bluuebunny/crossref_metadata_2025_split huggingface dataset:
The α7 nicotinic receptor positive allosteric modulator, PNU‐120596, reverses lipopolysaccharide‐induced depressive‐like behavior in mice
Authors: Alzarea Sami , Rahman Shafiqur 
Published: June 2021

Previous evidence indicates that neuroinflammation plays a critical role in the pathophysiology of major depressive disorder (MDD) involving microglial α7 nicotinic acetylcholine receptors (nAChRs). The objective of the present study was to determine the effects of PNU‐120596, an α7 nAChR positive allosteric modulator (PAM), on depressive‐like behavior and expression of ionized calcium binding adaptor molecule 1 (Iba‐1), microglial marker, in mice following lipopolysaccharide (LPS) administration, an animal model for depressive‐like behavior. Forced swim test (FST) and tail suspension test (TST) were used to determine the effects of PNU‐120596 on depressive‐like behavior. We also examined the effects of PNU‐120596 on Iba‐1 expression by using Western blot analysis and immunofluorescence staining in the hippocampus and prefrontal cortex, brain regions implicated in MDD. Administration of LPS (1 mg/kg, i.p.) significantly increased immobility time during FST and TST. The PNU‐120596 (1 or 4 mg/kg, i.p.) dose‐dependently decreased the LPS‐induced immobility time during FST and TST. Similarly, the PNU‐120596 (4 mg/kg, i.p.) significantly decreased the LPS‐induced increased Iba‐1 expression in the hippocampus and prefrontal cortex. In conclusion, these results suggest that PNU‐120596 reduces LPS‐induced depressive‐like behavior likely by targeting microglial α7 nAChRs in the hippocampus and prefrontal cortex in mice. Therefore, α7 nAChR PAMs could be developed as potential therapeutic utility for the treatment of MDD in humans.Support or Funding InformationSupported in part by grant from Saudi Arabian Cultural Mission, USAThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
AMPA receptor modulators selective for the accessory protein TARP‐g8
Authors: Maher Michael P. , Wu Nyantsz , Ravula Suchitra , Ameriks Michael K. , Savall Brad M. , Lord Brian , Seierstad Mark , Carruthers Nicholas I. , Lovenberg Timothy W. 
Published: June 2021

BackgroundThe α‐amino‐3‐hydroxyl‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) subtype of ionotropic glutamate receptors mediates the majority of fast synaptic transmission within the mammalian brain. The ubiquitous expression of the primary subunits of AMPA receptors (AMPARs), and the lack of pharmacological selectivity amongst them, preclude regional or neuronal subtype specificity. In vivo, AMPARs comprise a variety of accessory proteins. Of particular interest, TARP‐γ8 is highly expressed in the hippocampus, a region of the brain implicated in several psychiatric and neurological disorders.MethodsWe used high‐throughput screening to discover compounds that selectively modulate AMPARs containing TARP‐γ8. Subsequent medicinal chemistry efforts were used to improve potency and pharmacokinetics of the hits. Assays were developed to measure target occupancy and functional effects of the compounds in vivo.ResultsThese compounds possess a novel mechanism‐of‐action consistent with a partial attenuation of the interaction between the TARP and the pore‐forming subunits of the channel. Lead molecules with oral bioavailability and high brain penetration allowed demonstration of a strong relationship between pharmacokinetics and pharmacodynamics. The compounds show anticonvulsant and anxiolytic profiles in rodent. Molecules in this class provide large safety margins in preclinical species relative to non‐specific AMPAR antagonists due to the improved regional specificity of TARP‐γ8 modulators.ConclusionsAMPAR modulators selective for TARP‐γ8 have the potential to be novel treatments for anxiety/depression, bipolar disorder, temporal lobe epilepsy, and/or prodromal schizophrenia. This project also represents proof‐of‐concept for pharmacological targeting of accessory proteins and small‐molecule modulation of protein‐protein interactions.Support or Funding InformationAuthors are employees of Janssen R & D, LLCThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Ligand Binding to the β2‐Adrenergic Receptor is Dependent Upon its Oxidation State
Authors: Rambacher Kalyn M. , Moniri Nader H. 
Published: June 2021

