scholarly journals Comparative biochemical and pharmacological investigations of various newly developed opioid receptor ligands

2021 ◽  
Author(s):  
Edina Szűcs
Keyword(s):  
Rat Brain ◽  
G Protein ◽  
Antinociceptive Effect ◽  
Peptide Ligand ◽  
Selectivity Ratio ◽  
Binding Assays ◽  
Brain Membrane ◽  
Activity Measurements ◽  
Competition Binding

Bentley analogues: In vitro competition binding experiments all derivatives showed low subnanomolar affinity to MOR. For DOR the ligands showed comparable binding affinities than the selective DOR agonist Ile5,6-deltorphin II peptide ligand except 8 (Ki > 3000 nM). In the KOR binding assays the analogues still displayed nanomolar affinities. In G-protein activity measurements compound 1f, 2a, 2b had antagonistic; 1e, 2c, 8 had partial agonistic and 2d, 4, 5, 7 had full agonistic effects. Ligands were examined in G-protein activation tests in rat brain membranes, the selectivity could not be observed as the receptor selective antagonists such as Cyp, NTI, nor-BNI and the selective agonists such as DAMGO, Ile5,6-deltorphine II, U-69,593 are not able to inhibit the effects of the extremely potent Bentley analogues. In vivo tests in osteoarthritis inflammatory model the thevinol derivatives showed a significant antiallodynic effect, while orvinol derivatives, except for 2c, did not display this effect. Oligopeptides: In competition binding assays the KYNA‐containing peptide, KA1 bound selectively to the MOR with a low Ki value and a high selectivity ratio, the other oligopeptides also showed selectivity to MOR, except K3, which bound to MOR and DOR with similar affinity. In the G-protein activition tests the EM-2 containing compounds, K2 and K3 stimulated G-protein with low efficacy, compound KA1, K4, K5 behaved as full agonists, while K6 had efficacy and potency higher than those of the reference compound DAMGO. In functional binding assays all oligopetides were inhibited by Cyp (MOR) and NTI (DOR) in rat brain membrane. In guinea pig brain membrane K4 and K6 stimulated G-protein, the efficacy of K4 was inhibited by nor-BNI, while the effect of K6 was not. K6 exhibited a strong antinociceptive effect in formalin test.

scholarly journals Phosphorylation-dependent interaction of the synaptic vesicle proteins cysteine string protein and synaptotagmin I

2002 ◽  
Vol 364 (2) ◽  
pp. 343-347 ◽  
Author(s):  
Gareth J.O. EVANS ◽  
Alan MORGAN
Keyword(s):  
Rat Brain ◽  
Direct Interaction ◽  
Secretory Vesicle ◽  
Brain Membrane ◽  
Synaptotagmin I ◽  
Phosphorylated Form ◽  
Order Of Magnitude ◽  
And Function

The secretory vesicle cysteine string proteins (CSPs) are members of the DnaJ family of chaperones, and function at late stages of Ca2+-regulated exocytosis by an unknown mechanism. To determine novel binding partners of CSPs, we employed a pull-down strategy from purified rat brain membrane or cytosolic proteins using recombinant hexahistidine-tagged (His6-)CSP. Western blotting of the CSP-binding proteins identified synaptotagmin I to be a putative binding partner. Furthermore, pull-down assays using cAMP-dependent protein kinase (PKA)-phosphorylated CSP recovered significantly less synaptotagmin. Complexes containing CSP and synaptotagmin were immunoprecipitated from rat brain membranes, further suggesting that these proteins interact in vivo. Binding assays in vitro using recombinant proteins confirmed a direct interaction between the two proteins and demonstrated that the PKA-phosphorylated form of CSP binds synaptotagmin with approximately an order of magnitude lower affinity than the non-phosphorylated form. Genetic studies have implicated each of these proteins in the Ca2+-dependency of exocytosis and, since CSP does not bind Ca2+, this novel interaction might explain the Ca2+-dependent actions of CSP.

scholarly journals Comparative Effectiveness of Calabadion and Sugammadex to Reverse Non-depolarizing Neuromuscular-blocking Agents

2015 ◽  
Vol 123 (6) ◽  
pp. 1337-1349 ◽  
Author(s):  
Friederike Haerter ◽  
Jeroen Cedric Peter Simons ◽  
Urs Foerster ◽  
Ingrid Moreno Duarte ◽  
Daniel Diaz-Gil ◽  
...  
Keyword(s):  
Dose Response ◽  
Urine Analysis ◽  
Ex Vivo ◽  
Neuromuscular Blocking ◽  
Response Relationship ◽  
Binding Assays ◽  
Dose Response Relationship ◽  
Competition Binding ◽  
Relationship Of

