bluuebunny/crossref_metadata_2025 huggingface dataset:
The Catalytic α Subunit of cAMP‐Dependent Protein Kinase Regulates Formoterol‐Induced Gene Expression in BEAS‐2B Human Bronchial Epithelial Cells
Authors: Hamed Omar , Yan Dong , Newton Robert , Giembycz Mark Anthony
Published: November 2023

Rationale

The β2‐adrenoceptor is a GPCR that classically couples to Gsα, the activation of adenylyl cyclases and the formation of cAMP. Through distinct signalling complexes often containing cAMP‐dependent protein kinase (PKA), cAMP mediates a myriad of functional responses including the regulation of gene expression. We have shown previously that in the BEAS‐2B human airway epithelial cell line, long acting β2‐adrenoceptor agonists (LABAs) induce cAMP response element (CRE) dependent transcription, which we suggest contributes to their therapeutic activity in obstructive lung diseases. In the current study we have extended those findings by identifying the PKA catalytic subunit responsible for mediating gene expression induced by the LABA, formoterol.

Methods

Microarray, RT‐PCR, western blotting and gene silencing were used to identify and implicate PKA catalytic subunits in the regulation of LABA‐induced gene expression. A CRE reporter construct stably transfected into BEAS‐2B cells was used for this purpose together with an assessment of CREB phosphorylation and an analysis of a panel of cAMP‐inducible genes (NR4A3, CRISPLD2, RGS2, NR4A2, CD200 and IL‐6).

Results

Microarray‐based gene expression profiling indicated that BEAS‐2B cells express mRNA transcripts for PRKACA (Cα), PRKACB (Cβ) and PRKX (Cx) whereas PRKACG (Cg) was absent. These data were confirmed at the protein level by western blotting. siRNAs were optimized to achieve 80 to 90% knockdown of PRKACA, PRKACB and PRKX, which was reproduced at the protein level. Under these conditions, silencing PRKACA was associated with a significant (~50%) abrogation of formoterol‐induced CRE reporter activation, CREB phosphorylation and gene expression. No additional effect was seen when siRNAs that target PRKACB or PRKX were used in combination with those directed towards PRKACA.

Conclusions

These data show that Cα is an essential cAMP signalling component controlling formoterol‐induced gene transcription in BEAS‐2B cells. The incomplete inhibition produced under conditions of 80–90% Cα knockdown implies that this PKA catalytic subunit is present in significant excess of that required to maximally mediate cAMP‐induced gene expression. This is an initial first step at identifying cAMP signalling components (adenylyl cyclases, scaffold proteins, phosphodiesterases and effectors) that control LABA‐induced functional responses in airway structural cells. We propose that identifying these components and their assembly into multiprotein signalling complexes could provide new opportunities for the development of novel therapies for asthma and chronic obstructive pulmonary disease.

Support or Funding Information

Alberta Lung Association (ALA)
Cellular Compensation for Chronic Decreases in Cyclic AMP Concentration: Gαs‐Deficient Dendritic Cells as a Model and Implications for Therapeutics for Allergic Disorders
Authors: Chinn Amy M. , Lee Jihyung , Raz Eyal , Insel Paul A.
Published: November 2023

Dendritic cells (DCs), the main antigen‐presenting cells, are involved in the induction of type II helper T cell (Th2) immune responses, but the mechanism by which this occurs is not fully elucidated. Mice that have a CD11c (DC)‐specific deletion of the gene Gnas (CD11cΔGnas), which encodes the α subunit of the heterotrimeric (αβγ) G protein Gαs, display very low basal cyclic AMP (cAMP) concentrations and an allergic phenotype; the decrease in cAMP and protein kinase A (PKA) activation biases DCs to provoke a Th2 response ex vivo and in vivo (Lee et al PNAS 112: 1529–34 [2015]). By comparing the mRNA expression of cAMP pathway components in WT and CD11cΔGnas bone marrow‐derived DCs (BMDCs), we assessed how cells compensate for a chronic decrease in cAMP/PKA signaling. Using quantitative real‐time PCR (qPCR), we found that BMDCs from CD11cΔGnas mice have >2‐fold increased expression of adenylyl cyclase (AC) 3, decreased expression of numerous cyclic nucleotide phosphodiesterase (PDE) isoforms that degrade cAMP, but no overall increase in Gαi proteins or the cAMP transporter MRP4/ABCC4. We also assessed the expression of G protein‐coupled receptors (GPCRs; assayed by qPCR‐based Taqman GPCR arrays), including those that regulate cAMP formation. A greater number and larger increase in expression occurs for GPCRs that couple to Gαi (rather than other Gα proteins) in CD11cΔGnas BMDCs compared to BMDCs from WT mice. Additionally, expression of multiple PKA subunits (Cα, RIα, RIIα, RIIβ) is decreased at least 30% in CD11cΔGnas BMDCs. A‐kinase anchor protein (AKAP)6 and AKAP9 have an increase and decrease, respectively, in those BMDCs. Together, these data indicate that DCs with lower cAMP levels have altered expression of “upstream” cAMP signaling components (GPCRs and AC3) and of “downstream” components that regulate cAMP levels (PDE isoforms) and actions (PKA subunits, AKAPs). Such changes in expression of GPCRs and post‐G protein components may reflect withdrawal of “basal”, tonic effects of cAMP, may contribute to the greater Th2 responses and allergic asthma of CD11cΔGnas mice, and may be novel therapeutic targets for allergic asthma.

