Discovery and characterization of AMPA receptor modulators selective for TARP-γ8

67 Pages·2016·2.83 MB·English

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Title Page
Discovery and characterization of AMPA receptor modulators
γ
selective for TARP- 8
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, Timothy W. Lovenberg D
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Janssen Research & Development, LLC, Neuroscience Therapeutic Area, San Diego, CA 92121, USA from
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(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.) et.a
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Janssen Research & Development, LLC, Neuroscience Therapeutic Area, Beerse, B-2340, Belgium rna
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(L.V.D., T.S.) rg
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Running Title Page
γ
TARP- 8-selective AMPAR modulators
Corresponding author:
Michael P. Maher, Ph.D.
Janssen Research & Development, LLC
Neuroscience Therapeutic Area
3210 Merryfield Row
San Diego, CA 92121

Phone: [phone]
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Email: [email protected] lo
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Document Information: jp
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Number of pages: 59 t.a
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Number of tables: 2 p
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Number of figures: 8 tjou
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Number of references: 71 a
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Words in Abstract: 205 .o
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Introduction: 664 a
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Discussion: 2758 S
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Abbreviations: ACSF, artificial cerebrospinal fluid; AMPA, α-amino-3-hydroxyl-5-methyl-4- rna
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isoxazole-propionic acid; AMPAR, AMPA receptor; ARG, autoradiography; BBB, blood-brain barrier; o
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BCA, bicinchoninic acid; CNIH, cornichon family AMPA receptor auxiliary protein; CNS, central nervous Ja
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system; CT, carboxyl terminus; CTZ, cyclothiazide; DNMTP, Delayed Non-Match to Position; EEG, a
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electroencephalogram; EMG, electromyogram; eEPSC, evoked excitatory post-synaptic currents; EX, 1
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extracellular domain; fEPSP, field excitatory postsynaptic potentials; GluA, protein monomer of ionotropic , 2
0
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glutaβmate receptor AMPA βsubtype; GRIA, gene encoding ionotropic glutamate receptor AMPA subtype; 3
HP- -CD, hydroxypropyl- -cyclodextrin; HPMC, hydroxypropyl methylcellulose; IC , 50% inhibitory
50
concentration; IN, intracellular domain; i.v., intravenous; mM, millimolar; mOs, milliosmolar; MEA,
multielectrode array; MES, maximal electroshock; MWM, Morris Water Maze; NMDA, N-methyl-D-
aspartate; NT, amino terminus; PAM, positive allosteric modulator; pIC , negative log of the 50%
50
inhibitory concentration, expressed in molar; p.o., per os; PTZ, pentylenetetrazole; RED, Rapid
Equilibrium Dialysis; SEM, standard error of the mean; TARP, Transmembrane AMPA receptor
Regulatory Protein; TM, transmembrane domain.
Recommended section assignment: Neuropharmacology

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Abstract
Members of the AMPA-subtype of ionotropic glutamate receptors mediate the majority of fast synaptic
transmission within the mammalian brain and spinal cord, representing attractive targets for therapeutic
intervention. Here, we describe novel AMPA receptor modulators that require the presence of the
accessory protein CACNG8, also known as TARP-γ8. Using calcium flux, radioligand binding, and
electrophysiological assays of wild-type and mutant forms of TARP-γ8, we demonstrate that these
compounds possess a novel mechanism-of-action consistent with a partial disruption of the interaction D
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between the TARP and the pore-forming subunit of the channel. One of the molecules, JNJ-55511118, had loa
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excellent pharmacokinetic properties and achieved high receptor occupancy following oral administration. fro
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This molecule showed strong, dose-dependent inhibition of neurotransmission within hippocampus, and a t.a
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strong anti-convulsant effect. At high levels of receptor occupancy in rodent in vivo models, JNJ-55511118 u
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showed a strong reduction in certain bands in EEG, transient hyperlocomotion, no motor impairment on rg
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rotarod, and a mild impairment in learning and memory. JNJ-55511118 is a novel tool molecule for S
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reversible AMPA receptor inhibition, particularly within hippocampus, with potential therapeutic utility as ou
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an anticonvulsant or neuroprotectant. The existence of a molecule with this mechanism-of-action o
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demonstrates the possibility of pharmacological targeting of accessory proteins, increasing the potential ary
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number of druggable targets. , 20
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Introduction
Glutamate is the primary excitatory neurotransmitter in mammalian brain. The α-amino-3-hydroxyl-5-
methyl-4-isoxazole-propionic acid (AMPA) subtype of glutamate receptors are ligand-gated ion channels
expressed primarily on postsynaptic membranes of excitatory synapses in the central nervous system.
