Identification and pharmacological profile of SPP1, a ... - BPS - Wiley

Identification and pharmacological profile of SPP1, a potent, functionally selective and brain penetrant M1 muscarinic acetylcholine receptor agonist. Running title: Functionally selective, brain penetrant M1 agonist

Lisa M. Broad1*, Helen E. Sanger1, Adrian J. Mogg1, Ellen M. Colvin1, Ruud Zwart1, David A. Evans1, Francesca Pasqui1, Emanuele Sher1, Graham N. Wishart1, Vanessa N. Barth2, Christian C. Felder2, Paul J. Goldsmith1.

1

Eli Lilly and Company Ltd., Lilly Research Centre, Erl Wood Manor, Windlesham, Surrey

GU20 6PH, UK and 2Eli Lilly & Co. Ltd., Lilly Corporate Center, Indianapolis, IN 46285, USA.

List of Hyperlinks for Crosschecking Muscarinic acetylcholine receptors http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=2 Nicotinic acetylcholine receptor http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=76 M1 muscarinic receptor http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=13

*Corresponding author:

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/bph.14510 This article is protected by copyright. All rights reserved.

Lisa M. Broad Eli Lilly and Company Ltd, Lilly Research Centre, Erl Wood Manor, Windlesham, Surrey GU20 6PH. Tel: +44 (0) 1276 483016, Fax: +44 (0) 1276 483525 E-mail: [email protected] Number of text pages: 31 Number of tables: 3 Number of figures: 10 Number of references: 54 Number of words in Abstract: 249 Number of words in Introduction: 804 Number of words in Results: 1777 Number of words in Discussion: 1527

List of non-standard abbreviations: SPP, spiropiperidine; AD, Alzheimer’s disease; SAR, structure activity relationship; mAChR, muscarinic acetylcholine receptor; aCSF, artificial CSF; KP,uu, unbound plasma concentration ratio; AChEi, acetylcholinesterase inhibitor; PAM, positive allosteric modulator

Acknowledgements Human tissue samples were from Randy Woltjer at the Oregon Alzheimer’s Disease Center. The Oregon Alzheimer’s Disease Center is supported by NIH grant P30AG008017. We thank Deanna Koger, Anne Need, Megan Johnson for their technical assistance. We are grateful to Ralf Jaeger and John Huxter for help with statistical analysis.

This article is protected by copyright. All rights reserved.

Abstract

Background and Purpose: The goal of this work was to identify and develop novel, selective muscarinic M1 receptor agonists as potential therapeutics for the symptomatic treatment of Alzheimer’s disease.

Experimental Approach: We developed and utilized a novel M1 receptor occupancy assay to drive a structure activity relationship in a relevant brain region, whilst simultaneously tracking drug levels in plasma and brain to optimize for central penetration. Functional activity was tracked in relevant native in vitro assays allowing translational (rat-human) benchmarking of SAR molecules to clinical comparators.

Key Results: Using this paradigm, we identified a series of M1 selective molecules displaying desirable in vitro and in vivo properties and optimised key features, such as central penetration, whilst maintaining selectivity and a partial agonist profile. From this work, we identified the spiropiperidine SPP1. In vitro, SPP1 is a potent, partial agonist of cortical and hippocampal M1 receptors with activity conserved across species. SPP1 displays high functional selectivity for M1 receptors over native M2 and M3 anti-targets and over a panel of other targets. Assessment of central target engagement by receptor occupancy reveals SPP1 significantly and dose-dependently occupies rodent cortical M1 receptors.

Conclusions and Implications: In summary, we report the discovery of SPP1, a novel, functionally selective, brain penetrant partial M1 orthosteric agonist identified through the use of a novel receptor occupancy assay. SPP1 is amenable to in vitro and in vivo study and provides a valuable research tool to further probe the role of the M1 receptor in physiology and disease.

Keywords: muscarinic acetylcholine receptors, M1, Alzheimer’s Disease


Introduction The hallmarks of Alzheimer’s disease (AD) include Amyloid plaques, neurofibrillary tangles and memory loss. There are no treatments currently available to prevent disease progression, however symptomatic treatments are available to aid cognitive function. The most broadly utilised symptomatic treatments are the acetylcholinesterase inhibitors (AChEi), which include donepezil and rivastigmine. AChEs confer a modest improvement on cognitive symptoms (NICE, 2011) and are associated with undesired adverse effects (e.g. gastrointestinal side effects), which are dose-dependent (FDA Website, 2012, Lockhart et al., 2009) AD is associated with a loss of cholinergic input to forebrain regions and reduced cholinergic transmission (Douchamps and Mathis, 2017). As AChEs block the enzyme responsible for the break-down of ACh, they act to ameliorate the ACh deficit, thereby providing cognitive benefit. The extent of AChE inhibition correlates with cognitive improvements (Bohnen et al., 2005, Rogers and Friedhoff, 1996), however in vivo PET studies performed in subjects with AD report only modest inhibition (22-27%) of cortical AChE at clinically utilised doses of donepezil (Bohnen et al., 2005, Kuhl et al., 2000) suggesting it should be possible to boost central cholinergic transmission and potentially therefore cognitive benefits further. Whilst a higher dose donepezil (23mg) has been marketed, it is associated with significant adverse effects. ACh mediates its effects through activation of two classes of ACh receptors, muscarinic (mAChR) and nicotinic receptors. Clinical data demonstrates that mAChRs are important targets mediating the pro-cognitive effects of ACh released within hippocampal and cortical circuits. Firstly, non-selective mAChR antagonists, such as scopolamine, promote cognitive deficits in man (Rasmusson and Dudar, 1979, Robbins et al., 1997, Potter et al., 2000), whilst a direct acting non-selective mAChR agonist Xanomeline provided some evidence of cognitive benefit in patients with AD and Schizophrenia (Bodick et al., 1997, Shekhar et al., 2008). At the efficacious dose, Xanomeline was not well tolerated in AD patients, in common with high doses of AChEs gastrointestinal tolerability issues were a major issue (Bodick et al., 1997). Of the five isoforms of mAChRs (M1-5), M1 receptors are the most abundant in regions relevant for cognition (Scarr et al., 2016, Oki et al., 2005) and are strongly implicated in mediating, at least in part, the pro-cognitive effects of ACh. Use of selective M1 receptor agonists and PAMs (positive allosteric modulators) and M1 knock-out (KO) mice revealed that M1 modulates neuronal signaling and excitability, synaptic transmission and network function This article is protected by copyright. All rights reserved.