Support or Funding Information

Supported by NIH grants R56AI110505 and T32GM007752.
Environmental Phthalate Diesters as hMT1 and hMT2 Melatonin Receptor Ligands
Authors: Jones Anthony J , Dubocovich Margarita L , Rajnarayanan Rajendram V
Published: November 2023

Phthalates esters are commonly used plasticizers that have been linked to endocrine disruption and developmental alterations. Humans are exposed to these ubiquitous synthetic compounds via inhalation of ambient air, dermal contact with consumer products, and ingestion through food and drinking water. It is estimated from urinary metabolite levels that in the US, median adult daily exposure values for phthalate levels are about 8.5–16.8 μg/kg bw/day (Wittassek et al., Mol Nutr Food Res. 55:7–31, 2011). Using integrated pharmacoinformatics‐molecular docking approaches we discovered that several phthalate diesters show structural similarities to melatonin receptor ligands and bind with relatively high docking scores to melatonin binding pockets on in silico hMT1 and hMT2 melatonin receptor models. An assortment of phthalates including three monoesters and ten diesters with alkyl and/or aromatic substituents were tested for competition with 2‐[125I]‐iodomelatonin (100 nM) binding to hMT1 and hMT2 receptors stably expressed in CHO cells to assess binding affinity (Ki) as well as apparent intrinsic efficacy in the presence of GTP (100 μM). In GPCR binding assays, GTP decouples G proteins from receptors causing decreased affinity (rightward shift) for agonists but not for antagonists. Phthalate diesters with aromatic substituents (BBzP: Benzyl Butyl Phthalate; DPP: Diphenyl Phthalate) exhibited the highest affinities toward both hMT1 (BBzP Ki: 2.6 ± 0.3 μM, n=8; DPP Ki: 2.9 ± 0.7 μM, n=8) and hMT2 (BBzP Ki: 1.3 ± 0.2 μM, n=9; DPP Ki: 0.66 ± 0.10 μM, n=9) receptors. Among phthalates with alkyl substituents, four carbon (C4; DBP: Dibutyl Phthalate) and six carbon (C6; DHP: Dihexyl Phthalate) chain length diesters best competed for binding to hMT1 receptors with affinities ranging from 5 to 8 μM. hMT2 receptors did not show any preference to chain lengths of C6 or shorter (DEP: Diethyl Phthalate; DMP: Dimethyl Phthalate; DPrP: Dipropyl Phthalate; DBP; DHP), as all Ki values were between 2 and 7 μM. DBP and BBzP bound to melatonin receptors with affinities in the low micromolar range, however their respective monoester metabolites, MBP (Monobutyl Phthalate) and MBzP (Monobenzyl Phthalate), did not compete for binding to either receptor. The presence of GTP caused a leftward shift of the binding competition curves for both BBzP and DPP at hMT1 melatonin receptors (BBzP KiGTP/KiControl: 3.8; DPP KiGTP/KiControl: 2.6) suggesting inverse agonist efficacy. Little or no shift (BBzP KiGTP/KiControl: 1.3; DPP KiGTP/KiControl: 1.5) on competition curves for hMT2 melatonin receptors suggests antagonist efficacy. Together these data suggest that phthalate diesters could interact with hMT1 and hMT2 receptors to disrupt melatonin receptor‐mediated signaling processes in target tissues.

Support or Funding Information

Supported by ES 023684 to MLD and RVR.
Acetate, an Ethanol Metabolite Increases Neuroinflammation and Neuronal Death: Implications in Ethanol Neurodegeneration
Authors: Chapp Andrew D. , Driscoll Kyle M. , Behnke Jessica , Shan Zhiying , Chen Qinghui
Published: November 2023

Upregulation of neuroinflammatory signaling molecules following ethanol consumption has been identified as a key cellular mechanism to contribute to neuronal excitotoxicity. Evidence indicates that ethanol excitotoxicity occurs during ethanol withdrawal/clearance, times at which acetate concentrations are still elevated. Studies have demonstrated that acetate is a very active and powerful metabolite of ethanol, which may be one of the key components to underlie the mechanisms of neuronal excitotoxicity/apoptosis induced by ethanol consumption. Here, we tested the hypothesis that acetate increases neuroinflammatory cytokines, which may contribute to neuronal excitotoxicity/apoptosis and enhanced cellular death. Whole brain neuronal cultures incubated with acetate (2 mM) for 3 hrs. increased mRNA levels significantly (p < 0.05 vs control) of tumor necrosis factor alpha (TNFα), interleukin‐1β (IL‐1β) and fos related antigen 1 (fosl1); 2 fold, 8 fold and 2 fold respectively. Furthermore, 24 hr. treatment with acetate (2 mM) in whole brain neuronal cultures followed by staining with propidium iodide and analyzed via flow cytometry (1000 neurons, triplicates) showed significant neuronal death (p < 0.05 vs control), acetate (62.3 %) vs control (25.5 %). In a separate treatment, whole brain neuronal cultures were incubated with acetate (2 mM) for 24 hrs. and then stained with Fluo‐4AM (calcium dye) and analyzed via flow cytometry (1000 neurons, triplicates) and showed a significant 5 % increases in cytosolic calcium (p < 0.05 vs control). This data suggests that physiological circulating levels of acetate (2 mM) following ethanol consumption and metabolism lead to increased neuroinflammation which enhances whole brain cellular apoptosis/death and may be the underlying cause of alcohol associated neurodegeneration.

Support or Funding Information

AHA 16PRE27780121, Andrew Chapp, AHA 11SDG7420029, Zhiying Shan; R15‐HL122952, Qinghui Chen
The Neuroprotective Effect of Dimethyl Fumarate in MPTP‐mouse Model of Parkinson's Disease via ROS/NF‐κB/Nrf‐2
Authors: Casili Giovanna , Campolo Michela , Biundo Flavia , Crupi Rosalia , Cordaro Marika , Cuzzocrea Salvatore , Esposito Emanuela
Published: November 2023

