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 that GPR56 robustly activates serum GPR56 7TM (encodes Met-Thr383 to Ile693), 25 ng of GPR56 A386M response element (SRE) or serum response factor luciferase 7TM (encodes Ala386Met to Ile687), or 200 ng of GNA13Q226L gene reporters (Iguchi et al., 2008; Kim et al., 2010; Wu et al., pcDNA3.1 plasmids; 100 ng of the SRE-luciferase reporter; and 1 ng 2013; Stoveken et al., 2015; Kishore et al., 2016). We adapted of phRLuc (Stoveken et al., 2015). Total DNA levels were balanced our construct that produces the full tethered-agonist-activated with empty pcDNA3.1. Cells were serum starved for 10 hours the at ASPET Journals on October 2, 2021 GPR56 CTF for use as an SRE luciferase human embryonic following day; DHM or synthetic-peptide agonist was added to the cell kidney 293 (HEK293) cell high-throughput inhibitor screening medium at 2 and 4 hours, respectively, during the serum-starving platform (Stoveken et al., 2015). A counterscreen was developed phase. Cells were harvested in culture medium by trituration, washed in Tyrode’s solution (137 mM NaCl, 2.7 mM KCl, 1 mM MgCl , 1.8 mM in which each library compound was tested for inhibition of 2 CaCl , 0.2 mM Na HPO , 12 mM NaHCO , and 5.5 mM D-glucose), – 2 2 4 3 constitutively active Ga13-Q226L activated SRE luciferase. and lysed in firefly luciferase reagent (NanoLight Technologies, Inc., Since G13 activation is the first step downstream of GPR56, all Pinetop, AZ). Firefly luminescence was quenched using Renilla lu- compound hits that inhibited the GPR56 CTF, but not Ga13- ciferase buffer containing 3 mM coelenterazine H (Dyer et al., 2000). Q226L, are likely to act at the receptor level and not at points Luminescence was read using a TriStar2 plate reader (Berthold, downstream in the pathway. Sixty-six compounds from the Wildbad, Germany). All firefly luciferase data were normalized to the ∼2000-compound Spectrum Collection chemical library Renilla luciferase signal and expressed as fold increase over the signal inhibited GPR56-CTF–dependent luciferase activity, 63 of obtained from cells transfected with SRE-luciferase only. which also inhibited Ga13-Q226L and were therefore elimi- Insect Cell Culture, Baculovirus Generation, and Adhesion nated. The identified compounds were dihydromunduletone GPCR Membrane Preparation. Spodoptera frugiperda 9 and High-Five insect cells were maintained as described (Stoveken et al., (DHM), a derivative of the natural-product mundulone, and two 2015). Baculoviruses were generated per the manufacturer’s protocol structurally related natural products that we have temporarily (Bac-to-Bac manual; Invitrogen). For adhesion GPCR membrane “ ” “ ” termed H.M.S.1 and H.M.S.2, pending chemical syntheses preparations, High-Five insect cells were infected with a 1/50 dilution (Burrows et al., 1959; Ollis and Sutherland, 1961). (1° amplification) or a 1/100 dilution (2° amplification) of recombinant Here, we present evidence that DHM is a bona fide selective adhesion GPCR baculovirus stocks for 48 hours prior to cell harvest antagonist for a subset of aGPCRs. This is the first identifi- and preparation of native and urea-treated adhesion GPCR mem- cation of a small-molecule aGPCR inhibitor that has potential branes as described (Stoveken et al., 2015). to uncover novel aGPCR biology and represents a first step in Adhesion GPCR:G Protein Reconstitution Assays. Recombi- the development of a therapeutic strategy targeting aGPCRs. nant heterotrimeric G protein a subunits (Gai1,Ga13, Gas, Gaq) were purified from High-Five insect cells using the GST-Ric-8A or B

association method (Chan et al., 2011). Recombinant Gb1g2 was Materials and Methods purified from High-Five membranes using the Gai-His6 association method (Kozasa and Gilman, 1995). Adhesion GPCR membranes (1–5 Reagents and Antibodies. The GPR56 C-terminal antibody was mg) were reconstituted with purified G proteins (100 nM Ga of interest a gift from Dr. Randy Hall (Emory University, Atlanta, GA) (Paavola and 500 nM Gb1g2) in preincubation buffer [50 mM Hepes (pH 7.4), et al., 2011). Dihydromunduletone, isorotenone, mundulone, and 1 mM dithiothreitol, 1 mM EDTA, and 3 mg/ml bovine serum albumin] deguelin were from the Spectrum Collection chemical library and with 0 or 20 mM GDP. Compounds (DHM, deguelin, mundulone, 216 Stoveken et al. isorotenone, rotenone), synthetic-peptide agonist, or DMSO vehicle compound concentration, and curve fitting was performed using control (,5% v/v) was added to the membrane/G protein mixture and GraphPad Prism (GraphPad Software, La Jolla, CA). incubated for 30 minutes at 25°C. An equal volume of 59-3-O-(thio)- triphosphate (GTPgS) binding buffer (preincubation buffer containing 35 10 mM MgCl2 and 50 mM NaCl) with 2 mM GTPgS, [ S]GTPgS Results (20,000–50,000 cpm/pmol), and the indicated concentrations of com- Identification of GPR56 Small-Molecule Inhibitors pounds or synthetic-peptide agonist was added to initiate the kinetic assay (compounds or peptides were included in both incubation through High-Throughput Screening. Isolated adhesion mixtures to maintain the final concentrations). Triplicate samples GPCR CTFs/7TM domains are highly active due to the were removed from the reaction mixtures and quenched at the engaged state of the tethered-peptide agonist (Paavola indicated time points with 20 mM Tris (pH 7.7), 100 mM NaCl, et al., 2011, 2014; Liebscher et al., 2014; Stoveken et al.,