Inhaled β2‐adrenergic receptor (β2AR) agonists are the gold standard treatment of an acute asthma attack and function by eliciting rapid bronchial dilation. Asthmatic tissue is characterized by increased expression of NADPH Oxidase (NOX) and subsequent increased levels of reactive oxygen species (ROS) compared to healthy tissue. We and others have previously showed that agonism of β2AR with ‐isoproterenol (ISO) leads to activation of NOX, and generation of intracellular ROS. We have also previously shown that these ROS are capable of oxidizing cysteine residues on the β2AR itself, a process known as cysteine‐S‐sulfenation. Emerging evidence has established cysteine‐S‐sulfenation as a functionally significant post translational protein modification, which thus far has not been evaluated for its effects on β2AR ligand binding. Utilizing isolated plasma membrane and whole cell radioligand binding, the current study evaluates oxidation state‐dependent alterations to ligand binding to the β2AR in HEK293 cells over‐expressing β2AR and in immortalized human lung epithelial cells. Specific ligand binding is significantly increased in the oxidized receptor state in both cell lines and in both membrane and whole cell assays. This effect is reversed by receptor labeling with the sulfenic acid selective small molecule dimedone, indicating increased ligand binding is specific to the presence of cysteine sulfenic acids on the β2AR. Furthermore, isoproterenol can fully displace radioligand binding in all redox states to the same extent in a monophasic manner, indicating redox state either directly or indirectly modifies the β2AR's orthosteric binding pocket. Given the oxidative environment native to asthmatic tissue, the findings presented here have implications for asthma treatment with β2AR agonists, and present a need to better understand how β2AR may interact differently with ligands in asthmatic tissue compared to healthy models.Support or Funding InformationThis work was funded by the NIH/NHLBI grant HL138603 to NHM.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 Assisted Design of Small Molecule KCa Channel Modulators
Authors: Wulff Heike , Nguyen Hai M. , Brown Brandon M. , Shim Heesung , Yarov‐Yarovoy Vladimir 
Published: June 2021

Pharmacological modulation of ion channels offers tremendous opportunities for the design of new drugs for neurological and cardiovascular diseases. However, in contrast to other therapeutic targets such as protein kinases, ion channels have not been popular with medicinal chemists in the pharmaceutical industry. One reason is the absence of co‐crystals of medically relevant channels with bound drug molecules, a prerequisite for structure based drug design. Using the Rosetta molecular modeling suite we generated homology models of the pore of the Ca2+‐activated K+ channel KCa3.1 and tested the models by first confirming previously mapped binding sites and visualizing the mechanism of TRAM‐34, senicapoc and NS6180 inhibition at the atomistic level. All three compounds block ion conduction directly by fully or partially occupying the site that would normally be occupied by K+ before it enters the selectivity filter. We then challenged the model to predict the receptor sites and mechanisms of action of the dihydropyridine nifedipine and an isosteric 4‐phenyl‐pyran. Rosetta predicted receptor sites for nifedipine in the fenestration region and for the 4‐phenyl‐pyran in the pore lumen, which could both be confirmed by site‐directed mutagenesis and electrophysiology. We further used Rosetta modeling to explain the KCa3.1 selectivity of the positive allosteric gating modulator SKA‐121, a compound that binds at the interface of the calmodulin N‐lobe and the calmodulin binding domain of these voltage‐independent Ca2+‐activated channels, where it “facilitates” mechanical opening (= increase open channel probability) at a given Ca2+ concentration. Rosetta Ligand provides a plausible explanation for why KCa channel activators in general are 5–10‐fold more potent in activating KCa3.1 than KCa2 channels. The presence of R362 creates an extensive “background” hydrogen‐bond network in the KCa3.1 interface pocket that stabilizes the main contacts NH2‐substituted KCa activators make with M51 and E54 in calmodulin. The three KCa2 channels have shorter N or S residues in the corresponding position and therefore cannot form this hydrogen‐bond network.Overall these examples illustrate that ion channels are highly accessible to small molecule drugs and we suggest that both the KCa3.1 pore and KCa3.1‐CaM‐BD/CaM model could be used for structure assisted drug design.Support or Funding InformationThis work was supported by the CounterACT Program, National Institutes of Health Office of the Director (NIH OD), and the National Institute of Neurological Disorders and Stroke (NINDS), Grant Number U54NS079202 and R21NS101876 (to H.W.).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Soluble Epoxide Hydrolase/Phosphodiesterase Dual Inhibitor is Efficacious Against Inflammatory Pain
Authors: Wagner K. M. , Bloecher Rene , Reddy Gopireddy R. , Morisseau Christophe , Hammock Bruce D. 
Published: June 2021

