bluuebunny/crossref_metadata_2025_split huggingface dataset:
Expression of Multiple Sulfotransferase (SULT) 1C4 Transcript Variants in Developing Human Liver
Authors: Dubaisi Sarah , Fang Hailin , Gaedigk Roger , Vyhlidal Carrie A. , Kocarek Thomas A. , Runge‐Morris Melissa 
Published: June 2021

The cytosolic sulfotransferases (SULTs) are conjugating enzymes that catalyze the transfer of a sulfonate group from the cofactor 3′‐phosphoadenosine‐5′‐phosphosulfate to a variety of endogenous and xenobiotic substrates. The SULT1C subfamily includes three human genes, SULT1C2, SULT1C3, and SULT1C4, and relatively little is known about their expression and regulation or the functions of the enzymes. Previous studies have indicated that the human SULT1Cs are preferentially expressed during early life and are capable of metabolizing endogenous molecules such as thyroid and estrogen hormones, suggesting that these enzymes could play physiological roles during development. Several transcript variants (TVs) of SULT1C4 are indexed in GenBank, including the full‐length mRNA containing seven exons (TV1, NM_006588), a variant mRNA lacking exons 3 and 4 (TV2, NM_001321770), two non‐coding RNA variants (TV3, NR_135776 and TV4, NR_135779), and a predicted transcript variant (TVX1, XM_017003807). Using 5′‐RACE, RT‐PCR, and DNA sequencing, we detected TV1 and TV2, as well as a variant lacking only exon 3 (E3DEL) in Caco‐2 colorectal adenocarcinoma cells, HepaRG hepatic cells, and human liver specimens. The aim of the current study was to evaluate expression of the SULT1C4 TVs more thoroughly by (1) characterizing expression of individual SULT1C4 TVs in human liver specimens from prenatal, infant, and adult donors and (2) determining whether the individual mRNA variants can be translated into proteins. Using RT‐qPCR assays designed to quantify TV1, TV2, or E3DEL separately, all three variants were found to be primarily expressed in prenatal liver samples. The TV2 transcript lacking two exons was more abundant than the full‐length TV1, while E3DEL levels were minimal. In agreement with the RT‐qPCR data, RNA‐seq analysis of prenatal and pediatric liver specimens showed that TV1 and TV2 were both more abundant during early developmental stages. The RNA‐seq data also demonstrated that a non‐coding transcript variant (ENST00000494122.1) consisting of 2 exons only was expressed in the liver specimens and was predominantly expressed in prenatal liver. TV1 and TV2, but not E3DEL, were detected by western blot when plasmids expressing DDK‐tagged variants were transfected into HEK‐293T cells. These results indicate that at least four transcript variants of SULT1C4 are expressed in human liver, primarily during prenatal life, although only one of these (TV1) would encode a functional SULT enzyme. The physiological significance of this transcript diversity is unclear and will require further investigation.Support or Funding InformationSupported by NIH grants R01 ES022606 and P30 ES020957This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Regulation of Hepatic UGT1A4 by Liver X Receptor LXRα, and not LXRβ in hUGT1 Mice
Authors: Rettenmeier Eva , Yoda Emiko , van der Schoor Lori , Chen Shujuan , Barbier Olivier , Tukey Robert 
Published: June 2021

UDP‐glucuronosyltransferases (UGTs) are metabolic enzymes catalyzing the transformation of multiple xeno‐ and endobiotics into their more water‐soluble glucuronides. Among the UGT enzymes, UGT1A4 is known for the N‐glucuronidation of amine containing xenobiotics, including anticonvulsants, antidepressants and environmental mutagens. To examine the function of UGT1A4 and other genes associated with the human UGT1 locus, we have implemented humanized UGT1 mice (hUGT1). In this study we identify UGT1A4 as a positively regulated target gene of the nuclear receptor liver X‐receptor (LXR). LXR is a member of the nuclear receptor superfamily. Two isoforms of LXR exist, LXRα and LXRβ. The treatment of neonatal hUGT1 mice with the conventional LXR agonist T0901317 led to transcriptional activation of downstream LXR target genes, including UGT1A1, UGT1A4, Scd1 and Scd2. Since neonatal hUGT1 mice develop extreme hyperbilirubinemia, the induction of intestinal and liver UGT1A1 led to a dramatic reduction in total serum bilirubin. Repeating the induction patterns in