andrew , Yu Liping , Roman David L 
Published: November 2023

Regulator of G protein Signaling 17 (RGS17), as a GTPase accelerating protein (GAP), temporally controls G protein‐coupled receptor activity by accelerating the intrinsic rate of GTP hydrolysis of inhibitory Gα subunits, thus terminating signaling. Recent evidence has shown that RGS17 expression is drastically increased in malignancies in various tissues, including lung, breast, prostate, and liver, and inhibition of RGS17 via RNA interference is sufficient to slow cancer growth and progression. This makes RGS17 an attractive target for the development of small molecule inhibitors that could serve as chemotherapeutics with a novel mechanism of action. In order to identify novel druggable sites on the RGS homology domain of RGS17, we performed triple resonance NMR experiments using 15N, 13C doubly labeled protein samples to assign the protein. We then screened a library of 1000 fragments and assessed compound binding via chemical shift perturbation in protein‐detected 1H‐15N HSQC spectra. Seven hits were identified with affinities ranging from 530 μM to 5.2 mM and ligand efficiencies from 0.18 to 0.31. Interestingly, none of these identified hits bound directly to the region of RGS17 that forms the Gα interaction interface. Six fragments bound between α6 and α7 in the helical bundle domain, a site distant from the Gα interface. Additionally, one hit, an isothiazaolone, interacts with RGS17 in a covalent manner, showing that RGS17, like other RGS family members RGS4 and RGS8, is subject to inhibition via cysteine‐modification. Subsequent experimental and computational efforts will focus on structure‐activity relationship optimization of these hits to increase their affinities for RGS17 to further explore the druggabiliy of this allosteric site, as well as to facilitate the determination of the structure of the RGS17:fragment complex with atomic resolution.Support or Funding InformationThis work was supported by NIH 5R01CA160470 (DLR) and an American Foundation for Pharmaceutical Education Predoctoral Fellowship (MPH).