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 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.

scholarly journals Research fronts of Chemical Biology

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Shanshan Lv
Keyword(s):  
High Throughput Screening ◽  
Drug Targets ◽  
Chemical Biology ◽  
Rational Design ◽  
Future Research ◽  
Design Strategies ◽  
Binding Assays ◽  
Cellular Mechanisms ◽  
Diagnostic Technologies

Abstract Over the past decades, researchers have witnessed substantially increasing and ever-growing interests and efforts in Chemical Biology studies, thanks to the development of genome and epi-genome sequencing (revealing potential drug targets), synthetic chemistry (producing new medicines), bioorthogonal chemistry (chemistry in living systems) and high-throughput screening technologies (in vitro cell systems, protein binding assays and phenotypic assays). This report presents literature search results for current research in Chemical Biology, to explore basic principles, summarize recent advances, identify key challenges, and provide suggestions for future research (with a focus on Chemical Biology in the context of human health and diseases). Chemical Biology research can positively contribute to delivering a better understanding of the molecular and cellular mechanisms that accompany pathology underlying diseases, as well as developing improved methods for diagnosis, drug discovery, and therapeutic delivery. While much progress has been made, as shown in this report, there are still further needs and opportunities. For instance, pressing challenges still exist in selecting appropriate targets in biological systems and adopting more rational design strategies for the development of innovative and sustainable diagnostic technologies and medical treatments. Therefore, more than ever, researchers from different disciplines need to collaborate to address the challenges in Chemical Biology.

A novel small-molecule screening strategy identifies mitoxantrone as a RhoGTPase inhibitor

2013 ◽  
Vol 450 (1) ◽  
pp. 55-62 ◽  
Author(s):  
Aurélien Bidaud-Meynard ◽  
Daniela Arma ◽  
Said Taouji ◽  
Michel Laguerre ◽  
Jean Dessolin ◽  
...  
Keyword(s):  
Small Molecule ◽  
High Throughput Screening ◽  
Large Scale ◽  
Topoisomerase Ii ◽  
Screening Strategy ◽  
Filamentous Actin ◽  
Library Screen ◽  
Rac1 Gtpase ◽  
Gtpase Activation

RhoGTPases are GDP/GTP molecular switches that control a wide variety of cellular processes, thereby contributing to many diseases, including cancer. As a consequence, there is great interest in the identification of small-molecule inhibitors of RhoGTPases. In the present paper, using the property of GTP-loaded RhoGTPases to bind to their effectors, we describe a miniaturized and robust assay to monitor Rac1 GTPase activation that is suitable for large-scale high-throughput screening. A pilot compound library screen revealed that the topoisomerase II poison MTX (mitoxantrone) is an inhibitor of Rac1, and also inhibits RhoA and Cdc42 in vitro. We show that MTX prevents GTP binding to RhoA/Rac1/Cdc42 in vitro. Furthermore, MTX strongly inhibits RhoGTPase-mediated F-actin (filamentous actin) reorganization and cell migration. Hence, we report a novel biochemical assay yielding the identification of RhoGTPase inhibitors and we present a proof-of-concept validation with the identification of MTX as a novel pan-RhoGTPase inhibitor.

scholarly journals A High-Throughput Ligand Competition Binding Assay for the Androgen Receptor and Other Nuclear Receptors

2008 ◽  
Vol 14 (1) ◽  
pp. 43-48 ◽  
Author(s):  
Clémentine Féau ◽  
Leggy A. Arnold ◽  
Aaron Kosinski ◽  
R. Kiplin Guy
Keyword(s):  
Androgen Receptor ◽  
Nuclear Receptors ◽  
High Throughput ◽  
High Throughput Screening ◽  
Low Cost ◽  
Environmental Chemicals ◽  
Binding Assays ◽  
Receptor Ligands ◽  
Competition Binding ◽  
Nuclear Receptor Ligands

Standardized, automated ligand-binding assays facilitate evaluation of endocrine activities of environmental chemicals and identification of antagonists of nuclear receptor ligands. Many current assays rely on fluorescently labeled ligands that are significantly different from the native ligands. The authors describe a radiolabeled ligand competition scintillation proximity assay (SPA) for the androgen receptor (AR) using Ni-coated 384-well FlashPlates® and liganded AR-LBD protein. This highly reproducible, low-cost assay is well suited for automated high-throughput screening. In addition, the authors show that this assay can be adapted to measure ligand affinities for other nuclear receptors (peroxisome proliferation-activated receptor γ, thyroid receptors α and β). ( Journal of Biomolecular Screening 2009:43-48)

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 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.

scholarly journals Miniaturized Receptor Binding Assays: Complications Arising from Ligand Depletion

2007 ◽  
Vol 12 (2) ◽  
pp. 255-266 ◽  
Author(s):  
Clare M. Scaramellini Carter ◽  
Juliet R. Leighton-Davies ◽  
Steven J. Charlton
Keyword(s):  
High Throughput Screening ◽  
Free Ligand ◽  
Binding Assays ◽  
Simple Method ◽  
Ligand Affinity ◽  
Large Numbers ◽  
Single Target ◽  
Competition Binding ◽  
Ligand Depletion ◽  
Assay Volume

