Slow Agonist Dissociation Kinetics Drive Sustained A2A Responses
37
Chen JF, Eltzschig HK, and Fredholm BB (2013) Adenosine receptors as drug targets–
what are the challenges? Nat Rev Drug Discov 12:265–286.
are important in conferring stable binding interactions. For
example, 3ch is amphiphilic and charged but contains a highly
lipophilic biphenyl N6 substituent that appears to interact
favorably with the chemistry of the N6 subpocket to help
promote a sustained response. This highlights that high
lipophilicity logD7.4 is not required for slow dissociation and
that specific local interactions have more of an influence.
In summary, we show that slow dissociation kinetics can
control the duration of action of A2A agonists in a cellular
setting. We suggest that key regions within the A2A binding
pocket underlie this effect and that N6 and C2 substituted
compounds similar to UK-432,097 (3cd) achieve stable binding
by interacting with these. Whereas this stable binding effect is
clearly evident in UK-432,097, it is greatly enhanced by
modifying the C2 group for compounds such as 3cg and 3ch.
It is tempting to speculate that this could offer a means to
improve the clinical pharmacology of A2A ligands by extend-
ing duration of action. We anticipate that this SAR will prove
valuable information for drug discovery at this target by
informing the rational design of novel A2A agonists for
optimized effect duration and improved pharmacotherapeutic
profiles.
Chern Y, Chiou JY, Lai HL, and Tsai MH (1995) Regulation of adenylyl cyclase type
VI activity during desensitization of the A2a adenosine receptor-mediated cyclic
AMP response: role for protein phosphatase 2A. Mol Pharmacol 48:1–8.
Chern Y, Lai HL, Fong JC, and Liang Y (1993) Multiple mechanisms for de-
sensitization of A2a adenosine receptor-mediated cAMP elevation in rat
pheochromocytoma PC12 cells. Mol Pharmacol 44:950–958.
Copeland RA, Pompliano DL, and Meek TD (2006) Drug-target residence time
and its implications for lead optimization. Nat Rev Drug Discov 5:730–739.
de Lera Ruiz M, Lim YH, and Zheng J (2014) Adenosine A2A receptor as a drug
discovery target. J Med Chem 57:3623–3650.
Deflorian F, Kumar TS, Phan K, Gao ZG, Xu F, Wu H, Katritch V, Stevens RC,
and Jacobson KA (2012) Evaluation of molecular modeling of agonist binding in
light of the crystallographic structure of an agonist-bound A₂A adenosine receptor.
J Med Chem 55:538–552.
Ferrandon S, Feinstein TN, Castro M, Wang B, Bouley R, Potts JT, Gardella TJ,
and Vilardaga JP (2009) Sustained cyclic AMP production by parathyroid hormone
receptor endocytosis. Nat Chem Biol 5:734–742.
Field JJ, Lin G, Okam MM, Majerus E, Keefer J, Onyekwere O, Ross A, Campigotto
F, Neuberg D, Linden J, et al. (2013) Sickle cell vaso-occlusion causes activation of
iNKT cells that is decreased by the adenosine A2A receptor agonist regadenoson.
Blood 121:3329–3334.
Gao ZG and Jacobson KA (2011) Emerging adenosine receptor agonists: an update.
Expert Opin Emerg Drugs 16:597–602.
Gnad T, Scheibler S, von Kügelgen I, Scheele C, Kili ꢀc A, Glöde A, Hoffmann LS,
Reverte-Salisa L, Horn P, Mutlu S, et al. (2014) Adenosine activates brown
adipose tissue and recruits beige adipocytes via A2A receptors. Nature 516:
3
95–399.
Guo D, Hillger JM, IJzerman AP, and Heitman LH (2014) Drug-target residence
time–a case for G protein-coupled receptors. Med Res Rev 34:856–892.
Guo D, Mulder-Krieger T, IJzerman AP, and Heitman LH (2012) Functional efficacy
of adenosine A₂A receptor agonists is positively correlated to their receptor resi-
dence time. Br J Pharmacol 166:1846–1859.