Oxidative stress plays a key role in Parkinson's disease (PD) and nuclear transcription factor related to NF‐E2 (Nrf‐2) and nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NF‐κB) are involved in neuroprotection against PD. The aim of the present study was to investigate the neurotherapeutic action, NF‐κB/Nrf‐2 dependent, of DMF in a mouse model of PD and in vitro SHSY‐5Y cells. Daily oral gavage of DMF (10, 30 and 100 mg/kg) significantly reduced neuronal degeneration of dopaminergic tract and behavioral impairments induced by four injections of the dopaminergic neurotoxin 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP). Moreover, treatment with DMF prevented dopamine depletion increasing tyrosine hydroxylase (TH) and dopamine transporter (DAT) and also reduced α‐synuclein‐positive neurons. Furthermore, DMF treatment up‐regulated Nrf‐2 pathway, increasing NeuN+/Nrf‐2+ cells in the striatum and inducing activation of manganese superoxide dismutase (Mn‐SOD) and heme‐oxygenase‐1 (HO‐1), regulating glutathione levels too. Also, DMF reduced interleukin1 (Il‐1β), cyclooxygenase 2 (COX‐2), lowered nitrotyrosine (NT) and neuronal nitrite oxide synthase (nNOS) expression, modulated microglia activation, restored nerve growth factor (NGF) levels and preserved by microtubule‐associated protein 2 (MAP‐2) alterations. The protective effects of DMF treatment, via Nrf‐2, were confirmed in in vitro studies, trough the inhibition of Nrf‐2 by Trigonelline (TR). These findings underline that DMF, used in vivo at low dose (30mg/kg) and in vitro at the concentration of 30 μM, as NF‐κB/Nrf‐2 based therapy, offers novel cytoprotective modalities that further augment the natural antioxidant response in neurodegenerative and inflammatory disease models. These results support the thesis that DMF may constitute a promising therapeutic target for the treatment of PD.
Anti‐inflammatory and Neuroprotective Effects of Co‐ultraPEALut in a Mouse Model of Vascular Dementia
Authors: Siracusa Rosalba , Cordaro Marika , Impellizzeri Daniela , Crupi Rosalia , Esposito Emanuela , Cuzzocrea Salvatore
Published: November 2023

Vascular dementia, the second most common cause of cognitive impairment in the population, is a disease with a progressive course of cognitive impairments that results from reduction in regional cerebral blood flow and involves oxidative stress and inflammation. Co‐ultraPEALut is a new compound with beneficial effects, which include anti‐inflammatory and anti‐oxidant properties. Recently, co‐ultraPEALut has been shown to exhibit neuroprotective effects in models of Parkinson's disease, cerebral ischemia and Alzheimer's disease. However, its effects on vascular dementia remain unclear. Therefore, the present study examined the neuroprotective potential of co‐ultraPEALut containing N‐Palmitoylethanolamine (PEA) and the anti‐oxidant flavonoid luteolin (Lut) (10:1 by mass) in a mice model of vascular dementia using bilateral carotid arteries occlusion. At 24 hours after model induction, mice were orally administered 1 mg/kg co‐ultraPEALut daily and for 15 days. On the 15th days, brain tissues were processed for histological, immunohistochemical, Western blot and immunofluorescent analysis. Our results showed that co‐ultraPEALut improved learning, memory ability, locomotor activity and the reciprocal social interaction. Furthermore, mice subjected to vascular dementia and treated with co‐ultraPEALut showed reduced expression of pro‐inflammatory, pro‐apoptotic markers and of oxidative stress. These results confirmed that the neuroprotective effects of co‐ultraPEALut on vascular dementia were associated with its anti‐inflammatory and anti‐oxidant properties.
Factors Impacting the Estimation of Renal Clearance of N1‐methylnicotinamide, an Endogenous Probe for Renal Cation Transporters
Authors: Bergagnini‐Kolev Mackenzie C. , Hebert Mary F. , Easterling Thomas R. , Lin Yvonne S.
Published: November 2023

N1‐methylnicotinamide (1‐NMN) has been previously investigated as an endogenous probe for the activity of renal transporter organic cation transporters 2 (OCT2) and multidrug and toxin extrusion proteins 1 and 2‐K (MATE1 and MATE2‐K). In drug‐drug interaction studies, 1‐NMN renal clearance was shown to decrease after the administration of OCT2, MATE1, and MATE2‐K inhibitors. We have shown that pregnancy increases the renal clearance of 1‐NMN. We investigated the factors that may impact the estimation of renal clearance using 1‐NMN such as OCT2 genotype, as well as urine and plasma collection interval duration (8, 12 and 24 hours). In addition, we assessed whether 1‐NMN renal clearance as determined by spot urine/plasma measurements or longer collection intervals were comparable under basal and induced states. Secondary analysis was conducted on blood and urine samples collected from women prescribed metformin during early‐, mid‐, and late‐pregnancy and postpartum and healthy volunteers administered rifampin. Samples were analyzed for 1‐NMN using liquid chromatography‐mass spectrometry. The renal clearance of 1‐NMN was estimated and correlated with metformin renal clearance when data were available. 1‐NMN renal clearance was higher in both mid‐ (504 ± 293 mL/min, p < 0.01) and late‐pregnancy (557 ± 305 mL/min, p < 0.01) compared to postpartum (240 ± 106 ml/min). Metformin renal clearance and 1‐NMN renal clearance were positively correlated (rs = 0.68, p < 0.0001). OCT2 G808T genotype did not appear to affect 1‐NMN renal clearance. Plasma 1‐NMN levels were highly variable throughout the day, thus a single plasma measurement was not sufficiently representative of Css,1‐NMN to estimate 1‐NMN clearance. Studies looking at the impact of urine and plasma collection interval duration and spot sampling are ongoing. As an endogenous biomarker, 1‐NMN may be useful in assessing changes in renal OCT2, MATE1, and MATE2‐K activity, however sample collection and other subject‐specific factors (e.g., diet) may impact the results.