The advent of miniaturized assay formats has made possible the screening of large numbers of compounds against a single target, known as high-throughput screening. Despite this clear advantage, assay miniaturization also increases the risk of ligand depletion, where the actual concentration of free ligand is significantly lower than that added. This, in turn, complicates the interpretation of data from such assays, potentially introducing significant error if not recognized. In this study, the effects of reducing assay volume on radioligand Kd and competitor Ki values have been investigated, using the muscarinic M3 receptor as a model system. It was found that assay miniaturization caused dramatic effects, with up to a 30-fold underestimation of ligand affinity. A theoretical model was developed and shown to accurately predict both the degree of ligand depletion in any given assay volume and the effect of this depletion on affinity estimates for competing ligands. Importantly, it was found that in most cases, errors introduced by ligand depletion could be largely corrected for by the use of appropriate analysis methods. In addition to those previously described by others, the authors propose a simple method capable of correcting errors in competition binding experiments performed in conditions of ligand depletion.

scholarly journals Adaptive Focused Acoustics (AFA) Improves the Performance of Microtiter Plate ELISAs

2014 ◽  
Vol 19 (7) ◽  
pp. 1124-1130 ◽  
Author(s):  
David J. Green ◽  
Edwin A. Rudd ◽  
James A. Laugharn
Keyword(s):  
High Throughput Screening ◽  
Microtiter Plate ◽  
Antibody Binding ◽  
Binding Assays ◽  
Coefficients Of Variation ◽  
Assay Performance ◽  
Antigen Antibody ◽  
Lower Limits ◽  
Hiv 1

We investigated the use of Adaptive Focused Acoustics (AFA) technology to improve the performance of microtiter plate enzyme-linked immunosorbent assays (ELISAs). Experiments were performed with commercially available AFA instrumentation and off-the-shelf 96-well microtiter plate sandwich ELISAs. AFA was applied over a range of acoustic energies, temperatures, and durations to the antigen/antibody binding step of an ELISA for measuring HIV-1 p24 in tissue culture samples. AFA-mediated antigen/antibody binding was enhanced up to 2-fold over passive binding at comparable temperatures and was superior or comparable at low temperature (8–10 °C) to passive binding at 37 °C. Lower nonspecific binding (NSB), lower inter- and intra-assay coefficients of variation (CVs), higher Z′ factors, and lower limits of detection (LODs) were measured in AFA-mediated assays compared with conventional passive binding. In a more limited study, AFA enhancement of antigen/antibody binding and lower NSB was measured in an ELISA for measuring IGFBP-3 in human plasma. We conclude from this study that application of AFA to antigen/antibody binding steps in microtiter plate ELISAs can enhance key assay performance parameters, particularly Z′ factors and LODs. These features render AFA-mediated binding assays potentially more useful in applications such as high-throughput screening and in vitro diagnostics than assays processed with conventional passive antigen/antibody binding steps.

scholarly journals Neuronal Ceroid Lipofuscinoses Are Connected at Molecular Level: Interaction of CLN5 Protein with CLN2 and CLN3

2002 ◽  
Vol 13 (7) ◽  
pp. 2410-2420 ◽  
Author(s):  
Jouni Vesa ◽  
Mark H. Chin ◽  
Kathrin Oelgeschläger ◽  
Juha Isosomppi ◽  
Esteban C. DellAngelica ◽  
...  
Keyword(s):  
Molecular Level ◽  
Cell Types ◽  
Neuronal Ceroid Lipofuscinoses ◽  
Binding Assays ◽  
Intracellular Targeting ◽  
Disease Mutations ◽  
Vitro Binding ◽  
Multiple Cell ◽  
Membrane Bound

Neuronal ceroid lipofuscinoses (NCLs) are neurodegenerative storage diseases characterized by mental retardation, visual failure, and brain atrophy as well as accumulation of storage material in multiple cell types. The diseases are caused by mutations in the ubiquitously expressed genes, of which six are known. Herein, we report that three NCL disease forms with similar tissue pathology are connected at the molecular level: CLN5 polypeptides directly interact with the CLN2 and CLN3 proteins based on coimmunoprecipitation and in vitro binding assays. Furthermore, disease mutations in CLN5 abolished interaction with CLN2, while not affecting association with CLN3. The molecular characterization of CLN5 revealed that it was synthesized as four precursor forms, due to usage of alternative initiator methionines in translation. All forms were targeted to lysosomes and the longest form, translated from the first potential methionine, was associated with membranes. Interactions between CLN polypeptides were shown to occur with this longest, membrane-bound form of CLN5. Both intracellular targeting and posttranslational glycosylation of the polypeptides carrying human disease mutations were similar to wild-type CLN5.

Increased Protein Synthesis by Human Platelets during Phagocytosis of Latex Particles in Vitro

1976 ◽  
Vol 35 (02) ◽  
pp. 350-357 ◽  
Author(s):  
Hana Bessler ◽  
Galila Agam ◽  
Meir Djaldetti
Keyword(s):  
Protein Synthesis ◽  
Fold Increase ◽  
Human Platelets ◽  
Polystyrene Latex ◽  
Latex Particles ◽  
The Third ◽  
Platelet Protein ◽  
Dose Dependent ◽  
Phagocytotic Activity

SummaryA three-fold increase of protein synthesis by human platelets during in vitro phagocytosis of polystyrene latex particles was detected. During the first two hours of incubation, the percentage of phagocytizing platelets and the number of latex particles per platelet increased; by the end of the third hour, the first parameter remained stable, while the number of latex particles per cell had decreased.Vincristine (20 μg/ml of cell suspension) inhibited platelet protein synthesis. This effect was both time- and dose-dependent. The drug also caused a decrease in the number of phagocytizing cells, as well as in their phagocytotic activity.

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