10 mM MgCl2, 1 mM GTP, and 0.08% (m/v) deionized polyoxyethylene 2015). We exploited this property of the GPR56 7TM to 10 lauryl ether C12E10. Quenched samples were filtered through develop a high-throughput inhibitor screening assay using BA85 nitrocellulose filters (GE Healthcare Chicago, IL), dried, and the SRE-luciferase gene reporter. GPR56 activates G13 counted by liquid scintillation counting as described (Stoveken et al., robustly and thus, Ga13-sensitive SRE- or serum response 2015). factor–luciferase reporters (Shashidhar et al., 2005; Iguchi Mitochondrial Complex I (NADH:Ubiquinone Oxidoreduc- et al., 2008; Stoveken et al., 2015; Kishore et al., 2016). For tase) Activity Assay. Complex I activity was measured from frozen C57BL/6J mouse heart mitochondria isolated by differential centri- the high-throughput screen and counterscreen, HEK293 Downloaded from fugation (Wojtovich et al., 2011). Mitochondria (25 mg/ml) were cells were cotransfected with an SRE-luciferase reporter Q226L suspended in assay buffer (25 mM K2HPO4/KH2PO4, 10 mM MgCl2, plasmid and GPR56 7TM or GNA13 pcDNA3.1 plas- 2.5 mg/ml bovine serum albumin, 1 mM KCN, 75 mM NADH, pH 7.2, mids, respectively. Vehicle-treated cells were the positive 37°C) in the presence of the indicated isoflavone compounds. After the controls (top limit of the assays), and cells treated with addition of coenzyme Q1 (0.1 mM), complex I activity was measured as latrunculin B, which disrupts the signaling axis downstream the rotenone-sensitive rate (nmol/min/mg) of NADH oxidation (« 5 of both GPR56 and Ga13, were the negative controls (bottom 21 molpharm.aspetjournals.org 6180 M at 340 nm) and expressed as percentage of inhibition limit of the assays) (Evelyn et al., 2007). The Z9 score was 0.67 (Nadtochiy et al., 2007). for the screening assay and 0.8 for the counterscreening Rho GTPase Activation Assay. HEK293T cells were plated at assay (Supplemental Fig. 1) (Zhang et al., 1999). 4 Â 106 cells per 10-cm plate. Twenty-four hours later, cells were transfected with 6.5 mg GPR56 A386M 7TM pcDNA3.1 and 1 mg The 2000-compound Spectrum Collection chemical library ∼ – 3XHA-RhoA pcDNA3.1 (cDNA.org) using the polyethylenimine trans- (estimated 3 5 mM each) was then screened and coun- fection method (Oner et al., 2013). The next day, cells were trypsi- terscreened. In the screen, 66 compounds were identified that nized, pooled, and plated at 4 Â 106 cells per 10-cm plate. The seeded inhibited activity .90% ($3 standard deviations above con- transfected cells were allowed to attach for 8 hours and then serum trol), and 63 of these compounds also inhibited activity in the starved overnight. Serum-starved cells were treated with 10 mM DHM counterscreen, and were thus eliminated (Fig. 1, A and B). The or DMSO vehicle for 15 minutes. P7 synthetic peptide agonist or three hits were DHM and two compounds we have termed at ASPET Journals on October 2, 2021 DMSO vehicle was added to the cells and incubated for an additional H.M.S.1 and H.M.S.2. Since DHM was highly efficacious and 1 5 minutes. Cells were washed once with phosphate-buffered saline commercially available, we decided to characterize this mol- protease inhibitor mixture (23 mg/ml phenylmethylsulfonyl fluoride, ecule in detail. 21 mg/ml Na-p-tosyl-L-lysine-chloromethyl ketone, 21 mg/ml L-1-p- The concentration dependence of DHM inhibition of GPR56 tosylamino-2-phenylethyl-chloroketone, 3.3 mg/ml leupeptin, and 3.3 Q226L mg/ml lima bean trypsin inhibitor) and lysed in 1 ml of lysis buffer 7TM or GNA13 was evaluated using an independent dual SRE-luciferase assay (Fig. 1D). At concentrations of DHM up [25 mM Hepes (pH 7.2), 150 mM NaCl, 5 mM MgCl2, 1% Nonidet P-40, 5% glycerol, protease inhibitor mixture]. Lysates were cleared by to 5 mM, GPR56 7TM activity was inhibited and Ga13-QL centrifugation at 21,000g, and the total protein concentration of each activity was not, confirming the results obtained in the lysate sample was determined by amido black protein assay screening assay. The library was then scanned for compounds (Schaffner and Weissmann, 1973). Equal amounts of protein from structurally related to DHM to evaluate performance in the each lysate were added to tubes containing 60 mg of Rhotekin-RBD screen and counterscreen, even if subpar. Four isoflavonoid agarose (Cytoskeleton, Inc., Denver, CO) and tumbled for 45 minutes. compounds were found that are structurally related to DHM, Rhotekin-RBD agarose was washed three times with lysis buffer, and the isoflavone mundulone and the rotenoids isorotenone and beads were suspended in 50 mlof2Â sample buffer [200 mM Tris (pH deguelin, all of which inhibited GPR56 7TM activity more 6.8), 20% glycerol, 4% SDS, 200 mM dithiothreitol, 0.02% bromophe- nol blue] (Laemmli, 1970). Relative levels of active Rho recovered from than the Ga13-QL activity (Fig. 1E), although each result Rhotekin agarose were compared with levels of total Rho obtained constituted a single trial due to the nature of high throughput from input samples by immunoblotting with the HA antibody. screening. The fourth compound, the rotenoid, rotenone PicoGreen Cell Detachment Assay. HEK293T cells were plated inhibited activity in the screen only marginally better than at 2.5 Â 104 cells/well in 96-well plates in DMEM 1 10% FBS (v/v). it did in the counterscreen (n 5 1) (Fig. 1E). Twenty hours later, compounds (DHM, deguelin, isorotenone, A potential indirect mode of DHM action would be to mundulone, and rotenone) or DMSO vehicle control was serially negatively regulate GPR56 biosynthesis or trafficking or both. 1 diluted in DMEM 10% FBS from 50 mM to 100 nM, and the cell To rule out this possibility, GPR56 7TM High-Five insect cells medium was exchanged. Culture medium was replaced 24 hours later were cultured in the presence or absence of 5 mM DHM for with 100 ml TE buffer [10 mM Tris-HCl (pH 7.7) and 1 mM EDTA) for 24 hours. The cells were collected and the relative amounts of 1 hour to induce hypotonic cell lysis. Quant-iT PicoGreen double stranded DNA reagent was diluted 1:200 in TE, and 100 ml was added cell-surface receptor levels were found to be identical (Sup- to each well for 15 minutes to allow dye/DNA complex formation. The plemental Fig. 2; Supplemental Method 1). However, when fluorescent dye was excited at 485 nm using 0.1% lamp energy, and rotenone, isorotenone, deguelin, or mundulone (5 mM) was emission was measured at 535 nm using the Berthold TriStar2 Plate included in an overnight High-Five insect cell culture, the cells Reader. Relative fluorescent units were plotted as functions of appeared to die or arrest growth, and thus relative GPR56 cell A Class-Selective Small-Molecule Adhesion GPCR Antagonist 217

