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 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 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.

Incidence of decay in creosote-treated Scots pine poles in Ireland

Holzforschung ◽  
2018 ◽  
Vol 72 (12) ◽  
pp. 1079-1086 ◽  
Author(s):  
Jed Cappellazzi ◽  
Karl Maguire ◽  
Rob Nelson ◽  
Jeffrey J. Morrell
Keyword(s):  
Scots Pine ◽  
White Rot ◽  
White Rot Fungus ◽  
Simple Method ◽  
Decay Fungi ◽  
Moisture Management ◽  
Large Numbers ◽  
Phlebiopsis Gigantea ◽  
Utility System ◽  
Sistotrema Brinkmannii

AbstractAir-seasoning is a simple method for moisture management in utility poles prior to treatment, but it involves the risk of fungal invasion during drying. These fungi can be eliminated by heat treatment, but fungi surviving in the installed poles are a quality problem. In this context, the incidence of decay fungi was investigated in 963 creosote-treated Scots pine (Pinus sylvestris) poles of varying ages in a utility system in Ireland. Thirty-seven percent of increment cores removed from the poles contained at least one viable basidiomycete. There was no relationship between pole age or distance above the groundline and fungal isolations.Phlebiopsis gigantea, a white rot fungus, was the most common isolate followed byNeolentinus lepideusandSistotrema brinkmannii. The results highlight the importance of including a sterilizing process during treatment and maintaining quality controls when purchasing large numbers of poles.

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.

Determination of Kinase Inhibitor Potencies in Cell Extracts by Competition Binding Assays and Isobaric Mass Tags

2011 ◽  
pp. 141-155 ◽  
Author(s):  
Carsten Hopf ◽  
Dirk Eberhard ◽  
Markus Boesche ◽  
Sonja Bastuck ◽  
Birgit Dümpelfeld ◽  
...  
Keyword(s):  
Kinase Inhibitor ◽  
Binding Assays ◽  
Cell Extracts ◽  
Competition Binding

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.

scholarly journals Circumsporozoite proteins of malaria parasites contain a single immunodominant region with two or more identical epitopes.

1983 ◽  
Vol 157 (6) ◽  
pp. 1947-1957 ◽  
Author(s):  
F Zavala ◽  
A H Cochrane ◽  
E H Nardin ◽  
R S Nussenzweig ◽  
V Nussenzweig
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Specific Binding ◽  
Polyclonal Antibodies ◽  
Binding Assays ◽  
Malaria Parasites ◽  
Immunodominant Region ◽  
Additional Finding ◽  
Single Region ◽  
Competition Binding

We have used panels of monoclonal antibodies to circumsporozoite (CS) proteins of Plasmodium falciparium, P. vivax, and P. knowlesi to determine the number of topographically independent epitopes of these antigens. The results of competition binding assays indicated that single regions of the CS molecules were recognized by the homologous monoclonal antibodies. Competition binding assays were also used to study the specificity of antibodies contained in the sera of humans and monkeys that had developed sterile immunity after immunization with irradiated, intact sporozoites. We found that single monoclonal antibodies inhibited 70-95% of the specific binding of the polyclonal antibodies to crude extracts of sporozoites. It appears, therefore, that CS proteins are among the most immunogenic constituents of sporozoites, and that a single region of these molecules contains most of the immunogenic activity. An additional finding was that the immunodominant region of CS molecules is multivalent with regard to the expression of a single epitope. This was demonstrated by the ability of monomers of CS proteins to bind simultaneously two or more molecules of the same monoclonal antibody.

Cell-Free Ligand Binding Assays for Nuclear Receptors

2003 ◽  
pp. 53-71 ◽  
Author(s):  
Stacey A Jones ◽  
Derek J Parks ◽  
Steven A Kliewer
Keyword(s):  
Ligand Binding ◽  
Nuclear Receptors ◽  
Free Ligand ◽  
Binding Assays
Sign in / Sign up

Export Citation Format

Share Document