and exerts pre-clinical pro-cognitive effects (e.g. Betterton et al., 2017, Shirey et al., 2009, Uslaner et al., 2013, Lange et al., 2015, Puri et al., 2015, Vardigan et al., 2015, Butcher et al., 2016, Dennis et al., 2016). High M1 expression in cortex and hippocampus is conserved across species, including in brains from human AD patients (Overk et al., 2010, Bradley et al., 2017). As a consequence of their pre- and postsynaptic localization, pan activation of opposing muscarinic and nicotinic receptors by AChEs evokes a ‘net effect’ which may self-limit procognitive potential of the cholinergic system. The promise of selectively targeting key procognitive cholinergic receptors, such as the M1 receptor, is improved efficacy and reduced adverse effects. M2 and M3 are the major sub-types expressed in peripheral tissues and are proposed to underlie the undesirable gastrointestinal and cardiovascular side effects of nonselective agonists such Xanomeline. However, M1 receptors are implicated in addition to M3 receptors in mediating sweating and salivation (Felder et al., 2018), hence the desire for M1 agonists that are highly brain penetrant with a partial agonist efficacy profile to minimise peripheral load and M1 mediated adverse effects. Recent efforts to develop selective M1 ligands have focused on developing PAMs, allosteric agonists or orthosteric M1 agonists (Felder et al., 2018). Allosteric and orthosteric agonists have in common the ability to directly activate M1 receptors, albeit through interaction at distinct sites. In contrast, M1 PAMs act primarily to potentiate the action of the endogenous orthosteric agonist, ACh, at concentrations that do not activate M1 receptors in the absence of agonist. Several of these molecules have successfully advanced to the clinic including MK7622 a selective M1 PAM, GSK1034702, an allosteric M1 agonist, as well as AZD-6088 and HTL-9936, which are orthosteric M1 agonists (Brown et al, 2013; Felder et al., 2018). These next generation orthosteric M1 agonists differentiate from first generation counterparts, such as Xanomeline, on functional selectivity, but lack the high degree of brain penetration desirable to optimizing the benefit/risk profile. We report the discovery of a novel, selective, brain penetrant spiropiperidine (SPP) partial M1 orthosteric agonist, SPP1. This molecule was identified through the use of a novel receptor occupancy assay used to drive a structure activity relationship (SAR) in a relevant brain region whilst simultaneously tracking drug levels in plasma and brain to optimize for central penetration. M1 functional activity was tracked in relevant native in vitro assays to allow translational (rat-human) bench marking of SAR molecules to clinical comparators.


Methods In Vivo Animal Experiments All studies involving animals are reported in accordance with the ARRIVE guidelines for reporting experiments involving animals (McGrath et al, 2010). For occupancy experiments, male Sprague-Dawley rats (177-235 g) and wild type C57Bl/6J mice (17-25 g) were purchased from Harlan (Indianapolis, IN, USA,). Muscarinic M1 (line#1781) KO mice (15-47 g) were purchased from Taconic (Hudson, NY, USA. All animals were group-housed and provided with food and water ad libitum. A normal 12 h/12 h light/dark cycle was applied with lights on at 7:00 AM. The total number of rats used was approximately 20. All animal experiments were carried out either in accordance with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the U.S. National Institutes of Health, and were approved by Eli Lilly’s Animal Care and Use Committee. Ex Vivo Animal Experiments For Xenopus oocyte experiments adult female Xenopus laevis frogs which were purchased from Nasco (Fort Atkinson, WI, U.S.A.). Frogs were kept in the laboratory in a climate-controlled (20-23ºC) and light-regulated room with a 12 hours light/12 hours dark cycle. The animals were fed twice a week with trout pellets and once a week they were given earthworms. The care and use of the frogs complied with the guidelines of the U.K. Animals Scientific Procedures Act (1986) and associated guidelines. Eight frogs were used in this study. Frogs were anaesthetized by immersion in 0.5% 3-aminobenzoic acid ethyl ester until the animals became unresponsive to toe pinch. Toads were then decapitated and ovarian lobes were harvested and defolliculated by incubation in 2 mg/ml collagenase (Type 1 C-0130, SigmaAldrich, UK) in Ca2+-free Barth’s saline at room temperature. Defolliculated stage V-VI oocytes were selected and injected with 5 ng of muscarinic M1 receptor cDNA. For GTPS and radioligand binding experiments male Sprague Dawley rats (200-300 g), were obtained from Charles River (Harlow, UK). For electrophysiological experiments wild type male C57Bl/6J and M1 KO (same line as above) mice were obtained from Envigo (Loughborough, UK. For functional atrial & ileal assays male or female Wistar rats (375-425 g) were used. All animals were group-housed and provided with food and water ad libitum. A normal 12 h/12 h light/dark cycle was applied with lights on at 7:00 AM. The total number of rats and mice (WT & M1 KO) used was approximately 20 and 20 respectively. Animals were This article is protected by copyright. All rights reserved.

sacrificed by exposure to CO2 followed by cervical dislocation. For electrophysiological experiments mice were exposed to isoflurane followed by decapitation. All procedures were reviewed by local ethics committee and complied with the U.K. Animals Scientific Procedures Act (1986). Monkey brain tissue was obtained from 2 rhesus monkeys (1 male/1 female) from Covance, USA and shipped to the UK under CITES (Convention on International Trade in Endangered Species). Samples of cortex were dissected from fresh frozen brain slices and homogenized as per the protocol below. Tissue from the male monkey was used for radioligand binding experiments and tissue from the female monkey was utilized in GTPγ[35]S experiments. Human Brain Tissues Human brain tissue from post mortem healthy and AD patients was provided to Eli Lilly from the Oregon Alzheimer’s Disease Center with appropriate consent and utilized in experiments in the U.K. under the Human Tissue Act 2004. AD tissue was from subjects in Braak stage 5/6 as determined by number of amyloid plaques and neocortical tangles. Details of the demographic and histopathological status of the samples used in this study are included in Supplementary Table 2 Materials SPP1

(cyclopropyl

4-[3-methyl-1-(m-tolyl)-4-oxo-1,3,8-triazaspiro[4.5]decan-8-

yl]piperidine-1-carboxylate), SPP2 (cyclopropyl 4-[1-(3-chloro-4-fluoro-phenyl)-3-methyl-4oxo-1,3,8-triazaspiro[4.5]decan-8-yl]piperidine-1-carboxylate), SPP3 (isopropyl 4-[3-methyl1-(m-tolyl)-4-oxo-1,3,8-triazaspiro[4.5]decan-8-yl]piperidine-1-carboxylate),

SAR

and

reference standards were prepared by Eli Lilly and Company (Lilly Research Centre, Windlesham, UK). [3H]LSN3172176 (specific activity 1.295 TBq/mmol) was synthesized by Quotient Bioscience, Cardiff, UK. [3H]-n-methylscopolamine ([3H]NMS;specific activity 3.16 TBq/mmol) and Chinese hamster ovary (CHO) cell membranes expressing human M1-M5 mAChR were obtained from Perkin Elmer (Boston, MA, USA). All other materials/compounds were obtained from standard commercial sources. Receptor Occupancy Live Phase: Male Sprague Dawley rats (N=4 per dose group) from Charles Rivers were used in the rat studies. Male C57Bl/6J mice from Harlan (N=3-4 per dose group) were used in the


