Supplemental material to this article can be found at: http://molpharm.aspetjournals.org/content/suppl/2016/09/28/mol.116.105015.DC1 1521-0111/90/6/715–725$25.00 http://dx.doi.org/10.1124/mol.116.105015 MOLECULAR PHARMACOLOGY Mol Pharmacol 90:715–725, December 2016 Copyright ª 2016 by The American Society for Pharmacology and Experimental Therapeutics Role of the Second Extracellular Loop of the Adenosine A1 Receptor on Allosteric Modulator Binding, Signaling, and Cooperativity s Anh T. N. Nguyen, Elizabeth A. Vecchio, Trayder Thomas, Toan D. Nguyen, Luigi Aurelio, Peter J. Scammells, Paul J. White, Patrick M. Sexton, Karen J. Gregory, Lauren T. May, and Arthur Christopoulos Monash Institute of Pharmaceutical Sciences (A.T.N.N., E.A.V., T.T., L.A., P.J.S., P.J.W., P.M.S., K.J.G., L.T.M., A.C.), Monash Downloaded from e-Research Centre (T.D.N.), and Department of Pharmacology (A.T.N.N., E.A.V., P.M.S., K.J.G., L.T.M., A.C), Monash University, Victoria, Australia Received May 2, 2016; accepted September 27, 2016 ABSTRACT molpharm.aspetjournals.org Allosteric modulation of adenosine A1 receptors (A1ARs) offers a positive cooperativity between PD81723 and NECA was reduced novel therapeutic approach for the treatment of numerous central on alanine substitution of a number of ECL2 residues, including and peripheral disorders; however, despite decades of research, E170ECL2 and K173ECL2, whereas mutation of W146ECL2 and there is a relative paucity of structural information regarding the W156ECL2 decreased VCP171 cooperativity with NECA. Molec- A1AR allosteric site and mechanisms governing cooperativity with ular modeling localized a likely allosteric pocket for both modu- orthosteric ligands. We combined alanine-scanning mutagenesis lators to an extracellular vestibule that overlaps with a region used of the A1AR second extracellular loop (ECL2) with radioligand by orthosteric ligands as they transit into the canonical A1AR binding and functional interaction assays to quantify effects on orthosteric site. MD simulations confirmed a key interaction allosteric ligand affinity, cooperativity, and efficacy. Docking and between E172ECL2 and both modulators. Bound PD81723 is molecular dynamics (MD) simulations were performed using an flanked by another residue, E170ECL2, which forms hydrogen at ASPET Journals on September 29, 2021 A1AR homology model based on an agonist-bound A2AAR bonds with adjacent K168ECL2 and K173ECL2. Collectively, our structure. Substitution of E172ECL2 for alanine reduced the data suggest E172ECL2 is a key allosteric ligand-binding de- affinity of the allosteric modulators PD81723 and VCP171 for terminant, whereas hydrogen-bonding networks within the extra- the unoccupied A1AR. Residues involved in cooperativity with the cellular vestibule may facilitate the transmission of cooperativity orthosteric agonist NECA were different in PD81723 and VCP171; between orthosteric and allosteric sites. Introduction (May et al., 2007b). One promising approach for achieving greater selectivity is to target topographically distinct alloste- Gprotein–coupled receptors (GPCRs) account for ap- ric sites on GPCRs (Christopoulos, 2002; May et al., 2007b; proximately 30% of the targets of prescription medicines Wootten et al., 2013). Allosteric ligands have the potential to (Overington et al., 2006). Most of these medicines act at the modulate the binding and/or signaling properties of an endogenous agonist (orthosteric) site; however, in many cases, orthosteric ligand and/or modulate GPCR activity even in orthosteric-based drug discovery remains suboptimal owing to the absence of orthosteric ligand (May and Christopoulos, a lack of sufficient selectivity between related GPCR subtypes 2003; Christopoulos, 2014). Allosteric regions typically display greater sequence divergenceacrosstheGPCRsubtypes, This work was funded by the National Health and Medical Research Council enabling greater subtype selectivity compared with the of Australia (NHMRC) [Program Grants APP1055134, APP1084487, APP1084246]. A.T.N.N. is a recipient of an Australian Endeavour scholarship orthosteric domains. An additional advantage is that many and fellowship. L.T.M. is a recipient of an Australian Research Council allosteric modulators can selectively “tune” tissue responses Discovery Early Career Researcher Award (DECRA), A.C. is a senior principal research fellow, and P.M.S. is a principal research fellow, of the NHMRC. either up or down when and where the endogenous agonist is K.J.G. is an NHMRC Overseas Biomedical Postdoctoral Training Fellow. present. This type of spatial and temporal specificity of action dx.doi.org/10.1124/mol.116.105015. s This article has supplemental material available at molpharm@ that can be achieved with allosteric modulators