hUGT1/Lxrα−/− and hUGT1/LXRβ−/− mice, expression of Scd1 and Scd2 displayed specificity for LXRα. There was significant induction of these genes in hUGT1/Lxrβ−/− mice with limited activation in hUGT1/Lxrα−/− mice. In contrast, the induction of UGT1A1 and the UGT1A1 gene by T0901317 is regulated by both activated LXRα and LXRβ. However, like Scd1 and Scd2, induction of the UGT1A4 gene was selective towards LXRα, with greatly reduced expression in hUGT1/Lxrα−/− mice. Induction of the UGT1A4 gene was comparable in hUGT1/Lxrβ+/− and hUGT1/LXRβ−/− mice. Clearly, LXRα and LXRβ can display fine selectivity for downstream target gene activation, such as the UGT1A4 gene.Support or Funding InformationUSPHS grants GM086713 and ES010337, DFG (German Research Foundation)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Positive Allosteric Modulators of the D1 Dopamine Receptor Act at Diverse Binding Sites
Authors: Luderman Kathryn D. , Conroy Jennie L. , Free R. Benjamin , Southall Noel T. , Ferrer Marc , Aubé Jeffrey , Lane J. Robert , Frankowski Kevin , Sibley David R. 
Published: June 2021

The D1 dopamine receptor (D1R) and its signaling is associated with several important neurological processes and neuropsychiatric disorders. Enhancing D1R signaling in the prefrontal cortex is associated with increased cognition and therefore is an appealing treatment strategy for the cognitive decline observed in schizophrenia, Alzheimer's disease, and other disorders. Because of the clinical liability inherent with D1R agonists, positive allosteric modulators of the D1R have been proposed as an alternative, due to their potential for high selectivity and larger therapeutic windows. Currently, the location and characteristics of potential binding site(s) for allosteric modulators on the D1R is unknown. We have identified two structurally diverse D1R positive allosteric modulators, MLS1082 and MLS6585, via a high throughput screen of the NIH Molecular Libraries Program. The compounds potentiate dopamine‐stimulated G‐protein‐ (cAMP stimulation, 3–5 fold) and β‐arrestin‐mediated (6–8 fold) signaling pathways and increase the binding affinity of dopamine for the D1R (3–6 fold). Neither compound displayed agonist activity in the absence of dopamine. Taking advantage of our structurally distinct potentiators, G‐protein signaling and β‐arrestin recruitment experiments using maximally effective concentrations of MLS6585 and MLS1082 in combination were used to determine if the compounds act at similar or separate sites. In combination, MLS1082 + MLS6585 caused an additive potentiation of the potency of dopamine beyond that caused by either compound alone for both β‐arrestin recruitment (11–20 fold) and cAMP accumulation (4–6 fold). This suggests that the two compounds are acting at separate sites on the receptor. We also observed similar results using analogs of the two compounds, with analogs of MLS6585 having additive activity with MLS1082 and vice‐versa. However, analogs are not additive with their parent compound, meaning the parent compounds and their analogs act at similar sites. The combination experiments were repeated with Compound B, a known D1R positive allosteric modulator. Compound B was additive with MLS6585 but not MLS1082, further suggesting that the D1R has two separate positive allosteric modulator binding sites. Point mutation studies are currently underway to identify the location of these putative sites as well as whether these sites may contribute separately to potentiation of signaling efficacy versus dopamine binding affinity.Support or Funding InformationNINDS IRPThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identification of small molecule ligands targeting GPR83, a G‐protein coupled receptor activated by the abundant neuropeptide PEN
Authors: Lueptow Lindsay M. , Gomes Ivone , Gupta Achla , Sanchez Roberto , Devi Lakshmi 
Published: June 2021

