Data from studies using muscarinic acetylcholine receptor (mAChR) subtype 5 knock-out (M5-KO) mice suggest that M5 is the sole mediator of ACh-induced vasodilation in the cerebral vasculature, and thereby, may have therapeutic relevance for cerebrovascular diseases or acute ischemic stroke. M5-KO mice have also been found to exhibit deficits in long-term potentiation (LTP) at the hippocampal mossy fiber-CA3 synapse and show deficits in hippocampal-dependent behavioral cognitive tests. In light of these and other related findings, activation of M5 has been suggested as a potential target for treatment of Alzheimer's disease, perhaps in combination with M1 activation. However, due to the scarcity of selective compounds, a detailed understanding of the precise neurobiological roles of each subtype in various central nervous system (CNS) disorders has thus remained challenging. Recently, a number of novel highly subtype-selective allosteric ligands for M1 and M4 have emerged from functional cell-based screening efforts. However, no ligands have been reported to date as being highly M5-preferring or selective. Relative to the other mAChRs, little is known about M5, which is expressed at very low levels in the CNS and peripheral tissues. The currently identified probe ML129 (CID-42633508) possesses unprecedented M5 selectivity versus M1, M2, M3 and M4, as well as selectivity against a large panel of GPCRs, ion channels and transporters. The probe is not centrally penetrant, and so would need to be administered i.c.v. to study the role of central M5 activation in vivo. The probe also displays reasonable solubility in acceptable vehicles. Thus, ML129 can be used for in vitro molecular pharmacology and electrophysiology experiments to study, for the first time, the role of selective M5 receptor activation.
Assigned Assay Grant #: MH077607-01
Screening Center Name & PI: Vanderbilt Screening Center for GPCRs, Ion Channels and Transporters, C. David Weaver
Chemistry Center Name & PI: Vanderbilt Specialized Chemistry Center for Accelerated Probe Development, Craig W. Lindsley
Assay Submitter & Institution: P. Jeffrey Conn, Vanderbilt University
PubChem Summary Bioassay Identifier (AID): AID-2416
Probe Structure & Characteristics
1-(4-methoxybenzyl)-5-(trifluoromethoxy)indoline-2,3-dione MW = 351.2, logP = 3.6, tPSA = 55.8
Recommendations for the scientific use of this probe
This probe (CID 42633508) can be used for in vitro molecular pharmacology and electrophysiology experiments to study, for the first time, the role of selective M5 receptor activation. This probe possesses unprecedented selectivity versus M1, M2, M3 and M4, as well as a large panel of GPCRs, ion channels and transporters. CID 42633508 is not centrally penetrant, and so would need to be administered i.c.v. to study the role of central M5 activation in vivo. CID 42633508 also displays reasonable solubility in acceptable vehicles (>5 mg/mL) in 20% β-cyclodextrin and >100 μM in DMSO.
To identify small molecule positive allosteric modulators (PAMs) and/or allosteric agonists of the M1 muscarinic acetylcholine receptor that are cell permeable, possess submicromolar potency and show greater than 10-fold selectivity over the other mAChRs (M1, M2, M3 and M4) employing a functional HTS approach. Out of this effort aimed at M1, which afforded a highly selective M1 antagonist (CID 24768606) and a highly selective M1 allosteric agonist (CID 25010775), we also identified and optimized the first M5 ligand, an M5 PAM described herein. Another MLSCN screening effort identified a highly selective M4 PAM (CID 864492); thus, a toolkit containing highly selective mAChR ligands are available from the MLPCN to study individual mAChR function.
The five cloned muscarinic acetylcholine receptor subtypes (mAChR1-5 or M1-5) are known to play highly important and diverse roles in many basic physiological processes. (1–3) Correspondingly, muscarinic agonists and antagonists targeting one or more subtypes have been used preclinically and clinically for research and treatment of a wide range of pathologies. (3,4) Based on the high sequence homology of the mAChRs across subtypes, and particularly within the orthosteric acetylcholine (ACh) binding site, discovery of truly subtype-selective compounds has proven historically difficult. Due to the scarcity of selective compounds, a detailed understanding of the precise roles of each subtype in neurobiology and in various central nervous system (CNS) disorders has thus remained challenging (3,4). Recently, a number of novel highly subtype-selective allosteric ligands for M1 and M4 have emerged from functional cell-based screening efforts (5,6). However, no ligands have been reported to date as being highly M5-preferring or selective. Relative to the other mAChRs, little is known about M5, which is expressed at very low levels in the CNS and peripheral tissues (2–4).