Fig. 1. A cell-based high-throughput screen and counter- screen to identify candidate small-molecule GPR56 in- hibitors. HEK293T cells were cotransfected with the SRE-luciferase reporter plasmid and full tethered- agonist-activated GPR56 7TM pcDNA3.1 (A) or constitu- Downloaded from tively active GNA13Q226L pcDNA3.1 (B). Transfected cells were incubated overnight in 384-well format with ∼2000 individual compounds (5 mM each) from the Spectrum Collection. SRE reporter activity was measured in re- sponse to each compound and reported as percentage of inhibition over DMSO control for the screen and counter- screen. Top hits for individual compounds are represented as red, single dots, and compounds structurally similar to molpharm.aspetjournals.org the top hits are represented as green dots. The 90% inhibition threshold is shown with a light-blue line. (C) Compounds 1 (DHM), 2 (H.M.S.1), and 3 (H.M.S.2) were identified as top hits from the GPR56 7TM inhibitor screen that did not appreciably inhibit the constitutively active (CA) Ga13-Q226L activity in the counterscreen. (D) Pur- chased dihydromunduletone was tested for concentration- dependent GPR56 7TM or Ga13-Q226L inhibition using a distinct, directed HEK293T cell SRE-luciferase assay. Error bars are the average 6 thes.d.ofthreeexperimen- tal replicates. (E) DHM-related flavonoid compounds

present in the Spectrum Collection [4 (mundulone), 5 (iso- at ASPET Journals on October 2, 2021 rotenone), 6 (rotenone), and 7 (deguelin)] all inhibited GPR56 7TM–activated SRE luciferase to a greater extent than Ga13-Q226L–activated SRE luciferase.

