Supplemental material to this article can be found at: http://molpharm.aspetjournals.org/content/suppl/2016/08/05/mol.116.104828.DC1 1521-0111/90/3/214–224$25.00 http://dx.doi.org/10.1124/mol.116.104828 MOLECULAR PHARMACOLOGY Mol Pharmacol 90:214–224, September 2016 Copyright ª 2016 by The American Society for Pharmacology and Experimental Therapeutics Dihydromunduletone Is a Small-Molecule Selective Adhesion G Protein–Coupled Receptor Antagonist s Hannah M. Stoveken, Laura L. Bahr, M. W. Anders, Andrew P. Wojtovich, Alan V. Smrcka, and Gregory G. Tall Department of Pharmacology and Physiology (H.M.S., L.L.B., M.W.A., A.P.W., A.V.S.), and Department of Anesthesiology (L.L.B., A.P.W.), University of Rochester Medical Center, Rochester, New York; and Department of Pharmacology, University of Michigan, Ann Arbor, Michigan (G.G.T.) Received April 21, 2016; accepted June 10, 2016 Downloaded from ABSTRACT Adhesion G protein–coupled receptors (aGPCRs) have emerging response element (SRE)-luciferase–based screening approach roles in development and tissue maintenance and is the most to identify GPR56/ADGRG1 small-molecule inhibitors. A 2000- prevalent GPCR subclass mutated in human cancers, but to compound library comprising known drugs and natural products date, no drugs have been developed to target them in any was screened for GPR56-dependent SRE activation inhibitors molpharm.aspetjournals.org disease. aGPCR extracellular domains contain a conserved that did not inhibit constitutively active Ga13-dependent SRE subdomain that mediates self-cleavage proximal to the start of activation. Dihydromunduletone (DHM), a rotenoid derivative, the 7-transmembrane domain (7TM). The two receptor proto- was validated using cell-free aGPCR/heterotrimeric G protein mers, extracellular domain and amino terminal fragment (NTF), guanosine 59-3-O-(thio)triphosphate binding reconstitution as- and the 7TM or C-terminal fragment remain noncovalently says. DHM inhibited GPR56 and GPR114/ADGRG5, which have bound at the plasma membrane in a low-activity state. We similar tethered agonists, but not the aGPCR GPR110/ADGRF1, recently demonstrated that NTF dissociation liberates the 7TM M3 muscarinic acetylcholine, or b2 adrenergic GPCRs. DHM N-terminal stalk, which acts as a tethered-peptide agonist inhibited tethered peptide agonist-stimulated and synthetic permitting receptor-dependent heterotrimeric G protein activa- peptide agonist-stimulated GPR56 but did not inhibit basal tion. In many cases, natural aGPCR ligands are extracellular activity, demonstrating that it antagonizes the peptide agonist. at ASPET Journals on October 2, 2021 matrix proteins that dissociate the NTF to reveal the tethered DHM is a novel aGPCR antagonist and potentially useful agonist. Given the perceived difficulty in modifying extracellular chemical probe that may be developed as a future aGPCR matrix proteins to create aGPCR probes, we developed a serum therapeutic. Introduction event in which the P19 residue becomes the N terminus of the ∼17–26 amino acid tethered-agonist stalks (Lin et al., 2004; – Adhesion G protein coupled receptors (aGPCRs) are a Arac et al., 2012; Liebscher et al., 2014; Stoveken et al., 2015). – 33-member subclass of family B G protein coupled receptors Self-cleavage is thought to be constitutive, and the resultant (GPCRs) that have critical roles in tissue specification and receptor protomers or fragments remain noncovalently bound replenishment and human cancers (Langenhan et al., 2013; while in residence at the plasma membrane. In this state, the Hamann et al., 2015). It was recently discovered that the N terminus of the postcleaved tethered-agonist stalk binds extracellular peptide stalks emanating from the first firmly within a hydrophobic b-strand network that would transmembrane-spanning helix of multiple adhesion GPCRs preclude it from engaging the carboxy-terminal fragment are tethered-peptide agonists that activate aGPCR-mediated (CTF)/seven-transmembrane domain (7TM) orthosteric site. heterotrimeric G protein signaling (Liebscher et al., 2014; A current hypothesis which we favor that describes the Demberg et al., 2015; Stoveken et al., 2015; Wilde et al., 2016). dynamism of receptor activation consists of two components: 1) Tethered-agonist regulation is intimately linked to the ability anchored protein ligands bind aGPCR N-terminal fragment of aGPCRs to execute a precise autocatalytic, self-cleavage (NTF) binding determinants, and 2) the action of shear force created by cell movement serves to dissociate the NTF from the This work was supported by a PhRMA Foundation predoctoral fellowship in CTF to release or decrypt the tethered agonist (Karpus et al., pharmacology to H.M.S. The production of recombinant G proteins used for this work was supported by the National Institutes of Health National 2013; Langenhan et al., 2013; Scholz et al., 2015; Stoveken et al., Institute of General Medical Sciences [Grant GM088242 to G.G.T.]