Characterization of a Novel M1 Muscarinic Acetylcholine Receptor Positive Allosteric Modulator Radioligand, [3H]PT-1284

Home , Oxotremorine, Receptor modulator, Xanomeline

1521-0111/90/3/177–187$25.00 http://dx.doi.org/10.1124/mol.116.104737 MOLECULAR PHARMACOLOGY Mol Pharmacol 90:177–187, September 2016 Copyright ª 2016 by The American Society for Pharmacology and Experimental Therapeutics

Deborah L. Smith, Jennifer E. Davoren, Jeremy R. Edgerton, John T. Lazzaro, Che-Wah Lee, Sarah Neal, Lei Zhang, and Sarah Grimwood Neuroscience and Pain Research Unit (D.L.S., J.R.E., S.N., S.G.) and Worldwide Medicinal Chemistry (J.E.D., L.Z.), Pfizer Inc., Cambridge, Massachusetts; Primary Pharmacology Group (J.T.L.) and Worldwide Medicinal Chemistry (C.-W.L.), Pfizer Inc., Groton, Connecticut

Received April 14, 2016; accepted June 30, 2016 Downloaded from

ABSTRACT 3 3 Selective activation of the M1 muscarinic acetylcholine recep- of ACh required to inhibit [ H]N-methylscopolamine ([ H]NMS) tor (mAChR) via a positive allosteric modulator (PAM) is a new binding to M1, left-shifting the ACh Ki approximately 19-fold at approach for the treatment of the cognitive impairments asso- 10 mM. Saturation analysis of a human M1 mAChR stable cell line 3 molpharm.aspetjournals.org ciated with schizophrenia and Alzheimer’s disease. Herein, showed that [ H]PT-1284 bound to M1 mAChR in the presence of we describe the characterization of an M1 PAM radioligand, 1mMAChwithKd, 4.23 nM, and saturable binding capacity (Bmax), 8-((1S,2S)-2-hydroxycyclohexyl)-5-((6-(methyl-t3)pyridin-3-yl)- 6.38 pmol/mg protein. M1 selective PAMs were shown to inhibit methyl)-8,9-dihydro-7H-pyrrolo[3,4-hour]quinolin-7-one [3H]PT-1284 binding in a concentration-responsive manner, whereas 3 ([ H]PT-1284), as a tool for characterizing the M1 allosteric M1 allosteric and orthosteric agonists showed weak affinity (.30 mM). 2 binding site, as well as profiling novel M1 PAMs. 8-((1S,2S)-2- A strong positive correlation (R 5 0.86) was found to exist Hydroxycyclohexyl)-5-((6-methylpyridin-3-yl)methyl)-8,9- between affinity values generated for nineteen M1 PAMs in the 3 dihydro-7H-pyrrolo[3,4-hour]quinolin-7-one (PT-1284 (1)) was [ H]PT-1284 binding assay and the EC50 values of these ligands shown to potentiate acetylcholine (ACh) in an M1 fluorometric in a FLIPR functional potentiation assay. These data indicate that imaging plate reader (FLIPR) functional assay (EC50, 36 nM) and there is a strong positive correlation between M1 PAM binding carbachol in a hippocampal slice electrophysiology assay affinity and functional activity, and that [3H]PT-1284 can serve as at ASPET Journals on September 26, 2021 (EC50, 165 nM). PT-1284 (1) also reduced the concentration a tool for pharmacological investigation of M1 mAChR PAMs.

Introduction hippocampus, striatum, and cerebral cortex (Wall et al., 1991; Levey 1996; Porter et al., 2002) and activation of these Muscarinic acetylcholine receptors (mAChRs) are mem- receptors in regions known for memory and cognitive func- bers of the superfamily of G protein-coupled receptors (GPCR). tion are expected to bestow a procognitive effect (Caulfield Five mammalian subtypes have been identified and are re- et al., 1983; Hagan et al., 1987; Messer et al., 1990). ferred to as M –M , with distribution in the central nervous 1 5 The orthosteric agonist xanomeline achieved proof of con- system as well as the periphery. mAChR subtypes M1,M3,and cept in human clinical trials for improvements in positive, M5 couple to Gq/11 proteins, which results in IP3 production and negative, and cognitive symptoms associated with schizophre- subsequent calcium mobilization, whereas subtypes M2 and M4 nia (Shekhar et al., 2008), as well as improvements in cognitive couple to Gi/o proteins, thereby inhibiting adenylyl cyclase function associated with Alzheimer’s disease (Bodick et al., activity (Bonner et al., 1987; Caulfield 1993; Caulfield and 1997). Although reported as an M1/M4 mAChR-preferring Birdsall, 1998). The M1 subtype is highly expressed in the agonist, xanomeline caused peripherally mediated adverse effectssuchasnausea,vomiting, salivation, and gastroin- dx.doi.org/10.1124/mol.116.104737. testinal distress, which led to a high dropout rate in these

ABBREVIATIONS: ACh, acetylcholine; Bmax, saturable binding capacity; BQCA, 1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid, benzyl quinolone carboxylic acid; CHO, Chinese hamster ovary; CNS, central nervous system; compound 9, 8-((1S,2S)-2-hydroxycyclohexyl)- 5-((6-methylpyridin-3-yl)methyl)-8,9-dihydro-7H-pyrrolo[3,4-h]quinolin-7-one; compound 10, (2-((1S,2S)-2-hydroxycyclohexyl)-5-((6-methylpyridin- 3-yl)methyl)-1,2-dihydro-3H-benzo[e]isoindol-3-one; compound 12, 5-((6-chloropyridin-3-yl)methyl)-8-((1S,2S)-2-hydroxycyclohexyl)-8,9-dihydro- 7H-pyrrolo[3,4-hour]quinolin-7-one; compound 13,8-((1S,2S)-2-hydroxycyclohexyl)-5-((6-(tributylstannyl)pyridin-3-yl)methyl)-8,9-dihydro-7H- pyrrolo[3,4-hour]quinolin-7-one; DMF, dimethylformamide; FBS, fetal bovine serum; FLIPR, fluorometric imaging plate reader; Fu,b, fraction 3 unbound in brain; GPCR, G protein-coupled receptors; hM1, human M1; HPLC, high-performance liquid chromatography; [ H]PT-1284, 8-((1S,2S)- 2-hydroxycyclohexyl)-5-((6-(methyl-t3)pyridin-3-yl)methyl)-8,9-dihydro-7H-pyrrolo[3,4-h]quinolin-7-one; mAChR, muscarinic acetylcholine receptor; MPO, multiparameter optimization; NMS, N-methylscopolamine; 1H NMR, proton nuclear magnetic resonance; PAM, positive allosteric modulator; PET, positron emission tomography; PQCA, 1-{[4-cyano-4-(2-pyridinyl)-1-piperidinyl]methyl}-4-oxo-4H-quinolizine-3-carboxylic acid; PT-1284 (1), 8-((1S,2S)-2-hydroxycyclohexyl)-5-((6-methylpyridin-3-yl)methyl)-8,9-dihydro-7H-pyrrolo[3,4-h]quinolin-7-one; 13C NMR, carbon-13 nuclear magnetic resonance.