Abstract Background The authors evaluated the comparative effectiveness of calabadion 2 to reverse non-depolarizing neuromuscular-blocking agents (NMBAs) by binding and inactivation. Methods The dose–response relationship of drugs to reverse vecuronium-, rocuronium-, and cisatracurium-induced neuromuscular block (NMB) was evaluated in vitro (competition binding assays and urine analysis), ex vivo (n = 34; phrenic nerve hemidiaphragm preparation), and in vivo (n = 108; quadriceps femoris muscle of the rat). Cumulative dose–response curves of calabadions, neostigmine, or sugammadex were created ex vivo at a steady-state deep NMB. In living rats, the authors studied the dose–response relationship of the test drugs to reverse deep block under physiologic conditions, and they measured the amount of calabadion 2 excreted in the urine. Results In vitro experiments showed that calabadion 2 binds rocuronium with 89 times the affinity of sugammadex (Ka = 3.4 × 109 M−1 and Ka = 3.8 × 107 M−1). The results of urine analysis (proton nuclear magnetic resonance), competition binding assays, and ex vivo study obtained in the absence of metabolic deactivation are in accordance with an 1:1 binding ratio of sugammadex and calabadion 2 toward rocuronium. In living rats, calabadion 2 dose-dependently and rapidly reversed all NMBAs tested. The molar potency of calabadion 2 to reverse vecuronium and rocuronium was higher compared with that of sugammadex. Calabadion 2 was eliminated renally and did not affect blood pressure or heart rate. Conclusions Calabadion 2 reverses NMB induced by benzylisoquinolines and steroidal NMBAs in rats more effectively, i.e., faster than sugammadex. Calabadion 2 is eliminated in the urine and well tolerated in rats.

α1-Adrenergic receptor involvement in norepinephrine–ethanol inhibition of rat brain Na+-K+ ATPase and in ethanol tolerance

1985 ◽  
Vol 63 (9) ◽  
pp. 1075-1079 ◽  
Author(s):  
N. Rangaraj ◽  
H. Kalant ◽  
F. Beaugé
Keyword(s):  
Rat Brain ◽  
Inhibitory Effect ◽  
Dependent Manner ◽  
Receptor Antagonists ◽  
Concentration Dependent Manner ◽  
Brain Membrane ◽  
Hypothermic Effect ◽  
Wb 4101 ◽  
Number Of Binding Sites

Norepinephrine (NE) sensitization of rat brain Na+–K+ ATPase to ethanol (EtOH) inhibition appears to be mediated by α1-adrenoreceptors, since it was reversed by prazosin and WB-4101 (α1-receptor antagonists) in a concentration-dependent manner, but not by yohimbine and piperoxan (α2-receptor antagonists). In addition, clonidine (α2-agonist) and methoxamine (central receptor type uncertain) produced very little sensitization. Chronic EtOH administration to rats for 3 weeks produced tolerance to the hypothermic effect of test doses of EtOH (3 g/kg, i.p.) and a decreased inhibitory effect of NE + EtOH on the enzyme in vitro. This inhibition was still prevented by prazosin and WB-4101. However, the binding of tritiated WB-4101 and prazosin to brain membrane preparations from control and EtOH-tolerant rats revealed that the maximum number of binding sites (Bmax) and the dissociation constant (KD) of α1-adrenoreceptors were decreased after tolerance development. These changes in numbers and binding properties of α1-adrenoreceptors probably account for the decreased NE sensitization of the ATPase to EtOH inhibition in preparations from EtOH-tolerant rats.

Interaction of putative anxiolytic agents with central adenosine receptors

1981 ◽  
Vol 59 (8) ◽  
pp. 897-900 ◽  
Author(s):  
Michael Williams ◽  
Edwin A. Risley ◽  
Joel R. Huff
Keyword(s):  
Rat Brain ◽  
Adenosine Receptors ◽  
Molecular Mechanisms ◽  
Binding Assays ◽  
Receptor Level ◽  
Brain Membranes ◽  
Rat Brain Membranes ◽  
Adenosine Antagonist ◽  
Benzodiazepine Anxiolytics