. 2015). We reconstituted aGPCRs, GPR56 (ADGRG1) and dx.doi.org/10.1124/mol.116.104828. s This article has supplemental material available at molpharm. GPR110 (ADGRF1), with purified G protein heterotrimers and aspetjournals.org. provided a biochemical demonstration that experimentally ABBREVIATIONS: aGPCR, adhesion G protein–coupled receptor; CTF, carboxy-terminal fragment; DHM, dihydromunduletone; DMEM, Dulbecco’s modified Eagle’s medium; DMSO, dimethylsulfoxide; ETC, electron transport chain; FBS, fetal bovine serum; GPCR, G protein–coupled receptor; GTPgS, 59-3-O-(thio)triphosphate; HA, hemagglutinin; HEK293, human embryonic kidney 293; NTF, amino terminal fragment; PAR, protease activated receptors; [35S]GTPgS, 59-O-(3-[35S]thio)triphosphate; SRE, serum response element; 7TM, seven-transmembrane domain. 214 A Class-Selective Small-Molecule Adhesion GPCR Antagonist 215 induced NTF dissociation dramatically enhanced aGPCR- were purchased individually as powders from MicroSource Discovery mediated G protein activation (Stoveken et al., 2015). The known Systems, Inc. (Gaylordsville, CT). Rotenone and polyclonal hemag- protein ligands of aGPCRs are fibrillar extracellular matrix glutinin (HA) antibody were purchased from Sigma-Aldrich (St. Louis, proteins or, in some instances, proteins presented by neighboring MO). Latrunculin B was from Calbiochem (San Diego, CA). Quant-iT cells (Hamann et al., 1996; Sugita et al., 1999; Stacey et al., 2003; PicoGreen double stranded DNA reagent was from Invitrogen (Carls- bad, CA). Streptavidin Sepharose High Performance was from GE Xu et al., 2006; Bolliger et al., 2011; Chiang et al., 2011; Luo et al., ’ Healthcare (Chicago, IL). Sulfo-NHS-Biotin was from Thermo Scien- 2011; Silva et al., 2011; Boucard et al., 2012; O Sullivan et al., tific (Waltham, MA). 59-O-(3-[35S]thio)triphosphate ([35S]GTPgS) and 2012; Paavola and Hall, 2012; Paavola et al., 2014; Petersen the phRLuc-N1 plasmid were from PerkinElmer (Waltham, MA). The et al., 2015; Scholz et al., 2015). Based on the proposed two- pGL4.33 [luc2/SRE/Hygro] plasmid was purchased from Promega component mode of ligand-mediated aGPCR activation, we (Madison, WI). anticipate that natural NTF ligands or ligand-based NTF- High-Throughput Screen. HEK293T cells were maintained in binding mimetics will be of limited, stand-alone pharmacological Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Grand Island, use to manipulate receptor activities. Indeed, application of dilute, NY) supplemented with 10% (v/v) fetal bovine serum (FBS). Cells were transiently transfected with GPR56 7TM (Met-Thr383 to Ile693) soluble extracellular matrix protein ligands to aGPCR cell Q226L culture–based signaling assays most likely does not recapitulate pcDNA3.1 or GNA13 pcDNA3.1 (cDNA.org) and the SRE- luciferase reporter using a polyethylenimine transfection method authentic aGPCR pharmacology. Hence, there is a critical need (Wang et al., 2010; Oner et al., 2013; Stoveken et al., 2015). Five to develop small-molecule modulators that can bypass the hours after transfection, cells were trypsinized, counted, and seeded at Downloaded from two-component ligand-mediated regulation process and directly 15,000 cells per well in 384-well plates in 20 ml of phenol red–free modulate receptor activities. A number of adhesion GPCR small- DMEM buffered with 25 mM Hepes, pH 7.4, and incubated overnight molecule screening efforts are ongoing, but to our knowledge, only with ∼3–5 mM compounds in dimethylsulfoxide (DMSO); Spectrum a few candidate small-molecule activators were proposed to Collection, at the URMC High Throughput Screening Core (Micro- regulate GPR97 (Gupte et al., 2012; Southern et al., 2013). source Discovery Systems, Inc. Gaylordsville, CT). The next morning, We conducted a chemical-screening effort to identify small- 20 ml of SteadyLite luciferase reagent (PerkinElmer) was robotically molpharm.aspetjournals.org molecule inhibitors of GPR56, an attractive therapeutic target dispensed into each well and incubated for 15 minutes at room due to its proposed roles in neurogenesis, neuromaintenance, temperature. Luminescence was read on a PerkinElmer Envision Plate Reader. and cancer progression (Piao et al., 2004; Shashidhar et al., Directed Dual SRE-Luciferase Assay. HEK293T cells main- 2005; Xu et al., 2006, 2010; Yang et al., 2011; Saito et al., 2013; tained in DMEM 110% (v/v) FBS were transiently transfected in Hamann et al., 2015). GPR56 signals through G13 and multiple 24-well format using the polyethylenimine reagent with 200 ng of laboratories showed