mouse studies. Test compounds were administered by oral gavage at a single screening dose of 10 mg/kg. SPP1 was administered at doses of 0.03, 0.1, 0.3, 1, 3 and 10 mg/kg for generation of dose a response and at 3 mg/kg for the time-course study. Animals received either vehicle alone (1% Hydroxyethlycellulose, 0.25% Polysorbate 80, 0.05% Antifoam in purified water) or test compound in a dose volume of 10 ml/kg. In the dose response studies, rats or mice received i.v. administration of non-labeled tracer (LSN3172176, 10 g/kg, 0.5 ml/kg dose volume for rats and 5 ml/kg dose volume for mice) in the lateral tail vein 30 minutes after vehicle or compound administration. In the time-course study, rats received i.v. administration of non-labeled tracer (same conditions) in the lateral tail vein 30 minutes after vehicle administration and 30, 60, 120, 240, 480 or 1440 minutes after administration of SPP1. Animals were sacrificed by cervical dislocation 20 minutes after tracer administration. Brains were removed and dissected. Frontal cortex and cerebellum were used for the tracer measurement and the remaining brain and plasma used for compound exposure analysis. In the time-course study CSF was also collected. The receptor occupancy is considered to be measured at the time of tracer administration (t) but the SPP1 exposure is measured at the time of sacrifice (t plus 20 min). Studies were performed at Covance Alnwick or Greenfield. Tissue Preparation and Tracer Analysis: Cortex and cerebellar samples were weighed and placed in conical centrifuge tubes on ice. Four volumes (w/v) of acetonitrile containing 0.1% formic acid was added to each tube. Samples were then homogenized using an ultrasonic probe and centrifuged using a benchtop centrifuge at 14,000 RPM for 20 minutes. Supernatant was diluted by adding 50 µL to 150 µL sterile water in 96-well plates for LC/MS/MS analysis. Analysis of LSN3172176 was carried out using an API 4000 mass spectrometer. Chromatographic separation employed an Agilent Zorbax Eclipse XDB-C18 column (2.1 X 50 mm) and a gradient mobile phase consisting of 15% to 90% acetonitrile in water with an overall 0.1% formic acid content. Detection of LSN3172176 was accomplished by monitoring the precursor to product ion transition with a mass to charge ratio (m/z) of 386.3 to 128.0. Standards were prepared by adding known quantities of the tracer to brain tissue samples from non-treated rats or mice and processing as described above. Receptor Occupancy Determinations: Receptor occupancy was calculated using the ratio method. The level of tracer was measured in each cortical and cerebellar sample. A ratio of cortical levels (total binding) to cerebellar levels (nonspecific binding) was generated for each animal. Vehicle ratios represent 0% occupancy and a ratio of 1, where the binding in the


cortex is equal to the binding in the cerebellum, represents 100% occupancy. The ratios from the SPP1 pretreated groups were interpolated linearly between the ratio in the vehicle-treated animals (0% occupancy) and 1 (100% occupancy) in order to determine the percent M1 receptor occupancy. For the SPP1 dose response, a curve was fitted to a 4-parameter logistic function with the bottom and top fixed at 0% and 100%, respectively using GraphPad Prism version 6.0 and the dose achieving 50% receptor occupancy (RecOcc50) was calculated by the software. Values are given as mean ± SEM. For conversion of total plasma or brain levels to unbound levels, the % values for SPP1 free in the plasma (4.4%) and brain (10.1%) were used. The unbound brain to unbound plasma concentration ratio is the KP,uu, where Kp is the total brain to total plasma concentration ratio and uu stands for unbound brain and plasma. Native Tissue Membrane Preparation All procedures were performed at 4oC. Frozen human, rhesus monkey, rat or mouse cortices or hippocampi were homogenized in sucrose buffer (10 mM HEPES, 1 mM EGTA, 1 mM DTT, 10% sucrose and 1 tablet/50 ml Complete Protease Inhibitor Cocktail; pH 7.4) using an electric IKA RW20 homogenizer (800 rpm) with glass/Teflon homogenizer. Homogenate was centrifuged at 1000 x g for 10 min and supernatant collected, the pellet was re homogenized and centrifuged again, as above, and supernatant pooled and centrifuged at 11,000 x g for 20 min. The resulting pellet was suspended in a final storage buffer (10 mM HEPES, 1 mM EGTA, 1 mM MgCl2, 1 mM DTT; pH 7.4) and centrifuged at 27,000 x g for 20 min. Supernatant was removed and the final pellet suspended in a quantity of final storage buffer to give a suitable protein concentration. Protein concentration was measured using the Bradford method (Coomassie Plus, Bio-Rad protein assay kit) with BGG standards. Samples were then aliquoted and stored at –80 0C. GTP[35S] Binding Assays GTPγ[35S] binding in human, rat and mouse (WT and M1 KO) membranes was determined in replicates of 3 or more using an antibody capture technique in 96-well plate format (DeLapp et al., 1999). Membrane (15 µg/well for rat and mouse or 30 g/well for human) were incubated with test compound and GTPγ[35S] (500 pM/well) for 30 min (rat), 45 min (mouse) or 60 min (human). Labelled membranes were then solubilized with 0.27 % Nonidet P-40 plus Gqα antibody (E17, Santa Cruz) at a final dilution of 1:200 and 1.25 mg/well


of anti-rabbit scintillation proximity beads. Plates were left to incubate for 3 hours and then centrifuged for 10 mins at 2000 rpm. Plates were counted for 1 min/well using a Wallac MicroBeta Trilux scintillation counter (Perkin Elmer). All incubations took place at room temperature in GTP-binding assay buffer 20 mM HEPES, 100 NaCl mM, 5 mM MgCl2, pH 7.4. Data was converted to % response compared to oxotremorine M (100 M) or acetylcholine (100 M) before being transferred to GraphPad Prism software 6 to generate an EC50 (4 parameter curve fit, sigmoidal dose response, variable slope). A Furchgott analysis was performed on GTP[35S]-binding curves to evaluate the level of receptor reserve in rat and monkey hippocampus. For Furchgott analysis, binding was performed as above in the absence or presence of alkylating agent (60 nM Propylbenzyl choline mustard), as per Porter and colleagues (2002). Equally effective concentrations of oxotremorine-M (Oxo-M), SPP1 or Xanomeline in alkylated and unalkylated cortical membranes were analysed by double reciprocal plot. The resulting straight line was used to calculate a KA value using the equation: KA = (slope-1)/y-intercept. Percent occupancy at specific agonist concentrations was calculated from the KA using the equation: % occupancy = 100 . L/(L 1KA), where L is the agonist concentration. Functional M2 and M3 tissue assays Tissue studies using rat atria and ileum were run by Eurofins PanLabs, Taiwan following (Lambrecht et al., 1989). Briefly, left atria and strips of ileal longitudinal muscle (1.5 cm length) were isolated from Wistar rats and were set up under 0.5 g tension in 10 ml organ baths at 32ºC containing oxygenated (95% 02-5% CO2) McEwens (atrium) or Krebs (ileum) buffers. The tissue responses to the cumulative addition of compound were measured as changes in isometric (atrium) or isotonic (ileum) tension. A force-displacement transducer connected to a Hellige amplifier and a multichannel recorder were used for these measurements. For experiments involving atria, agonist induced negative inotropy is expressed relative to a 1 μM methacholine response and for antagonist responses inhibition of a 1 μM methacholine induced negative inotropic response is calculated. For experiments involving ileum, agonist induced contraction is expressed relative to a 1 μM methacholine response and for antagonist responses inhibition of a 1 μM methacholine induced contraction is calculated. Assay details also available on the Eurofins website (Eurofins PanLabs, 2017a, Eurofins PanLabs, 2017b).


Radioligand binding competition studies All experiments were performed in assay buffer of the following composition: 20 mM HEPES, 100 mM NaCl and 10 mM MgCl2, pH 7.5. Experiments using recombinant mAChR membranes used 10 µg of protein/well. Experiments using native tissues used 30 µg (mouse/rat) or 50 µg (monkey/human) protein/well with all membranes prepared as described for GTPγ[35S] binding above. Recombinant membranes were incubated with 0.2 nM [3H]-nmethylscopolamine

([3H]NMS).

