Binding Kinetics of ZM241385 Derivatives at the Human Adenosine A<sub>2A</sub> Receptor

Guo, Dong; Xia, Lizi; Veldhoven, Jacobus P. D. van; Hazeu, Marc D.; Mocking, Tamara A. M.; Brussee, Johannes; IJzerman, Adriaan P.; Heitman, Laura H.

doi:10.1002/cmdc.201300474

Cited by 48 publications

(52 citation statements)

References 125 publications

(130 reference statements)

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“…1 More recently, advances in cloning, cell-based assays, and purification of the target receptors, combined with improved knowledge of the molecular basis of diseases, has allowed a more direct measure of ligand-binding affinity, leading to the widely accepted practice of binding affinity optimization to guide early-stage drug discovery efforts. 1, 2 The success of this approach is predicated on the assumption that ligand-binding affinity is a suitable surrogate for in vivo efficacy, 1 yet off-rates for ligands and the resulting downstream signaling both are critical for eliciting drug effects. Further, binding kinetics and drug efficacy are rarely measured during the early stages of the drug discovery efforts, 3, 4 as they tend to be harder to measure experimentally.…”

Section: Introductionmentioning

confidence: 99%

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A2AR Binding Kinetics in the Ligand Depletion Regime

et al. 2017

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Ligand binding plays a fundamental role in stimulating the downstream signaling of membrane receptors. Here, ligand-binding kinetics of the full-length human adenosine receptor A2A (A2AR) reconstituted in detergent micelles were measured using a fluorescently labeled ligand via fluorescence anisotropy. Importantly, to optimize the signal to noise ratio, these experiments were conducted in the ligand depletion regime. In the ligand depletion regime, the assumptions used to determine analytical solutions for one-site binding models for either one or two ligands in competition are no longer valid. We therefore implemented a numerical solution approach in order to analyze kinetic binding data as experimental conditions approach the ligand depletion regime. By comparing the results from the numerical and the analytical solutions, we highlight the ligand-receptor ratios at which the analytical solution begins to lose predictive accuracy. Using the numerical solution approach, we determined the kinetic rate constants of the fluorescent ligand, FITC-APEC, and those for three unlabeled ligands using competitive association experiments. The association and dissociation rate constants of the unlabeled ligands determined from the competitive association experiments were then independently validated using competitive dissociation data. Based on this study, a numerical solution is recommended to determine kinetic ligand-binding parameters for experiments conducted in the ligand-depletion regime.

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Section: Introductionmentioning

confidence: 99%

“…1, 2, 4 Awareness and experimental evidence of the importance of binding kinetics are increasing and there is great interest in developing assays to measure ligand-binding kinetics, based on radioligand binding, fluorescent ligand binding, and surface plasmon resonance of unlabeled ligands to surface-tethered receptors. 3, 5–8 …”

Section: Introductionmentioning

confidence: 99%

A2AR Binding Kinetics in the Ligand Depletion Regime

et al. 2017

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“…In this study, we describe our efforts to obtain a covalent antagonist probe for the hA 2A AR, as a logical extension of our previous research on long residence time antagonists, i.e., compounds that dissociate only slowly from the receptor [19]. We used the antagonist ZM241385 as the starting point in our design efforts and synthesized a fluorosulfonyl derivative of it, LUF7445.…”

Section: Introductionmentioning

confidence: 99%

A covalent antagonist for the human adenosine A2A receptor

Yang

Guo

Michiels

et al. 2016

Purinergic Signalling

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The structure of the human A2A adenosine receptor has been elucidated by X-ray crystallography with a high affinity non-xanthine antagonist, ZM241385, bound to it. This template molecule served as a starting point for the incorporation of reactive moieties that cause the ligand to covalently bind to the receptor. In particular, we incorporated a fluorosulfonyl moiety onto ZM241385, which yielded LUF7445 (4-((3-((7-amino-2-(furan-2-yl)-[1, 2, 4]triazolo[1,5-a][1, 3, 5]triazin-5-yl)amino)propyl)carbamoyl)benzene sulfonyl fluoride). In a radioligand binding assay, LUF7445 acted as a potent antagonist, with an apparent affinity for the hA2A receptor in the nanomolar range. Its apparent affinity increased with longer incubation time, suggesting an increasing level of covalent binding over time. An in silico A2A-structure-based docking model was used to study the binding mode of LUF7445. This led us to perform site-directed mutagenesis of the A2A receptor to probe and validate the target lysine amino acid K153 for covalent binding. Meanwhile, a functional assay combined with wash-out experiments was set up to investigate the efficacy of covalent binding of LUF7445. All these experiments led us to conclude LUF7445 is a valuable molecular tool for further investigating covalent interactions at this receptor. It may also serve as a prototype for a therapeutic approach in which a covalent antagonist may be needed to counteract prolonged and persistent presence of the endogenous ligand adenosine.Electronic supplementary materialThe online version of this article (doi:10.1007/s11302-016-9549-9) contains supplementary material, which is available to authorized users.

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“…[17][18][19] Firstly, substituted arylpiperazines were alkylated with 2-(2-bromoethyl)isoindoline-1,3-dione (1) or 2-(4-bromobutyl)isoindoline-1,3-dione (2) 20 to give the phthalimide derivatives 3a-e and 4c-i. The amine functionality of the compounds was liberated using hydrazine hydrate.…”

Section: Chemistrymentioning

confidence: 99%