177 178 Smith et al. clinical trials. Alleviating these adverse effects and preserving Pfizer animal care facilities that supported this work are fully biologic efficacy by developing subtype-selective orthosteric accredited by the Association for Assessment and Accreditation of agonists has proven to be challenging (Messer, 2002) owing to Laboratory Animal Care (AAALAC) International. complete homology of the muscarinic receptors at the orthosteric acetylcholine (ACh) site (Kruse et al., 2013). PT-1284 (1) Synthesis A new approach for achieving subtype selectivity is the All solvents and reagents were obtained from commercial sources development of allosteric modulators that bind at sites distinct and were used as received. All reactions were monitored by thin layer from the orthosteric binding site (Kruse et al., 2013). Unlike the chromatography (TLC plates F254; Merck) or ultra high-performance orthosteric binding site, allosteric binding sites display hetero- liquid chromatography–mass spectrometry analysis (Waters Acquity, geneity across mAChR subtypes, providing opportunities for ESCi Multi-Mode Ionization Source). Proton nuclear magnetic reso- achieving desired subtype selectivity (Ma et al., 2009; Kruse nance (1H NMR) and carbon-13 nuclear magnetic resonance (13C et al., 2013). Therefore, by targeting allosteric binding sites, NMR) spectra were obtained using deuterated solvent on a Varian selective positive allosteric modulators (PAMs) could be iden- 400 MHz instrument (LabX, Midland, ON, Canada). All 1HNMR tified to enhance both the affinity and intrinsic activity of the shifts are reported in d units (ppm) relative to the signals for chloroform (7.27 ppm) and methanol (3.31 ppm). All 13C shifts are endogenous agonist, ACh, at the M1 mAChR without activation reported in d units (ppm) relative to the signals for chloroform (77.0 of other mAChR subtypes. This profile could potentially lead to 1 ppm) and methanol (49.1 ppm) with H-decoupled observation. All Downloaded from therapeutics with improved safety and toleration profiles, coupling constants (J values) are reported in hertz. NMR abbrevia- compared with nonselective mAChR activators. In 2009, tions are as follows: br, broadened; s, singlet; d, doublet; t, triplet; q, BQCA, a highly selective M1 PAM, was disclosed by Merck quartet; p, pentuplet; m, multiplet; dd, doublet of doublets; ddd, (Kenilworth, NJ; Ma et al., 2009). As a follow-on to BQCA, the doublet of doublet of doublets. second generation M1 PAM PQCA (Kuduk et al., 2011) was Compound 12. 6-Chloropyridin-3-yl)methyl)zinc(II) chloride shown to improve cognition in animal models designed to (12 ml, 6.0 mmol, 0.5 M in tetrahydrofuran) was added to a solution measure recognition memory, spatial working memory, and of compound 11 (1.8 g, 5.0 mmol), which was prepared according to molpharm.aspetjournals.org executive function (Uslaner et al., 2013; Lange et al., 2015; Puri previously reported procedures (Kuduk et al., 2012), with palladium et al., 2015; Vardigan et al., 2015). tris-tert-butylphosphine (128 mg, 0.25 mmol). The resulting mixture Availability of a potent and selective radioligand that binds was stirred at 60°C under a nitrogen atmosphere for 18 hours. The mixture was cooled to room temperature and concentrated under to the M allosteric site will facilitate the understanding of M 1 1 reduced pressure. The crude material was purified by flash chroma- PAM pharmacology and the characterization of M1 selective tography through silica using 5% methanol in dichloromethane to PAMs (Trankle et al., 1996). Herein, we describe our efforts in afford a brown solid, which was washed with methanol (10 ml) to 1 the identification and in vitro pharmacology profiling of an afford compound 12 as a white solid. HNMR(400 MHz, CDCl3), d 8.97 M1-selective PAM radioligand lead, 8-((1S,2S)-2-hydroxycy- (d, J52.76 Hz, 1 H), 8.33 (d, J52.26 Hz, 1 H), 8.28 (d, J57.28 Hz, 1 H), clohexyl)-5-((6-methylpyridin-3-yl)methyl)-8,9-dihydro-7H- 7.80 (s, 1 H), 7.50 (dd, J58.66, 4.14 Hz, 1 H), 7.34 (dd, J58.28, 2.51 Hz, at ASPET Journals on September 26, 2021 pyrrolo[3,4-hour]quinolin-7-one [PT-1284 (1)], derived from a 1 H), 7.20 (d, J58.28 Hz, 1 H), 4.83–5.05 (m, 2 H), 4.44 (s, 2 H), 4.14– chemotype described by Merck (Kuduk et al., 2012), in human 4.26 (m, 1 H), 3.70–3.83 (m, 1 H), 3.50 (d, J54.77 Hz, 1 H), 2.39 M (hM ) stable cell line assays and a native hippocampal (d, J56.53 Hz, 1 H), 2.22 (d, J511.54 Hz, 1 H), 2.01 (d, J512.30 Hz, 1 1 5 – – brain slice assay, as well as the evaluation of the binding 1 H), 1.86 (d, J 12.30 Hz, 2 H), 1.66 1.76 (m, 1 H), 1.32 1.56 (m, 2 H). Compound 13. Bis(tributyltin; 1.1 g, 1.84 mmol) was added to a characteristics of [3H]PT-1284. solution of compound 12 [150 mg, 0.37 mmol) with tetrakis (triphenylphosphine) palladium (0); 43 mg, 0.037 mmol] in toluene (1 ml). The Materials and Methods resulting mixture was heated at 110°C for 18 hours. The reaction mixture was cooled to room temperature, filtered to remove solids, and All chemicals and reagents were purchased from Sigma-Aldrich concentrated under reduced pressure. The crude material was (St. Louis, MO) unless otherwise specified. All test compounds were purified by flash chromatography through a finely ground mixture of synthesized at Pfizer, Inc. (Groton, CT) unless otherwise noted. silica and potassium carbonate (9:1 by weight) using a gradient of 0%

Chinese hamster ovary (CHO) cell lines stably expressing hM1 to 10% methanol in dichloromethane to afford compound 13 as a clear 3 1 ([ H]NMS saturable binding capacity (Bmax) 5 3069 fmol/mg protein) oil. HNMR(400 MHz, CDCl3) d 8.96 (d, J51.56 Hz, 1 H), 8.70 (s, 1 H), 3 and human M3 ([ H]NMS Bmax 5 976.88 fmol/mg protein) were obtained 8.36 (d, J57.81 Hz, 1 H), 7.82 (s, 1 H), 7.68 (dd, J512.10, 7.02 Hz, 2 H), from Wyeth Research (Princeton, NJ). Human embryonic kidney 293 7.53–7.59 (m, 1 H), 5.31 (s, 2 H), 4.84–5.03 (m, 2 H), 4.44 (s, 1 H), 4.13– 3 cell lines stably expressing human M2 ([ H]NMS Bmax 5 139.80 fmol/mg 4.25 (m, 1 H), 3.71–3.82 (m, 1 H), 2.37 (d, J56.24 Hz, 1 H), 2.22 3 protein) and human M4 ([ H]NMS Bmax 5 167.59 fmol/mg protein) were (d, J512.88 Hz, 1 H), 2.01 (d, J512.88 Hz, 1 H), 1.86 (d, J511.32 Hz, purchased from Promega (Madison, WI). [3H]NMS (85.5 Ci/mmol; 2 H), 1.25–1.71 (m, 18 H), 1.01–1.19 (m, 6 H), 0.86 (t, J 5 7.41 Hz, 6 H). 1 mCi/ml), Whatman GF/B filter plates, MicroScint-20, and Ultima PT-1284 (1). Iodomethane (177 mg, 0.08 mmol) was added Gold MV were purchased from PerkinElmer (Waltham, MA). Whatman to a solution of compound 13 [80 mg, 0.12 mmol) with tris(diben- GF/B filter sheets were purchased from Brandel (Gaithersburg, MD). zylideneacetone)palladium (0); 6 mg, 0.006 mmol], tri(o-tolyl)- F12 nutrient media and penicillin/streptomycin was purchased from phosphine (4 mg, 0.012 mmol), and cesium fluoride (55 mg, Gibco/Thermo Fisher Scientific (Grand Island, NY). Fluo-8-AM dye was 0.36 mmol) in dimethylformamide (DMF) (1 ml). The resulting purchased from Molecular Probes (Grand Island, NY). [3H]PT-1284 mixture was stirred at room temperature for 45 minutes. The mixture (79.5 Ci/mmol; 1 mCi/ml) was custom synthesized and purchased from was filtered and concentrated under reduced pressure. The crude PerkinElmer. The Pierce BCA protein assay kit was purchased from material was purified by HPLC [column: Atlantis dC18 5-mm, 4.6 Â