Support or Funding Information

This work was supported in part by the Eunice Kennedy Shriver National Institute of Child Health & Human Development [Grant U10HD047892]; NIH National Center for Advancing Translational Sciences through the Clinical and Translational Science Awards Program (CTSA) [Grant UL1TR000423]; and the NIH National Institute of General Medical Sciences [Grant T32GM 007750]. The content is solely the responsibility of the authors and does not necessarily represent the official views of the Eunice Kennedy Shriver National Institute of Child Health & Human Development or the National Institutes of Health.
Prioritizing Pharmacokinetic Drug Interaction Precipitants in Natural Products: Application to OATP Inhibitors in Grapefruit Juice
Authors: Johnson Emily J , Won Christina S , Köeck Kathleen , Paine Mary F
Published: November 2023

Background and objective

Herbal and other natural products typically contain multiple constituents that can precipitate adverse pharmacokinetic interactions with conventional drugs. Streamlined approaches are needed to prioritize candidate natural product precipitants during early investigation of these complex interactions. Grapefruit juice is a well‐characterized natural product that increases systemic exposure to several drugs via inhibition of organic anion‐transporting polypeptide (OATP) uptake in the intestine. Using grapefruit juice as a model natural product, a streamlined approach was developed to prioritize OATP inhibitors as precipitants of pharmacokinetic grapefruit juice‐drug interactions.

Methods

OATP2B1‐expressing MDCKII cells and the probe substrate estrone 3‐sulfate were used to assess the inhibitory effects of candidate constituents representative of the flavanone (naringin, naringenin, hesperidin), furanocoumarin (bergamottin, 6′,7‐dihydroxybergamottin), and polymethoxyflavone (nobiletin, tangeretin) classes contained in grapefruit juice. IC50s were recovered via nonlinear least‐squares regression analysis. Intestinal absorption simulations based on physiochemical properties were used to estimate the unbound intracellular enterocyte concentration (Ient) of each constituent. Two ratios were determined for each constituent: Ient/IC50 and maximum reported concentration in grapefruit juice to IC50 (IGFJ/IC50). Constituents were ranked in order of drug interaction liability according to pre‐defined cutoff values (Ient/IC50, 0.1; IGFJ/IC50, 10).

Results

Nobiletin was the most potent OATP2B1 inhibitor (IC50, 3.7 μM); 6′,7‐dihydroxybergamottin, naringin, naringenin, and tangeretin were moderately potent (IC50, 22–39 μM); and hesperidin and bergamottin were the least potent (IC50 >300 μM). The Ient/IC50 for naringin, naringenin, nobiletin, and tangeretin exceeded 0.1. The IGFJ/IC50 for naringenin and naringin exceeded 10.

Conclusions

Both Ient/IC50 and IGFJ/IC50 correctly prioritized the flavanones, and excluded the furanocoumarins, as precipitants of intestinal OATP‐mediated grapefruit juice‐drug interactions in humans. The more sensitive Ient/IC50 identified two polymethoxyflavones as potential additional inhibitors. A similar approach could be applied to other natural products for initial prioritization of constituents as potential precipitants of pharmacokinetic natural product‐drug interactions.

Support or Funding Information

This work was supported by the National Institutes of Health National Institute of General Medical Sciences [Grant R01 GM077482] and National Center for Advancing Translational Sciences [Grant UL1TR001111]. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.
Analysis of Liver Toxicity Markers in Danio rerio following Ethanol Exposure
Authors: Chiari John B , Charles Jamie M , Laperche Jacob M , McGinnis Courtney L
Published: November 2023

Zebrafish have been used for many years in developmental and reproductive biology, yet little is still known about the adult zebrafish liver. Additionally, many of the mammalian liver cell types have yet to be characterized in zebrafish, including Kupffer cells, oval cells, and stellate cells. Also, there is minimal information available regarding zebrafish liver transporters and nuclear receptors. Therefore, it is important to understand the mechanism of xenobiotic metabolism in zebrafish to support their use as a model organism for studying the fate and biological effects of xenobiotics. The value of zebrafish as a model organism is based on a broad set of gene similarities (among fish species and humans) involved in metabolism and transportation of endogenous and exogenous chemicals. While there are molecular similarities between zebrafish and humans, there are also differences that may impact how compounds are metabolized. Therefore, it is absolutely necessary to investigate and validate the assumptions that zebrafish possess conserved genes, enzymes, and processes involved in all stages of metabolism. This study investigates the impact of ethanol exposure on liver toxicity markers in adult Danio rerio. Liver tissue from sham and ethanol injected fish were collected, pooled and RNA was isolated for qPCR. Relative expression levels for each gene were determined and normalized to βactin. Of the seventeen genes that were assessed, our results demonstrate a significant upregulation of the following genes following ethanol treatment: abcb4 (mdr3), abcc1(mrp1), abcc4(mrp4), abcc9(sur2), abcg5, mate1, oatp1 and slc4a2(ae2). Alcohol metabolism increases acetate production, a precursor for cholesterol and fatty acid synthesis. Historically, this increase in acetate levels driven by alcohol, is a contributor of hepatic steatosis (fatty liver). In a previous study conducted in the lab, zebrafish chronically exposured to ethanol displayed hallmark signs of hepatic steatosis. An upregulation in slc4a2, a sterol and cholesterol transporter aimed to eliminate dietary cholesterol, was found, consistent with the digestion of high levels of acetate. Furthermore, upregulation of abcc4, a prostaglandin transporter, is indicative of oxidative stress within the liver, caused by ethanol metabolism. Additionally, the upregulation of the other liver transporters would be consistent with normal hepatic ion flow during xenobiotic methabolism.

