A2A and A2B adenosine receptors: The extracellular loop 2 determines high (A2A) or low affinity (A2B) for adenosine

Journal Pre-proofs A2A and A2B adenosine receptors: the extracellular loop 2 determines high (A2A) or low affinity (A2B) for adenosine Elisabetta De Filippo, Sonja Hinz, Veronica Pellizzari, Giuseppe Deganutti, Ali El-Tayeb, Gemma Navarro, Rafael Franco, Stefano Moro, Anke C. Schiedel, Christa E. Müller PII: DOI: Reference:

S0006-2952(19)30417-4 https://doi.org/10.1016/j.bcp.2019.113718 BCP 113718

To appear in:

Biochemical Pharmacology

Received Date: Accepted Date:

3 October 2019 13 November 2019

Please cite this article as: E. De Filippo, S. Hinz, V. Pellizzari, G. Deganutti, A. El-Tayeb, G. Navarro, R. Franco, S. Moro, A.C. Schiedel, C.E. Müller, A2A and A2B adenosine receptors: the extracellular loop 2 determines high (A2A) or low affinity (A2B) for adenosine, Biochemical Pharmacology (2019), doi: https://doi.org/10.1016/j.bcp. 2019.113718

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Biochemical Pharmacology

A2A and A2B adenosine receptors: the extracellular loop 2 determines high (A2A) or low affinity (A2B) for adenosine Elisabetta De Filippoa, Sonja Hinza, Veronica Pellizzarib, Giuseppe Deganuttib, Ali El-Tayeba, Gemma Navarroc, Rafael Francoc,d, Stefano Morob, Anke C. Schiedela, and Christa E. Müllera,*

a PharmaCenter

Bonn, Pharmaceutical Institute, Pharmaceutical & Medicinal Chemistry, University of Bonn, An der Immenburg 4, D-53121 Bonn, Germany. b Molecular

Modeling Section (MMS), Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Via Marzolo 5, Padua, Italy. c

Department of Biochemistry and Molecular Biomedicine, University of Barcelona, Avda Diagonal 643, 08028 Barcelona, Spain. d

Centro de Investigación en Red, Enfermedades Neurodegenerativas (CiberNed). Centro Nacional de Salud Carlos III. Madrid. Spain.

* corresponding

author:

C. E. Müller, [email protected], phone +49 228 732301; fax +49 228 732567

1

Abstract A2A and A2B adenosine receptors (ARs) are closely related G protein-coupled receptor subtypes, which represent important (potential) drug targets. Despite their almost identical binding sites for adenosine, A2AARs are activated by low (nanomolar) adenosine concentrations, while A2BARs require micromolar concentrations. In the present study, we exchanged the extracellular loop 2 (ECL2) of the human A2AAR for that of the A2BAR. The resulting chimeric A2A(ECL2-A2B)AR was investigated in radioligand binding and cAMP accumulation assays in comparison to the wildtype A2AAR. While the ribose-modified adenosine analog N-ethylcarboxamidoadenosine (NECA) and its 2-substituted derivative CGS-21680 did not exhibit significant changes, adenosine showed dramatically reduced potency and affinity for the A2A(ECL2-A2B)AR mutant displaying similarly low potency as for the wt A2BAR. Supervised molecular dynamics simulation studies predicted a metabinding site with high affinity for adenosine, but not for NECA, which may contribute to the observed effects.

Keywords Adenosine, cAMP, extracellular loop, GPCR, chimeric receptor, radioligand binding

2

1 Introduction G protein-coupled receptors (GPCRs), one of the largest superfamilies of transmembrane proteins, are involved in a number of physiological and pathological signaling pathways. Therefore, they represent major targets for drug development [1]. So far three-dimensional structures of more than 60 different GPCRs have been solved, providing more and more insights into protein function and their interaction with ligands or drugs. The majority of the solved structures belong to the rhodopsinlike class A GPCRs, in particular to the cluster of aminergic receptors, of which many represent current drug targets. Adenosine receptors (ARs) belong to the -branch of rhodopsin-like (class A) GPCRs; they are divided into four subtypes: A1, A2A, A2B, and A3, which are ubiquitously expressed in the human body and respond to the same endogenous ligand, adenosine (Fig. 1), but can activate different intracellular signalling pathways [2]. ARs are promising drug targets due to their involvement in a wide range of physiological conditions and diseases [3]. A2AR agonists have therapeutic potential because A2A receptor activation produces vasodilatation and reduces inflammatory processes [3]. Adenosine derivatives have been developed as diagnostic drugs for coronary artery disease (i.e. Regadenoson) and could be potential antiinflammatory drugs (e.g. for rheumatoid arthritis), however, hypotensive side-effects limit their application [2]. Prodrugs for the A2AAR have been developed to overcome these problems [4]. The A2AAR subtype mediates strong immunosuppressive effects [5] and therefore A2AAR antagonists are currently being developed for the immunotherapy of cancer [6]. Several A2AAR antagonists have been studied for the treatment of Parkinson’s disease (PD), Alzheimer’s disease (AD) and depression [7,8]; one of them, istradefylline, was approved in Japan as adjunct therapy of PD [2,9] and very recently by the US Food and Drug Administration (FDA) for the same purpose. The A2BAR is being investigated as a novel target for cancer therapy due to the direct antiproliferative effects of A2BAR antagonists on cancer cells in addition to their anti-metastatic, anti3

angiogenic, and immunostimulatory properties [10,11]. Moreover, A2BAR antagonists display analgesic, anti-inflammatory, anti-asthmatic, anti-diabetic effects [12], while A2BAR agonists are potential drugs for the treatment of coronary artery disease due to the A2BAR involvement in pre/postconditioning cardioprotection [13,14]. Despite the high sequence identity to its close homolog, the “high-affinity” A2AAR (59%), the A2BAR is known as the “low affinity” adenosine receptor because the endogenous ligand, adenosine, and its derivative NECA show significantly higher affinity for A2AAR. The A2AAR is activated under physiological conditions by adenosine at nanomolar concentrations, while the “low affinity” A2BAR requires much higher, micromolar concentrations to be activated [15]. Under hypoxic or inflammatory conditions, the intracellular level of adenosine increases up to micromolar concentrations and activates also A2BARs. Understanding the structural basis of this major difference in functionality between A2A- and A2BARs would be helpful for future drug development efforts. Interestingly, the A2BAR, when activated, seems to become the dominant receptors for adenosine because of its simultaneous ability to down-regulate A1AR-mediated responses [16] as well as the ligand binding and signaling through A2AARs through the formation of stable heteromers [17].

