Elsevier

Biochemical Pharmacology

Volume 105, 1 April 2016, Pages 34-41
Biochemical Pharmacology

Equilibrium and kinetic selectivity profiling on the human adenosine receptors

https://doi.org/10.1016/j.bcp.2016.02.018Get rights and content

Abstract

Classical evaluation of target selectivity is usually undertaken by measuring the binding affinity of lead compounds against a number of potential targets under equilibrium conditions, without considering the kinetics of the ligand–receptor interaction. In the present study we propose a combined strategy including both equilibrium- and kinetics-based selectivity profiling. The adenosine receptor (AR) was chosen as a prototypical drug target. Six in-house AR antagonists were evaluated in a radioligand displacement assay for their affinity and in a competition association assay for their binding kinetics on three AR subtypes. One of the compounds with a promising kinetic selectivity profile was also examined in a [35S]-GTPγS binding assay for functional activity. We found that XAC and LUF5964 were kinetically more selective for the A1R and A3R, respectively, although they are non-selective in terms of their affinity. In comparison, LUF5967 displayed a strong equilibrium-based selectivity for the A1R over the A2AR, yet its kinetic selectivity thereon was less pronounced. In a GTPγS assay, LUF5964 exhibited insurmountable antagonism on the A3R while having a surmountable effect on the A1R, consistent with its kinetic selectivity profile. This study provides evidence that equilibrium and kinetic selectivity profiling can both be important in the early phases of the drug discovery process. Our proposed combinational strategy could be considered for future medicinal chemistry efforts and aid the design and discovery of different or even better leads for clinical applications.

Introduction

Adenosine receptors (ARs) belong to the superfamily of G protein-coupled receptors (GPCRs), which represent the largest family of drug targets [1]. Four AR subtypes have been identified, namely A1R, A2AR, A2BR and A3R, according to their physiological effects in responding to adenosine, the endogenous ligand [2], [3]. The A1R and A3R couple to a Gi protein and inhibit the enzyme adenylate cyclase, whereas the A2AR and A2BR stimulate this enzyme via a Gs protein [4]. ARs are distributed throughout the body and involved in a wide range of (patho-)physiological responses, and may be promising drug targets [5]. However, the ubiquitous distribution of ARs challenges the discovery of new ligands. Over the years, many efforts have been undertaken to yield selective agonists and antagonists for the each AR subtype [6]. This selectivity is generally evaluated based on dose-dependent assessments of activities (i.e., EC50 or Ki values) performed under equilibrium conditions. However, such equilibrium in vitro is rarely met in the body and the binding selectivity evolves over the course of treatment as a function of the temporal binding between the drug and the main and secondary targets [7]. Thus, the binding kinetics of the drug–target interaction, in particular, residence time (RT = 1·koff−1), is gaining awareness, since it can provide detailed information under non-equilibrium situations [7]. Furthermore, accumulating evidence suggests that compounds with desired kinetic profiles can provide additional advantages. Compounds with long-lasting target occupancy may offer improved clinical efficacy, whereas compounds with fast dissociation kinetics from unwanted targets might show less side effects [8], [9]. Given this, designing drugs with desirable selectivity profiles should therefore not only require an appropriate tuning of binding selectivity but also the modulation of kinetic selectivity [10]. However, such notion has been rarely taken into account and only few studies have touched upon the concept of kinetic selectivity profiling before.

To obtain a complete profile of ligand–receptor selectivity, we examined a series of AR antagonists (Fig. 1) and extensively studied both their affinity and kinetics on different AR subtypes. The association rate (kon) and dissociation rate (koff) constants of these antagonists were determined using competition association assays at the A1R, A2AR and A3R. Furthermore, one compound with promising kinetic selectivity was further tested in a [35S]-GTPγS assay for functional evaluation.

Section snippets

Materials

[3H]-1,3-dipropyl-8-cyclopentylxanthine ([3H]-DPCPX, specific activity 120 Ci·mmol−1) and [3H]-4-(2-(7-amino-2-(furan-2-yl)-[1,2,4]triazolo[1,5-a][1,3,5]triazin-5-ylamino)ethyl)phenol ([3H]-ZM241385, specific activity 50 Ci·mmol−1) were purchased from ARC, Inc. (St. Louis, MO, USA). [3H]-(8R)-8-ethyl-1,4,7,8-tetrahydro-4-5H-imidazole[2,1-i]purin-5-one ([3H]-PSB-11, specific activity 56 Ci·mmol−1) was a gift from Prof. C.E. Müller (University of Bonn, Germany). Unlabeled PSB-11 and N6

Quantification of the association [kon (k1)] and dissociation rate constants [koff (k2)] of [3H]-DPCPX, [3H]-ZM241385 and [3H]-PSB-11 on the A1R, A2AR and A3R, respectively

The binding kinetics of [3H] -DPCPX, [3H]-ZM241385 and [3H]-PSB-11 on the human A1R, A2AR and A3R, respectively, were obtained from kinetic radioligand binding assays (Fig. 2). These experiments were conducted at a uniform temperature (i.e., 10 °C) for direct comparison of data on different ARs. At this temperature the binding kinetics of an unlabeled ligand can be accurately determined on three ARs. Lowering the assay temperature compromised the assay practicability (i.e., longer assay

Discussion

Classical target selectivity studies are largely based on evaluating a ligand’s potency or affinity for the primary and collateral targets. Identifying a compound that shows a big affinity margin has been one of the primary objectives pursued in drug design. Accumulating evidence suggests that drug–target binding kinetics is at least equally important as parameters of affinity and potency [9], [18]. In fact, the former kinetic parameters are perhaps more informative when testing a series of

Conflict of interest

None.

Author contributions

D.G. designed and conducted the experiments, analyzed the data and wrote the manuscript. G.S.D., T.V.D. and M.H. conducted the experiments and analyzed the data. L.H.H. and A.P.IJ. designed the experiments, assessed the results and wrote the manuscript.

Acknowledgements

The research described in this report is part of efforts of a larger consortium called “Kinetics for Drug Discovery (K4DD)”. This K4DD consortium is supported by the Innovative Medicines Initiative Joint Undertaking (IMI JU) under grant agreement n° [115366], resources of which are composed of financial contribution from the European Union’s Seventh Framework Programme (FP7/2007-2013) and EFPIA companies’ in kind contribution. More info: www.imi.europa.eu and www.k4dd.eu. The authors thank

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    Present address: Jiangsu Key Laboratory of New Drug Research and Clinical Pharmacy, Xuzhou Medical College, 209 Tongshan Road, Xuzhou 221004, Jiangsu, China.

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