Native

membranes

were

incubated

with

2

nM

[3H]LSN3172176 (Mogg et al., 2018). Binding was performed in the presence or absence (total binding) of 11 different concentrations of compound. Non-specific binding was determined in the presence of 10 µM atropine. All assay incubations were initiated by the addition of membrane suspensions and deep well blocks were shaken for 5 min to ensure complete mixing. Incubation was then carried out for 2 h at 21°C. Binding reactions were terminated by rapid filtration through GF/C filters pre-soaked with 0.5% w/v PEI for 1 h. Filters were then washed 3 times with ice-cold assay buffer. Dried filters were counted with Meltilex A scintillant using a Trilux 1450 scintillation counter (Perkin Elmer, Boston, MA, USA). The specific bound counts (dpm) were expressed as a percentage of the maximal binding observed in the absence of test compound (total) and non-specific binding determined in the presence of 10 µM atropine. Data analysis was accomplished using Excel and GraphPad Prism 6. The concentration–effect data were curve-fit using GraphPad Prism 6 to derive the potency (IC50) of the test compound. The equilibrium dissociation constant (Ki) of the test compound was then calculated by the Cheng–Prusoff equation: Ki=IC50/(1+([L]/Kd)) using the following Kd values M1 Kd = 196 pM; M2 Kd = 769 pM; M3 Kd = 642 pM; M4 Kd 143 pM; M5 Kd = 410 pM. Mouse Kd = 0.52 nM, rat Kd = 2.66 nM, monkey Kd = 1.33 nM, human Kd = 1.93 nM Xenopus Oocyte recordings The nucleotide sequence encoding the human M1 receptor was ligated into the pcDNA3.1 plasmid vector (Invitrogen, Carlsbad, CA). A volume of 18.2 nl of cDNA solution, with a cDNA concentration of 0.1 mg/ml, was injected into the nuclei of the oocytes using a Nanoject Automatic Oocyte Injector (Drummond, Broomall, PA). After injection, oocytes were incubated at 18°C for 4 days in a modified Barth’s solution containing 88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO3, 0.3 mM Ca(NO3)2, 0.41 mM CaCl2, 0.82 mM MgSO4, 15 mM HEPES and 5 mg/l neomycin (pH 7.6). Oocytes were placed in a 0.1 ml recording chamber and perfused with an oocyte recording solution (ORS) at a rate of 10 ml/min. ORS contained:


115 mM NaCl, 2.5 mM KCl, 1.8 mM CaCl2; MgCl2; and 10 mM HEPES. The pH of this solution was adjusted to 7.2 with NaOH. Four days after injection oocytes were placed in a recording chamber and impaled by two microelectrodes filled with 3M KCl (0.5-2.0 MΩ) and voltage-clamped at –60 mV using a Geneclamp 500B amplifier and PCLAMP 10 software (Axon Instruments, CA, U.S.A.). Traces were filtered at 100 Hz during recording and digitized at 500 Hz using the DigiData 1200 interface (Axon Instruments, CA, U.S.A.).

During the experiments oocytes were

continuously perfused at a rate of approximately 5 ml/min. Drugs were applied by switching the valves of the perfusion system (BPS8; ALA Scientific Inc., Westbury, NY). All experiments were carried out at room temperature. Experiments were performed on oocytes that had leakage currents of less than 100 nA at the holding potential of -60 mV.

Hippocampal slice electrophysiology Six weeks old, male C57Bl/6J mice (Charles River, UK) and M1KO C57Bl/6NTac male mice (Harlan, UK) were anaesthetized with isoflurane and killed by decapitation. The brain was removed and placed into ice-cold artificial CSF (aCSF) comprised of 87 mM NaCl, 75 mM sucrose, 2.5 mM KCl, 25 mM NaHCO3, 1.25 mM NaH2PO4, 7 mM MgCl2, 25 mM Dglucose, 0.5 mM CaCl2, continuously bubbled with 95% O2/5% CO2. Transverse slices (350 µm thick) were prepared from the hippocampi isolated from each cerebral hemisphere using a tissue slicer according to the manufacturer’s instructions. Slices were kept in a submerged holding chamber at room temperature for 30 min and then transferred to normal aCSF, comprised of 124 mM NaCl, 3 mM KCl, 26 mM NaHCO3, 1.25 mM NaH2PO4, 1 mM MgSO4, 10 mM glucose, 2 mM CaCl2 continuously bubbled with 95% O2/ 5% CO2. Slices were allowed to recover for at least another 30 min at room temperature before being transferred to a submerged recording chamber continuously perfused at ~3.5 ml/min with aCSF maintained at 30-32 °C by means of an inline heater (Scientifica, UK). Slices were weighed down with the aid of a slice anchor (made in house with a platinum-iridium wire). A glass recording electrode (1.5 mm OD, 0.86 mm i.d. glass with filament from Harvard, USA, filled with aCSF, and a resistance of 1-4 MΩ) and a bipolar stimulating electrode (made from Ni:Cr (80:20) formvar-coated wire (Advent, UK) were placed in the stratum radiatum within the CA1 area of the hippocampus. Responses were evoked by monophasic constant current stimulations of 0.1 ms duration at 30 seconds intervals. Data were filtered at 3 kHz and amplified 500x with an Axopatch 1D amplifier (Molecular Devices) and


digitized at 10 kHz. Averages of four successive trials were analyzed on- and off-line using WinLTP software (Anderson and Collingridge, 2007). The slope of the field excitatory postsynaptic potential (fEPSP) was used as a measure of synaptic strength. The stimulus intensity was set to a level which elicited about half of the maximal slope. After obtaining a stable baseline for at least 20 min, SPP1 was superfused onto the slices for 20 minutes at increasing concentrations (100 nM, 1 μM and 3 μM). SPP1 was prepared as a 3 mM stock solution in DMSO. Aliquots were stored at -20°C until required and diluted to their final concentration in aCSF just before use. The % change over baseline was calculated using the pooled data average of the last 5 point (10 min) for each drug concentration versus the pooled data average of the last 5 points (10 min) of the baseline. All data are expressed as mean ± SEM. Statistical significance was assessed using unpaired t test, by comparing WT and M1 KO mice using the average of the last 5 points (10 min) for each concentration applied. Data analysis was performed using Excel and graphs constructed using Excel and Prism. Cell Culture Chinese hamster ovary cells (CHO) cells expressing the human M1 muscarinic receptor were grown in DMEM/F12 (3:1) supplemented with 10% FBS, 10 mM HEPES, 2 mM Glutamax, 40 ug/ml proline, penicillin-streptomycin (PS), 250 ug/ml geneticin and seeded at 50K cells/well in PDL coated 96 well black/transparent bottom

plates for 24 h before the

experiment Ca2+ Influx Assays Receptor-mediated changes in intracellular calcium concentration were determined using a calcium-sensitive fluorescent dye, Fluo4-AM (Invitrogen) and a fluorimetric imaging plate reader (FLIPR Molecular Devices Corporation, Sunnyvale, CA, USA). Hank’s Balanced Salt Solution (HBSS) assay buffer, supplied by Invitrogen (Gibco 14025-050), was supplemented with 20 mM HEPES and adjusted to pH 7.4. At the start of the assay, growth media was removed from the 96-well cell plates by inversion and gentle tapping and media was replaced with assay buffer containing 4 M Fluo-4-AM/0.05% (v/v) pluronic F-127. Cell plates were stored in the dark at room temperature for 60 min,

to allow dye loading into cells.