Data from studies using mAChR5 knock-out (M5-KO) mice suggest that M5 is the sole mediator of ACh-induced vasodilation in the cerebral vasculature and thereby may have therapeutic relevance for cerebrovascular diseases or acute ischemic stroke (7,8). M5-KO mice have also been found to exhibit deficits in long-term potentiation (LTP) at the hippocampal mossy fiber-CA3 synapse and show deficits in hippocampal-dependent behavioral cognitive tests (8). In light of these and related findings, activation of M5 has been suggested as a potential target for treatment of Alzheimer’s disease, perhaps in combination with M1 activation (9). Consistent with the putative post-synaptic localization of M5 in the ventral tegmental area (VTA), other M5-KO data suggest this subtype plays an important role in regulation of mesolimbic dopamine transmission (3,9). Indeed, M5-KO mice exhibit decreased reward responses to morphine, decreased self-administration of cocaine, and less pronounced drug withdrawal symptoms, suggesting that M5 antagonists or negative modulators may have therapeutic value in treatment of illicit drug addiction (9–11). Further pharmacological exploration of these and related hypotheses greatly depends on the discovery of novel M5-preferring or selective small molecule tools.
Screening Center Information: Assay Implementation and Screening
PubChem Bioassay Name
Discovery of Novel Allosteric Modulators of the M1 Muscarinic Receptor: Agonist
List of PubChem bioassay identifiers generated for this screening project (AIDs)
PubChem Primary Assay Description
Chinese hamster ovary (CHO K1) cells stably expressing rat (r)M1 were purchased from the American Type Culture Collection (ATCC, Manassas, VA) and cultured according to their recommendations. CHO cells stably expressing human (h) M2, hM3, and hM5 were generously provided by A. Levey (Emory University, Atlanta, GA); rM4 cDNA provided by T. I. Bonner (National Institutes of Health, Bethesda, MD) was used to stably transfect CHO-K1 cells purchased from the ATCC using Lipofectamine 2000. To make stable hM2 and rM4 cell lines for use in calcium mobilization assays, cell lines were cotransfected with a chimeric G protein (Gqi5) using Lipofectamine 2000. hM2, hM3, and hM5 cells were grown in Ham’s F-12 medium containing 10% heat-inactivated fetal bovine serum, 2 mM GlutaMax I, 20 mM HEPES, and 50 μg/mL G418 sulfate. hM2-Gqi5 cells were grown in the same medium supplemented with 500 μg/mL hygromycin B. Stable rM4 cells were grown in Dulbecco’s modified Eagle’s medium containing 10% heat-inactivated fetal bovine serum, 2 mM GlutaMax I, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 20 mM HEPES, and 400 μg/mL G418 sulfate; rM4-Gqi5 cells were grown in the same medium supplemented with 500 μg/mL hygromycin B. CHO cells stably expressing rM1, hM3, or hM5 were plated at a seeding density of 50,000 cells/100 μL/well. CHO cells stably coexpressing hM2/Gqi5 and ratM4/Gqi5 were plated at a seeding density of 60,000 cells/100 μL/well. For calcium mobilization, cells were incubated in antibiotic-free medium overnight at 37°C/5% CO2 and assayed the next day.