The G‐protein coupled receptor GPR83 is expressed in numerous brain regions, including striatum and hypothalamus, as well as in the immune system, including the thymus and spleen, of mice. Previously, GPR83 was classified as an orphan GPCR with no known endogenous ligand; we identified the neuropeptide PEN, derived from the precursor ProSAAS, as a selective, high‐affinity endogenous ligand for GPR83. Previous studies using ProSAAS or GPR83 knockout models have shown both the neuropeptide and receptor to be important in modulation of stress and anxiety, as well as feeding and body weight regulation. Additionally, the expression of GPR83 on immune cells indicates a potential role in immune function. Therefore, development of small molecule ligands for GPR83 is necessary in order to further our understanding of the receptor in these biological processes. In order to identify potential agonists and antagonists, a homology model for GPR83, based on the crystal structure of phylogenetically related receptors, was generated and used to screen in silico with the eMolecules library, which consists of 7 million compounds. From these, the top fifty predicted hits were screened in a cell based, high‐throughput calcium‐release assay. The compounds that gave a large signal to noise ratio only in cells expressing GPR83 were further characterized using GTPγS, adenylyl cyclase, phospholipase C, and receptor trafficking assays. This led to the identification of CPD1 and CPD27as selective agonists. Those compounds that did not give an agonistic response were re‐screened in an antagonist mode (i.e., in the presence of PEN, the endogenous agonist). The compounds that blocked PEN‐mediated calcium increase were further tested in GTPγS, adenylyl cyclase and receptor trafficking assays in the antagonist mode. From these assays, we identified CPD25 as a GPR83‐selective antagonist. Currently, we are characterizing these compounds using the native receptors in the brain and immune cells, with tissues from mice lacking GPR83 as controls. Characterization of these small molecule compounds provides much needed tools for furthering our understanding of the precise role of GPR83 in these biological processes. Also, since GPR83 has been highly implicated in the regulation of stress, mood and reward‐related behaviors, as well as immune function, identification of GPR83 selective ligands opens the door to development of novel therapeutics as regulators of this neuropeptide‐receptor system.Support or Funding InformationFunding: NS026880 and DA019521 to L.A.D and T32 grant DA007135 to L.M.L.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structure‐activity relationships of a distinct family of selective agonists of α7‐nAChRs
Authors: Camacho‐Hernandez Gisela Andrea , Kaczanowska Katarzyna , Doan Lisa , Bindig Hanna , Stokes Clare , Papke Roger L. , Taylor Palmer 
Published: June 2021

Nicotinic acetylcholine receptors (nAChRs) are lead members of a pentameric ligand‐gated ion channel family; they are excitatory receptors, activated by the endogenous neurotransmitter, acetylcholine. nAChRs are present in the peripheral and central nervous system, where they mediate and modulate neuromuscular, autonomic and CNS functions. Receptor‐selective ligands that cross the blood‐brain barriers may form useful therapies for disorders of aging, such as Alzheimers disease and disorders of development, such as schizophrenia. In previous work, we developed a small sub‐group of 4,6 di‐substituted 2‐aminopyrimidines that interact with the acetylcholine binding protein (AChBP) and activate a7‐nAChRs. These agents show Kd and EC50 values for both AChBP binding and a7‐nAChR activation between 10 and 70nM. Moreover, they are structurally distinct from classical nicotinic agonists that are quaternary amines or typically contain a strong base separated from a weaker base or hydrogen bonding acceptor, often on separate ring systems. Selectivity for the a7‐AChR and high affinity for AChBP was achieved by substituting a dipicolyl group at the 4 position of the pyrimidine. In contrast to classical nicotinic agonists, the nitrogens on the pyrimidine and pyridine rings are weakly basic. Moreover, X‐ray crystallographic studies show that these ligands bind to the classical agonist‐antagonist site, but with a distinctive orientation of the core pyrimidine ring and the associated picolyl moiety. We have expanded our structural landscape of these AChBP binding and a7‐selective ligands, based on our previous leads by combining the pyrimidine system with the di‐picolyl substitution at the 4‐position. To examine receptor interactions of this family of compounds on nAChRs and related pentameric ligand‐gated ion channels, we employ HEK cells transfected with cDNA's