Subsequently, the dye solution was removed and replaced with assay buffer at which stage cell plates were transferred to the FLIPR for assay. Intracellular calcium levels were monitored before and after the addition of compounds, with data collection ranging from 1 image every second to 1 image every 3s. Responses were measured as the maximal peak height in Relative


Fluorescent Units (RFUs), and the assay window was defined as the maximal response obtained by agonist-stimulated wells. All RFU values were corrected for basal fluorescence in the absence of agonist. pERK Assays Chinese hamster ovary cells (CHO) cells stably expressing the human M1 muscarinic receptor were generated in house using the FLPin expression system. Cells were cultured and plated as for Ca2+ experiments described above. Cells were serum starved overnight prior to the experiment. Phospho-ERK1/2 (Thr202/Try204) levels after agonist stimulation (10 minutes at 37°C) were measured using a commercially available HTRF kit from CisBio (France) following the manufacturer’s instructions. Computational methods SPP1 was initially docked using the default Schrodinger induced fit procedure in Maestro 2012.1 into a homology model of the hM1 receptor built using the inactive state hM2 + hM3 x-ray structures (3UON + 4DAJ) as templates. The pose was further refined by minimizing the ligand coordinates into a homology model of hM1 built using the active state M2 structure 4MQS as template, subsequent to the release of these structures. Minimization and homology modelling were done with the Amber10:EHT force field in MOE 2013.


Data and Statistical Analysis The data and statistical analyses comply with the recommendations on experimental design and analysis in pharmacology (Curtis et al, 2015). For receptor occupancy experiments animals were randomly distributed between treatment groups and experimenters blinded for analysis purposes. Unless otherwise stated, values reported are means ± SEM. Comparisons between groups were made using ANOVA with Bonferroni’s or Dunnett’s post hoc tests, as appropriate, using GraphPad Prism software version 6.0 (GraphPad Software Inc., San Diego, CA, USA). Statistical significance was taken as P < 0.05. For calculation of Ki values for the native atrial and illeal assays the method of Cheng and Prusoff (1973) was used. EC50 values of methacholine were from Clague et al, 1985. For hippocampal slice electrophysiology experiments the following test was performed. Per animal the averages of the measurements at the applicable time points for baseline and at each of the treatments were determined. Then, per animal, the ratio of each of the three treatments’ values with the animals applicable baseline value were calculated as well as the ratio’s log10 transform. These normalized and log-transformed measurements of the animals across treatments were used to build a repeated measurements model (using SAS® proc mixed with unstructured covariance) with genotype (WT and KO) and treatment (100nM, 1 M and 3 M) as categorical factors, including a term for interaction. Type 3 tests for significance of the fixed effects were carried out as well as least-squares means estimation for each genotype. Nomenclature of Targets and Ligands Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Harding et al., 2018), and are permanently archived in the Concise Guide to PHARMACOLOGY 2017/18 (Alexander et al., 2017).


Results Use of in vivo receptor occupancy to drive an M1 agonist Structure Activity Relationship Assessment of central M1 receptor target engagement (receptor occupancy) was performed by monitoring in vivo block of an M1 tracer molecule, LSN3172176 (Mogg et al., 2018) administered 30 minutes after oral administration of SAR molecules. This time-point was selected as a suitable time-point from preliminary investigations. Initially molecules were screened at a single dose of 10 mg/kg in rats. Molecules displaying good occupancy levels, favorable unbound brain to unbound plasma concentration ratio (KP,uu) and an appropriate in vitro functional selectivity and efficacy profile at native rat M1-M3 receptors were subsequently screened to generate full dose-occupancy curves. Compounds were screened at doses ranging from 0.03-30 mg/kg and brain and plasma collected to allow tracking of the brain unbound concentrations required to achieve RecOcc50 and KP,uu, the goal being to drive down brain concentrations required to achieve RecOcc50 and drive up KP,uu towards highly brain penetrant molecules (Figure 1). The in vitro activity of SPP1, a representative molecule from the spiropiperidine series, was assessed in GTPγ[35S] binding studies using membranes prepared from hippocampus or cortex (Figure 2). In mouse hippocampal membranes SPP1 stimulated Gq mediated GTPγ[35S] signaling with an EC50 = 12.3 ± 2.1 nM and Emax = 44 ± 1.9 % (n=3), relative to 100 M OxoM. This potent, partial agonist activity (relative to the full agonist Oxo-M) was confirmed to be exclusively mediated by M1 receptor activation, as signals were absent in hippocampal membranes prepared from M1 KO mice (Figure 2A). In membranes prepared from rat cortex, SPP1 stimulated Gq mediated signaling with similar EC50 = 21.0 ± 5.1 nM and Emax = 31 ± 1.3% (n=4) values (data not shown). In studies using membranes prepared from human post mortem control or AD (Braak Stage 5/6) frontal cortex tissue the pharmacological profile of SPP1 was also conserved, with EC50 values of 10.4 ± 3.4 nM and 8.1 ± 2.5 nM and Emax values of 36 ± 7.6% and 25 ± 4.1%, respectively (n=3-5), Figure 2B. To bench mark the in vitro potency and efficacy profile of SPP1 and other SAR molecules to reference standards and ligands that have previously been, or are currently in clinical development, additional studies in native rat and human cortical tissue preparations were performed (Figure 3). The data reveals close alignment of potency and efficacy values across species. Of note, three orthosteric reference agonists which have advanced to the clinic,


Xanomeline, AZD-6088 and HTL-9936, similar to SPP1, displayed partial agonist profiles at native rat and human receptors in cortical membranes. The potency and partial efficacy profile of Xanomeline and AZD-6088 was comparable to SPP1 (EC50 9-37 nM, Emax 14-29%), while HTL-9936 was significantly less potent and more efficacious (EC50 2-2.7 M, Emax 45-77%) (Figure 3; Felder et al., 2018). Next the level of Gq/11-coupled M1 receptor reserve in the hippocampus was measured in vitro by GTPγ[35S] binding after receptor alkylation with propylbenzylcholine mustard (PrBCM) (Porter et al., 2002). The concentration of PrBCM which resulted in a 50% decrease in the efficacy of Oxo-M, SPP1 and Xanomeline was used to calculate the percentage of receptor occupancy at specific agonist concentrations. A maximal Oxo-M stimulated Gq/11 response was achieved at ~25% M1 receptor occupancy in rat and monkey hippocampal membranes, indicating a substantial level of spare receptors. Receptor reserve was estimated to be ~75% for Oxo-M (Figure 4;Supplementary Figure 2). Similar experiments performed to assess receptor reserve in rat hippocampus for SPP1 and Xanomeline, revealed that both agonists displayed a receptor reserve of ~40%, with maximal efficacy achieved at ~60% receptor occupancy (Figure 4;Supplementary Figure 2). The functional selectivity of SPP1 was assessed in vitro in relevant native tissue assays. In rat atrial (M2) and rat ileum (M3) functional assays, SPP1 was devoid of agonistic effects up to the highest concentration tested (Figure 5), but did block a subsequent challenge to a nonselective mAChR agonist, carbachol, with IC50 (Ki) values of 5.37 M (1.74 M) and 26.3 M (9.89 M) , respectively (Supplementary Figure 3). Potency and efficacy of clinically advanced mAChR agonists, Xanomeline, HTL-9936 and AZD-6088 was also assessed in these atrial and ileum tissue assays and has been reported (Felder et al., 2018). SPP1 also possessed high selectivity versus a panel of 29 other general cross-reactivity targets when assessed at 10 M (Supplementary table 1). The affinity and selectivity profile of SPP1 was further assessed in radioligand competition assays by assessing displacement of [3H]NMS by SPP1 from human recombinant M1-M5 receptors. In addition, we employed a tritiated version of a novel M1 selective ligand [3H]LSN3172176 (Mogg et al, 2018) to assess displacement from native M1 receptors from mouse, rat, monkey and human cortex (Table 1, Supplementary Figure 1). SPP1 displayed high binding affinity for human M1 receptors (Ki values of 21.3 ± 1.2 nM and 14.5 ± 2.5 nM on human recombinant and native M1 receptors, respectively), which was conserved across species (Ki values of 2.88 ± 0.36 nM, 8.67 ± 0.41 nM and 10.7 ± 1.3 nM on mouse, rat and This article is protected by copyright. All rights reserved.