Guo D, van Dorp EJ, Mulder-Krieger T, van Veldhoven JP, Brussee J, Ijzerman AP,
and Heitman LH (2013) Dual-point competition association assay: a fast and high-
throughput kinetic screening method for assessing ligand-receptor binding kinet-
ics. J Biomol Screen 18:309–320.
Acknowledgments
The authors thank Alastair Brown (Heptares Therapeutics and
AstraZeneca) for initiating and inspiring this project and Mark Timms
(
AstraZeneca) for assistance in obtaining and collating logD7.4 data.
Haskell-Luevano C, Miwa H, Dickinson C, Hadley ME, Hruby VJ, Yamada T,
and Gantz I (1996) Characterizations of the unusual dissociation properties of
melanotropin peptides from the melanocortin receptor, hMC1R. J Med Chem 39:
The authors are grateful to the scientists at WuXi PharmaTech and
BioDuro who synthesised the AstraZeneca proprietary compounds
used in this study.
4
32–435.
Hothersall JD, Bussey CE, Brown AJ, Scott JS, Dale I, and Rawlins P (2015) Sus-
tained wash-resistant receptor activation responses of GPR119 agonists. Eur J
Pharmacol 762:430–442.
Hothersall JD, Brown AJ, Dale I, and Rawlins P (2016) Can residence time offer a
useful strategy to target agonist drugs for sustained GPCR responses? Drug Discov
Today 21:90–96.
Koshiba M, Kojima H, Huang S, Apasov S, and Sitkovsky MV (1997) Memory of
extracellular adenosine A2A purinergic receptor-mediated signaling in murine
T cells. J Biol Chem 272:25881–25889.
Authorship Contributions
Participated in research design: Hothersall, Guo, Sarda, Chen,
Waring, IJzerman, Hill, Dale, and Rawlins.
Conducted experiments: Hothersall, Guo, Sarda, and Keur.
Contributed to reagents or analytic tools: Hothersall, Guo, Sarda,
Chen, and Waring.
Performed data analysis: Hothersall, Guo, Sarda, and Chen.
Wrote or contributed to the writing of the manuscript: Hothersall,
Guo, Sarda, Sheppard, Chen, Waring, IJzerman, and Rawlins.
Lebon G, Edwards PC, Leslie AG, and Tate CG (2015) Molecular Determinants of
CGS21680 Binding to the Human Adenosine A2A Receptor. Mol Pharmacol 87:
9
07–915.
Lebon G, Warne T, Edwards PC, Bennett K, Langmead CJ, Leslie AG, and Tate CG
(2011) Agonist-bound adenosine A2A receptor structures reveal common features
of GPCR activation. Nature 474:521–525.
Lee JY and Lyman E (2012) Agonist dynamics and conformational selection during
References
microsecond simulations of the A(2A) adenosine receptor. Biophys
J 102:
Abbracchio MP, Fogliatto G, Paoletti AM, Rovati GE, and Cattabeni F (1992)
Prolonged in vitro exposure of rat brain slices to adenosine analogues: selective
desensitization of adenosine A1 but not A2 receptors. Eur J Pharmacol 227:
2
114–2120.
Mantell S, Jones R, and Trevethick M (2010) Design and application of locally de-
livered agonists of the adenosine A(2A) receptor. Expert Rev Clin Pharmacol 3:
3
17–324.
5
5–72.
Adami M, Bertorelli R, Ferri N, Foddi MC, and Ongini E (1995) Effects of re-
peated administration of selective adenosine A1 and A2A receptor agonists on
Marumoto R, Yoshioka Y, Honjo M, and Kawazoe K (1974), inventors, Takeda
Chemical Industries Limited, assignee. 2,6-diaminonebularinderivate 2,6-
diaminonebularinderivate. Patent DE2359536 A1. 1973 Nov 29.