Since there is no A2BAR crystal structure available yet, some of the solved A2A structures have been used to construct homology models of the closely related A2BAR [18–20]. The accurate analysis of A2BAR models showed that the orthosteric binding pockets of the two close homologs are almost identical except for a single amino acid exchange in helix 6 (Leu249A2A/Val250A2B) [21]. Beyond the orthosteric pocket, it has been shown that the extracellular loops (ECLs), in particular, the ECL2, play a critical role in ligand binding and receptor activation. In class A GPCRs, the ECL2 is associated with ligand selectivity, which would explain their large variety [22]. The ECL2 is one of the least conserved regions between the A2A and A2B receptor subtypes, showing 34% identity and 4

46% similarity [23]. The ECL2 of the A2BAR is the longest of all four receptor subtypes. It possesses four cysteine residues, only one of which appears to be involved in disulfide bond formation [18]. The ECL2 might contribute to the specificity of ligand binding by directly forming part of the binding cavity. As several A2AAR crystal structures and mutagenesis studies showed, ECL2 residues can directly interact with both agonists and antagonists placed in the binding pocket (i.e. Phe1685.29 and Glu1695.30 form a π-stacking interaction and a hydrogen bond, respectively, Ballesteros Weinstein nomenclature [24], [25]. Furthermore, the ECL2 seems to be an important element conferring receptor subtype selectivity to ligands [26]. Previously, we investigated the hypothesis of the ECL2 as being critical for ligand recognition and receptor activation in ARs [23]. The complete ECL2 of the human A2BAR was exchanged for the corresponding loop in A2AAR subtype generating the A2B(ECL2-A2A) mutant. This loop exchange had significant effects on ligand binding and receptor function. The A2A-selective agonist CGS21680 was able to activate the A2B(ECL2-A2A) mutant but did not lead to any activation of the wildtype (wt) A2BAR. Moreover, the maximal effect of all agonists tested in cAMP accumulation assays of the Gscoupled receptor was significantly increased in the loop exchange mutant as compared to the wt receptor. These results showed that the ECL2 does not only contribute to ligand selectivity but, in addition, appears to stabilize agonist-bound active conformations of the receptor. The aim of the current study was to investigate the complementary mutant of the human A2AAR, in which the ECL2 was exchanged for that of the human A2BAR (A2A(ECL2-A2B)), using mutagenesis, functional and binding studies in combination with molecular dynamics (MD) simulations in order to get further insights into the role of the ECL2 in both AR subtypes.

5

2 Methods and Materials 2.1 Materials Cell culture media, Dulbecco’s Modified Eagle Medium (DMEM), DMEM-F12, penicillinstreptomycin solution, and all cell culture supplements were purchased from Invitrogen (Darmstadt, Germany). Fetal calf serum (FCS) and geneticin (G418) were obtained from Sigma-Aldrich (Taufkirchen, Germany). All other chemicals were purchased from Sigma-Aldrich (Taufkirchen, Germany), Roth (Karlsruhe, Germany) or Applichem (Darmstadt, Germany) unless otherwise noted. The enzymes and competent bacteria were purchased from New England Biolabs (Frankfurt, Germany). All primers and the vector pcDNA3.1(-) were obtained from Invitrogen (Darmstadt, Germany), while the retroviral vector pQCXIN was purchased from Clontech (Heidelberg, Germany). [3H]cAMP (3’,5’-cyclic adenosine monophosphate; specific activity 34 Ci/mmol) and [3H]MSX-2

(3-(3-hydroxypropyl)-7-methyl-8-(m-methoxystyryl)-1-propargylxanthine;

84

Ci/mmol) were obtained from Amersham-GE Healthcare (Frankfurt, Germany) while [3H]CGS21680 (36.05 Ci/mmol) was from Perkin Elmer (Rodgau, Germany). Adenosine deaminase (ADA) was purchased

from

Sigma-Aldrich

(Taufkirchen,

Germany),

Ro20-1724

(4-(3-butoxy-4-

methoxyphenyl)methyl-2-imidazolidone) from Hoffmann La Roche (Grenzach, Germany) and the scintillation cocktail LUMASAFE from Perkin Elmer (Rodgau, Germany). Adenosine and NECA were

obtained

from

Sigma-Aldrich

(Taufkirchen,

Germany),

carboxyethyl)phenethylamino-5′-N-ethylcarboxamidoadenosine)

and

from

CGS21680 Tocris

(2-p-(2-

(Wiesbaden-

Nordenstadt, Germany), and PSB-15826 (2-(4-(4-fluorophenyl)piperazin-1-yl)ethylthioadenosine) was synthesized at the Pharmaceutical Institute of the University of Bonn (El-Tayeb A., Müller C. E. et al. unpublished). 2.2 Mutagenesis and cloning

6

The second extracellular loop (ECL2) of the human A2AAR was replaced by the ECL2 of the human A2BAR by Gibson Assembly reaction mix (New England Biolabs – Frankfurt, Germany) [27]. Gibson Assembly combines multiple overlapping DNA fragments in a single-tube isothermal reaction. The master mix includes an exonuclease, a polymerase, and a DNA ligase. The human A2A and A2BAR were cloned into pcDNA3.1(-). Three sets of primers were designed to create three PCR products, corresponding to the ECL2 of A2BAR and ECL2 flanking regions of the A2AAR, with specific overlapping regions: f-a: 5’-ggaattgatccgcggccgcaccggtACCATGCCCATCATGGGCTCCTCGGTGTAC-3’, r-a: 5’-tgtctttactGTTCCAACCTAGCATGGGAGTCAGGCCGATG-3’, f-b: 5’-ctcccatgctaggttggaacAGTAAAGACAGTGCCACCAACAACTGCACAGAACC-3’, r-b: 5’-ccatgtagttcatggggaccacATTctcaaagagacacttcacaaggcagcagc-3’, f-c: 5’-aagtgtctctttgagaatgtggtccccatgaactacatggtgtacttcaac-3’, r-c: 5’-gggagaggggcggaattccggatccTCAGGACACTCCTGCTCCATCCTGGGCCAG-3’. The reaction was performed in accordance with the manufacturer's instructions. The DNA was transformed into Escherichia coli Top10, then plasmids were isolated and sequenced. The obtained mutant A2A(ECL2-A2B) receptor and the wildtype (wt) A2A and A2BAR were cloned into the vector pQCXIN with the human influenza haemagglutinin (HA) tag linked to the 5’-end of the coding region of the genes. 2.3 Cell culture and retroviral transfection GP+envAM12 packaging cells were cultured at 37 °C, 5% CO2 in DMEM supplied with 10% FCS, 100 U/ml penicillin G, 100 g/ml streptomycin, 1% ultraglutamine, 0.2 mg/ml hygromycin B, 15 g/ml hypoxanthine, 250 g/ml xanthine and 25 g/ml mycophenolic acid. Chinese hamster ovary (CHO) cells were cultured at 37 °C and 5% CO2 in DMEM-F12 supplemented with 10% FCS, 100 7

U/ml penicillin G and 100 g/ml streptomycin. A retroviral transfection system was used to generate CHO cells stably expressing the human A2AAR, the human A2BAR and the A2A(ECL2-A2B) receptor, as described before [23]. Only cells up to passage number 20 were used. 2.4 Membrane preparation Stably transfected CHO cells were seeded into dishes and grown until they reached 90% of confluency. One day before cell harvesting, a final concentration of 0.5 mg/ml of valproic acid was added to the cell medium in order to increase the exogenous protein expression [28]. After washing, cells were scraped off in ice-cold Tris buffer (5 mM Tris-HCl, 2 mM EDTA, pH 7.4). The cell suspension was homogenized on ice and centrifuged for 10 min at 1000 x g (4 °C). The supernatant was then centrifuged for 60 min at 37 000 x g. The obtained pellet was resuspended in 50 mM Tris buffer (pH 7.4) and centrifuged again under the same conditions. Aliquots of membrane preparations were stored at -80 °C. The protein concentration was determined by the Bradford assay. 2.5 Cell surface ELISA The plasma membrane expression of the human A2A, the human A2B and the mutant A2A(ECL2-A2B) receptors in CHO cells was determined by enzyme-linked immunosorbent assay (ELISA). The cells were seeded into 24-well plates (200000 cells/well) and cultured for 24 h. Then cells were washed with phosphate-buffered saline (PBS) and blocked with 1% bovine serum albumin (BSA)/PBS for 5 min. Cells were incubated with a hemagglutinin (HA) antibody solution (Covance, Munich, Germany), diluted 1:1000 in 1% BSA/PBS, for 1 h at room temperature. After washing 3 times with PBS, cells were fixed with a cold solution of 1:1 (v/v) methanol/acetone solution for 15 min at -20 °C, washed and blocked again for 10 min. The horseradish peroxidase-coupled secondary antibody (goat anti-mouse, Sigma, Munich, Germany) was diluted 1:5000 in 1% BSA/PBS and incubated with the cells for 1 h at room temperature. Then cells were washed 4 times and incubated for 50 min with 300 l of peroxidase substrate (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid; ABTS) 8