AMPA receptors (AMPARs) mediate the majority of fast synaptic transmission within the central nervous
system (CNS). Thus, inhibition or negative modulation of AMPARs is an attractive strategy for therapeutic
intervention in CNS disorders characterized by excessive neuronal activity. With the notable exception of D
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pore-blockers (which are selective for calcium-permeable AMPA receptors; see Stromgaard and Mellor, lo
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2004), no AMPAR inhibitors have been found to have selectivity amongst the AMPAR subtypes, or to fro
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exhibit regional specificity. Since AMPAR activity is ubiquitous within CNS, general antagonism results t.a
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in undesired effects, such as ataxia, sedation, and/or dizziness. In clinical use, AMPAR antagonists have tjou
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very narrow therapeutic dosing windows: the doses needed to obtain anti-convulsant activity are close to or .org
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overlap with doses at which undesired effects are observed (Rogawski, 2011). S
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Over the past two decades, investigations into the quaternary structure of native AMPA receptors have o
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revealed a remarkably large set of interaction partners. Although the pore-forming GluA subunits are o
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sufficient to form functional AMPA receptors in heterologous systems, full recapitulation of the trafficking, a
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localization, gating characteristics, and pharmacology of native AMPA receptors requires co-assembly with , 2
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a large and diverse set of accessory proteins (Jackson and Nicoll, 2011; Schwenk et al., 2012; Straub and
Tomita, 2012). These auxiliary subunits include cytoskeletal and anchoring proteins, other signaling
proteins, and several intracellular and transmembrane proteins with largely unknown functions. The wide
variety of proteins which can participate in AMPA receptor complexes vastly increases the ability of a
neuron to tune the response characteristics of its synapses. Here, we demonstrate that these accessory
proteins can be used as novel pharmacological targets.
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Members of the Transmembrane AMPA Receptor Regulatory Protein (TARP) family (CACNG2, 3, 4,
5, 7, and 8) are associated with most, if not all, AMPARs in the brain. These proteins were originally
discovered and named due to their homology to the gamma subunit of voltage-gated calcium channels
(Letts et al., 1998; Burgess et al., 1999; Klugbauer et al., 2000). TARPs were subsequently found to
associate with and to modulate the activity of AMPA receptors (Hashimoto et al., 1999; Tomita et al.,
2003). Several TARPs have distinct region-specific expression in the brain, leading to physiological
differentiation of the AMPA receptor activity. It has been theorized that targeting individual TARPs may
enable selective modulation of specific brain circuits without globally affecting synaptic transmission (Gill D
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and Bredt, 2011). The expression pattern of TARP-γ8 is particularly attractive in this respect. Based upon ad
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in situ hybridization studies, TARP-γ8 is the predominant TARP throughout the hippocampus, and is from
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expressed within essentially all neurons within stratum pyramidale and stratum granulosum. In addition, it s
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is expressed in a substantial proportion of neurons in amygdala, olfactory bulb, and olfactory nucleus, and rn
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in certain layers within frontal cortex. In contrast, TARP-γ8 shows very little expression within hindbrain, arg
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midbrain, or thalamus (Tomita et al., 2003; Lein et al., 2007; http://mouse.brain- E
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map.org/experiment/show/72108823). rn
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Negative modulation of AMPA receptors with a molecule selective for TARP-γ8 offers the possibility of J
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selectively reducing excitatory transmission within brain circuits associated with neuropsychiatric or 1
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neurological disorders. Such an agent could be a useful therapeutic in pathological conditions 23
characterized by hyperactivity within hippocampus, for example, temporal lobe epilepsy. This approach
should mitigate the side-effect profile attributed to non-selective AMPAR antagonists (Ko et al., 2015).