surface levels were not measured. This is not a surprising treated with increasing concentrations of compounds, and outcome given that rotenoids, predominantly rotenone, are 24 hours later, the cell medium and detached cells were used commercially as piscicides and insecticides. Upon appli- removed. The adherent cells that remained were measured by cation to a natural water source, rotenoids efficiently enter the a proxy assay in which the relative amounts of total DNA were bloodstream through fish gills, potently inhibit complex I of quantified by PicoGreen assay. All of the compounds induced the electron transport chain (ETC), and cause death (Chance some degree of HEK293 cell detachment, but DHM was the et al., 1963; Li et al., 2003; Ling, 2003; Tomlin, 2009). least potent and could safely be used in cultured HEK293 cell We were concerned that this known property of rotenoids to assays at concentrations up to 10 mM (Fig. 2A). Similarly, we induce cell/organism death through ETC inhibition might tested each compound to determine potency of ETC complex I prevent the usefulness of DHM as an adhesion GPCR in- inhibition in an isolated mitochondria assay. Again, DHM was hibitor. A comparative analysis was conducted on DHM, the least-potent complex I inhibitor, which correlated with its mundulone, and rotenoid inhibition of ETC complex I versus property as the least-potent mediator of HEK293 cell de- the ability to induce death of cultured cells. HEK293 cells were tachment (Fig. 2B). It is important to note that the apparently 218 Stoveken et al.

compound and reduced the rate at which GPR56 7TM activated G13 .75% (from 0.18 to 0.04 minute21) (Fig. 3B). The potency of DHM inhibition was determined by measur- ing the initial linear rates (0– to 3-minute time courses) of GPR56 7TM–stimulated G13 activation in response to in- creasing DHM concentrations. The IC50 of DHM inhibition was ∼21 mM, which is quite potent considering that DHM may interfere with the first-order action of a tethered-peptide agonist (Fig. 3C). DHM (50 mM) also inhibited intact full-length GPR56 activation of G13 4-fold (from 0.16 to 0.04 minute21) and full-length GPR56, in which we chemically dissociated the NTF with 7 M urea to induce the tethered- agonist-activated state (from 0.70 to 0.18 minute21) (Fig. 3D). The high constitutive activity (0.16 minute21) of the “intact” receptor may represent two populations: one that is truly intact and has the NTF bound to the CTF, and a second

population in which the NTF has been shed or dissociated Downloaded from from the CTF either spontaneously during live cell culture or procedurally during preparation of native receptor mem- branes, which involves multiple washing and Dounce homog- enization steps. The latter population would be active with a decrypted tethered-peptide agonist. These points are impor- tant when considering the potential mechanisms of DHM molpharm.aspetjournals.org inhibition as a competitive antagonist to the tethered agonist or as a negative allosteric modulator. DHM Inhibits GPR56-Related GPR114, but Not GPR110 or Class A GPCRs. GPR114/ADGRG5 is an ad- hesion GPCR that promotes cyclic AMP production in cells Fig. 2. The rank potency of flavonoid derivative–induced HEK293 cell and shares an identical amino acid sequence with GPR56 at detachment correlates with electron transport chain complex I inhibition. (A) the N-terminal tip of its tethered agonist (Gupte et al., 2012; Adherent HEK293 cells were grown for 24 hours in the presence of the Wilde et al., 2016) (Fig. 4A). We reconstituted the GPR114 indicated concentrations of the five flavonoids (DHM, mundulone, rotenone, 7TM domain with purified Gasshort and Gb1g2 and showed deguelin, isorotenone). Detached cells were removed and the relative levels at ASPET Journals on October 2, 2021 of adhered cells remaining were measured by proxy assay of PicoGreen that it robustly activates Gs GTPgS binding. At a concentra- quantification of total DNA. (B) Isolated mitochondria were supplied with tion of DHM that maximally inhibited GPR56 (50 mM), the NADH and an electron acceptor (Q1), and the loss of NADH over 5 minutes rate of GPR114 7TM–stimulated Gs activity was also at 340 nM was measured in the presence of the indicated amounts of each inhibited dramatically (Fig. 4B). GPR110/ADGRF1, a flavonoid. IC50 values were calculated from monoexponential association curves fitted using GraphPad Prism. R.F.U., relative fluorescent units. Error Gq-coupled group VI aGPCR, contains an amino acid sequence bars are the average 6 the s.d. of three experimental replicates. at the N-terminal tip of its tethered agonist that is distinct from GPR56/114 (Fig. 4A). When DHM (50 mM) was applied to very potent IC50 values of compound inhibition of complex I the GPR110 7TM, it failed to inhibit GPR110 stimulation of (range 5.6–146.9 nM) were generated using an assay with Gq GTPgS binding (Fig. 4C). To determine if DHM receptor isolated and permeabilized mitochondria. The nature of this inhibition was specific to select adhesion GPCRs, we tested it assay does not address questions of compound bioavailability with two class A GPCRs. The influence of DHM (50 mM) on to intact cells or organisms. In sum, DHM is the most receptor-stimulated G protein activation was measured for efficacious compound at inhibiting GPR56 in the cell-based the Gq-coupled M3 muscarinic acetylcholine receptor and the SRE-luciferase assays and the least-potent inhibitor of com- Gs-coupled b2 adrenergic receptor in the absence and pres- plex I and inducer of cultured cell death. ence of agonists (carbachol and isoproterenol, respectively). Secondary Assay Validation of DHM Inhibition of DHM was unable to inhibit G protein activation by either of GPR56 Activity. Our measurements of adhesion GPCR these receptors under basal or agonist-stimulated conditions activation of reconstituted G proteins provide an entirely (Fig. 4, D and E). independent, cell-free assay system to evaluate small- DHM Inhibits Peptide Agonist–Stimulated but Not molecule modulators obtained from cell-based screens Basal Receptor Activity. To delve into the mechanism by (Stoveken et al., 2015). Insect cell membranes expressing which DHM inhibits select aGPCRs, we tested DHM in- the recombinant adhesion GPCR of interest are prepared and hibition of a GPR56 7TM receptor with a partially defective reconstituted with purified, recombinant Ga and Gb1g2. tethered agonist. GPR56 F385M 7TM lacks the first two Assays are initiated by the addition of [35S]GTPgS, and the amino acids of the tethered agonist, and the third is mutated rates of aGPCR-stimulated G protein activation ([35S]GTPgS to an initiator methionine (TYFAVLM… to MAVLM…) (Fig. binding to Ga) are measured with or without the influence of 5A). The activity of this receptor is diminished by ∼80%, but added compounds. DHM and related compounds (deguelin, can be rescued by stimulation with the synthetic peptide mundulone, isorotenone, and rotenone; 50 mM each) inhibited agonist P7 (TYFAVLM) supplied in trans (Stoveken et al., the kinetics of GPR56 7TM–stimulated G13 GTPgS binding to 2015). To test if DHM is an aGPCR orthosteric antagonist, we varying degrees (Fig. 3A). DHM was the best inhibitory attempted a Schild analysis using the F385M receptor to A Class-Selective Small-Molecule Adhesion GPCR Antagonist 219