substrate (Thermo Scientific Pierce, Rockford, USA). The supernatant (170 l) was then transferred in a 96-well plate and the absorbance was measured at 405 nm. 2.6 Radioligand binding studies Competition experiments were performed with [3H]CGS21680 and [3H]MSX-2 in a final volume of 400 l. The vial contained 10 l of dissolved compound in DMSO, 190 l of Tris buffer (50 mM, pH 7.4), 100 l of radioligand solution (final radioligand concentration: 5 nM [3H]CGS21680 or 1 nM [3H]MSX-2) and 100 l of membrane preparation (70-150 g/vial, preincubated with 2 U/mg of adenosine deaminase for 20 min). Total binding was measured in the absence of test compound, while nonspecific binding was determined in the presence of 50 M NECA. In binding experiments with [3H]CGS21680, a final concentration of 10 mM MgCl2 was added. After an incubation time of 1 h for [3H]CGS21680 and 30 min for [3H]MSX-2, the assay mixture was filtered through GF/B glass fiber filters using a Brandel harvester (Brandel, Gaithersburg, USA). Filters used for binding studies with [3H]MSX-2 were pre-soaked in 0.3% aqueous polyethylenimine solution for 30 min. When adenosine was tested, adenosine deaminase was omitted and membranes were washed with 50 mM Tris buffer (pH 7.4) four times more to remove endogenous adenosine. We found that almost all adenosine could be removed by this procedure, and similar specific radioligand binding was observed as with ADA treatment. After harvesting, the filters were washed with ice-cold Tris buffer (50 mM, pH 7.4), transferred into vials and incubated for 6 h with 2.5 ml of scintillation cocktail. The vials were then counted in a liquid scintillation counter (Tricarb 2900TR, Perkin Elmer, Rodgau, Germany). Three independent experiments were performed in duplicates unless otherwise noted. The KD values were obtained by homologous competition binding experiments ([3H]CGS21680: A2AAR 72.1 ± 24.7 nM and A2A(ECL2-A2B) 194 ± 79 nM; [3H]MSX-2: A2AAR 5.26 ± 0.24 nM and A2A(ECL2-A2B) 29.9 ± 6.90 nM) and then used to determine Ki values. In a homologous competition binding experiment, using the Cheng and Prussoff equation, one assumes that the hot (tritium-labeled) 9

and the cold ligand have identical affinities so that KD and Ki values are the same: KD = Ki = IC50 – [radioligand].The Bmax value can be calculated from the homologous competition experiment by an equation implemented in GraphPad Prism 7.2 (La Jolla California, USA): Bmax = (Top/Bottom)/([Radioligand]/(KD + [Radioligand])).

2.7 cAMP accumulation assays CHO cells were seeded into 24-well plates 24 h before performing the assay. After removing the medium, cells were washed and incubated for 2 h at 37 °C and 5% CO2 with Hank’s Balanced Salt Solution (HBSS; 20 mM HEPES, 13 mM NaCl, 5.5 mM glucose, 5.4 mM KCl, 4.2 mM NaHCO3, 1.25 mM CaCl2, 1 mM MgCl2, 0.8 mM MgSO4, 0.44 mM KH2PO4 and 0.34 mM Na2HPO4, pH 7.4) with 1 U/ml of adenosine deaminase (ADA, Sigma-Aldrich, Taufkirchen, Germany). ADA was omitted in experiments where adenosine was tested as an agonist. Cells were then incubated for 15 min with the phosphodiesterase inhibitor Ro20-1724 (Hoffmann La Roche, Grenzach, Germany; final concentration 40 M) at 37 °C and 5% CO2. Different dilutions of agonists (adenosine, NECA, CGS21680 or PSB-15826) in 5% DMSO/HBSS buffer (final DMSO concentration: 1%) were added to the cells and incubated for 15 min under the same conditions described above. The supernatant was then removed and 500 l of hot lysis buffer (90 °C; 4 mM EDTA and 0.01% Triton X-100, pH 7.4) was added. After one hour of mixing on ice, the cAMP amount in the lysates was determined by competition radioligand binding experiments [4]. The lysate (50 l) was incubated with 30 l of [3H]cAMP solution in lysis buffer (final radioligand concentration 3 nM) and 40 l of cAMP binding protein (50 g/vial) [29]. For the cAMP standard curve, 50 l of different cAMP concentrations were measured instead of the cell lysate. Total binding was obtained by mixing radioligand solution and cAMP binding protein in lysis buffer, and the background was defined in the absence of binding protein. The mixture was incubated for 60 min on ice and then filtered through GF/B glass fiber filters 10

using a Brandel harvester. The filters were then washed with ice-cold Tris buffer (50 mM, pH 7.4), transferred into scintillation vials and incubated for 6 h with 2.5 ml of scintillation cocktail. The vials were then counted in a liquid scintillation counter. Three independent experiments were performed in duplicates unless otherwise noted. The cAMP amount was calculated by comparison with the standard curve and normalized to the maximal effect induced by 100 M forskolin (set as 100%). 2.8 Data analysis of pharmacological assays Data analysis and statistics (unpaired two-tailed t-test) were performed with GraphPad Prism version 7.2. 2.9 Homology model of the human A2A(ECL2-A2B) mutant receptor and supervised molecular dynamics The homology model of the human A2A(ECL2-A2B) mutant was obtained by superposing the intermediate active state A2BAR model obtained from Adenosiland [30] to the corresponding A2AAR template (PDB ID 2YDO). ECL2 residues of the A2BAR model were then inserted in the place of the A2AAR residues between Asn144ECL2 and Ala165ECL2. The newly introduced backbone bonds were minimized using the Amber99 force field using the Molecular Operating Environment (MOE, https://www.chemcomp.com/Products.htm). All computations were performed on a hybrid Central Processing Unit/Graphics Processing Unit (CPU/GPU) cluster. Molecular dynamic (MD) simulations were carried out with the Accelerating Biomolecular Dynamics (ACEMD) engine [31] on a GPU cluster equipped with four NVIDIA GTX 580, two NVIDIA GTX 680, three NVIDIA GTX 780, and four NVIDIA GTX 980. Trajectory analysis, figure and video generation have been performed using several functionalities implemented by Visual Molecular Dynamics [32], Wordom [33] and the Gnuplot graphic utility [34]. Ligand-receptor interaction energies were calculated by extrapolating the non-bonded energy interaction term of the CHARMM27 (Chemistry at Harvard Macromolecular Mechanics) Force Field [35] using Nanoscale Molecular Dynamics (NAMD) [36]. All molecular 11

dynamics simulations have been carried out using the ACEMD engine [31,37]. The numbering of the amino acids follows the scheme proposed by Ballesteros and Weinstein [24]: each amino acid identifier starts with the helix number (1-7), followed by a dot and the position relative to a reference residue among the most conserved amino acids in that helix, arbitrarily referred to as number 50.

