Miniaturized Receptor Binding Assays: Complications Arising from Ligand Depletion

Carter, Clare M. Scaramellini; Leighton-Davies, Juliet; Charlton, Steven J.

doi:10.1177/1087057106297788

Cited by 56 publications

(56 citation statements)

References 10 publications

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“…Ligand depletion occurs if more than 10% of the substrate binds to the protein (Motulsky and Christopoulous, 2003). When ligand depletion occurs, this assumption is invalidated and can, if not taken into account, cause errors in data analysis (Morton et al, 2002;Carter et al, 2007). This problem is commonly encountered in the centrifuge-based assay used to determine the kinetics of MT stabilizing agents.…”

Section: Discussionmentioning

confidence: 99%

Cell-Based and Biochemical Structure-Activity Analyses of Analogs of the Microtubule Stabilizer Dictyostatin

et al. 2007

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Compounds that bind to microtubules (MTs) and alter their dynamics are highly sought as a result of the clinical success of paclitaxel and docetaxel. The naturally occurring compound (Ϫ)-dictyostatin binds to MTs, causes cell cycle arrest in G 2 /M at nanomolar concentrations, and retains antiproliferative activity in paclitaxel-resistant cell lines, making dictyostatin an attractive candidate for development as an antineoplastic agent. In this study, we examined a series of dictyostatin analogs to probe biological and biochemical structure-activity relationships. We used a high-content multiparameter fluorescence-based cellular assay for MT morphology, chromatin condensation, mitotic arrest, and cellular toxicity to identify regions of dictyostatin that were essential for biological activity. Four analogs (6-epidictyostatin, 7-epi-dictyostatin, 16-normethyldictyostatin, and 15Z,16-normethyldictyostatin) retained low nanomolar activity in the cell-based assay and were chosen for analyses with isolated tubulin. All four compounds were potent inducers of MT assembly. Equilibrium binding constant (K i ) determinations using [14 C]epothilone B, which has a 3-fold higher affinity for the taxoid binding site than paclitaxel, indicated that 6-epidictyostatin and 7-epi-dictyostatin displaced Finally, we developed a set of quantitative structure-activity relationship equations correlating structures with antiproliferative activity. The equations accurately predicted biological activity and will help in the design of future analogs.

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Section: Discussionmentioning

confidence: 99%

Cell-Based and Biochemical Structure-Activity Analyses of Analogs of the Microtubule Stabilizer Dictyostatin

et al. 2007

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“…Aliquots of [ 3 H]NMS were also quantified accurately to determine how much radioactivity was added to each well by using liquid scintillation spectrometry on an LS6500 scintillation counter (Beckman Coulter, Fullerton, CA). In all experiments, total binding never exceeded more than 10% of that added, limiting complications associated with depletion of the free radioligand concentration (Carter et al, 2007 D-glucose) at 37°C (physiological temperature and sodium concentration), final assay volume 1 ml. An identical procedure was followed to determine the kinetic parameters of [ 3 H]NMS at the rat M 3 receptor by using rat CHO cell membranes (20 g/well Ϫ1 ) in HBSS at 37°C.…”

Section: Methodsmentioning

confidence: 99%

The Influence of Receptor Kinetics on the Onset and Duration of Action and the Therapeutic Index of NVA237 and Tiotropium

