scholarly journals The Influence of AA29504 on GABAA Receptor Ligand Binding Properties and Its Implications on Subtype Selectivity

2021 ◽  
Author(s):  
Sylvia Sikstus ◽  
Ali Y. Benkherouf ◽  
Sanna L. Soini ◽  
Mikko Uusi-Oukari
Keyword(s):  
Radioligand Binding ◽  
Acid Ethyl Ester ◽  
Hek293 Cells ◽  
Allosteric Modulator ◽  
Carbamic Acid ◽  
Binding Properties ◽  
Knock Out ◽  
Recombinant Receptors ◽  
Subtype Selectivity ◽  
The Impact

AbstractThe unique pharmacological properties of δ-containing γ-aminobutyric acid type A receptors (δ-GABAARs) make them an attractive target for selective and persistent modulation of neuronal excitability. However, the availability of selective modulators targeting δ-GABAARs remains limited. AA29504 ([2-amino-4-(2,4,6-trimethylbenzylamino)-phenyl]-carbamic acid ethyl ester), an analog of K+ channel opener retigabine, acts as an agonist and a positive allosteric modulator (Ago-PAM) of δ-GABAARs. Based on electrophysiological studies using recombinant receptors, AA29504 was found to be a more potent and effective agonist in δ-GABAARs than in γ2-GABAARs. In comparison, AA29504 positively modulated the activity of recombinant δ-GABAARs more effectively than γ2-GABAARs, with no significant differences in potency. The impact of AA29504's efficacy- and potency-associated GABAAR subtype selectivity on radioligand binding properties remain unexplored. Using [3H]4'-ethynyl-4-n-propylbicycloorthobenzoate ([3H]EBOB) binding assay, we found no difference in the modulatory potency of AA29504 on GABA- and THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol)-induced responses between native forebrain GABAARs of wild type and δ knock-out mice. In recombinant receptors expressed in HEK293 cells, AA29504 showed higher efficacy on δ- than γ2-GABAARs in the GABA-independent displacement of [3H]EBOB binding. Interestingly, AA29504 showed a concentration-dependent stimulation of [3H]muscimol binding to γ2-GABAARs, which was absent in δ-GABAARs. This was explained by AA29504 shifting the low-affinity γ2-GABAAR towards a higher affinity desensitized state, thereby rising new sites capable of binding GABAAR agonists with low nanomolar affinity. Hence, the potential of AA29504 to act as a desensitization-modifying allosteric modulator of γ2-GABAARs deserves further investigation for its promising influence on shaping efficacy, duration and plasticity of GABAAR synaptic responses.

scholarly journals Synthesis and biological evaluation of novel N-substituted nipecotic acid derivatives with tricyclic cage structures in the lipophilic domain as GABA uptake inhibitors

2020 ◽  
Author(s):  
Heinrich-Karl A. Rudy ◽  
Georg Höfner ◽  
Klaus T. Wanner
Keyword(s):  
Alkyl Substituent ◽  
Acid Ethyl Ester ◽  
Biological Evaluation ◽  
Secondary Amines ◽  
Gaba Uptake ◽  
Nipecotic Acid ◽  
Spacer Length ◽  
Gaba Transporters ◽  
Subtype Selectivity ◽  
The Impact

AbstractA new class of GABA reuptake inhibitors with sterically demanding, highly rigid tricyclic cage structures as the lipophilic domain was synthesized and investigated in regard to their biological activity at the murine GABA transporters (mGAT1–mGAT4). The construction of these compounds, consisting of nipecotic acid, a symmetric tricyclic amine, and a plain hydrocarbon linker connecting the two subunits via their amino nitrogens, was accomplished via reductive amination of a nipecotic acid derivative with an N-alkyl substituent displaying a terminal aldehyde function with tricyclic secondary amines. The target compounds varied with regard to spacer length, the bridge size of one of the bridges, and the substituents of the tricyclic skeleton to study the impact of these changes on their potency. Among the tested compounds nipecotic acid ethyl ester derivates with phenyl residues attached to the cage subunit showed reasonable inhibitory potency and subtype selectivity in favor of mGAT3 and mGAT4, respectively.

scholarly journals Calcium phosphate phase transition in the presence of Aminosilane

2014 ◽  
Vol 70 (a1) ◽  
pp. C67-C67
Author(s):  
Babak Mostaghaci ◽  
Brigitta Loretz ◽  
Robert Haberkorn ◽  
Guido Kickelbick ◽  
Claus-Michael Lehr
Keyword(s):  
Phase Transition ◽  
Calcium Phosphate ◽  
Blood Compatibility ◽  
Transfection Efficiency ◽  
Hek293 Cells ◽  
Step Method ◽  
Calcium Phosphate Nanoparticles ◽  
Amine Groups ◽  
The Impact