is unattain- aspetjournals.org able with orthosteric ligands, which continuously stimulate or ABBREVIATIONS: [3H]DPCPX, 8-cyclopentyl-1,3-dipropylxanthine, [dipropyl-2,3-3H(N)]; A1AR, adenosine A1 receptor; AR, adenosine receptor; ATL525, (2-amino-4,5,6,7-tetrahydro-benzo[b]thiophen-3-yl) biphenyl-4-yl-methanone); CHO, Chinese hamster ovary; ECL2, second extracellular loop; GPCR, G protein–coupled receptor; HA, human influenza hemagglutinin; MD, molecular dynamics; NECA, 59-N-ethylcarboxamidoadenosine; PAM, positive allosteric modulator; PBS, phosphate-buffered saline; PD81723, 2-amino-4,5-dimethyl-3-thienyl)-[3-(trifluoromethyl)phenyl]methanone; 3xHA-A1AR, human A1AR containing an amino terminal triple human influenza hemagglutinin epitope tag; TM, transmembrane; VCP171, (2-amino-4-(3-(trifluoromethyl)phenyl)thiophen-3-yl)(phenyl)methanone; WT, wild-type. 715 716 Nguyen et al. inhibit receptor function where and when they are present as important in the actions of the subtype selective 2-amino- (Christopoulos and Kenakin, 2002; May et al., 2007b). 3-benzoylthiophene A1AR allosteric modulators, as this The endogenous nucleoside adenosine acts via four adeno- region is the least conserved in both sequence and length sine GPCRs (ARs): A1AR, A2AAR, A2BAR, and A3AR across the AR subtypes. (Fredholm et al., 2001). The highly subtype selective A1AR In our accompanying article (Nguyen et al., 2016), we report allosteric ligand, PD81723 (2-amino-4,5-dimethyl-3-thienyl)- our work demonstrating that ECL2 can significantly influence [3-(trifluoromethyl)phenyl]methanone), was the first syn- orthosteric agonist and antagonist pharmacology. Further- thetic positive allosteric modulator (PAM) of GPCR agonist more, ECL2 is involved in the “transit” of agonists into the function identified (Fig. 1) (Bruns and Fergus, 1990; Bruns orthosteric site within the transmembrane (TM) domains. In et al., 1990; May et al., 2010; Hill et al., 2014). The utility of the present study, we assessed the influence of select A1AR- A1AR PAMs has been demonstrated in the kidney (Park et al., ECL2 alanine mutations on the affinity, cooperativity, 2012), heart (Bruns and Fergus, 1990; Valant et al., 2010), and and efficacy of the well characterized allosteric enhancer neurons (Pan et al., 2001; Imlach et al., 2015), suggesting that PD81723 and a more recent derivative, VCP171 ((2-amino-4- A1AR PAMs provide a promising therapeutic avenue to treat (3-(trifluoromethyl)phenyl)thiophen-3-yl)(phenyl)methanone) renal and myocardial ischemia-reperfusion injury and chronic (Fig. 1) (Aurelio et al., 2009b). We have found that ECL2 neuropathic pain. Given their therapeutic potential, numer- contributes to a common allosteric pocket in the extracellular Downloaded from ous studies have investigated the structure-activity relation- vestibule that overlaps with the transit pocket used by ships of A1AR PAMs, predominantly exploring derivatives orthosteric ligands, identifies E172ECL2 as a vital residue based on the 2-amino-3-benzoylthiophene scaffold (Romagnoli for modulator affinity, and highlights how different residues et al., 2015). Despite extensive derivation and interrogation of can mediate cooperativity and direct agonism in a manner this core scaffold for more than two decades; however, existing that can vary with the nature of the allosteric or orthosteric A1ARs PAMs display largely low affinity, low cooperativity, ligand under investigation. and generally poor solubility. molpharm.aspetjournals.org Alternative approaches, such as structure-based studies, are thus required to assist the design of more potent, selective, Materials and Methods and efficacious A1AR modulators. The recent high-resolution Materials. VCP171 was synthesized as described previously A2AAR crystal structures in both antagonist- and agonist- (Aurelio et al., 2009b; Valant et al., 2014); PD81723 was synthesized bound conformations have facilitated the generation of A1AR as described previously (Aurelio et al., 2009a). All other reagents were homology models (Jaakola et al., 2008; Doré et al., 2011; Lebon obtained from suppliers described in the accompanying article et al., 2011; Xu et al., 2011); however, there remains a relative (Nguyen et al., 2016). paucity of information regarding the location of the A1AR Receptor Mutagenesis, Transfection, Cell Culture, and allosteric site, reflecting the key challenges associated with Receptor Expression. Receptor mutagenesis, transfection, cell at ASPET Journals on September 29, 2021 structure-function analyses of allostery, because the observed culture, and measurement of cell surface expression of wild-type effect of any modulator in the presence of orthosteric ligand (WT) and mutant human A1ARs containing a triple human influenza reflects