Thermo Fisher Scientific. 50 mm; retention time: 1.7 minutes; gradient: 95% H2O/5% acetoni- Animals used were Sprague Dawley male rats aged 7–8 weeks trile (CH3CN) linear to 5% H2O/95% CH3CN over 4 minutes, hold at purchased from Charles River Laboratories (Wilmington, MA). All 5% H2O/95% CH3CN for an additional 1 minute (flow: 2.0 ml/min)] to 1 procedures performed on animals in this study were in accordance afford PT-1284 (1) as a white solid. H NMR (400 MHz, methanol-d4) with established guidelines and regulations, and were reviewed and d 8.98 (d, J53.01 Hz, 1 H), 8.65 (d, J58.53 Hz, 1 H), 8.35 (s, 1 H), 7.76 approved by the Pfizer Institutional Animal Care and Use Committee. (s, 1 H), 7.66 (dd, J58.78, 4.27 Hz, 1 H), 7.58 (dd, J58.03, 2.01 Hz, 1 H), 3 [ H]PT-1284, a Novel M1 mAChR PAM Radioligand 179

Fig. 1. Analysis of known literature M1 PAMs comparing CNS PET multiparameter optimization (MPO) with reported potency data. A database of 280 compounds were compiled from the literature and their potencies were correlated with CNS PET MPO. Initial leads were limited to compounds with CNS PET MPO . 3andM1 FLIPR calcium mobilization EC50 , 20 nM. Downloaded from molpharm.aspetjournals.org

7.25 (d, J58.03 Hz, 1 H), 4.91–5.05 (m, 2 H), 4.60 (s, 3 H), 4.03–4.14 (m, poly-D-lysine coated plates in Dulbecco’s Modified Eagle Medium media 1 H), 3.83–3.92 (m, 1 H), 2.50 (s, 3 H), 2.18–2.12 (m 1 H), 1.71–1.97 (m, supplemented with 10% FBS, 1% penicillin streptomycin, 500 mg/ml 4 H), 1.54 – 1.41 (m, 3 H). genetecin, 200 mg/ml hygromycin B, and 1% Glutamax for 24 hours at

37°C with 5% CO2. The culture media was replaced with equilibration Membrane Preparation media (88% CO2-independent media, 10% FBS, and 2% GloSensor cAMP reagent stock) for 2 hours at room temperature away from light. Stably transfected CHO cells overexpressing the human M mAChR 1 Following the 2-hour incubation, a mixture was added containing ACh subtype (M1 WYE clone 200-101, Wyeth Research) were grown at its EC20 and isoproterenol at its EC80 (concentrations were adjusted adherently in M Dulbecco’s Modified Eagle Medium growth medium at ASPET Journals on September 26, 2021 1 for each experiment) to the cell plate, which was then incubated for containing 10% fetal bovine serum (FBS), 1% nonessential amino acid, 10 minutes at room temperature. The plate was then read by Envision 1% penicillin/streptomycin, and 500 mg/ml genetecin. The cells were (PerkinElmer). harvested by gently scraping in a physiologic phosphate-buffered saline solution. The collected cells were homogenized on ice using a Dounce glass/Teflon homogenizer. The homogenate was centrifuged at Hippocampal Slice Electrophysiology 1000g for 10 minutes and the pelleted fraction was collected and stored Acute hippocampal slices were prepared from 7–10 week old rats at –80°C in 50 mM Tris (pH 7.4) at a protein concentration of whose brains were removed and immersed in ice-cold cutting solution approximately 2 mg protein/ml, determined using the Pierce BCA (206 mM sucrose, 3 mM KCl, 1.25 mM NaH2PO4, 7 mM MgCl2,26mM protein assay kit. Rat, nonhuman primate, and dog brain membranes NaHCO3,10mMD-glucose, 0.5 mM CaCl2,1mML-ascorbate, 1 mM were also prepared as described earlier. sodium pyruvate, bubbled continuously with 95% O2/5% CO2). Co- ronal slices (300 mm), containing the dorsal hippocampus were cut Fluorometric Imaging Plate Reader Functional Potentiation Assay

CHO cells expressing the hM1,hM3,andhM5 mAChR (HD Bio- science, Shanghai, China) were seeded at a density of 10K cells per well in black-wall, clear-bottomed 384-well plates in F12 nutrient media supplemented with 10% FBS and 1% penicillin/streptomycin. The cells were grown overnight at 37°C in the presence of 5% CO2. The cell media was subsequently removed and replaced with loading solution, containing 2 mM Fluo-8-AM dye, 2 mM probenecid, 1Â acid red 1, in Hanks’ balanced salt solution, and the plate was incubated for 1 hour at 37°C in the dark. For PAM potentiation measurements, the cells were preincu- bated with test compound for 10 minutes before being stimulated with an EC20 concentration of ACh (the actual concentration was adjusted for each experiment, but was found to be between 1 and 3 nM). Ca21 modulation was measured using a FLIPR Tetra fluorometric imaging plate reader (Molecular Devices, Sunnyvale, CA). cAMP Assay Fig. 2. Selection of PT-1284 (1). Incorporating a heteroaryl-N at the Human embryonic kidney 293 cells expressing hM2 and hM4 mAChR naphthalene core led to a quinoline analog PT-1284 (1) with comparable (cells expressed with Promega GloSensor cAMP technology) were M1 PAM potency, much reduced lipophilicity, and a 4-fold higher Fu,b than seeded and cultured at a density of 25K cells per well in white 384-well compound 10. 180 Smith et al.

Fig. 3. Synthesis of PT-1284 (1). Pd(t- Bu)3, palladium tris-tert-butylphosphine; (Bu3Sn)2, Bis(tributyltin); Pd(PPh3)4,tetrakis (triphenylphosphine) palladium; Mel, Iodomethane; Pd2dba3, tris (-dibenzylideneacetone) palladium; CsF, cesium floride. Downloaded from

with a vibratome (VT1000S or 1200S; Leica, Buffalo Grove, IL) temperature. DMF (0.5 ml) was added and immediately followed by molpharm.aspetjournals.org and allowed to recover in artificial cerebrospinal fluid (ACSF; introduction of [3H]-methyl iodide in DMF (1000 mCi at 85 Ci/mmol,

124 mM NaCl2, 3 mM KCl, 1.25 mM NaH2PO4, 1.3 mM MgCl2,26mM 0.0116 mM, 1 ml toluene). The reaction was stirred at room 3 NaHCO3,10mMD-glucose, 2 mM CaCl2,1mML-ascorbate, 1 mM temperature for 2 hours. Unreacted [ H]-methyl iodide was removed sodium pyruvate, bubbled continuously with 95% O2/5% CO2) at 32°C under vacuum. The residue was filtered and concentrated under for a recovery period of at least 1 hour. After recovery, slices were reduced pressure. The dark-green residue was analyzed by radio-high- transferred individually to MED-P515A 64-electrode arrays (Auto- performance liquid chromatography (HPLC) analysis. Approximately Mate Scientific, Inc., Berkeley, CA) and positioned with the CA1 90% of the radioactivity coeluted with the reference standard. stratum pyramidale directly over one row of eight electrodes and Purification to a radiochemical purity of 97.8% was achieved using a perfused continuously with recirculating ACSF warmed to 32°C. Luna C-18 column, 5-mm, 250 Â 10 mm (Phenomenex P/N 00G-4252-

Electrophysiological signals were high-pass filtered at 0.1 Hz and NO, S/N 529990-1). Fractions were collected, analyzed for radiochem- at ASPET Journals on September 26, 2021 digitized at 20 kHz using MED64 Software Mobius QT (WitWerx, Inc., ical purity, pooled, rotary-evaporated to dryness, and dissolved in Tustin, CA). Extracellular action potentials were detected offline ethanol to 1 mCi/ml. The radiochemical purity and the identity were using custom-written Matlab scripts. CA1 neuron spiking activity was measured by analytical HPLC (Phenomenex Luna C-18 column, 5-mm, measured as the multiunit firing rate for each electrode contacting the 250 Â 4.6 mm) at a flow rate of 1 ml/min and ultraviolet at 254 nm, stratum pyramidale. Compound activity was assessed alone (“agonist using 85% of 1% triethylammonium acetate (pH 4) and 15–40% mode”) or in the presence of an EC20 concentration (100 nM) of CH3CN and a 20-minute linear gradient and then 10 minutes at carbachol (“PAM mode”). 100% CH3CN. Identity was confirmed by coelution with the reference standard. Specific activity was calculated by mass spectral analysis to [3H]N-Methylscopolamine Binding Assay be 79.5 Ci/mmol. [3H]N-methylscopolamine ([3H]NMS) displacement studies were conducted using 96-well microtiter plates in a total volume of 250 ml containing 0.2 nM [3H]NMS with increasing concentrations of the orthosteric ligand ACh in the absence or presence of PT-1284 (1) (0.1, 0.32, 1, 3.2, or 10 mM). The binding assay was initiated by the addition of 30 mg of CHO hM1 membranes in binding buffer (50 mM Tris, 2 mM MgCl2, pH 7.4) and incubated at room temperature for 60 minutes. Nonspecific binding was defined with 10 mM atropine sulfate. The reaction was terminated by rapid vacuum filtration through presoaked (0.5% polyethylenimine) Whatman GF/B filter plates using the Packard FilterMate Harvester 196 (PerkinElmer). The filters were washed with iced-cold 50 mM Tris, pH 7.4, and allowed to dry prior to adding MicroScint-20 scintillation cocktail. Filter-bound radioactivity was measured using the TopCount NXT scintillation counter (PerkinElmer).