Calcium Mobilization Assay
Cells were loaded with calcium indicator dye [2 μM Fluo-4 acetoxymethyl ester (50 μL/well) prepared as a stock in DMSO and mixed in a 1:1 ratio with 10% Pluronic acid F-127 in assay buffer (1xHanks’ balanced salt solution supplemented with 20 mM HEPES and 2.5 mM probenecid, pH 7.4)] for 45 min at 37 °C. Dye was removed and replaced with the appropriate volume of assay buffer. All compounds were serially diluted in assay buffer for a final 2x stock in 0.6% DMSO. This stock was then added to the assay plate for a final DMSO concentration of 0.3%. Acetylcholine (EC20 concentration or full dose-response curve) was prepared at a 10x stock solution in assay buffer before addition to assay plates. Calcium mobilization was measured at 25 °C using a FLEXstation II (Molecular Devices, Sunnyvale, CA). Cells were preincubated with test compound (or vehicle) for 1.5 min before the addition of the agonist, acetylcholine. Cells were then stimulated for 50 s with a submaximal concentration (EC20) or a full dose-response curve of acetylcholine. The signal amplitude was first normalized to baseline and then as a percentage of the maximal response to acetylcholine.
Summary of Screen
This screen was performed in the pilot phase, the MLSCN, when the MLSMR compound collection at Vanderbilt only contained 65K compounds. From the primary M1 screen of 65K compounds, ~12 putative M1 PAMs were identified with an average Z’ score of 0.70±0.09. The confirmation screen (singles at 10 μM) produced two lead compounds, one of which is still in chemical lead optimization. The other, CID 3008304, represented a unique, and never before seen, pharmacological profile (Figure 1) in that it was a PAM of all the Gq-coupled mAChRs (M1 EC50 =6.1 μM, M3 EC50 =6.4 μM and M5 EC50 = 4.1 μM), but devoid of activity at the Gi/o-coupled M2 and M4. (12) This led us to predict it would be possible to dial-in or dial-out different mAChR subtypes and potentially develop an M1 selective PAM, an M3 selective PAM and/or an M5 selective PAM from this non-selective lead through chemical optimization. (13)
Probe Chemical Lead Optimization Strategy
For the lead optimization of CID 3008304, we employed a 2-dimensional library approach, evaluating 8 diverse isatins (1–8) in combination with 12 (9–20) functionalized benzyl halides to afford 92/96 analogs of CID 3008304 (21–112) (Scheme 1). Yields were generally good providing the desired products in yields ranging from 20–95%. (13) In order to evaluate the analogs expeditiously, we screened them in a single point format at a 30 μM final concentration in Ca2+ mobilization assays using M5 and M1 cells receiving a fixed submaximal concentration (~EC10) of ACh (Figure 2). This method efficiently triaged analogs displaying high M1 vs. M5 or M5 vs. M1 preference. Interestingly, some analogs displayed robust potentiation effects at M5 (i.e. elevation of ACh ~EC10 to >50–60% of maximum ACh response) with absent or weak potentiation at M1, thus exhibiting strong preference for M5 versus M1 activity (subject of this report). (13) Other analogs proved to be highly M1–preferring (subject of future probe report). A similar exercise with M3 cells afforded no M3-preferring PAMs thus far.
In terms of maximal potentiation efficacy, SAR from the initial 30 μM screen of the entire library (21–112) suggested that 5-OCF3 substitution of the isatin core (R1) was generally favored for increased M5 versus M1 activity, while halogen substitutions at the 4- position conferred a more dual M1/M5 activity (Figure 2). Indeed, most of the 5-OCF3 substituted compounds chosen from the initial screen possessed 1–5 μM potencies at M5 and >30 μM potencies at M1 (Table 1). Numerous benzylic substitutions (R2) were tolerated for M1 and M5 activity, depending on the isatin core (R1). Methoxybenzyls were generally favored for M5 versus M1 activity whereas trifluoromethylbenzyls were generally favored for dual M1/M5 potentiation activity (Table 1). Most other simple congeners, including those with various methyl substitutions at R1 or R2 displayed weak or negligible potentiation at either receptor. In light of the high M5 versus M1 potentiation preference displayed by CID 42633508 (1.16 μM M5 EC50 and >30 μM M1 EC50) in these assays, the compound was further profiled across the rtemaing subtypes. As shown in Figure 3A, the full subtype-selectivity profile of this compound was obtained in similar Ca2+ assays using M2, M3, and M4 cells. CID 42633508 had no effect on M2 and M4, and afforded only modest activation of M1 and M3 at 30 μM (~30%); thus, CID 42633508 provides >30x selectivity for M5 versus the other four subtypes, thus representing the first highly M5-preferring muscarinic ligand ever reported. (13)