2.9.1 Ligand parameterization Adenosine and NECA parameters were derived from the CHARMM General Force Field for organic molecules [38]. 2.9.2 Receptor membrane embedding and system preparation The mutant receptor was embedded in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid bilayer (95x95 Å wide) using an insertion method and according to the suggested orientation reported in the “Orientations of Proteins in Membranes (OPM)” database [39] for the A2AAR in complex with the endogenous agonist adenosine (PDB ID: 2YDO [40]). Initial POPC atom coordinates were constructed through the Visual Molecular Dynamics (VMD) membrane builder plugin and, after proteins insertion in the bilayers, overlapping lipids (within 0.45 Å) were removed. The prepared systems were solvated with TIP3Pwater [41] using the program Solvate 1.0 [42] and neutralized by Na+/Cl- counter-ions to a final concentration of 0.154 M. Membrane MD simulations were carried out on a GPU cluster with the ACEMD program using the CHARMM27 Force Field3 [35] and periodic boundaries conditions. The systems were equilibrated using a stepwise procedure. (1) To reduce steric clashes due to the manual setting up of the membrane-receptor systems, a 2000 steps conjugate-gradient minimization was performed; (2) to allow phospholipid fatty acid chains to reach equilibrium, water molecules to diffuse into the protein cavity and to avoid ligand-receptor interaction in the equilibration phase, protein, ligand and POPC phosphorus atoms were restrained for the first 5 ns by a force constant of 1 kcal/mol·Å2; (3) in order to permit the POPC lateral 12

diffusion inside the lipid bilayer the constraint applied to the phosphorus atoms were removed for the next 5 ns; (4) the force constant was gradually reduced to 0.1 kcal/mol·Å2 for the next 10 ns. The temperature was maintained at 298 K using a Langevin thermostat with a low damping constant of 1 ps-1, and the pressure was maintained at 1 atm using a Berendensen barostat. Bond lengths involving hydrogen atoms were constrained using the M-SHAKE algorithm [43] with an integration timestep of 2 fs. 2.9.3 Supervised molecular dynamics (SuMD) After the equilibration, harmonical restraints were removed and Supervised MD (SuMD) was conducted in an NVT ensemble (number of particles, volume and temperature are constant). Longrange Coulomb interactions were handled using the particle mesh Ewald summation method (PME) [44] with grid size rounded to the approximate integer value of cell wall dimensions. A non-bonded cutoff distance of 9 Å with a switching distance of 7.5 Å was used. As previously reported [45], SuMD is a standard MD simulation during which the distance between the center of masses of the ligand atoms and the residues composing the orthosteric binding site of the GPCR is monitored over a fixed time window (e.g. 600 ps). An arbitrary number of distance points per each checkpoint trajectory are collected in real-time and a linear function is fitted on the distance points, at the end of the checkpoint time. A supervision tabu-like algorithm is applied to increase the probability to produce ligand-receptor binding events without introducing bias to the MD simulation: if the slope of the linear function has a negative value, the ligand-receptor distance is likely to be shortened over the checkpoint time and classic MD simulation is restarted from the last produced set of coordinates, otherwise the simulation is restored from the original set of coordinates and atomic random velocities are reassigned coherently to the NVT ensemble. The tabu-like supervision algorithm is perpetuated until ligand-receptor distance is less than 5 Å. 2.9.4 SuMD trajectories analysis 13

To analyze the adenosine-mutant receptor recognition processes in a statistically acceptable manner, each SuMD simulation was repeated at least five times. Considering the specific aim of trying to rationalize experimental data concerning adenosine and its analog NECA towards the mutant receptor, two different analyses, available from the SuMD trajectory analyzer tool [46] have been specifically selected to analyze each adenosine receptor pathway: a) Recognition Pathway (Pollicino) Analyzer: the hypothetical receptor-approaching route of adenosine was monitored during the SuMD trajectory. The presence of a meta-binding site (a metastable intermediate state) is represented by a sphere whose radius is proportional to the residence time of adenosine in that specific meta-binding site; b) Energy Landscape Profile: the ligand-receptor interaction energy (based on the potential energy contributions derived by the force field) was monitoring during the SuMD trajectory.

3 Results 3.1 Generation and characterization of loop exchange mutant A2A(ECL2-A2B) The entire extracellular loop 2 (ECL2) of the human A2AAR was exchanged for the corresponding loop of the human A2BAR using the Gibson Assembly method [27] to obtain the A2A(ECL2-A2B) mutant receptor. Chinese hamster ovary (CHO) cells were stably transfected either with the loop mutant, the wildtype (wt) human A2A- or the wt human A2BAR. The human influenza hemagglutinin (HA) tag was linked to the N-terminus of the coding sequence of each gene; we have previously confirmed that the tag does not interfere with ligand binding and function of these GPCRs. Using an intact cell-ELISA with an antibody against the HA tag of the recombinantly expressed receptors, we then measured the expression levels of the cell surface ARs. The surface expression level of the wt A2AAR was set at 100% (see Fig. 2 and Table 1), and all three receptors were found to be expressed at similar levels. The wt A2AAR and the A2A(ECL2-A2B) mutant were subsequently analyzed in 14

homologous competition binding assays using the A2A agonist radioligand [3H]CGS21680 and the A2AAR antagonist radioligand [3H]MSX-2 to determine KD (affinity) and Bmax (maximal number of binding sites) values (Table 1; for curves see Fig. 3A and Fig. 4C). The affinity of the mutant receptor for the A2A-selective agonist CGS21680 (Fig. 1) appeared to be slightly decreased in comparison to the wt A2AAR but the difference did not reach statistical significance. The Bmax values of the mutant and the wt A2AAR were nearly identical, in agreement with the results obtained by determination of the receptor cell surface expression by ELISA (see Methods). The affinity of the mutant receptor for the antagonist MSX-2 was decreased by 6-fold in comparison to the wt A2AAR.

3.2 Radioligand binding studies Radioligand competition binding studies were performed to further characterize the consequences of the loop exchange. Structurally diverse A2A and A2B agonists were investigated: the endogenous agonist adenosine, its close ribose-modified analog NECA, the A2A-selective agonist CGS21680 that bears a long acidic substituent in the 2-position of NECA, and PSB-15826, a 2-substituted adenosine derivative (Fig. 1). For binding assays membrane preparations of cells stably expressing either the wt or the mutant receptor were employed. Adenosine deaminase (ADA) was included to remove endogenous adenosine, except for experiments with adenosine. In a first set of experiments, the compounds were evaluated versus the A2A agonist radioligand [3H]CGS21680. Adenosine showed 22-fold reduced affinity at the loop mutant in comparison to the wt A2AAR (Fig. 4 A and E), while its analog NECA displayed similar affinity for both, the wt and the mutant receptors (Fig. 4 B and F, Ki values in Table 2). In order to evaluate the effect of ADA on agonist affinities, the adenosine analog NECA was additionally tested in the absence of ADA. NECA showed nearly identical affinities for the wt A2AAR and for the mutant receptor in the presence and absence of ADA (wt A2AAR: 37.3 nM with ADA and 78.2 nM without ADA; mutant: 24.7 nM with ADA and 46.8 nM 15

