Supplemental material to this article can be found at: http://jpet.aspetjournals.org/content/suppl/2016/03/17/jpet.115.231712.DC1 1521-0103/357/2/394–414$25.00 http://dx.doi.org/10.1124/jpet.115.231712 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS J Pharmacol Exp Ther 357:394–414, May 2016 Copyright ª 2016 by The American Society for Pharmacology and Experimental Therapeutics Discovery and Characterization of AMPA Receptor Modulators Selective for TARP-g8 s Michael P. Maher, Nyantsz Wu, Suchitra Ravula, Michael K. Ameriks, Brad M. Savall, Changlu Liu, Brian Lord, Ryan M. Wyatt, Jose A. Matta, Christine Dugovic, Sujin Yun, Luc Ver Donck, Thomas Steckler, Alan D. Wickenden, Nicholas I. Carruthers, and Timothy W. Lovenberg Janssen Research and Development, LLC, Neuroscience Therapeutic Area, San Diego, California (M.P.M., N.W., S.R., M.K.A., B.M.S., C.L., B.L., R.M.W., J.A.M., C.D., S.Y., A.D.W., N.I.C., T.W.L.); and Janssen Research and Development, a Division of Janssen Pharmaceutica NV, Neuroscience Therapeutic Area, Beerse, Belgium (L.V.D., T.S.) Downloaded from Received December 23, 2015; accepted March 11, 2016 ABSTRACT Members of the a-amino-3-hydroxyl-5-methyl-4-isoxazole- pharmacokinetic properties and achieved high receptor occu- propionic acid (AMPA) subtype of ionotropic glutamate recep- pancy following oral administration. This molecule showed jpet.aspetjournals.org tors mediate the majority of fast synaptic transmission within strong, dose-dependent inhibition of neurotransmission within the mammalian brain and spinal cord, representing attractive the hippocampus, and a strong anticonvulsant effect. At high targets for therapeutic intervention. Here, we describe novel levels of receptor occupancy in rodent in vivo models, JNJ- AMPA receptor modulators that require the presence of the 55511118 showed a strong reduction in certain bands on electro- accessory protein CACNG8, also known as transmembrane encephalogram, transient hyperlocomotion, no motor impairment AMPA receptor regulatory protein g8 (TARP-g8). Using calcium on rotarod, and a mild impairment in learning and memory. JNJ- flux, radioligand binding, and electrophysiological assays of 55511118 is a novel tool for reversible AMPA receptor in- wild-type and mutant forms of TARP-g8, we demonstrate that hibition, particularly within the hippocampus, with potential at ASPET Journals on September 27, 2021 these compounds possess a novel mechanism of action therapeutic utility as an anticonvulsant or neuroprotectant. The consistent with a partial disruption of the interaction between existence of a molecule with this mechanism of action demon- the TARP and the pore-forming subunit of the channel. One strates the possibility of pharmacological targeting of acces- of the molecules, 5-[2-chloro-6-(trifluoromethoxy)phenyl]-1,3- sory proteins, increasing the potential number of druggable dihydrobenzimidazol-2-one (JNJ-55511118), had excellent targets. Introduction postsynaptic membranes of excitatory synapses in the central nervous system. AMPA receptors (AMPARs) mediate the Glutamate is the primary excitatory neurotransmitter in majority of fast synaptic transmission within the central mammalian brain. The a-amino-3-hydroxyl-5-methyl-4- nervous system (CNS). Thus, inhibition or negative modula- isoxazole-propionic acid (AMPA) subtype of glutamate recep- tion of AMPARs is an attractive strategy for therapeutic tors are ligand-gated ion channels expressed primarily on intervention in CNS disorders characterized by excessive neuronal activity. With the notable exception of pore blockers (which are selective for calcium-permeable AMPA receptors; dx.doi.org/10.1124/jpet.115.231712. s This article has supplemental material available at jpet.aspetjournals.org. see Stromgaard and Mellor, 2004), no AMPAR inhibitors have ABBREVIATIONS: ACSF, artificial cerebrospinal fluid; AMPA, a-amino-3-hydroxyl-5-methyl-4-isoxazole-propionic acid; AMPAR, AMPA receptor; ANOVA, analysis of variance; CHO, Chinese hamster ovary; CP-465022, 3-(2-Chlorophenyl)-2-[2-[6-[(diethylamino)methyl]-2-pyridinyl]ethenyl]-6- fluoro-4(3H)-quinazolinone hydrochloride; CNS, central nervous system; CT, carboxyl terminus; DMSO, dimethylsulfoxide; DNMTP, delayed non-match to position; EC50, half-maximal effective concentration; ED50, half-maximal effective dose; EEG, electroencephalogram; EMG, electromyogram; EPSC, excitatory postsynaptic current; EX, extracellular domain; FAM, familiar arm; fEPSP, field excitatory postsynaptic potential; f u, unbound fraction; GluA, AMPA subtype of ionotropic glutamate receptor; GYKI-53655, 1-(4-Aminophenyl)-3-methylcarbamyl-4- methyl-3,4-dihydro-7,8-methylenedioxy-5H-2,3-benzodiazepine hydrochloride; HABG, HibernateA supplemented with B27 and