Support or Funding Information

Quinnipiac University: College of Arts and Sciences
In Silico Molecular Dynamics Analysis of Dopamine Transporter Conformational Preference as a Function of Occupancy with Inhibitors of Low Abuse Potential
Authors: Wasko Michael J. , Madura Jeffry D. , Hong Weimin C. , Katz Jonathan L. , Surratt Christopher K.
Published: November 2023

The plasmalemmal dopamine transporter (DAT) protein removes dopamine from the extracellular space via electrogenically‐driven uptake, and is a primary target of the abused psychostimulants cocaine and the amphetamines. Certain “atypical” high‐affinity DAT inhibitors such as benztropine and GBR12909 do not share cocaine's abuse potential, and it has been postulated that a preference for the inward‐ (cytoplasmic‐) facing DAT conformation during the substrate translocation cycle correlates with low abuse potential. The objective of this study was to characterize for in vitro and in silico conformational preference several N‐substituted benztropine analogs (GA169, GA250, GA299, JHW013) that have been characterized as atypical because they bind the DAT inhibit dopamine uptake but have behavioral effects that differ from those of cocaine. A cysteine accessibility assay probed each drug's preference for the outward‐facing conformation of DAT. Computational models of the human DAT in outward‐ or inward‐facing conformations were created in MOE using the Drosophila DAT crystal structure 4M48 template. DAT inhibitors were docked to the optimized DAT models, followed by 400 ns molecular dynamics (MD) simulations using ACEMD software to ascertain a conformational preference for each ligand. Whereas cocaine showed its characteristic preference for the outward‐facing conformation in the cysteine accessibility assay, benztropine as well as the (R)‐2″‐amino‐3″‐methyl‐n‐butyl (GA250), indole‐3″‐ethyl (GA169), and cyclopropylmethyl (JHW013) analogs showed preference for the inward‐facing conformation, in keeping with their atypical effects. In contrast, the 2″‐aminoethyl analog (GA299) showed no conformational preference despite its atypical effects. Analysis of the MD simulations, using the external and internal gate residues as a reference, confirmed that cocaine and benztropine achieved their preferred conformation. The initial outward‐facing DAT simulation formed an occluded structure with benztropine in the S1 pocket, while the external gate remained open during the cocaine simulation. The outward‐facing simulation with GA299 shared characteristics with cocaine, while GA169 displayed similarities with benztropine bound to the inward‐facing structure. In summary, enough exceptions were observed to question the proposed correlation between a DAT inhibitor's abuse potential and its preference for the inward‐ or outward‐facing DAT conformation. This work suggests that in silico studies on DAT protein‐inhibitor complexes provide a new tool for analysis of physiologically‐relevant drug receptor conformations and for guiding therapeutic drug design.

Support or Funding Information

Funding: ZIA DA000103‐26 (IRP, NIDA), DA027806, XSEDE MCB060069, Butler Faculty Startup Fund, Holcomb Research Award.
Role of breast cancer resistance protein in obesity associated chronic low grade inflammation
Authors: Mishra Jayshree , Kumar Narendra
Published: November 2023

Objective

The major immunological manifestation in diabetes/obesity is characterized by chronic low grade inflammation that leads to altered drug metabolism and disposition. Alteration in drug metabolism has profound impact on the pharmacotherapy of widely used clinically relevant medications in terms of safety and efficacy and increased toxicity. Altered drug metabolism and disposition is associated with the alteration in the expression and activity of the several key drug metabolizing enzymes (DMEs) and transporters. However, the role of inflammation in regulation of drug transporters in diabetes/obesity remains unclear. Breast cancer resistance protein (BCRP) is a principal drug transporter protein expressed in intestine, liver and brain extrudes a broad range of xenochemicals and to protect the organism against xenotoxicity by facilitating excretion, and limiting absorption of potentially toxic substrate molecules, including many chemotherapeutic drugs, the possible involvement of BCRP in obesity is poorly understood. It is critical to understand the mechanisms that regulate mucosal detoxification mediated by BCRP under normal physiological conditions and under the chronic low grade inflammation as seen in obesity. A better understanding of the mechanistic details underlying the regulation of BCRP in obesity will contribute towards development of powerful in vitro and in vivo tools in predicting the drug response and opt for better drug design and development.

Materials and Methods

Freshly dissected human colon tissue from normal and obese were immunostained and analyzed for BCRP (Novus). Phosphorylation status of this protein in both normal and obese were assessed through immunoprecipitation. Functional activity of BCRP was measured using Efflux id Gold kits from Enzo Life sciences.

Summary of Results

Our results show that colonic epithelial cells of normal subject express BCRP, however there is a substantial decrease in expression of BCRP in obese patients. Also in obese patients there is a substantial decreases in the tyrosine phosphorylation status of BCRP. Expression and activity of BCRP was found to be significantly reduced in the induced with high fat diet.

Conclusion

Our data suggest that colonic BCRP plays an important role in obesity. Further, our goal is to facilitate a better understanding of the mechanistic role of BCRP in the process of chronic low grade inflammation and associated chronic diseases.

Support or Funding Information

Health‐ SBIR (GM109528)
Polarization of Adipose‐Derived Mesenchymal Stem Cells into Immunomodulating Phenotypes
Authors: Rivera‐Cruz Cosette M. , Shearer Joseph J , Figueiredo Marxa Leao
Published: November 2023

Mesenchymal stem cells (MSCs) have been studied often to assess their potential use in cell therapy because of their capacity to differentiate into non‐hematopoietic cell lineages, modulate immune responses, and promote tissue repair and regeneration. In addition, MSCs have been found to have roles in the promotion or repression of tumor growth and tumor cell migration. However, the mechanisms by which MSC affect tumor cells are not fully understood. Studies with bone marrow‐derived MSCs have shown that these cells can be polarized by Toll‐like receptor (TLR) signaling into two phenotypes with opposite effects on tumor development1. TLR4 signaling induces an immune activating MSC1 population, whereas TLR3 signaling induces an immune suppressive MSC2 population2. Adipose‐derived mesenchymal stem cells (ASCs) are important constituents of tumor microenvironments. Their ability to migrate into tumors make these cells a potential candidate for delivering immunotherapy against tumor progression. Although the MSC1 and MSC2 duality has not been reported for ASCs, we hypothesized that due to their similarity to bone marrow MSCs, that ASC may share the polarization phenotype. In order to test this hypothesis, we conducted preliminary experiments using ASC priming with lipopolysaccharide (LPS) or polyinosinic:polycytidylic acid (Poly (I:C)) and assessed gene expression changes that would be consistent with polarization using real time PCR. These experiments have shown expression of target TLRs in human ASCs from two independent donors. Further studies are in progress, including priming with TLR agonists, gene expression analysis to confirm priming (IL‐6 for ASC1; CCL5 for ASC2), and assays to assess whether ASC1 or ASC2 conditioned media promote or inhibit prostate tumor cell colony formation.