encoding one of three requisite receptor subtypes: α7‐nAChR, α4β2‐nAChR and 5HT3AR, along with a fluorescent reporter. To assess rapid activation of lead candidates we use an oocyte system, revealing an agonist behavior distinguishing them from silent agonists and ago‐PAM's. To examine the molecular determinants and ligand poses involved, we have crystallized the higher affinity complexes with AChBP. Accordingly, we have a structure‐guided approach to novel ligand design for nAChRs. In conclusion, we find: 1) a di‐picolyl moiety in the 4‐position is fundamental for the activation of the a7‐nAChR, 2) the 2‐amino group tolerates only certain structural changes controlled by steric and bonding constraints, 3) substitutions at the 6‐position affect both the potency and receptor subtype selectivity of the ligands.Support or Funding InformationCONACYT Mexico graduate fellowship to GACHThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
PEG‐peptide Scavenger Receptor Inhibitors for Non‐viral Gene
Authors: Allen Rondine , Rice Kevin , Mathew Basil 
Published: June 2021

The rapid capture and degradation of viral and non‐viral gene delivery nanoparticles by scavenger receptors (SR) on Kupffer cells and fenestrated endothelial cells in the liver results in a decreased half life of all nanoparticles in an in vivo system, as well as a lower percentage of the dose available for delivery to the target organ. Current attempts to achieve SR inhibition involves co‐administering high molecular weight, polyanionic inhibitors. Polyinosinic acid (Poly‐I), the most widely used and potent inhibitor, competes for SR binding and successfully inhibits the uptake and metabolism of viral gene delivery nanoparticles in the liver. However, Poly‐I also activates the immune system, resulting in toxicity in mice, making Poly‐I clinically unacceptable.We have discovered peptide based SR inhibitors that block SR uptake of DNA nanoparticles and improves their metabolic half‐life by forming protein nanoparticles in the blood.Radio‐iodinated PEG‐peptides were used to study the pharmacokinetics and biodistribution to understand the structural properties which influence transfection competency and potency. We hypothesized that the in vivo potency and activity is influenced by the stability of protein nanoparticles formed in the blood. Results show that peptides featuring both hydrophobic and positively charged residues are able to potently inhibit scavenger receptor uptake of non viral gene delivery systems.Support or Funding InformationThis work was supported by the Iowa Biosciences Academy and University of Iowa Pharmacological Sciences T32 training grant. The Iowa Biosciences Academy is supported by the National Institute Of General Medical Sciences of the National Institutes of Health under Award Number R25GM058939, the University of Iowa (UI) Office of the Vice President for Research and the UI Chief Diversity Office, the Pharmacological Sciences Training grant T32 GM067795 and NIH grant R01 GM087653.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Mechanistic Study on the Inhibition of Nicotine Metabolism by Trans‐Cinnamaldehyde
Authors: Espiritu Michael , Black Stephen , Johnston Casey , Chan Jeannine , Harrelson John 
Published: June 2021

Trans‐cinnamaldehyde, a natural product found in cinnamon oil, has previously shown to selectively inhibit the major nicotine metabolizing enzyme cytochrome P450 2A6 (CYP2A6) via metabolism based inactivation using assays involving coumarin as a reporter substrate. In this study we aimed to investigate the mechanism by which CYP2A6 is inactivated by trans‐cinnamaldehyde, with the ultimate goal of developing trans‐cinnamaldehyde as a smoking cessation agent. We have also determined the level of inhibition of nicotine metabolism when nicotine is incubated with trans‐cinnamaldehyde, human liver microsomes and cytosol. CYP2A6 activity and heme loss were monitored in the presence of trans‐cinnamaldehyde and NADPH in vitro using human liver microsomes (HLM) and reconstituted CYP2A6 respectively. The effect of reactive oxygen species (ROS) generation on CYP2A6 activity and heme loss was studied using the reactive oxygen scavengers: N‐acetylcysteine, glutathione, catalase and superoxide dismutase. In an attempt to determine if heme loss was observed due to heme destruction