measure DHM/P7 competitive action/binding (Schild, 1949; Arunlakshana and Schild, 1959; Bindslev, 2008). As the peptide-agonist concentration response was performed in response to increasing concentrations of DHM, the sigmoidal curves exhibited clear rightward shifts. However, sufficient synthetic peptide agonist could not be supplied to achieve maximal efficacies, as concentrations above 100–150 mM typically decreased activity by an unknown means (Fig. 5B). These results clearly demonstrate the effectiveness of DHM as an aGPCR inhibitor and leave open the possibility that DHM is an orthosteric antagonist. To test antagonism in another manner, we used the GPR56 A386M 7TM receptor, which has a more severely compromised tethered agonist that essentially renders the receptor a model of basal activity (Fig. 5A) (Stoveken et al., 2015). The sensitivity of our G protein reconstitution assay was increased

by the use of five times more GPR56 A386M 7TM receptor and Downloaded from by not including 20 mM GDP in the assay to promote the most efficient G13:receptor precoupling. Under these conditions, GPR56 A386M 7TM provided modest G13 activation. How- ever, DHM (50 mM) failed to inhibit this basal, tethered- agonist-independent activity (Fig. 5B). DHM did not inhibit basal signaling of GPR56 A386M 7TM when standard assay molpharm.aspetjournals.org conditions were used (Fig. 5D). The synthetic P7 peptide agonist TYFAVLM (100 mM) was then used as a surrogate to the tethered agonist to markedly stimulate GPR56 A386M 7TM activation of G13. When DHM (50 mM) and P7 (100 mM) were coapplied to GPR56 A386M 7TM, DHM provided nearly complete inhibition of the synthetic peptide-agonist-induced receptor activation (Fig. 5D). To verify GPR56 inhibition by DHM in cells, Rho GTPase activation assays were performed to test acute receptor inhibition. GPR56 activates G13 to stimulate Rho guanine at ASPET Journals on October 2, 2021 nucleotide exchange factor-mediated Rho-GTP production (Iguchi et al., 2008; Luo et al., 2011; Paavola et al., 2011). HEK293T cells transfected with HA-tagged RhoA and GPR56 A386M 7TM were treated with DHM (10 mM) or vehicle. RhoA-GTP production was stimulated by application of P7 peptide agonist (10 mM) or vehicle to the cell culture medium for 5 minutes. RhoA-GTP was isolated from each treated cell group by Rhotekin pull-down assay and visualized by anti-HA immunoblotting (Fig. 5E). Treatment with DHM alone did not affect basal RhoA-GTP levels. P7 treatment alone provided an increase in the amount of RhoA-GTP produced, but P7 peptide activation was clearly blocked by DHM. DHM inhibition of the P7 peptide response was then recapitulated using GPR56 SRE-luciferase assays. Cells transfected with GPR56 A386M 7TM were incubated with increasing concentrations of DHM. P7 peptide agonist was added, and SRE-luciferase activity was measured. DHM inhibited the P7 peptide–induced luciferase activity in a concentration-dependent manner (Fig. 5F). Cells were also