without ADA; Fig. 5 A and B; Table 3). The 2-substituted NECA derivative CGS21680 also exhibited no significant difference in affinity for the wt A2AAR as compared to the A2A(ECL2-A2B)AR mutant (Fig. 4 C and G). In contrast, the 2-substituted adenosine derivative PSB-15826 was 11-fold less potent at the mutant than at the wt A2AAR (Fig. 4 D and H). As a next step, the agonists were evaluated versus the A2AAR antagonist radioligand [3H]MSX-2. [3H]MSX-2 showed a KD value of 5.26 ± 0.24 nM at the wt A2AAR, while its KD value was reduced to 29.9 ± 6.9 nM (6-fold) at the mutant A2A(ECL2-A2B) receptor. The same shift was seen in homologous competition binding assays of MSX-2 versus [3H]MSX-2 (Fig. 3). The competition binding curves of the agonists versus the antagonist radioligand showed a biphasic behavior (Fig. 6), and the proportion of high- and low-affinity binding sites was altered in the A2A(ECL2-A2B) mutant as compared to the wt A2AAR. Adenosine, NECA and CGS21680 displayed somewhat higher affinity for the high-affinity site of the loop mutant than for that of the wt A2AAR (Fig. 6 A-C and E-G, Ki values in Table 2) but no significant change in affinity for the low-affinity site. In contrast, the bulky 2-substituted adenosine derivative PSB-15826 (Fig. 6 D and H) displayed a decreased affinity for the low-affinity site in the mutant in comparison with the wt A2AAR and unaltered affinity for the highaffinity binding site. The percentage of low- and high-affinity sites recognized by the structurally diverse agonists differed, adenosine, NECA and CGS21680 recognizing more high-affinity sites at the wt A2AAR, while the opposite was observed for PSB-15826 (Fig. 6). The proportion of high- and low-affinity sites changed in the A2A(ECL2-A2B) mutant depending on the structure of the agonist. The percentage of high-affinity sites recognized by adenosine decreased dramatically from 86% in the wt receptor to 29% in the loop mutant. In contrast, for PSB-15826 the proportion of high-affinity sites increased from 29% in the wt A2AAR to 49% in the mutant, while for NECA and CGS21680 no significant changes were observed.

16

In order to better understand the nature of ligand interactions, competition binding of the antagonist MSX-2 (unlabelled) versus the agonist radioligand [3H]CGS21680 was performed (Fig. 3 B). While a monophasic curve was observed for the wt A2AAR (Ki 14.2 nM), a clearly biphasic curve was obtained for the A2A(ECL2-A2B) mutant with Ki values of 2.30 nM and 6720 nM (ca. 50% each).

3.3 Functional studies (cAMP accumulation) Agonist-induced cAMP accumulation was determined to investigate the activation of the wt A2A- and A2BARs in comparison to the A2A(ECL2-A2B) mutant receptor (Fig. 7). Adenosine was significantly more potent at the high-affinity A2AAR (EC50: 170 nM) as compared to the low-affinity A2BAR (EC50: 12000 nM; Fig. 7 A; see Table 4) displaying similar efficacy at both receptor subtypes. Adenosine showed at the loop mutant a large rightward shift in comparison to the wt A2AAR, resulting in a similarly high EC50 value as at the wt A2BAR (EC50: 15300 nM, Table 4). Compared to the wt receptors, the efficacy for adenosine at the loop mutant was significantly increased (Fig. 7A) indicating that adenosine bound to the mutant receptor induced a receptor conformation that coupled more efficiently to the Gs protein than adenosine bound to the wt A2AAR. The adenosine derivative NECA was 10-fold more potent at the A2AAR as compared to the A2BAR. No statistically significant differences were noted, neither in potency nor in efficacy, between the wt A2AAR and the loop mutant (Fig. 7 B and values in Table 4). For evaluating the ADA effect on agonist potency, cAMP accumulation assays were also determined in the absence of ADA showing that ADA had no significant effect on the EC50 values (wt A2AAR: 10.5 nM with ADA, 8.10 nM without ADA; mutant: 7.21 nM with ADA, 9.84 nM without ADA) and on the efficacies (wt A2AAR: 52% with ADA, 53% without ADA; mutant: 59% with ADA, 74% without ADA) (Fig. 5 C and D; Table 3).

17

The 2-substituted NECA-derivative CGS21680 (Fig. 7 C and Table 4) was a poor agonist at A2BARs (EC50 > >10,000 nM), while it showed similarly high potency and efficacy at the wt A2AAR (EC50 16.6 nM) and the A2A(ECL2-A2B) mutant (EC50 27.1 nM). The 2-substituted adenosine derivative PSB-15826 (Fig. 7 D and Table 4) was completely inactive at the wt A2BAR confirming its high selectivity, and showed similarly high potency at the wt A2AAR and the loop mutant (EC50 53.5 and 65.7 nM, respectively). PSB-15826 was significantly less efficacious at the loop mutant in comparison to the wt A2AAR (84 versus 58%).

3.4 Supervised molecular dynamics (SuMD) simulations of adenosine at the A2A(ECL2-A2B) mutant receptor SuMD simulations of adenosine (Video S1) highlight one main recognition pathway affecting receptor residues prevalently located at the ECL2A2B and secondarily at the receptor’s ECL3. We performed SuMD simulations on the loop mutant receptor. While replicas 1, 2, 4 and 5 are characterized by metastable sites associated to short adenosine residence times, during replica 3 the endogenous agonist engages with the extracellular loop 2 of the A2BAR (ECL2A2B) experiencing different metastable complexes with long residence time, as indicated by green spheres (Figure 8 A). The energetic stabilizations associated with metastable binding sites for adenosine are delineated in the energy profile plot (Figure 8 B). The analysis of the predicted energy interactions between adenosine and the receptor confirms for replica 3 the ligand’s inclination to establish highly stable intermediate complexes with ECL2A2B, gaining energetic stabilization values that are approximately double as compared to the adenosine orthosteric complex (which fluctuate between 8 and 42 kcal/mol).

18

One of the most stable adenosine - ECL2A2B intermediate complex is shown in Figure 8, panel C: the endogenous agonist establishes a hydrogen bond with the side-chain of Thr154 and engages Asn162 and Glu173 side-chains in electrostatic interactions through its ribose moiety.

3.5 SuMD simulations of NECA at the human A2A(ECL2-A2B) mutant receptor SuMD simulations of NECA (Video S2) show the agonist approaching the human A2A(ECL2-A2B) mutant receptor in a similar manner as adenosine: ECL2A2B and ECL3 define the extracellular vestibule involved in the NECA recognition pathway (Figure 8 D). Metastable sites, however, are not persistently occupied by the agonist, as shown by the restricted sphere diameters. The energetic landscape, associated with the six SuMD replicas performed (Figure 8 E), highlights numerous stable NECA-mutant receptor intermediate complexes. Predicted energetic stabilizations achieve values similar to those computed during adenosine SuMD trajectories. Interestingly, the NECA - receptor orthosteric complex is able to reach higher energetic stabilization (between 12 and 62 kcal/mol) compared to the endogenous agonist, decreasing the energetic gap between metastable and orthosteric complexes. A metastable NECA - ECL2A2B receptor complex is reported in Figure 8, panel F: the agonist engages with the side-chain of Glu173 in electrostatic interactions through its Nethylcarboxyribose moiety, while the purine scaffold is oriented towards ECL3.

