Novel Alexa Fluor-488 labeled antagonist of the A2A adenosine receptor: Application to a fluorescence polarization-based receptor binding assay
Graphical abstract
Introduction
The A2AAR is one of the four adenosine receptor (AR) subtypes of the G protein-coupled receptor (GPCR) family which mediates multiple physiological effects of extracellular adenosine, both in the central nervous system (CNS) and in peripheral tissues, activating several G protein-dependent and independent signaling pathways [1]. Increasing evidence relates selective A2AAR ligands to applications in cardiac stress testing [2], neurodegenerative disorders such as Parkinson's disease [3], [4] and inflammation [5], renewing the interest in these receptors, which are increasingly viewed as promising therapeutic targets [6].
GPCRs represent the single largest molecular target of therapeutic drugs and are also the most common focus in high-throughput screening assays designed to identify potential new drug candidates. A major fraction of these assays are now formatted as radioligand binding assays. Numerous agonists and antagonists have been developed by medicinal chemical methods either by empirical probing to establish a structure activity relationship (SAR) or in quantitative structure activity relationship (QSAR) studies [7]. Thus, many laboratories currently seek to identify novel leads for potent A2AAR agonists and antagonists. Typically, to achieve this aim they use a radioligand-based binding assay and receptor-specific functional assays.
An alternative and potentially more versatile approach to measure affinity in receptor binding is fluorescence polarization (FP). FP offers several advantages in comparison to radioligand assays, such as low cost and avoiding problems like health hazards of radiation exposure and radioactive waste disposal. Furthermore, FP is faster and easier experimentally, involving only a homogenous measurement without filtration or other additional steps. Fluorescent ligands for various GPCRs including the ARs have been reported [8], [9], but several analytical issues have hampered the development of receptor binding assays based on fluorescence intensity alone. These assays are usually heterogeneous and require a filtration step for the separation of the bound and unbound fractions of fluorescent ligand [10].
FP measurements are based on the assessment of the rotational motions of molecular species [9]. FP can be considered a competition between the molecular motion and the lifetime of fluorophores in solution. If linear polarized light is used to excite an ensemble of fluorophores, only those fluorophores that are aligned with the plane of polarization will be excited. There are two scenarios for the fluorescence emission. Provided the fluorescence lifetime of the excited fluorescent probe is much longer than the rotational correlation time of the molecule to which it is bound, e.g., when the fluorescent ligand is free in the solution, the molecules will randomize in solution during the emission process. As a result, the emitted light of the fluorescent probe will be depolarized. If the fluorescence lifetime of the fluorophore is much shorter than the rotational correlation time, e.g., when the fluorescent ligand is bound to a membrane receptor, the excited molecules will stay aligned during the process of emission, and the resulting emission will be polarized and detectable. This technique has been described as fast, sensitive, and inexpensive and is therefore a method of choice for high-throughput screening (HTS), although so far it has been applied to only a limited number of GPCRs mainly due to the difficulties in synthesizing an appropriate FP ligand [11], [12].
Now with an X-ray structure of the human (h) A2AAR available [13], it is possible to select in silico and rationally design many more potent antagonists of the receptor. Use of the 3D structure of the A2AAR in theoretical ligand docking has also been demonstrated. It should be highly beneficial to use a fluorescence-based HTS assay to measure the binding affinity of the novel analogues at the A2AAR. In this study, we have introduced a new fluorescent tracer (MRS5346; 5-((2-(2-(4-(3-(5-amino-2-(furan-2-yl)-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-7-yl)propyl)phenoxy)acetamido)-ethyl)-carbamoyl)-2-(6-amino-3-iminio-4,5-disulfonato-3H-xanthen-9-yl)benzoate, Fig. 1) for FP studies, which contains an Alexa Fluor (AF)488 moiety, and demonstrated its utility in pharmacological experiments.
Section snippets
Materials
AF 488 carboxylic acid 2,3,5,6-tetrafluorophenyl ester was purchased from Invitrogen (Carlsbad, CA). [3H]R-N6-(2-phenylisopropyl)adenosine ([3H]R-PIA, 42.6 Ci/mmol) was obtained from Moravek Biochemicals (Brea, CA). [125I]4-Amino-3-iodobenzyl-5’-N-methylcarboxamidoadenosine ([125I]AB-MECA, 2200 Ci/mmol), [3H]2-chloro-N6-cyclopentyladenosine and [3H]-2-[p-(2-carboxyethyl)phenylethylamino]-5′-N-ethylcarboxamidoadenosine ([3H]CGS21680, 40.5 Ci/mmol) were purchased from PerkinElmer (Waltham, MA).
Choice of FP ligand
In considering the design of an appropriate FP ligand for the A2AAR, first we had to choose a small ligand with a relatively high affinity at the receptor that was suitable for derivatization as a functionalized congener [17]. We selected the antagonist SCH442416, which has high affinity and selectivity for the A2AAR [18], as a lead molecule. The strategy for selection of the site for chain extension and fluorophore conjugation is based on the X-ray structure of the A2AAR and will be described
Discussion
The present study demonstrated that the novel fluorescent antagonist, MRS5346, can be successfully used as a tool in the FP A2AAR binding assay, which represents the first example in the AR field. This FP ligand should be applicable in many areas for studies related to the A2AAR, including kinetic analysis and tests of ligand binding affinity. MRS5346 appears to be suitable for both regular binding assay and high-throughput screening. The FP assay should be applicable to both the study of
Acknowledgements
This research was supported by the Intramural Research Program of the NIH, National Institute of Diabetes and Digestive and Kidney Diseases. Special thanks to Csaba Vizler, Ph.D. (BRC-HAS).
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