1521-0111/89/5/485–491$25.00 http://dx.doi.org/10.1124/mol.115.102657 MOLECULAR PHARMACOLOGY Mol Pharmacol 89:485–491, May 2016 Copyright ª 2016 by The American Society for Pharmacology and Experimental Therapeutics Molecular Basis of Ligand Dissociation from the Adenosine A2A Receptor Dong Guo,1 Albert C. Pan, Ron O. Dror,2 Tamara Mocking,3 Rongfang Liu, Laura H. Heitman, David E. Shaw, and Adriaan P. IJzerman Division of Medicinal Chemistry, Leiden Academic Centre for Drug Research, Leidorken University, RA Leiden, The Netherlands (D.G., T.M., R.L., L.H.H., A.P.I.J.); D. E. Shaw Research, New York, New York (A.C.P., R.O.D., D.E.S.); and Department of Biochemistry and Molecular Biophysics, Columbia University, New York, New York (D.E.S.) Received November 26, 2015; accepted February 11, 2016 Downloaded from ABSTRACT How drugs dissociate from their targets is largely unknown. We altered the ligand’s dissociation rate despite only marginally investigated the molecular basis of this process in the adenosine influencing its binding affinity, demonstrating that even receptor A2A receptor (A2AR), a prototypical G protein–coupled receptor features with little contribution to affinity may prove critical to the (GPCR). Through kinetic radioligand binding experiments, we dissociation process. Our results also suggest that ZM241385 molpharm.aspetjournals.org characterized mutant receptors selected based on molecular follows a multistep dissociation pathway, consecutively inter- dynamic simulations of the antagonist ZM241385 dissociating acting with distinct receptor regions, a mechanism that may also from the A2AR. We discovered mutations that dramatically be common to many other GPCRs. Introduction receptor. The identified residues, many of which were not in contact with the ligand in the crystal structure, were sub- – G protein coupled receptors (GPCRs) represent the largest sequently mutated, and the resulting mutant receptors were class of drug targets; they are home for one-third of all then subjected to experimental determination of the ligand’s at ASPET Journals on September 29, 2021 marketed drugs (Overington et al., 2006). Recent develop- affinity and dissociation kinetics. We observed that the ments in this field have yielded several crystal structures that E169ECL2Q, H2647.29A, and T2566.58A mutants accelerated provide an atomic view on the ligand-receptor interaction ZM241385’s dissociation from the receptors, and the I662.63A, (Katritch et al., 2013). This information is vital for molecular S672.64A, K153ECL2A, and L2677.32A mutants slowed down the understanding of the ligand-receptor interaction; however, process. Interestingly, these mutations only minimally influ- the knowledge gained is largely “frozen” in nature and reflects enced ZM241385’s binding affinity. Our results also suggest only the final interaction at the ligand-binding site. How a that ZM241385 follows a multistep dissociation pathway, ligand dissociates from its binding pocket and which residues consecutively interacting with topographically distinct re- are involved in this dynamic process are still largely unknown. gions of the receptor. We speculate that such a multistep Here, we chose the human adenosine A2A receptor (A2AR), a dissociation process may be common to other GPCRs as well. prototypical GPCR, for such a mechanistic exploration. We applied molecular dynamics (MD) simulations to the crystal Materials and Methods structure of the A2AR [Protein Data Bank (PDB) ID: 4EIY] 3 (Liu et al., 2012) to help in the selection of amino acid residues Chemicals and Reagents. [ H]-ZM241385 (specific activity × 21 that have potential interactions with the crystallographic 47.7 Ci mmol )waspurchasedfromARCInc.(St.Louis,MO). ZM241385 was a gift from Dr. S. M. Poucher (Astra Zeneca, Macclesfield, ligand, ZM241385, along its dissociation pathway from the UK). Adenosine deaminase was purchased from Boehringer Mannheim (Manheim, Germany). Bicinchoninic acid (BCA) and BCA protein assay This work was supported by the Innovational Research Incentive Scheme of reagent were obtained from Pierce Chemical Company (Rockford, IL). The Netherlands Research Organization [Grant 11188]. All other chemicals were of analytical grade and obtained from standard 1Current affiliation: Jiangsu Key Laboratory of New Drug Research and Clinical Pharmacy, Xuzhou Medical College, Xuzhou, Jiangsu, China. commercial sources. 