or adduct formation, predictive multiple reaction monitoring (MRM) was performed using an optimized liquid chromatography tandem mass spectrometry (LC‐MS/MS) protocol. In activity studies using HLM and coumarin all reactive oxygen scavengers protected against activity loss, with N‐acetylcysteine and catalase offering the most protection (20% respectively) which, using a two tailed t‐test was found to be statistically significant with 99% confidence. Heme monitoring using LC‐MS/MS resulted in a significant amount of heme loss (97%) with a standard deviation (SD) of 3% after 18 minute incubations with trans‐cinnamaldehyde and NADPH in comparison to enzyme only controls. Inclusion of glutathione resulted in approximately 46% (SD of 7%) protection of the heme. Incubations with an NADPH regenerating system yielded a 41% (SD of 9%) loss of heme in comparison to controls without trans‐cinnamaldehyde. Predictive MRM yielded no detectable alkylated or acylated heme adducts or their respective oxidized forms. Results indicated an average inhibition of nicotine metabolism of 74.68% in the presence of trans‐cinnamaldehyde. This study demonstrated for the first time that CYP2A6‐mediated nicotine metabolism is inhibited by trans‐cinnamaldehyde and that ROS generation and heme destruction are major contributors to this process.Support or Funding InformationResearch reported here was supported by the National Institute on Drug Abuse of the National Institutes of Health under award number R15DA042341. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
DISTRIBUTION, METABOLISM AND ELIMINATION OF BIA 10‐2474 IN THE RAT
Authors: Loureiro Ana I. , Fernandes‐Lopes Carlos , Moser Paul , Soares‐da‐Silva Patricio 
Published: June 2021

In January 2016 one person died and others suffered adverse neurological effects during a Phase I clinical trial of the fatty acid amide hydrolase (FAAH) inhibitor BIA 10‐2474. We have evaluated the distribution, metabolism and excretion of BIA 10‐2474 in the rat.BIA 10‐2474 distribution and excretion was evaluated in Wistar rats after oral administration (30 mg/kg) of [14C]‐BIA 10‐2474. The metabolic profiling of BIA 10‐2474 was evaluated, in the same species, after oral administration of 90 mg/kg [14C]‐BIA 10‐2474.The mean observed Cmax of total radioactivity in plasma occurred at 1 h and it was detectable up to 72h post‐dosing with 30 mg/kg [14C]‐BIA 10‐2474. The calculated apparent terminal half‐life of total radioactivity was 45h. Five days after oral dosing of Wistar rats, 97.6% (94% to 103%) of the administered dose had been excreted, with 65.2% (58.7% to 73.8%) of the radiolabelled material excreted in urine and 23.7% (20.8% to 29.1%) excreted in faeces. The amount of BIA 10‐2474 excreted in exhaled air was less than 0.4 %. The majority of the faecal excretion occurred in the first 24h post‐dose, representing about 83.1% of the excretion via this route. Similarly, the majority of urinary radioactivity was excreted in the first 24 h post‐dose representing at least 93.0% of the excretion via this route. The quantitative Whole‐Body Autoradiography (QWBA) studies in rat following single oral administration of [14C]‐BIA 10‐2474 (30 mg/kg), indicate that radioactivity was rapidly absorbed and distributed throughout the body. The high tissue to blood ratio of total radioactivity observed in the kidney medulla (9.46), pituitary gland (6.32), liver (5.70), kidney cortex (4.83), spleen (4.02), thymus (3.94), epididymis (3.81) and skeletal muscle (3.63) at 24 h post‐dose suggests a slower elimination of BIA 10‐2474 related radioactivity from these tissues. Following oral administration of 90 mg/kg [14C]‐BIA 10‐2474, the hydroxylation of the cyclohexyl ring of BIA 10‐2474 (BIA 10‐3827 and BIA 10‐2639), the N‐oxide reduced form of BIA 10‐2474 (BIA 10‐2445) followed by the hydroxylation in the cyclohexyl ring (BIA 10‐3764 and/or BIA 10‐2631) were identified. In brain homogenate samples, BIA 10‐2474, and metabolites BIA 10‐3764 and/or BIA 10‐2631, and BIA 10‐2445 have been identified.In conclusion, BIA 10‐2474 was found to be rapidly absorbed and primarily excreted via the urine in the rat. The hydroxylation of the cyclohexyl ring of BIA 10‐2474 and BIA 10‐2445 was identified as the main metabolic route in the rat.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.