The soluble epoxide hydrolase (sEH) is the principal regulator of epoxy fatty‐acids (EpFA) transforming them in to the less active diols. Inhibitors of sEH block this activity and maintain levels of EpFA in vivo which has demonstrated potent analgesia in several preclinical models of acute and chronic pain. However, sEH inhibitors have no observable action in the absence of a pain state [1]. It was therefore hypothesized that an additional factor was present in the painful state that was required for the antinociceptive action of the EpFA [2]. Investigation began with cyclic adenosine monophosphate (cAMP) and experiments demonstrated this was active but also that increasing cAMP with phosphodiesterase inhibitors increased EpFA as well. Phosphodiesterase enzymes hydrolyze the cyclic nucleotide into adenosine monophosphate altering its secondary messenger capacity. This led to the design and synthesis of several dual inhibitors of sEH and phosphodiesterase.Multi‐target ligands are designed to improve safety and efficacy in treatment of diseases. The use of drug combinations cannot be assumed to be safe given drug‐drug interactions. Additionally, the prediction of pharmacodynamic and pharmacokinetic relationships is less complicated with a single entity. The multi‐target ligands are selected to improve therapeutic efficacy by hitting multiple targets in physiological disorders. Thus, increasing the EpFA by halting their degradation and at the same time potentiating their activity with an increase in cAMP was the goal with synthesis of the dual inhibitor. We present the biochemical characterization as well as the in vivo efficacy in an inflammatory pain model for the selected dual inhibitor of sEH and PDE enzymes. The IC50 of RBH61 for sEH determined on the sEH human recombinant enzyme is 2.1 ± 0.5 nM. This is paired with a IC50 in the PDE assay of 8.1 ± 0.05 nM in human embryonic kidney (HEK293) cells. Evaluation of the dual inhibitor in a model of inflammatory pain in rats demonstrates its antinociceptive efficacy.Support or Funding InformationThis work was supported, in part, by National Institutes of Health grants: NIEHS R01 ES002710 and NIEHS/Superfund Research Program P42 ES004699, NINDS U54 NS079202 and Grants NIEHS T32ES007059, NIH 5T32DC008072‐05 and 4T32HL086350‐09.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterization of a Novel Inhibitor of Soluble Epoxide Hydrolase and Role in Ocular Neovascularization
Authors: Park Bomina , Pasha Sheik Pran Babu Sardar , Si Yubing , Meroueh Samy O. , Seo Seung‐Yong , Corson Timothy W. 
Published: June 2021