Calcium phosphate has been the point of interest for in vitro gene delivery for many years because of its biocompatibility and straight forward application. However, there are some limitations regarding in vivo administration of these particles mostly because of vast agglomeration of the particles and lack of strong bond between the particles and pDNA. We introduced a simple single step method to functionalize calcium phosphate nanoparticles with Aminosilanes having a different number of amine groups. The nanoparticles were characterized chemically and structurally and their toxicity and interaction with pDNA were studied as well. Results revealed that different crystalline phase of calcium phosphate nanoparticles (Brushite and Hydroxyapatite) with a size below 150 nm were prepared, depending on conditions of synthesis and phase, each with a narrow size distribution. The aminosilane agents caused oriented nucleation and growth of crystallites and can decrease the pH for producing hydroxyapatite phase. The phenomenon could be revealed with the presence of anisotropy in the structure of synthesized hydroxyapatite. The number of amine groups in the Aminosilane agent could change the phase transition pH. Brushite particles revealed to have stronger interaction with pDNA mostly because of their higher positive surface charge. Both particles showed blood compatibility and negligible toxicity. Transfection experiment revealed the capability of both brushite and hydroxyapatite particles to transfect A549 and HEK293 cells. The new modified nanoparticles can be stored in a dried state and re-dispersed easily at the time of administration. Moreover, the transfection efficiency is higher in comparison with conventional calcium phosphate. This study showed the impact of presence and type of the modifying agent on the crystal structure and the amount of surface functionalization of nanoparticles, which in consequence influenced their interaction with cells.

scholarly journals Developmental up-regulation of NMDA receptors in the prefrontal cortex and hippocampus of mGlu5 receptor knock-out mice

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Tiziana Imbriglio ◽  
Remy Verhaeghe ◽  
Nico Antenucci ◽  
Stefania Maccari ◽  
Giuseppe Battaglia ◽  
...  
Keyword(s):  
Prefrontal Cortex ◽  
Nmda Receptor ◽  
Metabotropic Glutamate Receptors ◽  
Adult Life ◽  
Developmental Time ◽  
Postnatal Life ◽  
Developmental Studies ◽  
Knock Out ◽  
Nmda Receptor Subunits ◽  
The Impact

AbstractmGlu5 metabotropic glutamate receptors are highly expressed and functional in the early postnatal life, and are known to positively modulate NMDA receptor function. Here, we examined the expression of NMDA receptor subunits and interneuron-related genes in the prefrontal cortex and hippocampus of mGlu5−/− mice and wild-type littermates at three developmental time points (PND9, − 21, and − 75). We were surprised to find that expression of all NMDA receptor subunits was greatly enhanced in mGlu5−/− mice at PND21. In contrast, at PND9, expression of the GluN2B subunit was enhanced, whereas expression of GluN2A and GluN2D subunits was reduced in both regions. These modifications were transient and disappeared in the adult life (PND75). Changes in the transcripts of interneuron-related genes (encoding parvalbumin, somatostatin, vasoactive intestinal peptide, reelin, and the two isoforms of glutamate decarboxylase) were also observed in mGlu5−/− mice across postnatal development. For example, the transcript encoding parvalbumin was up-regulated in the prefrontal cortex of mGlu5−/− mice at PND9 and PND21, whereas it was significantly reduced at PND75. These findings suggest that in mGlu5−/− mice a transient overexpression of NMDA receptor subunits may compensate for the lack of the NMDA receptor partner, mGlu5. Interestingly, in mGlu5−/− mice the behavioral response to the NMDA channel blocker, MK-801, was significantly increased at PND21, and largely reduced at PND75. The impact of adaptive changes in the expression of NMDA receptor subunits should be taken into account when mGlu5−/− mice are used for developmental studies.

scholarly journals Metabolomic and transcriptomic analysis reveals endogenous substrates and metabolic adaptation in rats lacking Abcg2 and Abcb1a transporters

PLoS ONE ◽  
2021 ◽  
Vol 16 (7) ◽  
pp. e0253852
Author(s):  
Samit Ganguly ◽  
David Finkelstein ◽  
Timothy I. Shaw ◽  
Ryan D. Michalek ◽  
Kimberly M. Zorn ◽  
...  
Keyword(s):  
Uric Acid ◽  
Rat Plasma ◽  
Drug Clearance ◽  
Transcriptomic Analysis ◽  
Metabolic Adaptation ◽  
Sprague Dawley ◽  
Knock Out ◽  
Primary Driver ◽  
Endogenous Substrates ◽  
The Impact