2 Current affiliation: Departments of Computer Science and of Molecular and Molecular Dynamics. MD simulations of the ZM241385-A2AR Cellular Physiology, and Institute for Computational and Mathematical complex were performed on a special-purpose machine, Anton (Shaw Engineering, Stanford University, Palo Alto, California. et al., 2009). The simulation system was prepared and equilibrated 3Current affiliation: Amsterdam Institute for Molecules, Medicines and Systems, VU University Amsterdam, Amsterdam. following the protocols detailed in Kruse et al. (2012). Briefly, the dx.doi.org/10.1124/mol.115.102657. system was prepared using the crystallized complex of PDB ID 4EIY ABBREVIATIONS: A2AR, adenosine A2A receptor; BCA, bicinchoninic acid; CV, collective variable; GPCR, G protein–coupled receptors; HEK293, human embryonic kidney 293 cells; MD, molecular dynamics; PBS, phosphate-buffered saline; PDB, Protein Data Bank; RT, residence time; TAMD, temperature accelerated molecular dynamics; WT, wild-type; ZM241385, 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl) phenol. 485 486 Guo et al. Site-Directed Mutagenesis. Site-directed mutants were con- structed by polymerase chain reaction mutagenesis using pcDNA3.1- hourA2AR with N-terminal HA and FLAG tags and C-terminal His tag as a template. The mutants E169A and E169Q were generated by Baseclear (Leiden, The Netherlands), and the other mutants were created in house as follows. Mutant primers for directional polymerase chain reaction product cloning were designed using the online Quickchange primer design program (Agilent Technologies, Santa Clara, CA), and primers were obtained from Eurogentec (Maastricht, The Netherland). All DNA sequences were verified by Sanger sequencing at LGTC (Leiden, The Netherlands). Cell Culture, Transfection, and Enzyme-Linked Immuno- sorbent Assay. We followed procedures as described previously (Lane et al., 2012). Briefly, human embryonic kidney (HEK) 293 cells were grown as monolayers in Dulbecco’s modified Eagle’s medium supplemented with stable glutamine, 10% newborn calf serum, streptomycin, and penicillin at 37°C in a moist, 7% CO2 atmosphere. The cells were transfected with plasmid DNA using a calcium Downloaded from phosphate method followed by 48-hour incubation before membrane preparation. For enzyme-linked immunosorbent assay, 24 hours after transfection, cells were split into 96-well poly-D-lysine-coated plates at a density of 1 Â 106 cells/well. After an additional 24 hours, the cells were fixed with 4% formaldehyde and then washed with phosphate- Fig. 1. MD simulation based on PDB entry 4EIY. A contact was identified buffered saline (PBS) before adding the primary antibody, monoclonal when either the oxygen atom in the furan ring (red atom) or the nitrogen M2-anti-FLAG antibody (1:1000) and incubating for 30 minutes at molpharm.aspetjournals.org atom of the exocyclic amine group (blue atoms) of ZM241385 (inset) was 37°C. Next, the antibody was removed and the cells were washed within 4 Å of the backbone nitrogen or carbonyl oxygen of a receptor with Dulbecco’s modified Eagle’s medium/25 mM HEPES before adding residue. The ZM241385-A2AR binding pathway passes through multiple the second antibody, monoclonal anti-Mouse-HRP 1:5000 and incubat- distinct consecutive steps, represented by three superimposed snapshots: red (initial pose, 0 ns), green (28 ns), and magenta (32 ns). This figure was ing for 30 minutes at 37°C. After removing the second antibody and generated with ICM Browser v3.8 (Molsoft) from snapshots exported from washing the cells with warm PBS, 3, 39,5,59-tetramethyl-benzidine was VMD v1.9.1 added and incubated for 5 minutes in the dark. The reaction was stopped with 1 M H3PO4, and absorbance was read at 450 nm using a Victor2 plate reader (PerkinElmer Life and Analytical Sciences). solvated in approximately 14,000 three-point water model molecules Membrane Preparation. Cells were detached from the plates (MacKerell et al., 1998), 28 sodium ions, 38 chloride ions, and 134 by scraping into PBS. Cells were collected and centrifuged at 700g at ASPET Journals on September 29, 2021 dipalmitoylphosphatidylcholine lipids in a 72-Å3 box consisting of (3000 rpm) for 5 minutes. Pellets from 10 plates (10 cm ø) were pooled approximately 65,000 atoms. The prepared system was minimized and resuspended in 8 ml ice-cold buffer containing 50 mM Tris-HCl, and equilibrated as previously described (Kruse et al., 2012). pH 7.4. Cell suspension was homogenized with an UltraThurrax We then ran 10 temperature-accelerated molecular dynamics homogenizer (Heidolph Instruments, Schwabach, Germany). Cell (TAMD) simulations (Maragliano and Vanden-Eijnden, 2006) of the suspension was centrifuged at 100,000g (31,000 rpm) in a Beckman ZM241385 ligand dissociating from the A2A receptor, starting from the Optima LE-80K ultracentrifuge at 4°C for 20 minutes. The pellet was equilibrated snapshot of the ligand-receptor complex with different resuspended in 4 ml of Tris buffer, and the homogenization and random initial velocities. Trajectories ended with the ligand com- centrifugation