monkey respectively, Table 1, Supplementary Figure 1). SPP1 displayed modest selectivity for binding to human M1 mAChRs over human M4>M2>M5>>M3 mAChR subtypes, as did AZD-6088. In contrast, HTL-9936 and Xanomeline did not show preferential binding to M1 mAChRs (Table 2, Supplementary Figure 1, Mogg et al., 2018). Recruitment of downstream signaling pathways was assessed in heterologous expression systems, in either CHO cells (Table 3) or Xenopus Oocytes (Figure 6) expressing recombinant human M1 Rs. In CHO-hM1 cells concentration response curves evoked by SPP1, AZD-6088, HTL-9936 or Xanomeline were assessed by measuring intracellular calcium responses using FLIPR or pERK signaling using ELISA (Table 3). All agonists were highly efficacious (>70% efficacy) in evoking calcium responses with a maximal efficacy comparable to ACh. In contrast, in the same cells the agonists showed significantly different maximal efficacies for pERK signaling, with AZD-6088 being the least efficacious agonist (11% compared to a maximal response to ACh) and Xanomeline demonstrating the highest efficacy at 55% (Table 3). In oocytes heterologously expressing M1 receptors, application of 10 M Oxo-M evoked an inward current (Figure 6A) mediated by endogenous calcium-dependent chloride channels activated by calcium release from intracellular stores on M1 receptor activation. The inward current shown in Figure 6A is the chloride current flowing through the activated calcium-dependent chloride channels which occurred a few seconds after Oxo-M was applied. Application of 10 M Xanomeline or SPP1 also resulted in the activation of calciumdependent chloride currents in oocytes (Figure 6B, but the amplitude of these were significantly smaller than those induced by Oxo-M (0.129 ± 0.043 A (n=8); 1.12 ± 0.32 A (n=7), and 5.25 ± 1.09 A (n=9) for SPP1, Xanomeline, and Oxo-M, respectively. This is consistent with SPP1 and Xanomeline being partial agonists of muscarinic M1 receptors as compared to OxoM in this expression system. Control experiments were performed in order to exclude a possible contribution of endogenous muscarinic receptors in the oocytes used for these experiments. Both the application of 10 M Oxo-M or 10 M SPP1 to un-injected oocytes from the same batch as those used for the M1 cDNA injected oocytes did not evoke a chloride current at all (not shown). SPP1 was then studied in in vitro slice electrophysiology experiments to assess effects on glutamatergic synaptic transmission in the CA1 region of the hippocampus (Figure 7). In slices from WT C57Bl/6J mice, a concentration dependent potentiation of synaptic transmission was observed (17%, 32% and 30% over baseline at 100 nM, 1 μM, and 3 μM, respectively; Figure 7A). In identical experiments performed in slices from M1KO This article is protected by copyright. All rights reserved.

C57Bl/6NTac mice, the effect of SPP1 on synaptic transmission was greatly reduced at all of the concentrations tested (Figure 7B). Therefore, consistent with previous findings for M1 allosteric agonist GSK-5 (Dennis et al., 2016), SPP1 potentiates glutamatergic synaptic transmission via interaction with M1 receptors. Moving to in vivo studies, assessment of central M1 receptor target engagement was performed by monitoring in vivo block of an M1 selective receptor occupancy ligand (Mogg et al., 2018) at various doses or time-points after oral administration of SPP1 (Figure 8). A doseoccupancy curve was generated in rats 30 minutes after administration of single doses of SPP1, ranging between 0.1-10 mg/kg, generating a RecOcc50 value of 1.1 mg/kg (95% CI 0.93-1.27), n=4 (Figure 8A). Conversion of SPP1 dose to brain unbound concentrations of SPP1 ([Br]u) led to an RecOcc50 value of 24.3 nM (Figure 8B). In a time course study, where the level of M1 receptor occupancy for SPP1 was measured in rats following a single oral dose of 3 mg/kg at 30, 60, 120, 240, 480, and 1440 minutes after oral administration, a time-dependent decrease in occupancy was observed. Occupancy fell from 64 ± 8% at 30 minutes to 2 ± 8% at 1440 minutes, n=4 (Figure 8C). The relationship between M1 receptor occupancy and unbound plasma, unbound brain and CSF exposures was also assessed (Figure 8C). In these experiments the KP,uu value for SPP1 of 0.62 was significantly higher than observed in the 10mg/kg screening assay (KP,uu ~0.2, Figure 1). The ratio for CSF to unbound plasma levels of SPP1 (0.83) also displayed favorable distribution to brain. Comparison of the potency of a large set of SAR molecules and reference ligands in the rat cortical receptor occupancy assay (RecOcc50 values, [Br]u, nM) and rat cortical membrane GTPS assay (EC50 values, nM) revealed close alignment between the in vivo and in vitro assays (Figure 9). In a system with substantial receptor reserve one may expect a non-linear relationship. However, the tracer used to assess RecOcc50 is itself an agonist and thus only binds to a subset of the total pool of available M1 receptors. Previously, we demonstrated that the Bmax for [3H]NMS binding measured in the same tissues/membranes was significantly higher than for LSN3172176, the tracer molecule used for the receptor occupancy studies (Mogg et al., 2018). Finally we sought to investigate the binding of SPP1 by docking it into the orthosteric site of an active state model of human M1 receptor (Figure 10). SPP1 could successfully be docked into the model and interactions with key residues are proposed to help gain an understanding as to how SPP1 is able to achieve functional selectivity despite binding to the


highly conserved orthosteric site. The only residue different in the orthosteric site (defined as being within 4.5Å of the modelled SPP1 pose) across the hM1-M4 receptors is Leu183 in hM1, which is homologous to a phenylalanine in hM2. The phenyl ring of the SPP scaffold is modelled to bind in the region around Leu183 and would clash with Phe181 in hM2, therefore an initial design hypothesis was that steric bulk in this region would maintain or enhance selectivity versus M2. The selectivity observed versus M3 is harder to explain, but we note that this residue sits in the extracellular loop between helix 4 and 5 where neighbouring residues are not conserved between muscarinic subtypes, and it is hypothesized that this region plays a role in the coupling between the orthosteric M1 site and receptor activation (Kruse et al., 2013, Abdul-Ridha et al., 2014).