Abcg2/Bcrp and Abcb1a/Pgp are xenobiotic efflux transporters limiting substrate permeability in the gastrointestinal system and brain, and increasing renal and hepatic drug clearance. The systemic impact of Bcrp and Pgp ablation on metabolic homeostasis of endogenous substrates is incompletely understood. We performed untargeted metabolomics of cerebrospinal fluid (CSF) and plasma, transcriptomics of brain, liver and kidney from male Sprague Dawley rats (WT) and Bcrp/Pgp double knock-out (dKO) rats, and integrated metabolomic/transcriptomic analysis to identify putative substrates and perturbations in canonical metabolic pathways. A predictive Bayesian machine learning model was used to predict in silico those metabolites with greater substrate-like features for either transporters. The CSF and plasma levels of 169 metabolites, nutrients, signaling molecules, antioxidants and lipids were significantly altered in dKO rats, compared to WT rats. These metabolite changes suggested alterations in histidine, branched chain amino acid, purine and pyrimidine metabolism in the dKO rats. Levels of methylated and sulfated metabolites and some primary bile acids were increased in dKO CSF or plasma. Elevated uric acid levels appeared to be a primary driver of changes in purine and pyrimidine biosynthesis. Alterations in Bcrp/Pgp dKO CSF levels of antioxidants, precursors of neurotransmitters, and uric acid suggests the transporters may contribute to the regulation of a healthy central nervous system in rats. Microbiome-generated metabolites were found to be elevated in dKO rat plasma and CSF. The altered dKO metabolome appeared to cause compensatory transcriptional change in urate biosynthesis and response to lipopolysaccharide in brain, oxidation-reduction processes and response to oxidative stress and porphyrin biosynthesis in kidney, and circadian rhythm genes in liver. These findings present insight into endogenous functions of Bcrp and Pgp, the impact that transporter substrates, inhibitors or polymorphisms may have on metabolism, how transporter inhibition could rewire drug sensitivity indirectly through metabolic changes, and identify functional Bcrp biomarkers.

scholarly journals Mechanism of Coup and Contrecoup Injuries Induced by a Knock-Out Punch

2020 ◽  
Vol 25 (2) ◽  
pp. 22 ◽  
Author(s):  
Milan Toma ◽  
Rosalyn Chan-Akeley ◽  
Christopher Lipari ◽  
Sheng-Han Kuo
Keyword(s):  
Cerebrospinal Fluid ◽  
Interaction Model ◽  
Brain Parenchyma ◽  
Primary Objective ◽  
Element Analysis ◽  
Knock Out ◽  
Particle Hydrodynamics ◽  
Smoothed Particle ◽  
The Impact ◽  
The Brain

Primary Objective: The interaction of cerebrospinal fluid with the brain parenchyma in an impact scenario is studied. Research Design: A computational fluid-structure interaction model is used to simulate the interaction of cerebrospinal fluid with a comprehensive brain model. Methods and Procedures: The method of smoothed particle hydrodynamics is used to simulate the fluid flow, induced by the impact, simultaneously with finite element analysis to solve the large deformations in the brain model. Main Outcomes and Results: Mechanism of injury resulting in concussion is demonstrated. The locations with the highest stress values on the brain parenchyma are shown. Conclusions: Our simulations found that the damage to the brain resulting from the contrecoup injury is more severe than that resulting from the coup injury. Additionally, we show that the contrecoup injury does not always appear on the side opposite from where impact occurs.

Binding Sites, Singly Bound States, and Conformation Coupling Shape GABA-Evoked Currents

2003 ◽  
Vol 89 (2) ◽  
pp. 871-883 ◽  
Author(s):  
Jerzy W. Mozrzymas ◽  
Andrea Barberis ◽  
Katarzyna Mercik ◽  
Ewa D. Z˙arnowska
Keyword(s):  
Binding Sites ◽  
Bound States ◽  
Time Course ◽  
Functional Coupling ◽  
Binding Properties ◽  
The Impact ◽  
Kinetics Of ◽  
Source Of Information ◽  
Respective Rate ◽  
Gating Scheme