[3H]PT-1284 Radiolabeling Fig. 4. PAM functional concentration-response curves for hM1-M5 mAChR. Measurement of functional responses for PT-1284 (1) in the 8-((1S,2S)-2-Hydroxycyclohexyl)-5-((6-(tributylstannyl)pyridin-3- presence of an EC20 concentration of ACh. Data are expressed as yl)methyl)-8,9-dihydro-7H-pyrrolo[3,4-hour]quinolin-7-one (compound percentage of functional effect, and the endpoints were generated using 13; MW 662.4, 60 mg, 0.09 mmol, yellow glue-like residue), tri FLIPR for M1,M3,andM5 and cAMP assays for M2 and M4. Open circles for M1 data were not fitted to the curve; this “inverted U” effect occurred at (dibenzylideneacetone)-dipalladium (0; 5 mg, 0.00546 mmol), tri- concentrations at which direct agonist activity was observed using agonist (o-tolyl)phosphine (2.76 mg, 0.009 mM), and cesium fluoride (50 mg, mode assay conditions. Data shown are representative of two (M2–M5)or 0.329 mM) were weighed into a 1-ml microflask and chilled to ice-cold fourteen (M1) separate experiments and expressed as the mean 6 S.E.M. 3 [ H]PT-1284, a Novel M1 mAChR PAM Radioligand 181

Saturation Binding Assay Autoradiographic Study of [3H]PT-1284 [3H]PT-1284 saturation studies were conducted using 96-well Male Sprague Dawley rat brains were rapidly removed and microtiter plates in a total volume of 250 ml containing various immediately placed on dry ice to freeze and then stored at –80°C. concentrations of [3H]PT-1284 (0.4-50 nM) in the presence of ACh The brains were mounted, and 20-mm cryostat-cut (Zeiss, Thornwood, (800 nM or 1 mM). The binding assay was initiated by the addition of NY) coronal sections were transferred onto gelatin-coated slides and either 100 mg of CHO hM1 mAChR membranes or brain membranes in stored at -80°C. Prior to the assay, the sections were thawed in a 37°C binding buffer (50 mM Tris, 2 mM MgCl2, pH 7.4) and incubated at incubator. Sections were then incubated for 30 minutes at room room temperature for 60 minutes. Nonspecific binding was defined temperature in assay buffer (50 mM Tris, 2 mM MgCl2, pH 7.4) with with 10 mM compound 9 (Kuduk et al., 2012). The reaction was 5nM[3H]PT-1284 in the presence of 1 mM ACh. Nonspecific binding terminated by rapid vacuum filtration through presoaked (0.5% was defined with 10 mM compound 9. Following the incubation period, polyethylenimine) Whatman GF/B filter sheets using the Brandel the sections were washed (2 Â 2 minutes) in ice-cold assay buffer ML 96-well Harvester (Brandel, Gaithersburg, MD). The filters were followed by a 30-second wash in ice-cold deionized water. The sections washed with ice-cold 50 mM Tris, pH 7.4, and allowed to dry prior to were dried rapidly under a cool air stream and further dried in a addition of Ultima Gold MV scintillation cocktail. Filter-bound desiccant-containing vacuum. Labeled sections were exposed to radioactivity was measured using the PerkinElmer Tri-Carb scintil- Kodak BioMax MR Film for 47 days before being scanned on a Bio- lation counter. Rad 800 densitometer. Sections were later analyzed using Quantity One 1-D Software (Bio-Rad, Hercules, CA). Downloaded from Displacement Binding Assay Data Analysis [3H]PT-1284 displacement studies were conducted in the presence of ACh (800 nM and 1 mM). A total volume of 250 ml containing All data were analyzed using GraphPad Prism 6.03 (GraphPad either 30 or 5 nM [3H]PT-1284 (corresponding to studies run with Software, La Jolla, CA). Displacement binding curves were best fit to a 800 nM ACh or 1 mM ACh, respectively) with increasing concen- one-site four-parameter model, where Ki values were determined from 5 1 trations of compound added to 96-well microtiter plates. The the Cheng-Prusoff relationship such that Ki IC50/1 [ligand]/Kd, binding assay was initiated by the addition of approximately where [ligand] is the concentration of the free radioligand used in the molpharm.aspetjournals.org

100 mgofCHOhM1 membranes in binding buffer (50 mM Tris, assay and Kd is the dissociation constant of the radioligand for the 2mMMgCl2, pH 7.4). Nonspecific binding was defined with 10 mM receptor (Cheng and Prusoff, 1973). Saturation curves were fitted compound 9. The assay was incubated at room temperature for using the one-site binding hyperbola model, where the Ki and Bmax 60 minutes. The reaction was terminated by rapid vacuum filtration through presoaked (0.5% polyethylenimine) Whatman GF/B filter plates. Filters were washed with ice-cold 50 mM Tris, pH 7.5, and allowed to dry prior to addition of MicroScint-20 scintillation cocktail. Radioactivity was measured using the Per- kinElmer TopCount NXT scintillation counter. Reported Ki values are all mean 6 S.E.M. at ASPET Journals on September 26, 2021