Niiya K, Jacobson KA, Silvia SK, and Olsson RA (1993) Covalent binding of a se-
lective agonist irreversibly activates guinea pig coronary artery A2 adenosine re-
ceptors. Naunyn Schmiedebergs Arch Pharmacol 347:521–526.
Nishiyama K and Hirai K (2015) In vitro comparison of duration of action of mela-
tonin agonists on melatonin MT receptor: possible link between duration of action
and dissociation rate from receptor. Eur J Pharmacol 757:42–52.
Ong S, Liu H, Qiu X, Bhat G, and Pidgeon C (1995) Membrane partition coefficients
chromatographically measured using immobilized artificial membrane surfaces.
Anal Chem 67:755–762.
pentylenetetrazole-induced convulsions in the rat. Eur
83–389.
J Pharmacol 294:
3
Andreassen KV, Hjuler ST, Furness SG, Sexton PM, Christopoulos A, Nosjean O,
Karsdal MA, and Henriksen K (2014) Prolonged calcitonin receptor signaling by
salmon, but not human calcitonin, reveals ligand bias. PLoS One 9:e92042.
Åstrand ABM, Lamm Bergström E, Zhang H, Börjesson L, Söderdahl T, Wingren C,
Jansson A-H, Smailagic A, Johansson C, Bladh H, et al. (2015) The discovery of a
selective and potent A2a agonist with extended lung retention. Pharmacol Res
Perspect 3:e00134.
Austin RP, Barton P, Bonnert RV, Brown RC, Cage PA, Cheshire DR, Davis AM,
Dougall IG, Ince F, Pairaudeau G, et al. (2003) QSAR and the rational design of
Palmer TM, Gettys TW, Jacobson KA, and Stiles GL (1994) Desensitization of the
canine A2a adenosine receptor: delineation of multiple processes. Mol Pharmacol
long-acting dual D2-receptor/beta 2-adrenoceptor agonists.
210–3220.
J Med Chem 46:
3
4
5:1082–1094.
Baines AE, Corrêa SA, Irving AJ, and Frenguelli BG (2011) Differential trafficking of
adenosine receptors in hippocampal neurons monitored using GFP- and super-
ecliptic pHluorin-tagged receptors. Neuropharmacology 61:1–11.
Bridges AJ, Moos WH, Szotek DL, Trivedi BK, Bristol JA, Heffner TG, Bruns RF,
and Downs DA (1987) N6-(2,2-diphenylethyl)adenosine, a novel adenosine receptor
agonist with antipsychotic-like activity. J Med Chem 30:1709–1711.
Palmgren JJ, Monkkonen J, Korjamo T, Hassinen A, and Auriola S (2006) Drug
adsorption to plastic containers and retention of drugs in cultured cells under
in vitro conditions. Eur J Pharm Biopharm 64: 369–378.
Pidgeon C, Ong S, Liu H, Qiu X, Pidgeon M, Dantzig AH, Munroe J, Hornback WJ,
Kasher JS, Glunz L, et al. (1995) IAM chromatography: an in vitro screen for
predicting drug membrane permeability. J Med Chem 38:590–594.
Sato N, Yuki Y, Shinohara H, Takeji Y, Ito K, Michikami D, Hino K, and Yamazaki H
(2010) inventors, Otsuka Pharmaceutical Co Ltd, assignee. A novel cyanopyr-
imidine derivative. Patent WO/2010/090299. 2010 Dec 8.
Sykes DA, Parry C, Reilly J, Wright P, Fairhurst RA, and Charlton SJ (2014) Ob-
served drug-receptor association rates are governed by membrane affinity: the
Cerqueira MD (2004) The future of pharmacologic stress: selective A2A adenosine
receptor agonists. Am J Cardiol 94: 33D–42D.
Charalambous C, Gsandtner I, Keuerleber S, Milan-Lobo L, Kudlacek O, Freissmuth
M, and Zezula J (2008) Restricted collision coupling of the A2A receptor revisited:
evidence for physical separation of two signaling cascades. J Biol Chem 283:
9
276–9288.