Here, we describe the in vitro and in vivo characterization of JNJ-55511118 and JNJ-56022486. These
compounds are potent negative modulators of AMPA receptors containing TARP-γ8. They show exquisite
selectivity, with no measurable effects upon AMPARs containing other TARPs, or upon TARP-less
receptors. Using chimeric proteins comprising various segments of TARP-γ8 and -γ4 followed by site-
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directed mutagenesis, we identified the specific amino acids responsible for this remarkable selectivity.
We demonstrate in vivo target occupancy using ex vivo autoradiography, and provide a preliminary
investigation of the in vivo pharmacological effects of TARP-γ8-selective AMPA receptor inhibition.

Methods
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CP-465022 (Menniti et al., 2000), GYKI-53655 (Bleakman et al., 1996), and Philanthotoxin-74 ow
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(Kromann et al., 2002) were purchased from Tocris (Bristol, United Kingdom). LY-395153 (Linden et al., e
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2001) was purchased from Diverchim (Roissy-en-France, France). Perampanel (Hanada et al., 2011) was jp
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purchased from Alsachim (Illkirch-Graffenstaden, France). Unless otherwise noted, all data analysis, p
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statistics, and data plots were performed using Origin 2015 or Origin Pro 2015 (OriginLab, Northampton, rna
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MA). Grubbs test was performed prior to statistical analysis; if identified, a single extreme outlier was a
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excluded from further analysis. Unless otherwise noted, averages are expressed as mean ± standard error of E
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the mean (SEM). Significance levels in figures are denoted as follows: (*) p < 0.05; (**) p < 0.01; and rn
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(***) p < 0.001. Unless otherwise noted, parameters from linear and non-linear least squares fitting J
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procedures are expressed as value ± standard error. 1
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Animal studies described in this article that were performed in the United States were in accordance 23
with the Guide for the Care and Use of Laboratory Animals (National Research Council, 2011). Studies
performed in Europe were in accordance with the European Communities Council Directive 2010/63/EU
(September 22, 2010) and local legislation on animal experimentation. Facilities were accredited by the
Association for the Assessment and Accreditation of Laboratory Animal Care (AAALAC). Animals were
allowed to acclimate for 7 days after receipt. They were housed in accordance with institutional standards,
received food and water ad libitum, and were maintained on a 12-hour light/dark cycle.
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Chemical synthesis
General synthetic methods. All reagents were purchased from Sigma-Aldrich, Strem Chemicals, or
Combi-Blocks and used without further purification, except where noted. Solvents were purchased from
EMD chemicals and dried by passing through activated alumina columns maintained under argon. All
reactions were conducted under a nitrogen atmosphere unless otherwise noted. Flash chromatography was
performed on Teledyne Isco CombiFlash systems using commercially available RediSep silica gel
cartridges. Reverse-phase HPLC purifications were performed on an Agilent 1100 Series system with a
Waters XBridge C18 OBD 5 μM preparative column, unless otherwise noted. NMR spectra were recorded D
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on a Bruker UltraShield-400, Bruker UltraShield-500, or Bruker UltraShield-600 spectrometer and were ad
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referenced to trimethylsilane (TMS). Chemical shifts are recorded in ppm relative to TMS and J values are m
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reported in Hz. Combustion analysis was performed at Intertek Pharmaceutical Services (Whitehouse, NJ). t.as
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Tritium labeling was conducted at Moravek Biochemicals (Brea, CA). The reaction scheme for the rn
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synthesis of JNJ-55511118 is shown in Supplemental Figure 1. The reaction scheme for the synthesis and rg
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tritiation of JNJ-56022486 is shown in Supplemental Figure 2. P
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Molecular Biology an
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Molecular cloning of GluA receptors and their accessory proteins from different species. cDNAs 12
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for human GluA1-FLIP, GluA1-FLOP, GluA2-FLOP, GluA2-FLIP, GluA3-FLOP, GluA4-FLOP, and 3
monkey, dog, mouse, rat GluA1-FLOP, as well as their accessory proteins including human CACNG2,
CACNG3, CACNG4, CACNG7, CACNG8, CNIH2, monkey CACNG8, mouse CACNG8, and CACNG8
were PCR amplified from brain cDNAs from respective species. A point mutation was introduced into the
GluA2 constructs at the Q/R editing site to allow calcium permeability in the expressed protein (Burnashev
et al., 1992). Dog CACNG8 was synthesized with codon optimization based on the published sequence
(Genbank accession No. KT749896). The sequences for PCR primers are listed in Supplemental Table 1.