4 Discussion Adenosine receptors (ARs) are important pharmacological targets for drug development due to their involvement in pathological processes [3]. In particular, antagonists for the A2A- and A2BAR subtypes have recently attracted enormous interest due to their potential application in cancer (immuno)therapy [6]. Moreover, the first A2AAR antagonist, istradefylline, was approved for the treatment of PD, and 19

further A2AAR antagonists are in development having great potential for the treatment of neurodegenerative diseases [47–49]. Despite the increasing number of solved GPCR crystal structures, many aspects of ligand-receptor interaction and receptor modulation still remain unclear. In the last decade, it has become obvious that not only the most highly conserved transmembrane domains (TMs) but also the extracellular loops (ECLs) play important roles in ligand binding and receptor activation. The ECLs, in particular the ECL2, show low sequence similarity even within GPCRs of the same subfamily. This diversity might be one of the explanations for the receptor subtype-selectivity of cognate agonists as well as synthetic ligands [22]. The closely related AR subtypes A2A and A2B show large differences in affinity for their cognate agonist adenosine, the molecular basis for which is still unclear. Previously we had generated a human A2BAR mutant receptor in which the ECL2 was exchanged for that of the A2AAR [23]. We showed in radioligand binding and functional studies that the ECL2 contributes to ligand affinity and selectivity. While the A2BAR is not activated by the A2A-selective agonist CGS21680, the loop mutant, containing the ECL2 of the A2AAR, gained affinity for this compound and was activated by it. In addition, we noticed that the ECL2 exchange also led to the stabilization of the receptor’s active conformation, resulting in significantly increased efficacies of all investigated agonists. In order to obtain further insights into the role of the ECL2 in A2A and A2BARs, we exchanged the whole ECL2 of the human A2AAR replacing it by the corresponding loop of the human A2BAR, yielding the mutant receptor A2A(ECL2-A2B). CHO cells were stably transfected with either the wt A2AAR, the wt A2BAR or the loop mutant, using retroviral transfection. The expression levels of the receptors were determined by ELISA in intact cells, which detects only the receptor population at the cell surface. In addition, homologous binding experiments estimating the number of receptor binding sites in the membrane preparations were performed. The loop mutant receptor was efficiently 20

transported to the cell surface and its expression level was similar to those of the wt A2A- and A2BAR. As expected, values obtained by ELISA were slightly lower than those determined by homologous competition binding experiments, since the ELISA was performed in intact cells detecting only receptors expressed in the cell membrane, while for radioligand binding membrane preparations including those of intracellular organelles were employed. The expression levels of the loop mutant, the wt A2BAR, and the wt A2AAR were not statistically different and therefore, the data obtained from the three cell lines can be compared. Our investigations included cAMP accumulation and radioligand binding studies using the A2A agonist radioligand [3H]CGS21680 which labels an active state of the receptor, and the antagonist radioligand [3H]MSX-2 which labels active and inactive receptor conformations [50]. The results for the investigated agonists differed significantly depending on their structures. They could be divided into two clusters: (i) N-ethylcarboxamido-ribose-modified adenosine derivative (NECA, CGS21680) and (ii) adenosine and PSB15826, its 2-substituted derivative.

4.2 NECA and its 2-substituted derivative CGS21680 The metabolically stable adenosine analog NECA was initially investigated in the presence of adenosine deaminase (ADA), an enzyme that removes endogenous adenosine, which might otherwise interfere with the assay [51]. In many preparations, significant levels of adenosine are present which bind to the adenosine receptors thereby affecting or even impeding radioligand binding and functional studies. Since previous studies had also demonstrated that ADA might act as an allosteric modulator of the A2AAR and other AR subtypes [52], we additionally performed functional and binding experiments with NECA in the absence of ADA (Fig. 5 and Table 3). Our results demonstrated that affinity and potency of NECA at both, the wt A2AAR and the mutant receptor, were not affected by ADA. 21

NECA did not show a significant difference between the wt A2AAR and the mutant A2A(ECL2-A2B) receptor in cAMP accumulation (Fig. 7B) as well as in binding studies versus the agonist radioligand [3H]CGS21680 (Fig. 4 B and F). The same was observed for the NECA derivative CGS21680 (Fig. 4C,G and Fig. 7C). Both NECA and CGS21680 share a common partial structure: the Nethylcarboxamido modification of the ribose moiety. The only differences observed on the loop mutant as compared to the wt A2AAR was the somewhat increased affinity for the high-affinity binding site detected in antagonist radioligand binding assays vs. [3H]MSX-2. These results indicate no direct binding of NECA to the ECL2 of the receptors. They also suggest a possibly less tight binding of the NECA-stabilized mutant receptor to the Gs protein as compared to the wt receptor.

4.3 Adenosine and its 2-substituted derivative PSB-15826 In contrast to NECA, adenosine itself and its derivative PSB-15826 displayed significantly altered profiles in the loop mutant as compared to the wt A2AAR. The curves for adenosine in functional and binding assays were strongly shifted to the right (90-fold increased EC50 value, 22-fold increased Ki value). Thus, the A2A(ECL2-A2B) loop mutant showed a similarly low affinity and potency for adenosine as the wt A2BAR with Ki and EC50 values in the micromolar range. Similar, but quantitatively smaller effects were observed for PSB-15826. While adenosine showed an increased efficacy in the loop mutant, the efficacy of PSB-15826 was decreased. Interestingly, the number of high-affinity sites recognized by adenosine in [3H]MSX-2 binding studies was significantly decreased in the loop mutant, while the opposite was observed for PSB-15826 (increase in high-affinity sites). These results strongl suggested a direct interaction of the ECL2 of the A2A- and/or A2BAR with adenosine and its 2-substituted derivative PSB-15826.

4.4 Supervised molecular dynamics studies 22

In order to better understand the reason for these dramatic differences between structurally similar agonists, adenosine and NECA, at the A2A(ECL2-A2B) loop mutant, SuMD simulation studies were performed. To this end, a homology model of the loop mutant receptor was established and supervised SuMD studies were performed for adenosine and NECA. The results suggested that adenosine can form a stable intermediate complex with the meta-binding site in the ECL2 which is energetically favorable in comparison with binding to the orthosteric site. Previous studies on several GPCRs suggested the presence of such a meta-binding site, also termed metastable binding site, in the extracellular region of the receptors (ECL2 and 3), to which ligands bind before their entry into the orthosteric binding pocket [53–55]. This mechanism was proposed to act as a selectivity filter for GPCRs. In contrast to adenosine, during SuMD simulations, NECA was able to bind to the meta-binding site with affinity comparable to the canonical orthosteric site (Figure 8E). Based on these results it can be speculated that adenosine forms highly stable complexes with the ECL2 of the A2BAR and that this negatively affects its entering into the orthosteric binding pocket. Thus, the affinity for the orthosteric binding site and the potency of adenosine to activate the A2BAR and the A2A(ECL-A2B) mutant are decreased in comparison to the wt A2AAR. On the contrary, NECA forms highly stable complexes with the orthosteric binding site, because of the carboxamido-modified ribose moiety which acts as an anchor in the binding pocket. Another explanation might be the formation of a ternary complex which has recently been proposed for A2AARs [56] based on computational studies. Two adenosine molecules might simultaneously bind to the receptor, one to the orthosteric and another one to the meta-binding site. The second adenosine molecule bound to the meta-binding site could allosterically modulate (decrease) the affinity of the orthosterically bound adenosine molecule.