Fig. 3. Secondary validation assays demonstrating that DHM and other DHM. The IC50 of DHM inhibition was derived from the semilog plot of a one- rotenoids inhibit GPR56. (A–C) Prepared membranes containing the full phase monoexponential association function using GraphPad Prism. Error tethered-agonist-activated GPR56 7TM receptor were reconstituted with bars are the average of each linear line slope (rate) 6 the s.d. of three technical purified G13 heterotrimer. The kinetics of G13 GTPgS binding were measured replicates. (D) Membranes containing full-length (Karpus et al., 2013) GPR56 in the presence of 50 mM isoflavonoids (DHM, deguelin, mundulone, isorote- receptor were untreated or treated with ice-cold 7 M urea to mimic the proposed none, or rotenone) or DMSO vehicle control (A), or 50 mMDHMorDMSO process of ligand-mediated receptor activation via N-terminal extracellular control with 20 mM GDP present (B). Error bars are the average 6 the s.d. of fragment dissociation. The kinetics of receptor-activated G13 GTPgSbinding three experimental replicates. (C) The initial rates of full tethered-agonist- were measured in the presence of 50 mM DHM or DMSO control. Error bars activated GPR56 7TM stimulation of G13 GTPgS binding were determined were frequently smaller than the plotted symbols and are the average 6 the from four point (t = 0,1,2,3 min.) linear functions for each concentration of s.d. of three experimental replicates. Full-length FL, full-length. 220 Stoveken et al.

treated with a fixed concentration of 3 mM DHM or vehicle and then stimulated with an increasing concentration of P7 peptide agonist. DHM treatment blunted P7 peptide activa- tion at each concentration (Fig. 5G). In conclusion, DHM antagonizes synthetic-peptide agonist and tethered-peptide agonist–mediated aGPCR activation in isolated membranes and HEK293T cell–based assays, but it does not inhibit basal receptor signaling. This strongly indicates that the mode of DHM inhibition is to act as an antagonist that interferes with peptide agonist binding, but does not preclude the possibility that DHM may act as a negative allosteric modulator.

Discussion We support a hypothesis where adhesion GPCR activation requires at least two separable events to occur in a prescribed order: 1) adhesion GPCR NTFs bind to anchored protein Downloaded from ligands, and 2) shear force generated by cell movement in relation to the anchored-ligand:NTF complex overcomes large energetic and entropic barriers that are required to dissociate the NTF from the CTF (Yona et al., 2008; Karpus et al., 2013; Langenhan et al., 2013; Scholz et al., 2015; Stoveken et al.,

2015). NTF dissociation results in decryption of the adhesion molpharm.aspetjournals.org GPCR tethered-peptide agonist, which rapidly and perhaps irreversibly binds its orthosteric site on the 7TM domain (CTF). This model nearly mandates that adhesion GPCR signaling deactivation follows an internalization/desensitiza- tion mechanism. Based on this model, the development of natural adhesion GPCR ligand mimetics seems to be a very unlikely means to create receptor modulators. It may be far better to develop synthetic-peptide modulators based on the

tethered-peptide agonists that activate multiple adhesion at ASPET Journals on October 2, 2021 GPCRs (Liebscher et al., 2014; Demberg et al., 2015; Stoveken et al., 2015; Wilde et al., 2016). Such peptides would act akin to protease activated receptors (PAR) agonist pep- tides that modulate protease-activated receptors (PAR GPCRs) (Vu et al., 1991; Scarborough et al., 1992). Alterna- tively, and perhaps even more effectively, small-molecule modulators could be identified that would bypass the two event-mediated activation processes and regulate adhesion GPCR activities directly. Using a cell-based high-throughput screening assay to uncover small-molecule inhibitors of tethered-agonist- activated GPR56, we identified DHM as a novel adhesion GPCR antagonist. In direct receptor-stimulated G protein– activation assays, DHM inhibited two of three adhesion GPCRs tested, but not two class A GPCRs, indicating that DHM has selective action for a subset of adhesion GPCRs. DHM inhibition of GPR56 was verified using a cell-based RhoA GTPase effector enzyme assay and by an independent, dual luciferase-gene-reporter assay. Mechanistically, DHM did not inhibit basal GPR56 activity, but did inhibit GPR56 activity stimulated by its tethered-peptide agonist or by a synthetic-peptide agonist. These data establish that the mode Fig. 4. DHM exhibits specificity for group VIII adhesion GPCRs, but does not inhibit GPR110 or agonist-stimulated b2 adrenergic or M3 muscarinic acetylcholine class A GPCRs. (A) Alignment of the tethered agonist regions of group VI and group VIII adhesion GPCRs. Membranes containing the incubated with Gq and the indicated combinations of 50 mM carbachol tethered-agonist-activated GPR114 7TM (B) or GPR110 7TM (C) receptors (CCh), 50 mM DHM, or vehicle prior to measurement of Gq GTPgS binding were reconstituted with purified Gs or Gq heterotrimers, respectively. The kinetics. (E) b2 Adrenergic receptor membranes were incubated with Gs kinetics of heterotrimeric G protein GTPgS binding were measured in the and the indicated combinations of 10 mM isoproterenol (ISO), 50 mM DHM, presence of DHM (50 mM) or an equivalent volume of DMSO vehicle or vehicle prior to measurement of Gs GTPgS binding kinetics. Error bars control. (D) M3 muscarinic acetylcholine receptor membranes were are the average 6 the s.d. of three experimental replicates. A Class-Selective Small-Molecule Adhesion GPCR Antagonist 221 Downloaded from molpharm.aspetjournals.org at ASPET Journals on October 2, 2021