3 Fig. 6. [ H]NMS binding to hM1 cell membranes. (A) PT-1284 (1) Fig. 5. Hippocampal slice electrophysiology. PT-1284 (1) increased spon- competes at the [3H]NMS orthosteric binding site. Inhibition of [3H]NMS taneous firing of CA1 neurons in rat hippocampal slices. When the binding to hM1 mAChR membranes by PT-1284 (1). (B) Inhibition of 3 compound was applied alone (agonist mode), CA1 firing rates increased [ H]NMS binding to hM1 mAChR membranes by ACh in the absence or in a concentration-dependent manner (EC50 =1.16 0.32 mM, max = 35 6 presence of various concentrations of PT-1284 (1). Data are expressed as % 6Hz;n = 8). When the compound was applied in the continuous presence of inhibition and analyzed for Ki values using the one-site heterologous with an EC20 concentration of carbachol (PAM mode), PT-1284 (1) increased depletion model (Swillens 1995). Data shown are representative curves spontaneous firing at much lower concentrations (EC50 =1656 44 nM, from three separate experiments performed in duplicate and expressed as max = 96 6 20 Hz, n = 9). Data are shown as mean 6 S.E.M. the mean 6 S.E.M. 182 Smith et al. were derived from the formula Y 5 (Bmax * X)/(Kd 1 X), where Y is the 10 (0.025) prompted us to synthesize and profile additional specific binding and X is the concentration of the ligand. To account for close-in analogs of compound 10 aiming for higher Fu,b, thus 3 ligand depletion in [ H]NMS saturation binding analyses for the lowering the risk of high nonspecific binding, at the same time 3 human M1,M3, and M4 mAChR cell lines and in [ H]PT-1284 maintaining favorable M1 PAM potency and selectivity. As saturation binding analysis for the human M1 mAChR cell line, a shown in Fig. 2, incorporation of a heteroaryl-N at the global-fit model with parameter-sharing was used (Motulsky and 3 naphthalene core led to a quinoline analog, PT-1284 (1), that Christopoulos, 2004). Also, owing to ligand depletion, [ H]NMS 5 displacement binding was best fit to a one-site heterologous binding had comparable M1 PAM potency [PAM EC50 of 36 nM (n 14) with depletion model (Swillens, 1995). Fluorometric imaging plate versus 18 nM for compound 10] with reduced lipophilicity reader (FLIPR) EC50 data were fitted to the compound percent effect (ELogD 1.83 versus 2.90 for compound 10; Lombardo et al., using four-parameter logistic fit models. 2001), which led to much improved Fu,b (0.10 versus 0.025 for compound 10). The synthesis of PT-1284 (1) can be seen in Fig. 3. It is worth noting that PT-1284 (1) was expected to have low Results brain permeability (Feng et al., 2008) as it is a P-glycoprotein substrate (MDR BA/AB 5 8.8; Johnson et al., 2014). [3H]PT- For the development of M -selective PAM radioligands, we 1 1284 showed no specific binding in vivo when dosed up to used a set of central nervous system (CNS) positron emission 100 mCi. However, it is fit-for-purpose to enable in-vitro or tomography (PET) ligand design-and-selection criteria that Downloaded from ex-vivo tissue radioligand binding assays for which brain our group had previously published, considering the similar permeability is not required and, subsequently can be used to property requirements between a PET ligand and a facilitate the development of novel M PAM in vivo radio- [3H] radioligand (Zhang et al., 2013). Specifically, we targeted 1 tracers and PET ligands. leads that possessed potent M PAM activity and high 1 FLIPR Functional Potentiation Activity. The effect of selectivity against other mAChR subtypes, and, importantly, PT-1284 (1) on functional activity was assessed for selectivity demonstrated low nonspecific binding levels. Toward this end, molpharm.aspetjournals.org among all mAChRs. PT-1284 (1) increased M calcium levels a database of approximately 280 patent compounds with 1 in a concentration-dependent manner whether applied alone reported M PAM potency values were compiled, and their 1 (agonist mode) or in the presence of a low concentration (EC ) CNS PET multiparameter optimization (MPO) values were 20 of the nonselective agonist ACh (PAM mode). In the presence calculated. Upon filtering by potency (M PAM EC , 20 nM) 1 50 of ACh at its EC , signaling endpoints were generated using and physicochemical property criteria (CNS PET MPO . 3), 20 the FLIPR assay for M ,M, and M , whereas the cAMP 18 leads were identified (Fig. 1). Notably, eight out of these 1 3 5 accumulation assay was used to generate data for M and M 18 leads shared the tricyclic lactam core exemplified by 2 4 (Fig. 4). In the agonist mode of our FLIPR assay, PT-1284 (1) compound 10 (Kuduk et al., 2012), and only three had a displayed transient partial agonist activity that ranged from synthetic handle for tritiation, among which compound 10 125 nM to .13 mM with variable efficacy. Though this activity at ASPET Journals on September 26, 2021 emerged as the most promising lead, with a reported M PAM 1 was noted, its relevance was uncertain and we were unable EC of 18 nM and good physicochemical properties [CNS PET 50 to quantify it. In response to an EC challenge with M MPO desirability of 3.15]. 20 1 endogenous agonist ACh, free-base PT-1284 (1) displayed One of the leading causes of radioligand failure is high robust potentiation of ACh with an EC of 36 nM 6 7 (mean nonspecific binding. Fraction unbound in brain (F ; Di et al., 50 u,b 6 S.E.M., n 5 14). There were no functional PAM responses 2011) has been shown to be a useful predictor for nonspecific observed for PT-1284 (1) at the M –M mAChR subtypes when binding and, specifically, we targeted F . 0.05 to minimize 2 5 u,b tested up to 10 mM. this risk (Zhang et al., 2013). Toward this end, compound 10 Hippocampal Slice Electrophysiology. M receptors was synthesized and its F was measured to monitor non- 1 u,b are expressed in hippocampal CA1 pyramidal neurons and specific binding risk. The relatively low F value of compound u,b interneurons, where they increase cell excitability (Langmead et al., 2008, Dasari and Gulledge, 2011). In acute hippocampal

TABLE 1 3 [ H]PT-1284 saturation binding to hM1 mAChR, rat, nonhuman primate, and dog brain membranes [3H]PT-1284 saturation binding values were obtained in the presence of 1 mM ACh unless otherwise noted. Data shown are the result of three separate experiments performed in triplicate unless otherwise noted and expressed as the mean 6 S.E.M.

Membrane Kd Bmax

nM fmol/mg hM1a 29 6 3.15 2479 6 415 hM1 4.23 6 0.17 6383 6 346 Rat Frontal Cortex 2.65 6 0.34 415 6 31 Rat Hippocampus 2.55 6 0.57 410 6 51 Rat Cerebellumb NDc NDc NHP Cortex 2.28 6 0.19 244 6 36 NHP Cerebellumb NDc NDc Dog Cortex 2.88 6 0.69 118.5 6 7.72 Fig. 7. ACh concentration-response curve in the [3H]PT-1284 binding 3 assay. [ H]PT-1284 (5 nM) binding to hM1 mAChR membranes by ACh. aSaturation binding in the presence of 800 nM ACh. Data shown are representative of four separate experiments performed in bn =2. duplicate and expressed as counts per minute (mean 6 S.E.M.). cNo specific binding detected. 3 [ H]PT-1284, a Novel M1 mAChR PAM Radioligand 183 Downloaded from molpharm.aspetjournals.org

3 Fig. 8. [ H]PT-1284 saturation curves and Scatchard analyses (inset graphs) for (A) hM1 cell membranes (total binding = 46,204 dpm and nonspecific binding = 2714 dpm at approximately 3 nM [3H]PT-1284), (B) rat frontal cortex membranes (total binding = 4329 dpm and nonspecific binding = 2559 dpm at approximately 3 nM [3H]PT-1284), (C) nonhuman primate cortex membranes (total binding = 1594 dpm and nonspecific binding = 735 dpm at approximately 3 nM [3H]PT-1284), (D) dog cortex membranes (total binding = 706 dpm and nonspecific binding = 360 dpm at approximately 3 nM [3H]PT- 1284). Saturation binding was measured in the presence of 1 mM ACh. Data shown are a representative of three separate experiments performed in triplicate and expressed as the mean 6 S.E.M. slices, this can be measured as an increase in the spontaneous- mode). However, the potency and efficacy of the compound were action-potential-firing rate of CA1 neurons (Langmead et al., both enhanced by the presence of carbachol, consistent with an at ASPET Journals on September 26, 2021 2008). Since acetylcholinesterase is present in rat brain slices agonist-PAM pharmacological profile. When the concentration- and has the ability to catalyze the breakdown of the mAChR response data from each slice were fitted individually, the endogenous ligand ACh (Lockhart et al., 2001), carbachol was agonist EC50 was 1.1 6 0.32 mM(mean6 S.E.M., n 5 8slices) used as the cholinergic agonist to define PAM mode for this and the maximal agonist effect was 35 6 6 Hz, whereas the assay. In acute slices of dorsal hippocampus from adult rats, PAM EC50 was 165 6 44 nM (mean 6 S.E.M., n 5 9 slices) and PT-1284 (1) increased spontaneous CA1 neuron firing in a the maximal PAM effect was 96 6 20 Hz (Fig. 5). concentration-dependent manner whether applied alone (ag- [3H]NMS Displacement. The effects of PT-1284 (1) on the 3 onist mode) or in the presence of a low concentration (∼EC10– affinity of ACh at the [ H]NMS orthosteric binding site were EC30) of the nonselective cholinergic agonist carbachol (PAM examined using CHO cells stably transfected with the hM1

TABLE 2 3 [ H]PT-1284 displacement binding to hM1 mAChR membranes by positive allosteric modulators and orthosteric or allosteric binding ligands 3 [ H]PT-1284 displacement binding values (pKi) were obtained in the presence of 1 mM ACh. Data shown are the result of three separate experiments performed in duplicate and expressed as the mean pKi 6 S.D.