Support or Funding Information

We acknowledge support from the Purdue PREP program (DTA Transformative Award) and statrt up funds from the BMS department
Inhibition of Neuropilin 1 signaling in glioma associated macrophages and microglia slows tumor progression
Authors: Miyauchi Jeremy T , Chen Danling , Choi Matthew , Shroyer Kenneth , Selwood David , Tsirka Stella
Published: November 2023

Gliomas are the most commonly diagnosed primary tumors of the central nervous system (CNS). Median times of survival are dismal regardless of the treatment approach, underlying the need to develop more effective therapies. Modulation of the immune system is a promising strategy as innate and adaptive immunity play important roles in cancer progression. Glioma associated microglia and macrophages (GAMs) can comprise over 30% of the cells in glioma biopsies. Gliomas secrete cytokines that suppress the anti‐tumorigenic properties of GAMs, causing them to secrete factors that support the tumor's spread and growth. Neuropilin 1 (Nrp1) is a transmembrane receptor that in mice both amplifies pro‐angiogenic signaling in the tumor microenvironment and affects behavior of innate immune cells. Using a Cre‐lox system, we generated mice that lack expression of Nrp1 in GAMs. We demonstrate, using an in vivo orthotopic glioma model, that tumors in mice with Nrp1‐deficient GAMs exhibit less vascularity, grow at a slower pace, and are populated by increased numbers of anti‐tumorigenic GAMs. Moreover, glioma survival times in mice with Nrp1‐deficient GAMs were significantly longer. Treating wild‐type mice with a small molecule inhibitor of Nrp1's b1 domain, EG00229, which we show here is selective for Nrp1 over Nrp2, yielded an identical outcome. Moreover, transplanting bone marrow from mice with Nrp1‐deficient macrophages to wild type recipient mice led to reductions in tumor growth, al‐be‐it not to the extent seen in knockout mice. Nrp1‐deficient or EG00229‐treated wild‐type microglia exhibited a shift towards anti‐tumorigenicity as evident by altered inflammatory marker profiles in vivo and decreased SMAD2/3 activation when conditioned in the presence of glioma‐derived factors. These results provide support for the proposal that pharmacological inhibition of Nrp1 constitutes a potential strategy for suppressing glioma progression.

Support or Funding Information

This work was funded by the following award from the NIH: NCI F30CA196110
Identification of ABCG2 Inhibitors as a New Combination Therapy to Improve Outcome in AML
Authors: Allcock Alice , Fukuda Yu , Lin Wenwei , Chen Taosheng , Schuetz John D.
Published: November 2023

Acute myeloid leukemia (AML) arises secondary to lesions that disrupt normal myelopoiesis. Despite recent advances in chemotherapy, the 5‐year survival rate is poor (~50%) due to relapse and/or refractory disease. The primary treatment regimen for AML relies upon the antineoplastic, mitoxantrone. An ATP‐binding cassette (ABC) transporter, ABCG2, exports multiple cytotoxic chemotherapeutics, including mitoxantrone, from cancer cells producing strong resistance. In AML, ABCG2 is expressed at higher levels in samples from relapse patients, and high ABCG2 levels are associated with poor survival in multiple AML cohorts. Currently available ABCG2 inhibitors are not suitable due to neurotoxicity, low specificity, or possess a high IC50 that is not clinically achievable. Therefore, we hypothesized that identification of chemotherapeutics that target pathways activated in AML, (e.g. Wnt or tyrosine kinase) but also inhibit ABCG2 function would have clinical impact when combined with a chemotherapy that includes mitoxantrone. To this end, we developed a high throughput screening assay that assessed ABCG2's ability to export a specific fluorescent substrate, pheophorbide a (PhA) from mouse embryonic fibroblasts (MEFs) which were engineered to express human ABCG2. We validated the assay using a previously reported ABCG2 inhibitor, lapatinib (tyrosine kinase inhibitor, TKI), as a control. Subsequently, using a single concentration of 10 μM, a library of ~12,000 bioactive compounds including FDA‐approved drugs was assessed for their ability to block the export of PhA. Compounds producing >80% inhibition, compared to lapatinib, were further subjected to a secondary dose responsive analysis. Chemotherapeutic agents displaying activity were further validated for their specificity and activity in wild‐type MEFs, MEFs lacking Abcg2(KO) or KO‐MEFs harboring human ABCG2. These compounds were evaluated for their ability to enhance the cytotoxicity of mitoxantrone in MEFs and AML cell lines. Several TKIs have been shown to inhibit ABCG2, but at high concentrations (>10 μM). We identified a TKI that inhibits ABCG2 with an IC50 < 1 μM, but was previously unknown. We also identified a Wnt pathway antagonist that potently inhibits ABCG2. In summary, these potent ABCG2 inhibitors we identified have a selective advantage in killing AML cells because their dual actions will disrupt a vital pathway in AML, but also increase the cytotoxicity of mitoxantrone, an approach that is likely to reduce relapse and increase durable remissions.

Support or Funding Information

This work was supported by NIH and by the ALSAC.
Impact of ABCB1 genetic polymorphisms on the interactions between novel dipyranone derivatives and P‐glycoprotein
Authors: Wang Yen‐Hsiang , Chen Chien‐Yu , Liu Nai‐Yu , Chang Chih‐Shiang , Hung Chin‐Chuan
Published: November 2023

Multidrug resistance (MDR) is one of the major obstacle in cancer treatment and over‐expression of P‐glycoprotein (P‐gp) is highly related to this issue. Genetic polymorphisms in P‐gp may influence clinical treatment outcomes. Therefore, this study aims to evaluate the effect of novel dipyranone derivatives on wild‐ and variant‐types human P‐gp efflux function and investigate underlying kinetic and molecular mechanisms.