Biphasic curves 23

Interesting results were obtained when the A2AAR agonists were evaluated in binding studies versus the A2AAR antagonist radioligand [3H]MSX-2. All A2AAR agonists showed biphasic competition curves at the wt A2AAR and at the A2A(ECL2-A2B) mutant. Previous studies at the adenosine A1AR [57] had shown that ADA has a direct effect on the receptor and could convert the low-affinity state of the receptor into two different affinity states. However, this hypothesis does not explain the results of our experiments since the biphasic behavior was found with all investigated A2AAR agonists including adenosine, which was studied in the absence of ADA. An interpretation would involve the presence of different receptor conformers able to bind the ligands with different affinities, since the biphasic curves were obtained with the A2AAR neutral antagonist [3H]MSX-2 which labels both the active and the inactive states of the receptor. However, the loop mutant receptor also showed a biphasic behavior when unlabelled MSX-2 was tested against the A2AAR agonist [3H]CGS21680, which is supposed to label only the high-affinity conformation of the receptor (Fig. 3 B). An explanation could be the presence of A2AAR di- or oligomers which may elicit allosteric modulation and cooperativity [58,59]. If dimer formation occurs, the binding of the first ligand molecule to the receptor induces a conformational change which will then modify the affinity of the second ligand to the second receptor protein involved in the dimer [59]. In this case, the loop exchange in the A2AAR producing the A2A(ECL2-A2B) mutant resulted in altered, more pronounced allosteric effects depending on the structure of the investigated ligand. The ECL2 may be responsible for specific conformational changes which influence the second receptor protein involved in the di-/oligomer and consequently the affinity of agonists (and antagonists) for the receptor proteins within the di- or oligomer. Previous studies on class A GPCRs already demonstrated that the ECL2 is involved in conformational changes, i.e. in the A2BAR loop mutant we found a stabilization of an active conformation [23], in the complement anaphylatoxin C5a receptor (C5aR) the ECL2 can stabilize an inactive conformation [60], and in the M3 muscarinic receptor the ECL2 stabilizes an active conformation [61], data which support our hypothesis. 24

4.3 Conclusions The most crucial finding of our study is the dramatic reduction in affinity and potency of adenosine upon exchanging the ECL2 of the A2AAR by that of the A2BAR. The ECL2-A2B was sufficient to switch the “high-affinity” A2AAR to a “low-affinity” A2B-like AR with regard to the endogenous agonist adenosine. In contrast, the structurally closely related agonist NECA was hardly affected by the loop exchange still showing high affinity for the mutant receptor. This shows once more that investigating the physiological ligands is essential because all observed effects may be strongly affected by small changes in ligand structure [62]. In summary, we have discovered that the ECL2 of ARs is important for adenosine recognition and for receptor subtype-selectivity. The presence of a meta-binding site for adenosine in the ECL2 may be responsible for or contribute to the observed effects.

Acknowledgments E.D.F., A.C.S., and C.E.M. were supported by the German Federal Ministery of Education and Research (BMBF project Bonn International Graduate School in Drug Sciences (BIGS DrugS)) and by the State of North-Rhine Westfalia (NRW International Research Graduate School BIOTECHPHARMA). The laboratory of M.M.S. is very grateful to Chemical Computing Group, OpenEye, and Acellera for the scientific and technical partnership, and gratefully acknowledges the support of NVIDIA Corporation for the donation of the Titan V GPU used for this research. R.F. was supported by a grant from the Spanish Ministerio de Ciencia, Innovación y Universidades (# 2019_RTI2018094204-B-I00; it may include EU FEDER funds). G.N. was supported by a grant from the Spanish Ministerio de Economía y Competitividad (MINECO #SAF2017-84117-R; it may include EU FEDER funds). 25

Conflict of interest No conflict of interest.

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Table 1. Expression levels (Bmax) and KD values of the human A2AAR and the human A2A(ECL2A2B) receptor determined by homologous competition binding versus [3H]CGS21680 and [3H]MSX-2. The cell surface expression levels determined by ELISA are shown for comparison. Data represent mean ± SD of three independent experiments, unless otherwise noted.

Receptor

KD ± SD (nM) Bmax ± SD (fmol/mg of protein) ELISA % expression level

A2AAR

A2A(ECL2-A2B)

[3H]CGS21680

[3H]CGS21680

72.1 ± 24.7

194 ± 79 a, ns

480 ± 70

799 ± 107 b, ns

100 ± 20

117 ± 38 c, ns

Results of two-tailed t test: ns not significantly different from the wt human A2AAR, ** p<0.01. a

p = 0.056

b

p = 0.179

c

n = 5.

35

Table 2. Affinities of adenosine A2A receptor agonists and A2B receptor partial agonist at the human A2AAR and the human A2A(ECL2-A2B) receptor mutant determined by radioligand binding studies versus [3H]CGS21680 (5 nM, upper panel) and [3H]MSX-2 (1 nM, lower panel). Data represent means ± SD of three independent experiments, unless otherwise noted. KD values were determined by homologous competition binding experiments (for CGS21680 see Table 1; for MSX-2: A2AAR KD = 5.26 ± 0.24 nM; A2A(ECL2-A2B) KD = 29.9 ± 6.9 nM). Ki values ± SD (nM) obtained vs. the agonist radioligand [3H]CGS21680 Receptor

Adenosine

NECA

CGS21680

PSB-15826

A2AAR

437 ± 26 a

37.3 ± 8.4

72.6 ± 23

33.2 ± 5.3

A2A(ECL2-A2B)

9540 ± 952 a, ***

24.7 ± 2.6 ns, p=0.379

204 ± 77 ns, p=0.119

384 ± 44 **

Ki values ± SD (nM) obtained vs. the antagonist radioligand [3H]MSX-2 Receptor

Adenosine

NECA

CGS21680

PSB-15826

high

low

high

low

high

low

high

low

A2AAR

1350 ± 325 a

> 10 000 a

99.2 ± 6.1

6610 ± 276

385 ± 239

> 10 000

6.96 ± 0.12

300 ± 164

A2A(ECL2-A2B)

298 ± 31 a,**

> 10 000 a

31.9 ± 1.5 ***

3790 ± 249 ns

40.7 ± 3.3 ns

> 10 000

169 ± 97 ns

> 10 000

“Low” refers to the Ki value of the biphasic curve at the lower agonist concentration, while “high” refers to the one at higher agonist concentration. Results of two-tailed t-test: ns not significantly different from A2AAR, ** p<0.01, *** p<0.001. a

The affinity of adenosine for the receptors may be underestimated because adenosine deaminase could not be used in the assay. 36

Table 3. Effects of adenosine deaminase on the binding and function of the agonist NECA at the human A2AAR and the human A2A(ECL2-A2B) receptor mutant. Competition binding assays of NECA vs [3H]CGS21680a,c NECA

Human A2AAR

Human A2A(ECL2-A2B)

Ki  SD

with ADA

37.3  8.4

24.7  2.6

(nM)

without ADA

78.2  21.8 ns

46.8  6.6 ns

cAMP accumulation assaysb,c NECA EC50  SD (nM) Efficacy  SD (%) a

Human A2AAR

Human A2A(ECL2-A2B)

with ADA

10.5  1.4

7.21  6.74

without ADA

8.10  3.01 ns

9.84  1.09 ns

with ADA

52  6

59  10

without ADA

53  11 ns

74  3 ns

Radioligand concentration: 5 nM, membrane preparations from CHO cells stably expressing the

relevant receptor. Data represent mean ± SD of three independent experiments. KD values for calculating Ki values were determined by homologous competition. b Data

are normalized to the effect of forskolin (100 µM), set as 100%; they represent means  SD of

three independent experiments. c

Statistical evaluation using the two-tailed t-test:

treated with ADA.