ObjectiveOcular neovascularization causes blindness in diseases such as proliferative diabetic retinopathy (PDR) and wet age‐related macular degeneration (AMD). Today, the effort to treat wet AMD is hampered by patients who are resistant and refractory to the current standard of care, anti‐vascular endothelial growth factor (VEGF) therapies. Therefore, identification of novel therapeutic targets and inhibitors is desirable to address the unmet needs in antiangiogenic treatment. Previously, we have identified soluble epoxide hydrolase (sEH) as a binding target of an antiangiogenic small molecule, SH‐11037. Here, we aimed to further investigate sEH expression in the eye and the inhibition of its activity by SH‐11037.MethodsThe expression and localization of sEH in retinal sections of laser‐induced choroidal neovascularization (L‐CNV) mice were evaluated by immunohistochemistry. The sEH enzymatic activity in ocular tissue homogenates of L‐CNV and control mice was assayed using trans‐stilbene oxide (t‐SO) as substrate. The sEH inhibition by SH‐11037 and mode of inhibition was assessed by recombinant sEH activity assay using a fluorogenic substrate, PHOME. Docking and molecular dynamics simulations were performed to predict the binding mode of SH‐11037 within the active site of sEH. In addition, intravitreal injections of sEH substrate 19,20‐epoxy docosapentaenoic acid (EDP) and product 19,20‐dihydroxy docosapentaenoic acids (DHDP) were given to L‐CNV mice, and effects were assessed by imaging and choroidal flatmount immunofluorescence.ResultssEH was upregulated in the rod photoreceptors in L‐CNV mice compared to control mice and it did not co‐localize with other retinal cell type markers. Correspondingly, sEH activity was increased in L‐CNV eyes and was normalized by SH‐11037 or a known sEH inhibitor. In a recombinant enzyme assay, SH‐11037 inhibited sEH activity in a dose‐dependent manner, with IC50 = 0.15 μM. Enzyme kinetics analysis demonstrated that increasing concentrations of SH‐11037 decreased Vmax and increased KM, revealing SH‐11037 as a mixed‐type inhibitor of sEH, with Ki = 1.73 ± 0.45 μM. Docking of SH‐11037 to sEH showed an energetically favorable binding mode with the ligand spanning the active site of the enzyme and interacting with key catalytic residues. Intravitreal 19,20‐EDP reduced CNV lesion volume compared to vehicle and 19,20‐DHDP, confirming antiangiogenic properties of this sEH substrate.ConclusionOur studies confirm the relevance of sEH for ocular neovascularization and reveal the mechanism for SH‐11037's inhibition of sEH. Further optimization of SH‐11037's structure could yield more potent, novel sEH inhibitors.Support or Funding InformationNIH/NEI R01EY025641, Retina Research Foundation, Research to Prevent Blindness, Inc.
Modulation of Arachidonic Acid Metabolism in the Rat Kidney by Quercetin: Implications for Regulation of Blood Pressure
Authors: Elbarbry Fawzy , Harrelson John , Espiritu Michael , Pham Therese , Ibrahim Khaled 
Published: June 2021

BackgroundWe investigated the effects of quercetin (QR), a plant polyphenol from the flavonoid group, on Arachidonic acid (AA) metabolism in the kidney and its effect on arterial blood pressure, using spontaneously hypertensive rats (SHR) as our animal model system.MethodsRats were treated for 7 weeks with QR (10, 30, or 60 mg/L) added to their drinking water; drinking water alone was used as our control. Mean arterial pressure (MAP) was measured at 7‐day intervals throughout the study. At the end of the treatment, rats were euthanized, followed by preparation of kidney microsomes and cytosols to measure the activity (using LC/MS/MS) and expression (using immunoblotting) of enzymes involved in regulation of vasoactive metabolites: CYP4A, the key enzyme in the formation of 20‐hydroxyeicosatetraenoic acid, and the soluble epoxide hydrolase (sEH), which is responsible for the degradation of the vasodilator metabolites such as epoxyeicosatetraenoic acids.ResultsWe found that treatment with QR lead to significant reductions in both the expression and activity of renal CYP4A isozyme. Only the high dose QR resulted in a significant reduction in the expression, but not the activity, of renal sEH. Consistent with these data, we have found that treatment with QR resisted the progressive rise in MAP in the developing SHR in a dose‐dependent manner.ConclusionThis is the first demonstration that QR modulates the metabolism of AA by both P450 enzymes and sEH in SHR rats. This may represent a novel mechanism by which QR protects SHR rats against the progressive rise in blood pressure.Support or Funding InformationPacific University Research Incentive GrantThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Alterations in the Eicosanoid Profile and Mitochondrial Injury in Human Ventricular Tissue Following Myocardial Infarction
Authors: Jamieson Lockhart , Shah Saumya , Edin Matthew , Zeldin Darryl , Oudit Gavin Y. , Seubert John M. 
Published: June 2021