The time course of GABA-evoked currents is the main source of information on the GABAAreceptor gating. Since the kinetics of these currents depends on the transitions between several receptor conformations, it is a major challenge to define the relations between current kinetics and the respective rate constants of the microscopic gating scheme. The aim of this study was to further explore the impact of different GABAA receptor conformations on the kinetics of currents elicited by ultra-fast GABA applications. We show that the rising phase and amplitude of GABA-evoked currents depend on desensitization and singly bound states. The occupancy of bound receptors depends not only on binding properties but also on opening/closing and desensitization. The impact of such functional coupling between channel states is critical in conditions of high non-equilibrium typical for synaptic transmission. The concentration dependence of the rising phase of the GABA-elicited current indicates positive cooperativity between agonist binding sites. We provide evidence that preequilibration at low GABA concentrations reduce GABA-evoked currents due to receptor trapping in a singly bound desensitized state.

scholarly journals Synthesis, Biological, and Computational Evaluation of Antagonistic, Chiral Hydrobenzoin Esters of Arecaidine Targeting mAChR M1

2020 ◽  
Vol 13 (12) ◽  
pp. 437
Author(s):  
Marius Ozenil ◽  
Jonas Aronow ◽  
Daniela Piljak ◽  
Chrysoula Vraka ◽  
Wolfgang Holzer ◽  
...  
Keyword(s):  
Binding Affinity ◽  
Acetylcholine Receptors ◽  
Radioligand Binding ◽  
Chinese Hamster ◽  
Muscarinic Acetylcholine Receptors ◽  
Ovary Cell ◽  
Computational Evaluation ◽  
Subtype Selectivity ◽  
Pet Tracer ◽  
Positron Emission

Muscarinic acetylcholine receptors (mAChRs) are a pivotal constituent of the central and peripheral nervous system. Yet, therapeutic and diagnostic applications thereof are hampered by the lack of subtype selective ligands. Within this work, we synthesized and chemically characterized three different stereoisomers of hydrobenzoin esters of arecaidine by NMR, HR-MS, chiral chromatography, and HPLC-logP. All compounds are structurally eligible for carbon-11 labeling and show appropriate stability in Dulbecco’s phosphate-buffered saline (DPBS) and F12 cell culture medium. A competitive radioligand binding assay on Chinese hamster ovary cell membranes comprising the human mAChR subtypes M1-M5 showed the highest orthosteric binding affinity for subtype M1 and a strong influence of stereochemistry on binding affinity, which corresponds to in silico molecular docking experiments. Ki values toward M1 were determined as 99 ± 19 nM, 800 ± 200 nM, and 380 ± 90 nM for the (R,R)-, (S,S)-, and racemic (R,S)-stereoisomer, respectively, highlighting the importance of stereochemical variations in mAChR ligand development. All three stereoisomers were shown to act as antagonists toward mAChR M1 using a Fluo-4 calcium efflux assay. With respect to future positron emission tomography (PET) tracer development, the (R,R)-isomer appears especially promising as a lead structure due to its highest subtype selectivity and lowest Ki value.

scholarly journals The Cell Envelope Stress Response of Bacillus subtilis towards Laspartomycin C

Antibiotics ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. 729
Author(s):  
Angelika Diehl ◽  
Thomas M. Wood ◽  
Susanne Gebhard ◽  
Nathaniel I. Martin ◽  
Georg Fritz
Keyword(s):  
Cell Wall ◽  
Bacillus Subtilis ◽  
Stress Response ◽  
Cell Envelope ◽  
Resistance Mechanisms ◽  
Detailed Knowledge ◽  
Knock Out ◽  
Lipid Ii ◽  
Envelope Stress ◽  
The Impact

Cell wall antibiotics are important tools in our fight against Gram-positive pathogens, but many strains become increasingly resistant against existing drugs. Laspartomycin C is a novel antibiotic that targets undecaprenyl phosphate (UP), a key intermediate in the lipid II cycle of cell wall biosynthesis. While laspartomycin C has been thoroughly examined biochemically, detailed knowledge about potential resistance mechanisms in bacteria is lacking. Here, we use reporter strains to monitor the activity of central resistance modules in the Bacillus subtilis cell envelope stress response network during laspartomycin C attack and determine the impact on the resistance of these modules using knock-out strains. In contrast to the closely related UP-binding antibiotic friulimicin B, which only activates ECF σ factor-controlled stress response modules, we find that laspartomycin C additionally triggers activation of stress response systems reacting to membrane perturbation and blockage of other lipid II cycle intermediates. Interestingly, none of the studied resistance genes conferred any kind of protection against laspartomycin C. While this appears promising for therapeutic use of laspartomycin C, it raises concerns that existing cell envelope stress response networks may already be poised for spontaneous development of resistance during prolonged or repeated exposure to this new antibiotic.

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