Sykes

Dowling

Leighton-Davies

et al. 2012

J Pharmacol Exp Ther

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112

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Studies under nonphysiological conditions suggest that long receptor residency time is responsible for the 24-h duration of action of the long-acting muscarinic antagonist (LAMA) tiotropium. Our aim was to determine how clinically relevant dissociation rates under more physiological conditions influence the differences in onset of action between tiotropium and 3-[(cyclopentylhydroxyphenylacetyl oxy]-1,1-dimethyl-pyrrolidinium bromide (NVA237), a once-daily dry-powder formulation of the LAMA glycopyrronium bromide in development for chronic obstructive pulmonary disease. In addition, we have investigated kinetic selectivity at each of the muscarinic receptor subtypes to determine whether the improved cardiovascular therapeutic index obtained with NVA237 in animal models is attributable to differences in kinetic rate constants. The binding of radioligand [ 3 H]N-methyl-scopolamine was measured in the presence/absence of several concentrations of unlabeled competitors, and data were analyzed using a competition kinetic model to provide on/off rates for the competitor. We found shorter dissociation half-lives for NVA237 and tiotropium under physiological (11.4 and 46.2 min, respectively) versus nonphysiological conditions (173 and 462 min, respectively). NVA237 had a more rapid onset of action (3-4.8 times) versus tiotropium, determined in an vitro calcium and rat tracheal strip assay. Simulations suggested that the more rapid onset of NVA237 action could be explained by differences in kinetic parameters. NVA237 had greater equilibrium binding and kinetic selectivity for muscarinic type 3 (M 3 ) versus muscarinic type 2 (M 2 ) receptors, with a faster off rate from M 2 versus M 3 receptors than tiotropium, potentially affording it a more favorable therapeutic index. This study suggests that the 24-h duration of action of NVA237 and tiotropium is not solely the result of their slow dissociation from the M 3 receptor and highlights the importance of conducting in vitro experiments in conditions reflecting those in vivo.

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“…In all experiments, total binding never exceeded more than 10% of that added, therefore limiting significant depletion of the free radioligand concentration (Carter et al, 2007). To ensure the experiment was at the described temperature, all components were prewarmed for $2 hours.…”

Section: H]-qaw039 and [mentioning

confidence: 99%

Fevipiprant (QAW039), a Slowly Dissociating CRTh2 Antagonist with the Potential for Improved Clinical Efficacy

et al. 2016

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Here we describe the pharmacologic properties of a series of clinically relevant chemoattractant receptor-homologous molecules expressed on T-helper type 2 (CRTh2) receptor antagonists, including fevipiprant (NVP-QAW039 or QAW039), which is currently in development for the treatment of allergic diseases. min 21 and 0.048 minute 21 , respectively. Importantly, the k off of QAW039 (half-life 5 14.4 minutes) was .7-fold slower than the slowest reference compound tested, AZD-1981. In functional studies, QAW039 behaved as an insurmountable antagonist of PGD 2 -stimulated [35 S]-GTPgS activation, and its effects were not fully reversed by increasing concentrations of PGD 2 after an initial 15-minute incubation period. This behavior is consistent with its relatively slow dissociation from the human CRTh2 receptor.In contrast for the other ligands tested this time-dependent effect on maximal stimulation was fully reversed by the 15-minute time point, whereas QAW039's effects persisted for .180 minutes. All CRTh2 antagonists tested inhibited PGD 2 -stimulated human eosinophil shape change, but importantly QAW039 retained its potency in the whole-blood shape-change assay relative to the isolated shape change assay, potentially reflective of its relatively slower off rate from the CRTh2 receptor. QAW039 was also a potent inhibitor of PGD 2 -induced cytokine release in human Th2 cells. Slow CRTh2 antagonist dissociation could provide increased receptor coverage in the face of pathologic PGD 2 concentrations, which may be clinically relevant.

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Miniaturized Receptor Binding Assays: Complications Arising from Ligand Depletion

Cited by 56 publications

References 10 publications

Cell-Based and Biochemical Structure-Activity Analyses of Analogs of the Microtubule Stabilizer Dictyostatin

Cell-Based and Biochemical Structure-Activity Analyses of Analogs of the Microtubule Stabilizer Dictyostatin

The Influence of Receptor Kinetics on the Onset and Duration of Action and the Therapeutic Index of NVA237 and Tiotropium

Fevipiprant (QAW039), a Slowly Dissociating CRTh2 Antagonist with the Potential for Improved Clinical Efficacy

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