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The PCR products were cloned into mammalian expression vectors as indicated: pCIneo (Promega),
pcDNA3.1(+) (Life Technologies), or pcDNA4/TO (Life Technologies). Cloning sites (highlighted in
shaded letters) were introduced into primers to facilitate the cloning process. The insert regions were
sequenced to confirm the sequence identities. FLOP and FLIP splice variants are designated with ‘o’ and
‘i’ suffixes, respectively (e.g the FLIP variant of GluA1 is designated ‘GluA1i’).
Generation of GluA1o-CACNG8 fusion protein expression constructs. To ensure a 1:1
stoichiometry of GluA1o and γ8 in the expressed channel, a fusion of the cDNAs for GRIA1o and
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CACNG8 was used. Following Shi et al. (2009), we fused the cDNA encoding the C-terminus of GluA1o o
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to the cDNA encoding the N-terminus of γ8. We inserted a linker sequence encoding ad
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QQQQQQQQQQEFAT between the two full-length cDNAs. The channels expressed with this construct m
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appear to have identical properties to channels formed by co-expression of GRIA1o with an excess of s
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CACNG8 (Shi et al., 2009). Human, mouse, and rat GluA1o-CACNG8 fusion protein expression rn
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constructs were generated by overlapping PCR followed by cloning into mammalian expression vectors. arg
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The human GluA1o-CACNG8 fusion protein expression DNA was cloned into pCIneo between EcoR1 and E
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Not1 sites, while the mouse and rat GluA1o-CACNG8 expression constructs were cloned into pcDNA4/TO rn
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between HindIII and Not1 sites. The primers, templates for the overlapping PCRs are listed in J
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Supplemental Table 2. All clones were sequenced and the identities were confirmed. DNA coding and ry
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predicted amino acid sequences for the fusion constructs are listed in Supplemental Table 3. 2
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Construction of chimeric proteins using TARPs γ8, γ4, and γ2. The sequences for the human
variants of each protein were aligned using the Uniprot alignment tool, which also predicted the
transmembrane segments of the proteins. The protein sequences were divided into 9 regions separated near
the borders of the predicted transmembrane sections; these nine regions were the N- and C-termini (NT and
CT), the four transmembrane domains (TM1-TM4), the two extracellular domains (EX1, EX2), and the
intracellular domain (IN1). The predicted topology of the TARP is shown in Figure 3A, and the splice
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points between the TARPs are shown in Supplemental Table 4. The chimeras were designated by a 9-
digit number; each digit indicates the TARP used for that section of the protein, starting from the N-
terminus. Graphical representations of the chimeric TARPs are shown in Supplemental Figure 3. The
chimeric expression constructs were generated using overlapping PCRs except those indicated otherwise.