The benzodiazepine anxiolytics flurazepam and diazepam and CL 218872, zopiclone and two β-carboline ethyl carboxyl esters, compounds which are potent displacers of specific [3H]diazepam binding from rat brain membranes, have little or no activity in displacing [3H]2-chloroadenosine ([3H]2-CADO) from central A1-adenosine receptors. Conversely, the purine agonists, 1-N6-phenylisopropyladenosine, N6-cyclohexyladenosine, 2-chloroadenosine. and the adenosine antagonist 8-phenyltheophylline have no significant effect on [3H]diazepam binding. Etazolate (SQ 20009) and Avermectin B1a which enhance [3H]diazepam binding in vitro were also without significant effect on [3H]2-CADO binding. The lack of correlation of the activities of the compounds examined in the two binding assays is discussed in relation to the hypothesis that purine-like compounds may be involved in the molecular mechanisms related to anxiolytic action at the receptor level.

scholarly journals Dissection of Complex Molecular Interactions of Neurofascin with Axonin-1, F11, and Tenascin-R, Which Promote Attachment and Neurite Formation of Tectal Cells

1998 ◽  
Vol 142 (4) ◽  
pp. 1083-1093 ◽  
Author(s):  
Hansjürgen Volkmer ◽  
Ute Zacharias ◽  
Ursel Nörenberg ◽  
Fritz G. Rathjen
Keyword(s):  
Alternative Splicing ◽  
Neurite Outgrowth ◽  
Molecular Interactions ◽  
Cell Attachment ◽  
Binding Assays ◽  
Ig Superfamily ◽  
Competition Binding ◽  
Neurite Formation ◽  
Additional Protein

Neurofascin is a member of the L1 subgroup of the Ig superfamily that promotes axon outgrowth by interactions with neuronal NgCAM-related cell adhesion molecule (NrCAM). We used a combination of cellular binding assays and neurite outgrowth experiments to investigate mechanisms that might modulate the interactions of neurofascin. In addition to NrCAM, we here demonstrate that neurofascin also binds to the extracellular matrix glycoprotein tenascin-R (TN-R) and to the Ig superfamily members axonin-1 and F11. Isoforms of neurofascin that are generated by alternative splicing show different preferences in ligand binding. While interactions of neurofascin with F11 are only slightly modulated, binding to axonin-1 and TN-R is strongly regulated by alternatively spliced stretches located in the NH2-terminal half, and by the proline-alanine-threonine-rich segment. In vitro neurite outgrowth and cell attachment assays on a neurofascin-Fc substrate reveal a shift of cellular receptor usage from NrCAM to axonin-1, F11, and at least one additional protein in the presence of TN-R, presumably due to competition of the neurofascin– NrCAM interaction. Thereby, F11 binds to TN-R of the neurofascin/TN-R complex, but not to neurofascin, whereas axonin-1 is not able to bind directly to the neurofascin/TN-R complex as shown by competition binding assays. In conclusion, these investigations indicate that the molecular interactions of neurofascin are regulated at different levels, including alternative splicing and by the presence of interacting proteins.

In vitro autoradiography of serotonin 5-HT2A/2C receptor-activated G protein: Guanosine-5?-(?-[35S]thio)triphosphate binding in rat brain

2000 ◽  
Vol 61 (6) ◽  
pp. 674-685 ◽  
Author(s):  
Mella Adlersberg ◽  
Victoria Arango ◽  
Shu-chi Hsiung ◽  
J. John Mann ◽  
Mark D. Underwood ◽  
...  
Keyword(s):  
Rat Brain ◽  
G Protein ◽  
In Vitro Autoradiography

scholarly journals Identification of A Novel Class of Benzofuran Oxoacetic Acid-Derived Ligands that Selectively Activate Cellular EPAC1

Cells ◽  
2019 ◽  
Vol 8 (11) ◽  
pp. 1425 ◽  
Author(s):  
Elizabeth M. Beck ◽  
Euan Parnell ◽  
Angela Cowley ◽  
Alison Porter ◽  
Jonathan Gillespie ◽  
...  
Keyword(s):  
Cyclic Nucleotide ◽  
High Throughput Screening ◽  
Partial Agonist ◽  
Fold Increase ◽  
Binding Assays ◽  
Camp Analogue ◽  
Competition Binding ◽  
Multiple Cell ◽  
Gtpase Activation