Fig. 5. DHM inhibits tethered- and synthetic-peptide agonist–stimulated GPR56, but does not inhibit basal receptor activity, a mode of action consistent with that of a neutral antagonist. (A) Alignment of stalk regions of intact tethered agonist and compromised tethered agonist GPR56 7TM domain receptors. The seven amino acid synthetic GPR56 agonist peptide is aligned underneath. (B) Partially compromised tethered agonist GPR56 F385M 7TM receptor membranes were reconstituted with G13, and P7 peptide concentration–dependent activation of G13 GTPgS binding was measured in response to increasing concentrations of DHM. Initial rates are plotted and were determined from four point (t = 0,1,2,3 min.) linear functions. Error bars are the average of each linear line slope (rate) 6 the s.d. of three technical replicates. (C) The ability to measure near-basal activity of the GPR56 A386M 7TM receptor with a negligibly active tethered agonist was enhanced by use of five times more receptor membranes and excluding GDP from the reconstitution assay. GPR56 A386M 7TM basal activation of G13 GTPgS binding was measured in the presence of 50 mM DHM or the DMSO vehicle control. Error bars are the average 6 the s.d. of three experimental replicates. (D) DHM inhibits P7 synthetic-peptide agonist stimulation of the compromised tethered agonist GPR56 A386M 7TM receptor. GPR56 A386M 7TM membranes were reconstituted with G13, and receptor-stimulated G13 GTPgS binding kinetics were measured in response to 100 mM P7 agonist peptide and/or 50 mM DHM in the presence of 20 mM GDP. Error bars are the average 6 the s.d. of three experimental replicates. (E) P7 agonist-peptide (10 mM) stimulation of the GPR56 A386M 7TM receptor in intact HEK293 cells activated RhoA 222 Stoveken et al.

DHM. Rotenone has a well established cellular target; it directly inhibits complex I of the mitochondrial ETC (Chance et al., 1963). Complex I inhibition explains the toxic effects of rotenone as an insecticide and potent piscicide (Chance et al., 1963; http://www.doc.govt.nz/documents/science-and-technical/ SFC211.pdf). Rotenone is less toxic to humans but can cause death if a large quantity is ingested (Wood et al., 2005). We directly compared the ability of all five compounds to inhibit ETC complex I versus toxicity toward cultured HEK293 cells using a proxy assay that measured compound ability to induce cell detachment over 24 hours (Fig. 2). DHM was clearly the least-potent inhibitor of complex I, which correlated with it being the poorest mediator of cell detachment. Importantly, DHM was the most efficacious inhibitor of GPR56 and GPR114. For practical considerations, we recommend use of DHM as a probe compound in cell-based adhesion GPCR studies at

concentrations ,10 mM to avoid cytotoxicity (Fig. 2A). Downloaded from Our future and ongoing optimization of DHM will use a dual structure-activity relationship approach to seek derivatives that widen the gap between higher-potency adhesion GPCR inhibition and lower-potency complex I inhibition (and asso- ciated cytotoxicity). A comparison of the chemical structures of the five isoflavonoid compounds provides insight into the key molpharm.aspetjournals.org structural features to focus on and may explain the different potencies of adhesion GPCR inhibition (Fig. 6). Rotenone, isorotenone, and deguelin consist of five joined hydrocarbon rings that share an isoflavanone-like core. The contiguous nature of these rings imparts a rigid, planar structural orientation. DHM and mundulone each possess a moiety that disrupts the contiguousness of the five hydrocarbon rings to confer increased flexibility. DHM and mundulone differ in that the former has a 2-phenylacetophenone core, and the at ASPET Journals on October 2, 2021 Fig. 6. Structural features of DHM and related compounds. The rotenoids latter has an isoflavone core. The 2-phenylacetophenone core (isorotenone, rotenone, and deguelin) are relatively rigid, planar com- in DHM would confer additional flexibility over mundulone, pounds with isoflavanone-like cores. In contrast, mundulone, a true isoflavone, and DHM both lack the isoflavanone core, which allows for and thus, DHM has the most flexibility of the five studied increased flexibility and the predicted ability to adopt multiple conforma- isoflavonoids. Examples of potential DHM conformers are tions. Mundulone may rotate around one central carbon-carbon bound, shown in Fig. 6. Additionally, the phenolic hydroxyl of DHM, indicated by the arrow. The core of DHM is a 2-phenylacetophenone that which is blocked in mundulone, could be important for would permit rotation about the three indicated carbon-carbon bonds, strongly suggesting that DHM is the most flexible compound of the five adhesion GPCR target interaction. compounds studied. Representative conformers of DHM are shown to The mode of DHM inhibitory action remains a partially open illustrate the predicted flexibility of the compound [conformational anal- question, although our data are consistent with those of a ysis was done with the Merck Molecular Force Field (MMFF) force field, iSpartan (www.wavefun.com); structures were drawn with ChemDraw Pro neutral antagonist that competes with a tethered-peptide (Perkin Elmer, Waltham, MA)]. agonist for binding to its orthosteric site. Still, the data do not eliminate the possibility that DHM may serve as a of DHM inhibition may be as a neutral antagonist that negative allosteric modulator. A Schild analysis was attemp- competes with the tethered-peptide agonist for binding to its ted to directly measure DHM competitive action to the GPR56 orthosteric site. If DHM indeed acts as an orthosteric antag- synthetic peptide agonist P7 (Fig. 4B) (Schild, 1949; onist, this provides a solidifying pharmacological argument Arunlakshana and Schild, 1959; Bindslev, 2008). When the supporting the emerging hypothesis that adhesion GPCRs are P7 peptide agonist concentration response was performed activated by tethered-peptide agonists (Liebscher et al., 2014; with increasing DHM concentrations, the response curves Stoveken et al., 2015). exhibited clear rightward shifts but could not be fit to full Four additional compounds in the Spectrum Collection that sigmoidal functions because maximum efficacy of P7- are structurally related to DHM were assayed by SRE- mediated activation (rate of G protein GTPgS binding) was luciferase and GPR56 reconstitution assays. Mundulone, unachievable. Activation of GPR56 with P7 peptide agonist rotenone, isorotenone, and deguelin all inhibited GPR56- concentrations .150 mM results in diminishing efficacy. The mediated G13 activation, but with lower efficacies than reason for this is unknown, although we speculate that use of