37

ns

not significantly different from the receptor

Table 4. EC50 values and maximal effects of selected agonists determined in cAMP accumulation assays at the human A2AAR, the human A2BAR and the mutant A2A(ECL2-A2B)AR. Data for the inverse mutant (A2B(ECL2-A2A) are included for comparison. Data are normalized to the effect of forskolin (100 M), set as 100%. Data are mean  SD from three to five independent experiments.a,b Agonist

a

Human A2AAR

Human A2A(ECL2 A2B)AR

Human A2BAR

EC50  SD (nM)

Efficacy  SD (%)

EC50  SD (nM)

Efficacy  SD (%)

EC50  SD (nM)

Efficacy  SD (%)

EC50  SEM (nM)

Efficacy  SEM (%)

Adenosine

170 ± 50

44 ± 6

15,300 ± 6420***

68 ± 10 **

12,000 ± 948 ***

40 ± 3 ns

1170 ± 120

58 ± 2

NECA

10.5 ± 1.4

52 ± 6

7.21 ± 6.74 ns

59 ± 10 ns

109 ± 7 **

51 ± 14 ns

199 ± 53

101 ± 9

CGS21680

16.6 ± 1.5

49 ± 8

27.1 ± 25.2 ns

64 ± 8 ns

31,100 ± 1860***

28 ± 10 *

47,000 ± 7700

105 ± 4

PSB-15826

53.5 ± 3.6

84 ± 7

65.7 ± 18.8 ns

58 ± 6 **

> 100,000 d

nd

nd

nd

Statistical evaluation was performed by a two-tailed t-test: ns not significantly different from the human A2AAR. * p < 0.05, ** p < 0.01,

*** p < 0.001. bnd, c

Human A2B(ECL2 A2A)AR c

not determined.

Seibt et al., 2013

d no

EC50 value could be determined since the agonist is inactive at the receptor.

38

Figre legends Fig. 1. Structures of investigated compounds.

Fig. 2. Cell surface expression levels of the human A2AAR, the human A2BAR and the A2A(ECL2A2B) receptor determined by ELISA. Values were normalized to the untransfected CHO cells, set as 0%, and to the A2AAR, set as 100%. Data represent mean values ± SD of three (A2BAR), four (A2AAR) or five (A2A(ECL2-A2B)) independent experiments performed in triplicates. Two-tailed t-test was performed comparing the A2AAR with the A2BAR and the A2A(ECL2-A2B): both comparisons were not significantly different (ns) from the A2AAR (p = 0.507 and p = 0.250, respectively).

Fig. 3. (A) Homologous competition binding [3H]MSX-2 (1 nM) versus MSX-2 at the human A2AAR and at the A2A(ECL2-A2B) receptor. Membrane preparations from CHO cells stably expressing the receptor were used. Data points represent means ± SD of three independent experiments performed in duplicates. The corresponding KD and Bmax values are listed in Table 1. (B) Competition binding studies at the human A2AAR and at the A2A(ECL2-A2B) receptor versus [3H]CGS21680 (5 nM) using MSX-2. The curves were performed using membrane preparations of Hela and CHO cells stably expressing the A2A and the loop mutant receptor, respectively. Data points represent means ± SD of two-three independent experiments performed in duplicates. For some points, the error bars are shorter than the height of the symbol thus, they are not visible. “Low” and “high” refer to the Ki values of the biphasic curve at the lower and higher cold ligand concentration, respectively.

Fig. 4. Competition binding studies at the human A2AAR and at the A2A(ECL2-A2B) receptor versus [3H]CGS21680 (5 nM) using (A) adenosine, (B) NECA, (C) CGS21680 and (D) PSB-15826. Membrane preparations from CHO cells stably expressing the receptors were used. Data points represent means ± SD of three independent experiments performed in duplicates. For some points, the error bars are shorter than the height of the symbol and thus, they are not visible. The bar diagrams indicate pKi ± SD values corresponding to the binding assay data of (E) adenosine, (F) NECA, (G) 39

CGS21680 and (H) PSB-15826 against [3H]CGS21680 at the human A2AAR and at the A2A(ECL2A2B) receptor. Two-tailed t-test was performed: ns not significantly different from the A2AAR, **p < 0.01, ***p < 0.001.

Fig. 5. Adenosine deaminase effect on function and binding of the agonist NECA at the human A2AAR and the A2A(ECL2-A2B) receptors. (A,B) Competition binding studies versus [3H]CGS21680 (5 nM) using NECA at the human A2AAR (C) and at the A2A(ECL2-A2B) receptor (D). Membrane preparations from CHO cells stably expressing the receptors were used in presence or absence of adenosine deaminase (ADA). Data points represent means ± SD of three independent experiments performed in duplicates. (C,D) NECA-induced cAMP accumulation studies at CHO cells stably expressing the human A2AAR (A) and the mutant A2A(ECL2-A2B) AR (B) with and without preincubation with adenosine deaminase (ADA). Data represent mean  SD from three independent experiments performed in duplicates. Data are normalized to the effect of 100 M forskolin, set as 100%. Corresponding EC50, maximal effects and corresponding Ki values are listed in Table 2.

Fig. 6. Competition binding studies at the A2AAR and the A2A(ECL2-A2B)AR versus [3H]MSX-2 (1 nM) using (A) adenosine, (B) NECA, (C) CGS21680 and (D) PSB-15826. Membrane preparations from CHO cells stably expressing the receptor were used. Data points represent means ± SD of three independent experiments performed in duplicates. For some points, the error bars are shorter than the height of the symbol and thus, they are not visible. The arrows located at the graph side correspond to the two different conformational states of the receptor (black: A2AAR and red: A2A(ECL2-A2B)AR). The bar diagrams indicates pKi ± SD values corresponding to the binding assay data of (E) adenosine, (F) NECA, (G) CGS21680 and (H) PSB-15826 against [3H]MSX-2 at the human A2AAR and at the A2A(ECL2-A2B)AR. “Low” refers to the higher Ki value calculated from the biphasic curve, while “high” refers to the lower Ki value. A two-tailed t-test was performed: ns not significantly different from the A2AAR, **p < 0.01, ***p < 0.001.

40

Fig. 7. Agonist-induced cAMP accumulation studies at CHO cells stably expressing the human A2AAR, the human A2BAR and the mutant A2A(ECL2-A2B)AR using (A) adenosine, (B) NECA, (C) CGS21680 or (D) PSB-15826 as agonist. Data represent means  SD from three to five independent experiments performed in duplicates. For some points, the error bars are shorter than the height of the symbol and thus, they are not visible. Data are normalized to the effect of 100 M forskolin, set as 100%. Corresponding EC50 and maximal effects are listed in Table 3.

Fig. 8. Supervised Molecular Dynamics (SuMD) of adenosine and NECA at the loop mutant homology model. (A) Adenosine Recognition Pathway (Pollicino) Analyzer, (B) adenosine Energy Landscape Profile and (C) adenosine - A2A(ECL2-A2B) receptor meta-stable complex. (D) NECA Recognition Pathway (Pollicino) Analyzer, (E) NECA Energy Landscape Profile and (F) NECA A2A(ECL2-A2B) receptor meta-stable complex. Each SuMD replica is associated to a color, both in the Recognition Pathway (Pollicino) Analyzer and Energy Landscape Profile. Images of metastable sites report lipid head tail atoms as gray spheres.

Author contributions E.D.F. performed the majority of the experiments, analyzed the data, and wrote the first draft of the manuscript supported by A.C.S.. S.H. performed some of the experiments. V.P., G.D. and S.M. performed the computational studies which were supervised by S.M.. G.N. and R.F. suggested some of the experiments and contributed to the interpretation of the data. A.E.T. synthesized and analyzed PSB-15826. C.E.M. and A.C.S. designed and supervised the study. C.E.M. analyzed the data and wrote the final version of the manuscript. All authors contributed to writing of the manuscript.

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