Compound ID Binding Site pKi Reference PT-1284 (1) PAM 5.97 6 0.19 WO2012003147 Compound 2 PAM 8.15 6 0.23 WO2011084368 Compound 3 PAM 8.09 6 0.02 WO2011084368 Compound 4 PAM 7.99 6 0.27 WO2012003147 Compound 5 PAM 7.51 6 0.07 WO2014077401 Compound 6 PAM 7.45 6 0.08 WO2011084368 Compound 7 PAM 7.28 6 0.19 WO2011084368 PF-06764427 PAM 6.92 6 0.23 Sigma #PZ0300 Compound 8 PAM 6.61 6 0.21 WO2013091773 AZD6088 Allosteric Agonist 5.53 6 0.19 WO2009034380 Danipon Allosteric Agonist ,5.00 (Sumiyoshi et al., 2013) GSK1034702 Allosteric Agonist ,5.00 (Budzik et al., 2010) TBPB Allosteric Agonist 5.57 6 0.05 Sigma no. SML0453 Xanomeline Orthosteric Agonist 5.58 6 0.15 Sigma no. X2754 Oxotremorine Orthosteric Agonist ,5.00 Tocris no. 0843 Carbachol Orthosteric Agonist ,5.00 Sigma no. C4382 184 Smith et al.

3 mAChR subtype ([ H]NMS: Kd 5 0.506 nM; Bmax 5 binding to M1 mAChR, left-shifting the EC50 of ACh by 3069 fmol/mg protein). Although PT-1284 (1) was shown to approximately 19-fold at 10 mM (Fig. 6B). compete with the nonselective muscarinic receptor antagonist Optimization of [3H]PT-1284 Binding Protocol. Pre- 3 3 [ H]NMS with weak affinity (Ki 5 7.84 6 1.42 mM; mean 6 liminary studies to ascertain assay conditions for [ H]PT-1284 S.E.M., n 5 3; Fig. 6A), there were no changes to the maximal binding to CHO cells stably expressing the hM1 mAChR were response of ACh, indicating that PT-1284 (1) did not compete performed at radiolabel concentrations below PT-1284 (1)’s with ACh for binding to M1 mAChR (Fig. 6B). PT-1284 (1) EC50 of 36 nM. Binding experiments were initially performed reduced the concentration of ACh required to inhibit [3H]NMS in the absence of the orthosteric agonist ACh and no specific Downloaded from molpharm.aspetjournals.org at ASPET Journals on September 26, 2021

Fig. 9. Structures of compounds used in [3H]PT-1284 displacement binding assay. Structures include mAChR allosteric agonists, orthosteric agonists, and M1 PAMs. 3 [ H]PT-1284, a Novel M1 mAChR PAM Radioligand 185

nonspecific binding determinations (mean 6 S.E.M. of three experiments). [3H]PT-1284 Saturation Binding. [3H]PT-1284 saturation experiments were performed in the presence of 800 nM or 1 mM ACh (Table 1; Fig. 8, A–D). Using CHO cells stably 3 expressing hM1 mAChR, [ H]PT-1284 bound with a higher affinity (Kd) when in the presence of 1 mM ACh (4.23 6 0.17 nM, mean 6 S.E.M., n 5 3) and the Bmax increased nearly 3-fold (6383 6 346 fmol/mg protein, mean 6 S.E.M., n 5 3) compared with saturation binding in the presence of 800 nM ACh (29 6 3.15 nM; 2479 6 415 fmol/mg protein, mean 6 S.E.M., n 5 3). [3H]PT-1284 bound to rat frontal cortex and hippocampus with similar affinity and saturable binding capacity [2.65 6 0.34 nM and 415 6 31 fmol/mg protein; 2.55 6 0.57 nM and 410 6 51 fmol/mg protein, respectively (mean 6 S.E.M., n 5 3)]. [3H]PT-1284 bound to nonhuman primate Downloaded from (NHP) and dog cortex with Kd and Bmax values of 2.28 6 0.19 nM; 244 6 36 fmol/mg protein and 2.88 6 0.69 nM; 118.5 6

3 7.72 fmol/mg protein, respectively (mean 6 S.E.M., n 5 3). Fig. 10. Correlation between hM1 functional and [ H]PT-1284 displacement binding data. Positive allosteric modulators show a correlation Scatchard plots for all saturation experiments could be fitted 3 3 between FLIPR calcium mobilization EC50 values and [ H]PT-1284 by a straight line, indicating that [ H]PT-1284 bound to a displacement binding. The dashed line indicates unity (R2 = 1.00). Solid – 2 single site in each case (Fig. 8, A D, inset graphs). M1 mAChR

line indicates best linear fit of the data generated (R = 0.86). Data shown molpharm.aspetjournals.org are the mean values of at least three separate experiments. specific binding was not detected in rat or nonhuman primate cerebellum when tested up to a [3H]PT-1284 concentration of 50 nM (data not shown). binding was detected (data not shown). A concentration- [3H]PT-1284 Displacement Binding. [3H]PT-1284 dis- response curve for ACh in this assay showed significantly placement studies were performed with a series of com- increased specific binding with a maximal effect at 1 mM (Fig. 7), pounds previously determined to be PAMs, allosteric indicating a positive cooperation between the orthosteric and agonists, or orthosteric agonists at the M1 mAChR (data allosteric binding sites. not shown). The rank order of potency was consistent with [3H]PT-1284 (5 nM)-specific binding levels were determined structural recognition of the [3H]PT-1284 binding site, in over a protein concentration range from 6 to 200 mg per well. that PAMs showed higher affinity than compounds known to at ASPET Journals on September 26, 2021 Specific binding increased with increasing concentrations of be allosteric or orthosteric agonists (Table 2; Fig. 9). M1 membranes and plateaued between 50 and 200 mg per well. mAChR PAM affinities were 10-fold weaker when the assay Kinetic studies showed the rate of association of [3H]PT-1284 was run in the presence of 800 nM ACh. For example, the (5 nM), which reached its equilibrium within 10 minutes, and affinity of compound 3 for the [3H]PT-1284 binding site remained stable for up to 120 minutes at room temperature (data decreased from 9.7 6 0.23 nM to 84 6 15 nM (mean 6 S.E.M., not shown). Subsequent experiments used a radioligand concen- n 5 3; data not shown), respectively. As shown in Fig. 10, for a tration of 5 nM, membrane concentration of 100 mgperwell,and cohort of 19 M1 PAMs previously reported by our group incubations were performed at room temperature for 60 minutes. (Davoren et al., 2016), a positive correlation (R2 5 0.86) Using these established conditions, in the presence of 1 mM was found between the EC50 values obtained using the 3 ACh, the percent specific binding of [ H]PT-1284 to hM1 M1 mAChR FLIPR PAM assay and the binding pKi values membranes was greater than 90%, with binding values of determined with the [3H]PT-1284 binding displacement 49,523 6 1659 dpm for total binding and 2067 6 389 dpm for assay.

Fig. 11. Autoradiographic analysis of representative rat brain coronal sections using [3H]PT-1284. (A) Cortex, caudate, and nucleus accumbens and (B) hippocampus repre- sent autoradiograms for total specific binding. (C) Inhibition of specific binding by 10 mM compound 9. All binding was done in the presence of 1 mM ACh. (D) Density scale in the range of 3.7–2000 mCi/g protein. 186 Smith et al.