Study objectiveMyocardial infarction (MI) accounts for a significant proportion of death and disability in the ageing population. n‐6 PUFAs linoleic acid and arachidonic acid have come under investigation for their role in mediating a cardiac response following ischemic injury. CYP450 metabolism of these fatty acids results in formation of numerous metabolites, called eicosanoids, which can be further metabolised by the enzyme soluble epoxide hydrolase (sEH). These lipid mediators exhibit a wide range of cellular effects including alterations to mitochondria structure and function which may impact cardiac performance. This study investigated alterations to CYP‐derived eicosanoids and mitochondria in heart tissue obtained from individuals who experienced a previous MI.MethodsSamples were obtained from male and female individuals included in the Human Explanted Heart Program and correlated to non‐failing control hearts (NFC) collected from unused transplant donors through the Human Organ Procurement and Exchange Program at the University of Alberta. Ventricular tissues were harvested from patients who had previously experienced a LAD infarct (≥2yrs), as determined by echocardiography, ECG and coronary angiograms. Protein expression was determined by immunoblotting techniques. Mitochondrial enzymatic activities were assessed by spectrophotometric methods while mitochondrial ultrastructure and cristae density were assessed by electron microscopy. A metabolic profile in ventricular tissue was obtained by LCMS.ResultsMarked differences in both LA and AA metabolic profiles were revealed in post‐MI tissues. There were significant increases in cardiotoxic metabolites correlating with decreased cardiac function and injury. Interestingly, there was a significant increase in both sEH activity and expression in post‐MI tissues compared to NFC. Expression of mitochondrial proteins remained unchanged, however enzymatic function declined in post‐MI tissues compared to NFC correlating with marked disruption in mitochondrial ultrastructure.ConclusionsThese data provide the first evidence demonstrating a marked shift in eicosanoid metabolism in post‐MI hearts that correlates with mitochondrial structural disruption and overall decreased mitochondrial function.Support or Funding InformationSupport for this project was provided by the Canadian Institutes of Health Research.KL Jamieson is supported by funding from Alberta Innovates‐Health Solutions.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
EETs exacerbate chronic hypoxia‐induced pulmonary hypertension
Authors: Kandhi Sharath , Alruwaili Norah , Froogh Ghezal , Yang Yang‐Ming , Deng Wensheng , Wolin Michael S. , Huang An , Sun Dong 
Published: June 2021

We have demonstrated that epoxyeicosatrienoic acids (EETs), metabolites of arachidonic acids that are degraded by soluble epoxide hydrolase (sEH), initiate increases in right ventricular systolic pressure (RVSP) by perhaps, increasing pulmonary vascular resistance. Also, EETs enhance acute hypoxic pulmonary vasoconstriction (HPV) by shifting the balance between constrictor and dilator prostaglandins towards constrictor prostanoids. Prolonged HPV promotes hypoxia‐induced pulmonary hypertension (HPH), we therefore, tested the hypothesis that deletion of sEH gene (sEH‐KO) promotes EET‐mediated potentiation of HPH.MethodsMale sEH‐KO (12‐weel old) and age matched C57BL/6J (WT) mice were exposed to 10% O2 for 21 days. Cardiac hemodynamics were assessed by echocardiography and right heart catheterization. Right ventricular hypertrophy (RVH) was expressed as the weight ratio of right ventricle (RV) to left ventricle (LV) with septum (RV/LV+S). sEH expression in pulmonary tissue was assessed by western blot. Pulmonary EET levels were measured by LC‐MS/MS analysis.ResultsPulmonary doppler flow echocardiography showed that hypoxia elicited decreases in pulmonary arterial acceleration time (PAAT), ejection time (ET), and PAAT/ET ratio. The reduction is significantly greater in sEH‐KO mice compared to that of WT mice. Baseline RVSP was comparable between WT and sEH‐KO mice. Hypoxia exposure significantly increased RVSP in both strains of mice, the increment however, was significantly greater, associated with greater RVH in sEH‐KO mice compared to their WT controls. This indicates that sEH‐KO mice developed more severe HPH. Although chronic hypoxia also downregulated pulmonary sEH expression in WT mice, sEH‐KO mice exhibited highest level of EETs that correlated to their greater increases in RVSP and more severe HPH. In conclusion, the deficiency of sEH promotes EET‐mediated exacerbation of HPH.Support or Funding Information(This work was supported by HL070653, HL115124 and HL129797).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.