Discussion We herein report the discovery of a novel highly functionally selective M1 partial agonist that is amenable to in vivo study. SPP1 potently stimulates native M1-mediated Gq signaling in regions relevant for cognition, displaying partial efficacy compared to the endogenous agonist, ACh. This profile is conserved across species (mouse, rat, human) and in post-mortem Alzheimer’s disease brain samples. SPP1 has high functional selectivity versus M2 (>100-fold) and M3 (>100-fold) anti-targets when assayed in relevant rat tissue assays. SPP1 displays high affinity for human M1 receptors, with no significant difference in binding affinity for M1 receptors across species and selectivity over a panel of cross-reactivity targets. SPP1 displays partial agonism at native and recombinant receptors in vitro and evokes changes in electrophysiological parameters in CA1 hippocampal neurons which are absent in tissue from M1 knock-out mice. In vivo SPP1 displays dose-dependent occupancy of M1 receptors in rat cortex when dosed orally. Use of an in vivo central M1 target engagement assay to drive the chemistry SAR proved to be an effective way to optimize desirable parameters. Traditional approaches would have relied more heavily on filtering first on in vitro parameters and subsequently collecting exposure and finally assessing efficacy of the most promising SAR molecules in a battery of in vivo behavioral assays. Our approach decreases reliance on behavioural assays, the reliability and translatability of which have been questioned. Receptor occupancy assays may also be less time consuming, requiring fewer animal numbers to generate highly reproducible results, aiding efficient drug discovery. The close correlation between the in vivo brain unbound


exposures achieved at the RecOcc50 and in vitro EC50 values in the rat cortex GTPγ[35S]-Gq assay for a range of M1 agonists, as well as the overlapping pharmacology observed at rat and human M1 receptors gives confidence in the robustness of the data and highlights the translational value of the assays. Bench-marking of SPP1 to M1 molecules that have advanced to the clinic within the same in vitro and in vivo assays allowed for relative comparisons to be drawn. Xanomeline displayed potent partial M1 agonist properties, similar to SPP1 in the GTPS] assay. Xanomeline also displayed high brain penetration, but displayed little functional selectivity for M1 over M3 receptors, and clinically was poorly tolerated at the efficacy dose in AD patients (Bodick et al., 1997). Selectivity over M2/M3 mAChRs has been a key differentiator for next generation drug discovery efforts and AZD-6088, similar to SPP1, achieves the desired high functional selectivity for M1 in spite of binding to the highly conserved orthosteric site (Felder et al., 2018). AZD-6088 however is a strong PGP substrate, displaying relatively low brain penetration and clinically AZD-6088 was terminated due to peripheral adverse-events (Felder et al., 2018). Hence minimizing peripheral load and optimization of central penetration appears to be an important goal even for highly selective M1 agonists. SPP1 displayed a KP,uu of between 0.2-0.8 (including CSF partitioning), which is a significant improvement over AZD6088 (KP,uu 0.13). Ideally KP,uu values should be ~1 and a number of molecules with values in this range were identified in the course of SAR screening. Restricting agonist efficacy (Emax) may also be an important feature for minimizing unwanted M1 mediated adverse effects, such as salivation or sweating, and was a key goal for the current work. Compared to Xanomeline, AZD-6088 and SPP1, HTL-9936 is a less potent but more efficacious M1 agonist, displaying relatively low brain penetration (Felder et al., 2018). Clinically, HTL-9936 was reported to evoke statistically significant changes in brain electrical activity measured using multiple electroencephalography biomarkers relevant to cognition (Felder et al., 2018, Heptares Therapeutics Website, 2016). The impact of the host system on the functional response profile of GPCR agonists is well documented (Kenakin, 2014a), hence our emphasis on assessing functional pharmacology in relevant native tissues for M1 (e.g. cortex, hippocampus), and M2/3 anti-targets (e.g. atrium, ileum). A general experimental observation is that cellular responses do not require 100% receptor occupancy to produce a maximal response, leading to a phenomenon coined receptor reserve (spare receptors not required for production of the maximal response). Previously,


~85% receptor reserve was reported for Oxo-M in mouse hippocampus, with activation of ~15% of the receptors generating a maximal response (Porter et al., 2002). Our data with OxoM in rat and monkey hippocampus closely replicates the findings of Porter and colleagues, confirming high receptor reserve in this brain region across species. Receptor reserve is a property of the tissue, the assay and the agonist (Kenakin, 2014a) and as expected, the partial agonists SPP1 and Xanomeline required higher fractional occupancy to achieve a maximal response compared to Oxo-M. However these agonists still displayed significant receptor reserve (~40%). This data supports the hypothesis that in vivo, pharmacodynamic responses for SPP1 might be observed at low M1 occupancy. Understanding the occupancy required to achieve an in vitro pharmacodynamic response in a relevant tissue is useful, however understanding the overall efficacy/receptor occupancy relationship at a systems level is the goal and this is important to define in order to set an appropriate clinical target efficacy dose range and to avoid over dosing. This is tricky as the translational value of building an efficacy/receptor occupancy relationship using pre-clinical behavioural assays has its limitations. Use of translational pharmacodynamic biomarkers relevant for cognition might be another option for further refining dose selection using a PET based strategy (Felder et al., 2018) and aligned to this, LSN3172176 has the potential to be used as a PET tracer in man (Jesudason et al., 2017, Nabulsi et al., 2017). Our data showing that the pharmacological profile of SPP1 is conserved in Alzheimer’s Disease cortical tissue is consistent with other data indicating that post-synaptic M1 receptors are still present and function is retained (Overk et al., 2010, Bradley et al., 2017, Nathan et al., 2013). This data confirms therefore that targeting these receptors in AD is a valid approach to boosting cholinergic signalling. Activation of Gq (assessed in the functional GTPS binding assays) is one of the most proximal events to M1 receptor activation. M1 receptors can also signal via non-Gq dependent means, such as through -arrestin recruitment, and recruit diverse downstream signaling cascades, demonstrating significant signal amplification and in some cases agonist dependentsignaling bias, whereby certain cellular pathways are activated to a greater extent than others (Kenakin, 2014b). Our data revealed considerable variability in the potency and efficacy profile of Xanomeline, SPP1, HTL-9936 and AZD-6088 across the various in vitro cellular assays studied. The is likely contributed to by cell and assay specific factors (e.g. receptor reserve, effector coupling, proximity to M1 receptor activation), but a degree of signaling bias is


suggested as the agonists do not retain the same rank order of potency or maximal efficacy across assays. For example, Xanomeline and SPP1 displayed similar efficacy in Gq-GTPγS functional binding, but markedly different efficacies in pERK signaling and oocyte studies. These studies highlight the complexity of GPCR and M1 receptor signaling and our lack of understanding of which signaling profile is desirable for maximizing efficacy and minimizing adverse effects. A role for -arrestin recruitment in M1 PAM mediated cognitive benefits has been suggested by Ma and colleagues (2009) and in some elegant studies of the M3 mAChR using genetically engineered mice to map physiological pathways, a compelling case has been put forward for pursuit of next generation GPCR therapies based on understanding and exploiting this complexity (Bradley et al., 2016, Bradley et al., 2014). Confirmation that SPP1 could alter hippocampal neuronal excitability, and that the effects are largely M1 receptor dependent, is a relevant finding for a potential therapeutic designed to alter hippocampal function in vivo. M1 receptors are abundant in human hippocampus (Levey et al., 1995) and physiologically activation of these receptors leads to the regulation of a range of ion channels that are co-expressed in the same neurons, including NMDA receptors (Markram and Segal, 1990, Marino et al., 1998, Calabresi et al., 1998, Zwart et al., 2017) and potassium channels (Buchanan et al., 2010, Shapiro et al., 2000, Zwart et al., 2016, Giessel and Sabatini, 2010). Currents through NMDA receptors are enhanced upon activation of M1 receptors, whereas currents through potassium channels are reduced. Both effects result in an increase in excitability of the post-synaptic neuron. For many years it was hypothesized that selectivity could not be achieved with molecules that bind to the orthosteric site, due to the high sequence identity between M1-5 receptors at this site. However, the new highly functionally selective M1 agonists reported by us and others suggests that functional selectivity is achievable, even if binding selectivity for these molecules is more modest. We speculate this may stem from the larger size of these next generation molecules compared to ACh and several first generation non-selective agonists, as well as the orientation in which they occupy the orthosteric site and affect allosteric receptor activation. In summary, we report the discovery of SPP1, a novel, functionally selective, brain penetrant partial M1 orthosteric agonist identified through the use of a novel receptor occupancy assay. SPP1 is amenable to both in vitro and in vivo study and should provide a


valuable research tool to further probe the role of the M1 receptor in physiology and pathophysiology.