Autoradiographic Analysis for [3H]PT-1284 Binding present. This, in turn, increased the affinity of [3H]PT-1284 to Rat Brain. Autoradiographic distribution of [3H]PT-1284 for the receptor, revealing subnanomolar affinity at maximal binding to coronal sections of rat brain is shown in Fig. 11. In concentrations of ACh. In this study, we showed that representative sections shown in (A) and (B), the highest cooperativity between our PAM ligand and the orthosteric 3 distribution of M1 PAM mAChR [ H]PT-1284 binding can be ligand ACh may be improved by increasing the number of G seen in the cortex (CTX), caudate (CPu), nucleus accumbens protein-bound receptors. In spite of the fact that differences (Abc), and the hippocampus (Hip). [3H]PT-1284 binding was in ACh levels have been reported across species (Fujii et al., displaced by 10 mM compound 9 (Fig. 11C). 1995), [3H]PT-1284 bound with similar affinity to native rat, dog, and NHP tissues when enhanced with the addition of Discussion ACh, which is important in that cross-species translation can be used to predict occupancy of a clinical compound in Although recent advancements have been made in the humans from rat in vivo binding or NHP PET receptor development of mAChR allosteric modulators to improve occupancy studies (Shaffer et al., 2014). Autoradiographic subtype selectivity, little has been reported on the develop- localization studies showed distribution in the cortex, cau- ment of radiolabeled modulators to study the actual allosteric date, nucleus accumbens, and hippocampus of the rat brain, binding pocket. With the characterization of the M2/M4 which correlates with the observed binding capacities like- mAChR allosteric modulator radioligand [3H]LY2119620, wise found in our saturation binding studies. Also, with Downloaded from great strides were taken to further the understanding of [3H]PT-1284 displacement studies, we observed greater mAChR allosteric binding sites (Schober et al., 2014). As with affinity of M1 PAM compounds in the presence of higher other GPCR PAM radioligands such as the mGluR4 PAM, and concentrations of ACh. The rank order of affinities was [3H]PAM2 (Le Poul et al., 2012), these authors showed consistent with structural recognition of the PT-1284 (1) positive cooperativity between the orthosteric and allosteric binding site, in that PAMs showed higher affinity compared molpharm.aspetjournals.org binding sites and further showed that selectivity between M2 with compounds known to be allosteric or orthosteric ago- and M4 mAChR subtypes was dependent on the allosteric nists. Taken together, these data showed cooperation be- ligand used. This is the first report on the development and tween the ACh orthosteric binding site and the PT-1284 (1) 3 characterization of a novel M1-selective mAChR PAM radio- binding site. To demonstrate [ H]PT-1284 as a tool in ligand that possesses high M1 PAM potency and broad characterizing novel M1 mAChR PAMs, a direct correlation 2 spectrum selectivity, and demonstrated high M1-specific of binding pKi and functional EC50 values (R 5 0.86) was binding in vitro. observed for a cohort of compounds. Herein, we presented evidence from several in vitro assays In summary, we have demonstrated PT-1284 (1)tobeaM1- to show the pharmacological profile of PT-1284 (1)asan selective PAM agonist that acts as a PAM when [3H]PT-1284 agonist-PAM. PT-1284 (1) was shown to have PAM charac- binding is performed using 5 nM. With the characterization at ASPET Journals on September 26, 2021 teristics in a FLIPR assay by potentiating M1 mAChR calcium of the radioligand and a direct correlation with our cal- 3 mobilization when in the presence of an EC20 concentration of cium mobilization assay, we have shown [ H]PT-1284 to be ACh. PT-1284 (1) also showed higher potency and induced a a tool in profiling future M1 mAChR PAMs. These findings greater response in hippocampal CA1 neurons when applied could enable improvements in the treatment of symptoms in the presence of carbachol. In the past, the only way to assess of schizophrenia and cognitive deficits associated with allosteric binding activity of a compound such as BQCA was Alzheimer’s disease. indirectly through the orthosteric binding site. Radioligand binding studies utilizing [3H]NMS, the nonselective orthos- Authorship Contributions teric mAChR ligand, showed a positive cooperativity between Participated in research design: Smith, Davoren, Lee, Zhang, PAM compounds and orthosteric agonists ACh, carbachol, or Grimwood. oxotremorine-M for M ,M, and M mAChR subtypes (Ma Conducted experiments: Smith, Edgerton, Lee, Neal. 1 2 4 Performed data analysis: Smith, Edgerton, Lazzaro, Neal. et al., 2009; Canals et al., 2012; Abdul-Ridha et al., 2014; Wrote or contributed to the writing of the manuscript: Smith, Schober et al., 2014). In line with literature values, the affinity Davoren, Edgerton, Lee, Zhang, Grimwood. of ACh in our [3H]NMS binding assay was 400 mM. In the current study, we also determined that PT-1284 (1) was References Abdul-Ridha A, Lane JR, Mistry SN, López L, Sexton PM, Scammells PJ, Christo- positively cooperative with ACh for the M1 mAChR showing 3 poulos A, and Canals M (2014) Mechanistic insights into allosteric structure- a 19-fold leftward shift of ACh’s activity at the [ H]NMS function relationships at the M1 muscarinic acetylcholine receptor. J Biol Chem binding site. 289:33701–33711. Bodick NC, Offen WW, Levey AI, Cutler NR, Gauthier SG, Satlin A, Shannon HE, It has been suggested that GPCR binding levels are directly Tollefson GD, Rasmussen K, and Bymaster FP et al. (1997) Effects of xanomeline, a proportional to the number of receptors in an active state, selective muscarinic receptor agonist, on cognitive function and behavioral symptoms in Alzheimer disease. Arch Neurol 54:465–473. reflected by the coupling of the GPCR to the G protein Bonner TI, Buckley NJ, Young AC, and Brann MR (1987) Identification of a family of (Christopoulos and El-Fakahany, 1999; Schober et al., 2014). muscarinic acetylcholine receptor genes. Science 237:527–532. 3 Budzik B, Garzya V, Shi D, Walker G, Woolley-Roberts M, Pardoe J, Lucas A, Tehan [ H]PT-1284 binds to the allosteric pocket of the M1 mAChR B, Rivero RA, and Langmead CJ et al. (2010) Novel N-Substituted Benzimidazo- and enhances G protein coupling and the affinity of ACh for lones as Potent, Selective, CNS-Penetrant, and Orally Active M1 mAChR Agonists. 3 ACS Med Chem Lett 1:244–248. this receptor. The [ H]NMS binding density for a CHO cell line Canals M, Lane JR, Wen A, Scammells PJ, Sexton PM, and Christopoulos A (2012) A overexpressing mAChR hM1 (3 pmol/mg protein) is in line Monod-Wyman-Changeux mechanism can explain G protein-coupled receptor (GPCR) allosteric modulation. J Biol Chem 287:650–659. with literature values ranging from 2 to 4 pmol/mg protein. At Caulfield MP (1993) Muscarinic receptors–characterization, coupling and function. 3 concentrations up to 50 nM, [ H]PT-1284 bound to a single, Pharmacol Ther 58:319–379. Caulfield MP, Higgins GA, and Straughan DW (1983) Central administration of the saturable site on human M1 receptors, with saturation muscarinic receptor subtype-selective antagonist pirenzepine selectively impairs binding levels increasing with higher concentrations of ACh passive avoidance learning in the mouse. J Pharm Pharmacol 35:131–132. 3 [ H]PT-1284, a Novel M1 mAChR PAM Radioligand 187