Glutamax; HEK-293, human embryonic kidney 293; HPMC, hydroxypropyl methylcellulose; J values, indirect dipole-dipole coupling constants; JNJ-55511118, 5-[2-chloro-6-(trifluoromethoxy)phenyl]-1,3-dihydrobenzimidazol-2-one; JNJ-56022486, 2-(3-chloro-2-(2-oxo-2,3-dihydro-1H-benzo[d]imidazol- 5-yl)phenyl)acetonitrile; LC-MS/MS, liquid chromatography–tandem mass spectrometry; LY-395153, N-[4-[1-(propan-2-ylsulfonylamino)propan- 2-yl]phenyl]benzamide; MES, maximal electroshock; MWM, Morris water maze; NEW, novel arm; NIH, National Institutes of Health; NMDA, N-methyl-D-aspartate;NREM,non-rapideyemovement;NSB,nonspecificbinding;PAM,positiveallostericmodulator;PCR, polymerase chain reaction; Philanthotoxin-74, (S)-N-[7-[(4-Aminobutyl)amino]heptyl]-4-hydroxy-a-[(1-oxobutyl)amino]benzenepropanamide dihydrochloride; p.o., per os; PTZ, pentylenetetrazole; RED, Rapid Equilibrium Dialysis; REM, rapid eye movement; SB, specific binding; TARP, transmembrane AMPA receptor regulatory protein; TB, total binding; TM, transmembrane domain. 394 TARP-g8–Selective AMPAR Modulators 395 been found to have selectivity among the AMPAR subtypes, or Here, we describe the in vitro and in vivo characterization of to exhibit regional specificity. Since AMPAR activity is 5-[2-chloro-6-(trifluoromethoxy)phenyl]-1,3-dihydrobenzimidazol- ubiquitous within the CNS, general antagonism results in 2-one (JNJ-55511118) and 2-(3-chloro-2-(2-oxo-2,3-dihydro-1H- undesired effects, such as ataxia, sedation, and/or dizziness. benzo[d]imidazol-5-yl)phenyl)acetonitrile (JNJ-56022486). These In clinical use, AMPAR antagonists have very narrow thera- compounds are potent negative modulators of AMPA receptors peutic dosing windows: the doses needed to obtain anticon- containing TARP-g8. They show exquisite selectivity, with no vulsant activity are close to or overlap with doses at which measurable effects upon AMPARs containing other TARPs, or undesired effects are observed (Rogawski, 2011). upon TARP-less receptors. Using chimeric proteins comprising Over the past two decades, investigations into the quater- various segments of TARP-g8and-g4 followed by site-directed nary structure of native AMPA receptors have revealed a mutagenesis, we identified the specific amino acids responsible for remarkably large set of interaction partners. Heterologous this remarkable selectivity. We demonstrate in vivo target expression of individual members of the AMPA subtype of occupancy using ex vivo autoradiography, and provide a pre- ionotropic glutamate receptor (GluA) is sufficient to form liminary investigation of the in vivo pharmacological effects of functional AMPA receptors. However, full recapitulation of TARP-g8–selective AMPA receptor inhibition. the trafficking, localization, gating characteristics, and pharmacology of native AMPA receptors requires coassem- bly with a large and diverse set of accessory proteins Materials and Methods Downloaded from (Jackson and Nicoll, 2011; Schwenk et al., 2012; Straub 3-(2-Chlorophenyl)-2-[2-[6-[(diethylamino)methyl]-2-pyridinyl]ethenyl]- and Tomita, 2012). These auxiliary subunits include cyto- 6-fluoro-4(3H)-quinazolinone hydrochloride (CP-465022; Menniti et al., skeletal and anchoring proteins, other signaling proteins, 2000), 1-(4-Aminophenyl)-3-methylcarbamyl-4-methyl-3,4-dihydro- and several intracellular and transmembrane proteins with 7,8-methylenedioxy-5H-2,3-benzodiazepine hydrochloride (GYKI-53655; largely unknown functions. The wide variety of proteins Bleakman et al., 1996), and (S)-N-[7-[(4-Aminobutyl)amino]heptyl]- which can participate in AMPA receptor complexes vastly 4-hydroxy-a-[(1-oxobutyl)amino]benzenepropanamide dihydrochlo- increases the ability of a neuron to tune the response ride (Philanthotoxin-74; Kromann et al., 2002) were purchased from jpet.aspetjournals.org characteristics of its synapses. Here, we demonstrate that Tocris (Bristol, UK). N-[4-[1-(propan-2-ylsulfonylamino)propan- these accessory proteins can be used as novel pharmacological 2-yl]phenyl]benzamide (LY-395153; Linden et al., 2001) was pur- chased from Diverchim (Roissy-en-France, France). Perampanel targets. (Hanada et al., 2011) was purchased from Alsachim (Illkirch- Members of the transmembrane AMPA receptor regula- Graffenstaden, France). Unless otherwise noted, all data analyses, tory protein (TARP) family (CACNG2, 3, 4, 5, 7, and 8) are statistics, and data plots were performed using Origin 2015 or associated with most, if not all, AMPARs in the brain. These OriginPro 2015 (OriginLab, Northampton, MA). Grubbs’ test was proteins were originally discovered and named due to their performed prior to statistical analysis; if identified, a single extreme at ASPET Journals on September 27, 2021 homology to the gamma subunit of voltage-gated calcium