First, two separate PCR reactions (5’ end PCR and 3’ end PCR) that generated overlapping PCR products
were performed. Next, the 5’end and 3’ end PCR products were mixed to serve as the template for the PCR
reactions that generated the full length PCR product for molecular clonings. The primers and templates
used for PCR reactions are listed in Supplemental Table 5. DNA coding and predicted amino acid D
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sequences for the chimeric constructs are listed in Supplemental Table 6. a
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Generation of point mutations. All mutant expression constructs were generated by overlapping PCR from
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using the human wild type CACNG8 or CACNG4 cDNA as the templates. The primers used for generation t.a
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of the mutants are listed in Supplemental Table 7. DNA coding and predicted amino acid sequences for rn
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the chimeric constructs are listed in Supplemental Table 8. rg
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Calcium flux assay a
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A clonal cell line stably expressing the human GluA1o-γ8 fusion construct under geneticin selection in Ja
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HEK-293 was established for the primary calcium flux assay. All other combinations of GluA subunits and 12
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TARPs were performed using co-transfections of the respective plasmids into HEK-293F cells. AMPA 3
receptors formed by co-transfections are designated with the ‘plus’ symbol (e.g. GluA1i co-transfected with
TARP-γ8 is referred to as GluA1i+γ8).
For assays with transiently-transfected cells, cells were generated by bulk transfection. Prior to
transfection, 293-F cells were cultured in Freestyle-293 Expression Medium (Gibco, Grand Island, NY) at
0.5-2 million cells/mL in shaker flasks at 37°C and 8% CO at 120 rpm. At the time of transfection, cells
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were diluted to 1 million/mL with FreeStyle-293 medium. Cell viability was above 90% for transfections
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to be considered successful. Transfection was performed by combining equal amounts of pAdvantage
vector (Promega Corp., Madison WI) and target DNA. Total DNA was 50µg per 40mL transfection. The
DNA ratio of AMPA receptor to TARPs was 4:1. The transfection reagent was Freestyle MAX
(Invitrogen). Cells were seeded into 384-well poly-lysine-coated plates at 15k cells/ well at 16-24 hours
after transfection, and used for assays 24-48 after transfection.
The calcium flux assays were performed as follows. Cell plates were washed with assay buffer (135
mM NaCl, 4 mM KCl, 3 mM CaCl , 1 mM MgCl , 5 mM glucose, 10 mM HEPES, pH 7.4, 300 mOs)
2 2
using a Biotek EL405 plate washer. The cells were then loaded with a calcium-sensitive dye according to D
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the manufacturers’ instructions (Calcium-5 or Calcium-6; Molecular Devices, Sunnyvale, CA) combined a
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with the test compounds at a range of concentrations. Calcium flux following the addition of 15µM from
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glutamate was monitored using a FLIPR Tetra (Molecular Devices, Sunnyvale, CA). t.a
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The fluorescent response in each well was normalized to the response of negative and positive control rn
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wells. The negative control wells had no added compounds, and the positive control wells had been rg
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incubated with 50 µM CP-465022 (a non-subtype-selective AMPAR antagonist; Lazzaro et al., 2002). The P
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responses ( ) to glutamate as functions of the test compound concentrations ( ) were fitted to a four- u
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(cid:0) (cid:2) (cid:4) (cid:5) (cid:6)(cid:4) (cid:7)(cid:4) (cid:8)⁄(cid:6)1(cid:5)(cid:6)(cid:10)⁄(cid:10) (cid:8)(cid:4)(cid:8) ls o
parameter logistic function (cid:0) (cid:2) (cid:0) (cid:3) . The fitted parameter corresponding to n
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(cid:10) u
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the midpoint ( (cid:3)) was taken to be the potency of inhibition of the compound (IC ; 50% inhibitory ry
50 1
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pIC (cid:2) (cid:7)log (cid:6)IC (cid:18)M(cid:20)(cid:8) , 2
concentration). Potency is expressed as (cid:5)(cid:3) (cid:2)(cid:3) (cid:5)(cid:3) . 02
3

Knockout animals
The TARP-γ8 knockout mouse line Cacng8tm1Ran was originally described by Rouach et al. (2005). This
mouse line, generated by homologous recombination in embryonic stem cells to replace exons 2 and 3 with
a neomycin resistance gene, was re-derived by back-crossing into a C57BL/6J mouse line at Jackson
Laboratory (Bar Harbor, ME).
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