Caulfield MP and Birdsall NJ (1998) International Union of Pharmacology. XVII. Lombardo F, Shalaeva MY, Tupper KA, and Gao F (2001) ElogD(oct): a tool for Classification of muscarinic acetylcholine receptors. Pharmacol Rev 50:279–290. lipophilicity determination in drug discovery. 2. Basic and neutral compounds. J Cheng Y and Prusoff WH (1973) Relationship between the inhibition constant (K1) Med Chem 44:2490–2497. and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an Ma L, Seager MA, Wittmann M, Jacobson M, Bickel D, Burno M, Jones K, Graufelds enzymatic reaction. Biochem Pharmacol 22:3099–3108. VK, Xu G, and Pearson M et al. (2009) Selective activation of the M1 muscarinic Christopoulos A and El-Fakahany EE (1999) Qualitative and quantitative assess- acetylcholine receptor achieved by allosteric potentiation. Proc Natl Acad Sci USA ment of relative agonist efficacy. Biochem Pharmacol 58:735–748. 106:15950–15955. Dasari S and Gulledge AT (2011) M1 and M4 receptors modulate hippocampal py- Messer WS, Jr (2002) The utility of muscarinic agonists in the treatment of Alz- ramidal neurons. J Neurophysiol 105:779–792. heimer’s disease. J Mol Neurosci 19:187–193. Davoren JE, O’Neil SV, Anderson DP, Brodney MA, Chenard L, Dlugolenski K, Messer WS, Jr, Bohnett M, and Stibbe J (1990) Evidence for a preferential in- Edgerton JR, Green M, Garnsey M, and Grimwood S et al. (2016) Design and volvement of M1 muscarinic receptors in representational memory. Neurosci Lett optimization of selective azaindole amide M1 positive allosteric modulators. Bioorg 116:184–189. Med Chem Lett 26:650–655. Motulsky HJ and Christopoulos A (2004) Fitting Models to Biological Data Using Di L, Umland JP, Chang G, Huang Y, Lin Z, Scott DO, Troutman MD, and Liston TE Linear and Nonlinear Regression. A Practical Guide to Curve Fitting. GraphPad (2011) Species independence in brain tissue binding using brain homogenates. Software, Inc., San Diego, CA Drug Metab Dispos 39:1270–1277. Porter AC, Bymaster FP, DeLapp NW, Yamada M, Wess J, Hamilton SE, Nathanson Feng B, Mills JB, Davidson RE, Mireles RJ, Janiszewski JS, Troutman MD, and de NM, and Felder CC (2002) M1 muscarinic receptor signaling in mouse hippo- Morais SM (2008) In vitro P-glycoprotein assays to predict the in vivo interactions campus and cortex. Brain Res 944:82–89. of P-glycoprotein with drugs in the central nervous system. Drug Metab Dispos 36: Puri V, Wang X, Vardigan JD, Kuduk SD, and Uslaner JM (2015) The selective 268–275. positive allosteric M1 muscarinic receptor modulator PQCA attenuates learning Fujii T, Yamada S, Yamaguchi N, Fujimoto K, Suzuki T, and Kawashima K (1995) and memory deficits in the Tg2576 Alzheimer’s disease mouse model. Behav Brain Species differences in the concentration of acetylcholine, a neurotransmitter, in Res 287:96–99. whole blood and plasma. Neurosci Lett 201:207–210. Schober DA, Croy CH, Xiao H, Christopoulos A, and Felder CC (2014) Development

Hagan JJ, Jansen JH, and Broekkamp CL (1987) Blockade of spatial learning by the of a radioligand, [(3)H]LY2119620, to probe the human M(2) and M(4) muscarinic Downloaded from M1 muscarinic antagonist pirenzepine. Psychopharmacology (Berl) 93:470–476. receptor allosteric binding sites. Mol Pharmacol 86:116–123. Johnson TW, Richardson PF, Bailey S, Brooun A, Burke BJ, Collins MR, Cui JJ, Deal Shaffer CL, Osgood SM, Smith DL, Liu J, and Trapa PE (2014) Enhancing ketamine JG, Deng YL, and Dinh D et al. (2014) Discovery of (10R)-7-amino-12-fluoro- translational pharmacology via receptor occupancy normalization. Neuropharma- 2,10,16-trimethyl-15-oxo-10,15,16,17-tetrahydro-2H-8,4-(metheno)pyrazolo[4,3-h] cology 86:174–180. [2,5,11]-benzoxadiazacyclotetradecine-3-carbonitrile (PF-06463922), a macrocyclic Shekhar A, Potter WZ, Lightfoot J, Lienemann J, Dubé S, Mallinckrodt C, Bymaster inhibitor of anaplastic lymphoma kinase (ALK) and c-ros oncogene 1 (ROS1) with FP, McKinzie DL, and Felder CC (2008) Selective muscarinic receptor agonist preclinical brain exposure and broad-spectrum potency against ALK-resistant xanomeline as a novel treatment approach for schizophrenia. Am J Psychiatry 165: mutations. J Med Chem 57:4720–4744. 1033–1039.

Kruse AC, Ring AM, Manglik A, Hu J, Hu K, Eitel K, Hübner H, Pardon E, Valant C, Sumiyoshi T, Enomoto T, Takai K, Takahashi Y, Konishi Y, Uruno Y, Tojo K, Suwa molpharm.aspetjournals.org and Sexton PM et al. (2013) Activation and allosteric modulation of a muscarinic A, Matsuda H, and Nakako T et al. (2013) Discovery of novel N-substituted oxin- acetylcholine receptor. Nature 504:101–106. doles as selective m1 and m4 muscarinic acetylcholine receptors partial agonists. Kuduk SD, Beshore DC, Yang Z, and Shu Y (2012) inventors. Merck Sharp & Dohme ACS Med Chem Lett 4:244–248. Corp., assignee. Fused isoindoline M1 receptor positive allosteric modulators and Swillens S (1995) Interpretation of binding curves obtained with high receptor con- their preparation and use for the treatment of M1-mediated diseases. U.S. patent centrations: practical aid for computer analysis. Mol Pharmacol 47:1197–1203. WO2012003147. 2012 Jan 5. Tränkle C, Kostenis E, Burgmer U, and Mohr K (1996) Search for lead structures to Kuduk SD, Chang RK, Di Marco CN, Pitts DR, Greshock TJ, Ma L, Wittmann M, develop new allosteric modulators of muscarinic receptors. J Pharmacol Exp Ther Seager MA, Koeplinger KA, and Thompson CD et al. (2011) Discovery of a 279:926–933. selective allosteric M1 receptor modulator with suitable development proper- Uslaner JM, Eddins D, Puri V, Cannon CE, Sutcliffe J, Chew CS, Pearson M, Vivian ties based on a quinolizidinone carboxylic acid scaffold. J Med Chem 54: JA, Chang RK, and Ray WJ et al. (2013) The muscarinic M1 receptor positive 4773–4780. allosteric modulator PQCA improves cognitive measures in rat, cynomolgus ma- Lange HS, Cannon CE, Drott JT, Kuduk SD, and Uslaner JM (2015) The M1 mus- caque, and rhesus macaque. Psychopharmacology (Berl) 225:21–30. carinic positive allosteric modulator PQCA improves performance on translatable Vardigan JD, Cannon CE, Puri V, Dancho M, Koser A, Wittmann M, Kuduk SD, tests of memory and attention in Rhesus monkeys. J Pharmacol Exp Ther 355: Renger JJ, and Uslaner JM (2015) Improved cognition without adverse effects: at ASPET Journals on September 26, 2021 442–450. novel M1 muscarinic potentiator compares favorably to donepezil and xanomeline Langmead CJ, Austin NE, Branch CL, Brown JT, Buchanan KA, Davies CH, Forbes in rhesus monkey. Psychopharmacology (Berl) 232:1859–1866. IT, Fry VA, Hagan JJ, and Herdon HJ et al. (2008) Characterization of a CNS Wall SJ, Yasuda RP, Hory F, Flagg S, Martin BM, Ginns EI, and Wolfe BB (1991) penetrant, selective M1 muscarinic receptor agonist, 77-LH-28-1. Br J Pharmacol Production of antisera selective for m1 muscarinic receptors using fusion proteins: 154:1104–1115. distribution of m1 receptors in rat brain. Mol Pharmacol 39:643–649. Le Poul E, Boléa C, Girard F, Poli S, Charvin D, Campo B, Bortoli J, Bessif A, Luo B, Zhang L, Villalobos A, Beck EM, Bocan T, Chappie TA, Chen L, Grimwood S, Heck and Koser AJ et al. (2012) A potent and selective metabotropic glutamate receptor SD, Helal CJ, and Hou X et al. (2013) Design and selection parameters to accel- 4 positive allosteric modulator improves movement in rodent models of Parkinson’s erate the discovery of novel central nervous system positron emission tomography disease. J Pharmacol Exp Ther 343:167–177. (PET) ligands and their application in the development of a novel phosphodies- Levey AI (1996) Muscarinic acetylcholine receptor expression in memory circuits: terase 2A PET ligand. J Med Chem 56:4568–4579. implications for treatment of Alzheimer disease. Proc Natl Acad Sci USA 93: 13541–13546. Lockhart B, Closier M, Howard K, Steward C, and Lestage P (2001) Differential Address correspondence to: Deborah L. Smith, 610 Main Street, Pfizer, inhibition of [3H]-oxotremorine-M and [3H]-quinuclinidyl benzilate binding to Inc., Neuroscience and Pain Research Unit, Cambridge, MA 02139. E-mail: muscarinic receptors in rat brain membranes with acetylcholinesterase inhibitors. [email protected] Naunyn Schmiedebergs Arch Pharmacol 363:429–438.