Cyclic AMP promotes EPAC1 and EPAC2 activation through direct binding to a specific cyclic nucleotide-binding domain (CNBD) within each protein, leading to activation of Rap GTPases, which control multiple cell responses, including cell proliferation, adhesion, morphology, exocytosis, and gene expression. As a result, it has become apparent that directed activation of EPAC1 and EPAC2 with synthetic agonists may also be useful for the future treatment of diabetes and cardiovascular diseases. To identify new EPAC agonists we have developed a fluorescent-based, ultra-high-throughput screening (uHTS) assay that measures the displacement of binding of the fluorescent cAMP analogue, 8-NBD-cAMP to the EPAC1 CNBD. Triage of the output of an approximately 350,000 compound screens using this assay identified a benzofuran oxaloacetic acid EPAC1 binder (SY000) that displayed moderate potency using orthogonal assays (competition binding and microscale thermophoresis). We next generated a limited library of 91 analogues of SY000 and identified SY009, with modifications to the benzofuran ring associated with a 10-fold increase in potency towards EPAC1 over SY000 in binding assays. In vitro EPAC1 activity assays confirmed the agonist potential of these molecules in comparison with the known EPAC1 non-cyclic nucleotide (NCN) partial agonist, I942. Rap1 GTPase activation assays further demonstrated that SY009 selectively activates EPAC1 over EPAC2 in cells. SY009 therefore represents a novel class of NCN EPAC1 activators that selectively activate EPAC1 in cellulae.

scholarly journals Cell-Free Assay of G-Protein-Coupled Receptors Using Fluorescence Polarization

2008 ◽  
Vol 13 (5) ◽  
pp. 424-429 ◽  
Author(s):  
Jessi Wildeson Jones ◽  
Tiffani A. Greene ◽  
Christine A. Grygon ◽  
Benjamin J. Doranz ◽  
Martha P. Brown
Keyword(s):  
G Protein ◽  
Fluorescence Polarization ◽  
G Protein Coupled Receptors ◽  
Peptide Ligand ◽  
Binding Assays ◽  
Free System ◽  
Molecular Binding ◽  
Soluble Cell ◽  
G Protein Coupled ◽  
Peptide Interaction

A recently developed nanotechnology, the Integral Molecular lipoparticle, provides an essentially soluble cell-free system in which G-protein-coupled receptors (GPCRs) in their native conformations are concentrated within virus-like particles. As a result, the lipoparticle provides a means to overcome 2 common obstacles to the development of homogeneous, nonradioactive GPCR ligand-binding assays: membrane protein solubilization and low receptor density. The work reported here describes the first application of this nanotechnology to a fluorescence polarization (FP) molecular binding assay format. The GPCR chosen for these studies was the well-studied chemokine receptor CXCR4 for which a peptide ligand (T-22) has been previously characterized. The EC50 determined for the CXCR4-T-22 peptide interaction via FP with CXCR4 lipoparticles (15 nM) is consistent with the IC50 determined for the unlabeled T-22 peptide via competitive binding (59 nM). ( Journal of Biomolecular Screening 2008:424-429)

scholarly journals Structure of the Lifeact–F-actin complex

2020 ◽  
Author(s):  
Alexander Belyy ◽  
Felipe Merino ◽  
Oleg Sitsel ◽  
Stefan Raunser
Keyword(s):  
Binding Pocket ◽  
Actin Binding ◽  
Binding Assays ◽  
Filamentous Actin ◽  
Activity Measurements ◽  
High Resolution Structure ◽  
Hydrophobic Binding ◽  
Binding Loop

AbstractLifeact is a short actin-binding peptide that is used to visualize filamentous actin (F-actin) structures in live eukaryotic cells using fluorescence microscopy. However, this popular probe has been shown to alter cellular morphology by affecting the structure of the cytoskeleton. The molecular basis for such artefacts is poorly understood. Here, we determined the high-resolution structure of the Lifeact–F-actin complex using electron cryo-microscopy. The structure reveals that Lifeact interacts with a hydrophobic binding pocket on F-actin and stretches over two adjacent actin subunits, stabilizing the DNase I-binding loop of actin in the closed conformation. Interestingly, the hydrophobic binding site is also used by actin-binding proteins, such as cofilin and myosin and actin-binding toxins, such as TccC3HVR from Photorhabdus luminescens and ExoY from Pseudomonas aeruginosa. In vitro binding assays and activity measurements demonstrate that Lifeact indeed competes with these proteins, providing an explanation for the altering effects of Lifeact on cell morphology in vivo. Finally, we demonstrate that the affinity of Lifeact to F-actin can be increased by introducing mutations into the peptide, laying the foundation for designing improved actin probes for live cell imaging.

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