Recombinant P‐gp‐expressing HEK293 cell lines were established with the common polymorphisms in human ABCB1 gene. Real‐time RT‐PCR was performed to confirm the mRNA expression. Influence of dipyranone derivatives on P‐gp function was evaluated by calcein‐AM uptake assay, rhodamine 123 efflux assay and doxorubicin efflux assay. MDR1 shift assay and ATPase assay were conducted to evaluate the interaction between P‐gp and dipyranone derivatives.

Among the tested compounds, compound A, B and C were the top three P‐gp inhibitors after primary screening. SRB assay showed that all of these three compounds had no cytotoxicity effect. However, wild‐type P‐gp mRNA expressions were reduced after treating compound B and C. Calcein‐AM uptake assay, rhodamine 123 efflux assay and doxorubicin efflux assay indicated that these three compounds significantly inhibited P‐gp efflux function. For rhodamine 123 efflux, compound A competitive inhibited wild‐type P‐gp, while compound B and C uncompetitive inhibited wild‐type P‐gp. In doxorubicin efflux, compound A and B modulated wild‐type P‐gp through uncompetitive inhibition, and compound C inhibited wild‐type P‐gp by competitive inhibition. Moreover, the common variants on ABCB1 gene would affect the P‐gp inhibition effect and interaction kinetics of compound B. Based on the results of MDR1 shift assay, these three compounds were not the substrates of P‐gp. However, compound A, B, and C influenced the P‐gp ATPase function and may interact with other P‐gp substrates as indicated by the ATPase assays.

This study characterized the novel dipyranone derivatives, compound A, B and C as potential P‐gp inhibitors. The inhibition potency were affected by the common polymorphism haplotypes in ABCB1 gene, which implied that genotypes may take into consideration for further in vivo studies.
Study of Jadomycin Pharmacokinetics in Balb/C Mice
Authors: Relja Nicholas , Hall Steven R. , Sleno Lekha , Bennett Leah , MacLeod Jeanna , Martinez‐Farina Camilo F. , Jakeman David L. , Goralski Kerry B.
Published: November 2023

Background

Jadomycins are natural product molecules derived from the soil bacteria Streptomyces venezuelae; jadomycins maintain their cytotoxicity in multidrug resistant (MDR) human and mouse breast cancer cell lines that overexpress ATP‐binding cassette (ABC) transporters. While effective in in vitro assays, jadomycins have not been studied in animal models.

Objective

To conduct the first in animal pharmacokinetic and safety studies of jadomycins B, S, and F in male and female Balb/C mice.

Methods

Jadomycins were administered intraperitoneally (IP) to male and female Balb/C mice at a 6mg/kg dose. In one group of mice (n = 5) up to four repeated saphenous vein blood samples (40 mL) were obtained after 15, 30, 60, and 90 minutes with terminal blood collection occurring at 120 minutes in order to characterize absorption and distribution phases. In a second group of mice saphenous vein blood samples were obtained at 2, 4, 6, 8 and 10 hours to characterize terminal elimination. Liver, kidney, lung and brains were collected after 2 hours and then at 8 or 10 hours (males and females respectively) to characterize jadomycin distribution to these tissues. Serum and tissue samples underwent extraction and jadomycin concentrations were then determined by mass spectrometry.

Results

The concentration versus time profiles of jadomycins B, S and F were similar. They were absorbed rapidly after IP injection with peak concentrations in the 1–10 mM range observed after 15 minutes. This was followed by a rapid distribution phase (t1/2a = 20 – 40 min) and a slower terminal elimination (t1/2b = 1.5 – 2 hours). Jadomycins accumulated in the liver > kidney > lung and were not detectable in the brain. No metabolites were detected and no acute toxicity responses were observed.

Conclusions

These 1st in vivo studies of jadomycins reveal that jadomycins are safe at a 6 mg/kg IP dose and achieve concentrations in mouse serum that are above the IC50s (1–4 μM) for human and mouse breast cancer cells in vitro. Our results indicate that jadomycins are rapidly eliminated as unchanged drug and that two to three times daily dosing will be required for future tumor studies.

Support or Funding Information

Cancer Research Training Program (CRTP) through Beatrice Hunter Cancer Research Institute, Canadian Breast Cancer Foundation Atlantic (CBCF), Nova Scotia Health Research Foundation (NSHRF), and Dalhousie University
Endothelial cell‐specific deletion of a single Nanog allele demonstrates its crucial role in the microenvironment of adult tissues
Authors: Baruah Jugajyoti , Wary Kishore
Published: November 2023

Rationale

Although Nanog is a known regulator of pluripotency, the functional significance of low level expression of Nanog in endothelial cells (ECs) remains incompletely understood.

Methods and results

We have created ROSAmT/mG::Nanogfl/+::Cdh5CreERT2 genetically engineered mouse model (GEMM) system. Administration of tamoxifen (TAM) to these adult mice induced EC‐specific loss of single Nanog allele. GFP lineage tracing combined with high‐resolution microscopy revealed GFP‐positive ECs in the microenvironment of adult tissues. Knockout (KO) or knockdown of single Nanog allele in ECs decreased expression of VEGFR2 and hTERT upon Wnt3a stimulation, suggesting a close relationship between Nanog and hTERT. In a chromatin immunoprecipitation (ChIP) assay, we identified Nanog binding sites on the promoter/enhancer region of the human hTERT in ECs. Elevated expression of Nanog increased hTERT‐promoter luciferase activity in ECs. Conversely, in Wnt3a stimulated ECs, Nanog‐depletion decreased hTERT protein which modulates EC proliferation. Importantly, re‐expression of hTERT in Nanog‐depleted ECs partially restored proliferation and angiogenesis.