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Authorship Contributions Participated in research design: LMB, PJG, HES, AJM, ES, VB, CC, GNW Conducted experiments: HES, AJM, EMC, DAE, FP, RZ Contributed new reagents or analytic tools: PJG Performed data analysis: PJG, HES, AJM, EMC, DE, FP, RZ, GNW Wrote or contributed to the writing of the manuscript: LMB, AJM, HES, DE, RZ, PJG

Conflicts of interest The authors declare no conflicts of interest. Eli Lilly does not sell any of the compounds or devices mentioned in this article.


Table 1: Comparison of SPP1 binding affinity to native mouse, rat, monkey or human M1 Receptors. Competition radioligand binding experiments measuring displacement of [3H]LSN3172176 were used to assess affinity (Ki) of SPP1 in cortical membranes from mouse, rat, monkey and human. Values represent the mean ± SEM of data from 3 individual experiments each containing 4 replicates for each condition Mouse Rat Monkey Human Mean Ki ± SEM Mean Ki ± SEM Mean Ki ± SEM Mean Ki ± SEM SPP1 2.88 ± 0.36 nM 8.67 ± 0.41 nM 10.7 ± 1.3 nM 14.5 ± 2.5 nM Table 2 Comparison of compound affinity at human recombinant M1-M5 mAChR subtypes. Competition radioligand binding experiments measuring displacement of [3H]NMS were used to assess affinity (Ki) of compounds in purified membranes from recombinant cells expressing M1-M5 mAChR receptor subtypes. Values represent the mean ± SEM of data from 3 individual experiments each with 4 replicates for each condition.

SPP1 HTL9936 AZD6088

M1 Mean Ki ± SEM

M2 Mean Ki ± SEM

M3 Mean Ki ± SEM

M4 Mean Ki ± SEM

M5 Mean Ki ± SEM

(Selectivity vs M1)

(Selectivity vs M1)

(Selectivity vs M1)

(Selectivity vs M1)

(Selectivity vs M1)

21.3 ± 1.2 nM

128 ± 9.0 nM (6-fold) 6502 ± 297 nM (0.7-fold) 125 ± 8.3 nM (2-fold)

7855 ± 1094 nM (370-fold) 150300 ± 31979 nM (16.5-fold) 11871 ± 4197 nM (189-fold)

115 ± 0.7 nM 5.4-fold) 3804 ± 319 nM (0.4-fold) 133 ± 48 nM (2.1-fold)

195 ± 12.6 nM (9.2-fold) 55490 ± 9391 nM (6.1-fold) 602 ± 75.4 nM (9.6 fold)

9093 ± 887 nM 62.7 ± 19.4 nM

Table 3 Comparison of downstream signaling in a recombinant hM1 receptor expressing cell line. Values represent the mean ± SD of data from ≥2 individual experiments.

SPP1

HTL-9936

AZD-6088 Xanomeline

hM1 Ca2+ mobilization (FLIPR)

hM1 pERK

Mean EC50 (Efficacy, % ACh Max)

Mean EC50 (Efficacy, % ACh Max)

175 ± 62.7 nM (75 ± 4%) n=2 56.2 ± 133 nM (111 ± 6%) n=4 12.2 ± 13.5 nM (92 ± 14%) n=4 96.6 ± 54.4 nM (112 ± 11%) n=4

16.9 ± 1.1 nM (20 ± 6%) n=2 1161 ± 115.3 nM (49 ± 9%) n=2 118.3 ± 60.3 nM (11 ± 5%) n=2 17.8 ± 2.1 nM (55 ± 9%) n=2


Figure 1 – Driving a structure-activity relationship using M1 receptor occupancy and unbound brain to unbound plasma concentration ratios. Compounds were screened at doses ranging from 0.03-30mg/kg to generate 6-point dose response curves. Brain and plasma was collected from each animal at each dose level to allow calculation of unbound brain and unbound plasma concentrations. Receptor occupancy was then plotted against unbound drug concentration to generate the unbound plasma and brain concentrations generating 50% occupancy (RecOcc50). This allowed tracking of the EC50 nM brain unbound required to achieve RecOcc50 compared to the unbound plasma concentrations and KP,uu, the goal being to drive down brain EC50 and drive KP,uu up towards 1 (highly brain penetrant).


Figure 2 – (A) M1 mAChR function was assessed in mouse hippocampal membranes from WT or M1-/- mice using GTPγ[35S] Gq antibody capture binding methodology. Responses were normalised to a maximally effective concentration of oxotremorine M (100 M). Points represent mean ± SEM from 3 individual experiments each containing 4 replicate conditions. (B) M1 mAChR function was assessed in native human post mortem frontal cortex from control or AD patients (Braak stage 5/6), with separate patient samples used in each individual experiment, using GTPγ[35S] Gq antibody capture binding methodology. Responses were normalised to a maximally effective concentration of acetylcholine (100 M). Points represent mean ± SEM from 3 individual experiments each containing 4 replicate conditions.



Figure 3 - Correlation plots for potency and efficacy values at native rat and human cortical M1 receptors for SAR molecules and reference standards. Native M1 mAChR function was assessed in rat and human cortex using GTPγ[35S] Gq antibody capture binding methodology as represented in Figure 2. Responses were normalised to a maximally effective concentration of oxotremorine M (100 M). Potency and efficacy values were correlated between the two species using Pearson’s correlation coefficient in GraphPad Prism 6 software.


Figure 4 - Receptor reserve of agonists was assessed using Furchgott analysis in rat or monkey hippocampus using GTPγ[35S] Gq antibody capture binding methodology. A dose response curve of oxotremorine-M (n=2 monkey and n=3 rat), SPP1 (n=3) or xanomeline (n=2) was assessed in the presence and absence of an alkylating agent (propylbenzyl choline mustard) at a concentration which irreversibly inhibited 50% of the response (please refer to Supplementary Figure 2 for raw data). Data from replicate experiments were pooled and from these curves Furchgott plots were generated.


Figure 5 - Functional native rat tissue selectivity data. Rat atrium and ileum tissue response assays measured as changes in isometric (atrium) or isotonic (ileum) tension. For experiments involving atria, agonist-induced negative inotropy is expressed relative to a 1 μM methacholine response. For experiments involving ileum, agonist induced contraction is expressed relative to a 1 μM methacholine response. Data points represent mean ± SEM, (n=4; SPP1, n=2; Xanomeline, each with 2 replicates).


Figure 6 - Effects of maximally-effective concentrations of Oxo-M, Xanomeline and SPP1 on Xenopus oocytes expressing recombinant human M1 receptors. A) 10 μM of the full agonist Oxo-M evokes large inward currents, 10 μM Xanomeline evokes much smaller inward currents, demonstrating that Xanomeline is a partial agonist at M1 receptors. 10 M SPP1 also evokes inward currents, but these are much smaller than Oxo-M or Xanomeline-induced inward currents. B) Bar graph showing significant (****; p