GTPase, an effect blocked by DHM (10 mM) (n = 3); shown is a representative immunoblot. (F) P7 agonist-peptide (10 mM) stimulation of the GPR56 A386M 7TM receptor in intact HEK293 cells activated SRE-luciferase. Increasing concentrations of DHM (1–4 mM) inhibited the P7 stimulus. Error bars are the average 6 the s.d. of three technical replicates. (G) Increasing P7 agonist peptide (up to 20 mM) activated the SRE-luciferase reporter in a concentration-dependent manner, and 3 mM DHM inhibited P7 activation. Error bars are the average 6 the s.d. of three experimental replicates. A Class-Selective Small-Molecule Adhesion GPCR Antagonist 223 very high adhesion GPCR synthetic peptide agonist concen- References trations results in nonproductive aggregation of an essential Araç D, Boucard AA, Bolliger MF, Nguyen J, Soltis SM, Südhof TC, and Brunger AT (2012) A novel evolutionarily conserved domain of cell-adhesion GPCRs mediates assay component. 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Supplemental Data

Dihydromunduletone is a Small-Molecule Selective Adhesion G Protein-Coupled Receptor Antagonist.

Hannah M. Stoveken, Laura L. Bahr, M.W. Anders, Andrew P. Wojtovich, Alan V. Smrcka, and Gregory G. Tall

Molecular Pharmacology

GPR56 7TM G13 Q226L Screen CounterScreen Z’ = 0.67 Z’ = 0.8 4 1006 Supplementary Figure 1. Dynamic Range of the High-Throughput Screen and 3 1006 Counter-screen. HEK293T cells transfected with GPR56 7TM (screen) or GNA13Q226L 2 1006 (counter-screen) and the SRE-luciferase reporter were treated with DMSO or 5 µM 1 1006 latrunculin B. The resulting luciferase activities were used to establish the Z’ factors

Relative Luciferase Units Luciferase Relative 0 for the high throughput screen and counter- 05 05 screen (n = 16). [Latrunculin B] (µM)

Supplementary Figure 2 A (A) Cell-surface biotin labeling and GPR56 Input Streptavidin pull-down titration experiment to (Cell Lysate) Pull-Down kDa demonstrate that the assay is not saturable based on the number of cells 50 used in relative quantification

37 experiments. High-Five insect cells in suspension culture were infected for 24 h with GPR56 7TM baculovirus. 0.5 x 106, 1.0 6 6 6 25 x 10 , 2.0 x 10 , or 4.0 x 10 cells were recovered from the suspension culture and 20 WB: GPR56 C-terminus subjected to intact cell (i.e., cell-surface) biotin labeling. The biotinylation reactions were quenched prior to cell lysis in detergent B buffer. Biotinylated proteins were isolated DMSO DHM 37 kDa with streptavidin Sepharose, eluted with 2x Laemmli sample buffer, and subjected to immunoblot analysis with the GPR56 C- terminal antibody alongside cell lysate input samples. The GPR56 7TM domain 25 kDa (predicted M.W. ~41 KDa) runs aberrantly low at ~25 kDa) (B) Relative cell-surface GPR56 7TM receptor levels in baculovirus- WB: GPR56 C-terminus infected High-Five cells remained unchanged after overnight cell culture with 5 µM DHM or an equivalent volume of vehicle (DMSO).

Supplemental Method 1

Adhesion GPCR Cell-Surface Levels High-Five cells were cultured in Sf900II medium and grown to mid-log phase. Cells were infected at 1/100 dilution with primary-amplified recombinant GPR56 7TM baculovirus. After 5 h of incubation with baculovirus, cells were divided to individual flasks and incubated overnight in the presence of 5 µM DHM or vehicle. Twenty-four hours after infection, intact cells were subjected to cell-surface biotinylation, and biotinylated receptors were isolated with streptavidin Sepharose and visualized using receptor-specific antibodies (1, 37)