These novel findings establish a key role for low level expression of Nanog in the regulation of EC homeostasis that is independent from its recognized function as a regulator of pluripotency.

Support or Funding Information

Studies were supported by the National Institute of Health (HL079356), American Heart Association (GRNT25710129) and by the University of Illinois at Chicago Center for Clinical and Translation Science Award (UL1RR029879) to K.K.W. J.B was supported by AHA pre‐doctoral fellowship mid‐west affiliate (15PRE22760004) and UIC‐CCTS‐PECTS fellowship (#1UL1TR002003).
Pharmacological inhibition of endothelial HDAC6 provides protection against atherogenesis through upregulation of cystathionine gamma‐lyase derived hydrogen sulfide
Authors: Leucker Thorsten Martin , Kim Jae Hyung , Nomura Yohei , Bhatta Anil , Wang Victor , Pandey Deepesh
Published: November 2023

Endothelial cystathionine gamma‐lyase (CSE) molecular mechanisms that control CSE gene expression in vascular endothelium during cardiovascular diseases are unclear. In this study, isolated murine aortic rings exposed to OxLDL (50mg/ml) for 24 hours exhibited impaired endothelial‐dependent vascular relaxation but were protected by adenoviral‐mediated increases in CSE expression. Cultured human aortic endothelial cells (HAECs) that were exposed to OxLDL and intact aortas from atherogenic ApoE knockout mice fed a high‐fat diet for 12 weeks both exhibited reduced CSE mRNA and protein expression. Utilizing class‐specific inhibitors of histone deacetylases (HDACs) class‐specific inhibitors and HDAC6 specific small molecule inhibitor and siRNA, we identified HDAC6 to be a critical regulator of endothelial CSE expression in response to injury stimulus such as OxLDL. HDAC6 mRNA and protein expression were both upregulated in OxLDL exposed HAECs. The reduced endothelial CSE expression/activity with OxLDL was reversed by an HDAC6 inhibitor, tubacin (10mM), and by HDAC6 siRNA (30nM). Further, OxLDL significantly impaired endothelial‐dependent vascular relaxation in mouse aortic rings and reduced CSE expression. The deleterious effect of OxLDL was significantly improved by adenoviral‐mediated overexpression of CSE in murine aortas and by inhibition of HDAC6 with tubacin. Our findings show that HDAC6 is a critical regulator of CSE expression in the vascular endothelium, and strategies to inhibit HDAC6 activity may improve endothelial function and prevent/reverse the development of atherosclerosis.

Support or Funding Information

This work was supported by American Heart Association Scientist Development Grant to D. Pandey (16SDG27340010), 2017 StAAR award from Department of Anesthesiology and Critical Care Medicine, Johns Hopkins School of Medicine to D. Pandey and National Institutes of Health‐National Heart, Lung, and Blood Institute training grant T32‐HL007227‐40 to T. Leucker.
Protection of Endothelial Function from Homocysteine‐induced Injury by Endothelial Nitric Oxide Synthase Enhancer in Human Internal Mammary Artery
Authors: Hou Hai‐Tao , Yang Qin , Wang Jun , Wang Zheng‐Qing , Chen Tie‐Nan , Liu Zhi‐Gang , Liu Xiao‐Cheng , He Guo‐Wei
Published: November 2023

Objective

Homocysteine (Hcy) is a sulfur‐containing amino acid formed during the metabolism of methionine. Hcy is an independent risk factor for endothelial dysfunction in cardiovascular diseases and atherosclerosis in the general population. We hypothesized that the eNOS transcription enhancer AVE3085 might improve the endothelial function altered by Hcy in the human internal mammary artery (IMA).

Methods

Cumulative concentration‐relaxation curves to acetylcholine (−10 to −4.5 log mol/L) were established in IMAs (n=28) from patients undergoing coronary artery bypass grafting in the precontraction induced by U46619 (−8 log mol/L) in the absence or presence of Hcy (100 μmol/L) with/without AVE3085 (30 μmol/L) in vitro in a myograph. Quantitative real‐time reverse transcription–polymerase chain reactions (RT‐qPCR) and enzyme‐linked immunosorbent assay (ELISA) were used to quantify the mRNA and protein levels of eNOS.

Results

Maximal relaxation induced by acetylcholine was significantly attenuated by Hcy in human IMA. Co‐incubation with AVE3085 protected endothelium from the impairment of Hcy. Exposure to Hcy for 24 h downregulated eNOS protein expression (P<0.05) whereas it upregulated the expression of eNOS at the mRNA level (P<0.05). The presence of AVE3085 in addition to Hcy significantly increased the eNOS protein (P<0.05) and slightly decreased the mRNA level.

Conclusions

Hcy caused endothelial dysfunction through the modulation of NO production associated with a downregulation of eNOS in human IMA. AVE3085 prevents endothelial dysfunction, which is attributed to upregulation of eNOS expression. These findings provide new insights into the protection of the endothelium of the coronary artery bypass grafting conduits and the possible improvement of the long‐term patency of the grafts. Therefore, the present study may have strong clinical implications regarding the long‐term results of coronary artery surgery.

Supported by National Natural Science Foundation of China (81641017), Zhejiang Provincial Natural Science Foundation (LY15H020008), Hangzhou Science and Technology Projects (20140633B12), & Tianjin Binhai Key Platform for Creative Research Program (2012‐BH110004), Binhai New Area Health Bureau (2016BWKZ003, 2016BWKY007, 2012BWKZ008)

Support or Funding Information

This study was supported by National Natural Science Foundation of China (81641017), Zhejiang Provincial Natural Science Foundation (LY15H020008), Hangzhou Science and Technology Projects (20140633B12), & Tianjin Binhai Key Platform for Creative Research Program (2012‐BH110004), Binhai New Area Health Bureau (2016BWKZ003, 2016BWKY007, 2012BWKZ008)