Subsequent experiments (Figure 3) were performed to determine if CID 42633508 was a true PAM. As shown in Figure 3B, CID 42633508 had no effect on M5 cells alone, but caused a dose-dependent increase in M5 receptor activation in the presence of a submaximal (EC20) concentration of ACh. At a fixed 30 μM concentration, CID 42633508 elicited a 14-fold leftward shift of the ACh concentration-response-curve (CRC) (Figure 3C). In [3H]-NMS binding experiments, CID 42633508 did not displace the orthosteric radio-ligand whereas atropine, an orthosteric antagonist, afforded complete displacement (Figure 3D). In order to address the mechanism of M5 PAM activity, we measured the effect of fixed 30 μM CID 42633508 on ACh competition with [3H]-NMS binding, and found a 10-fold increase in ACh affinity for M5 – a value close to the leftward fold-shift (14-fold). Thus, the mechanism of M5 potentiation by CID 42633508 is due in part to the enhancement of ACh binding. Given these data, CID 42633508 is a true PAM of M5, devoid of intrinsic agonist activity. (13)
We then evaluated pharmacokinetics and brain penetration for the M5 PAM to evaluate its ability to serve as an in vivo probe. Unfortunately, CID 42633508 is characterized by poor systemic absorption after intraperitoneal administration with maximum concentration in plasma (161.7 ng/mL) being achieved within 1 hour. However, it is slowly eliminated from systemic circulation and has elimination half life of 4.5 hours. Although quickly taken up in the brain, it exhibits poor brain penetration with AUCbrain/AUCplasma value of 0.25. (13) Future lead optimization will focus on improving brain penetration. At this point, the Lead Profiling Screen (68 GPCRs, ion channels and transporters) from MDS Pharma was performed on CID 42633508 to determine a broader ancillary pharmacology profile for this MLPCN probe. In addition to selectivity versus the mGluR family, CID 42633508 possessed reasonably clean ancillary pharmacology in a 68 Target MDS GPCR, Ion Channel and Transporter Lead Profiling Panel, displaying no significant activity (no inhibition >50% at 10 μM) for 59 of the 68 targets. CID 42633508 did have activity at 9 targets: A3A (65%@10 μM), A2A (99%@10 μM), NET (66%@10 μM), CB1 (84%@10 μM), D4 (61%@10 μM), H1 (62%@10 μM), H2 (55%@10 μM), MOP (70%@10 μM) and hERG (60%@10 μM). Of these, only two possessed Kis below 10 μM: A2A (760 nM) and CB1 (2.2 μM). (14)
Thus, CID 42633508 is a first-in–class M5 PAM, the first M5-selective ligand (>30-fold versus M1–M4) with generally clean ancillary pharmacology. CNS penetration is poor however, restricting CID 42633508 for use as an in vitro probe. (13) Future work will focus on developing a centrally penetrant M5 PAM devoid of any ancillary pharmacology for use as an in vivo probe.
Synthetic procedure (large scale) and spectral data for CID 42633508
CID 42633508, 1-(4-methoxybenzyl)-5-(trifluoromethoxy)indoline-2,3-dione [ML129]
To a vial containing ACN (15 mL) was added 5-trifluoromethyoxyisatin (1.00 g, 4.33 mmol), K2CO3 (8.66 mmol, 2.0 eq), KI (0.43 mmol, 0.1 eq), and 4-methoxybenzyl chloride (4.76 mmol, 1.1 eq). The reaction was stirred for ~24 hours at room temperature while monitoring by TLC. After judging complete, the reaction was partitioned between EtOAc and H2O, and the combined organics were dried over MgSO4, filtered, and then concentrated in vacuo to afford the pure 1-(4-methoxybenzyl)-5-(trifluoromethoxy)indoline-2,3-dione title compound as an orange solid (1.50 g, 4.26 mmol, 98%). 1H-NMR (400MHz, d6 - DMSO) δ 7.60 (m, 2H), 7.36 (d, J = 8.7, 2H), 7.04 (m, 1H), 6.89 (m, 2H), 4.84 (s, 2H), 3.71 (s, 3H). 13C-NMR (100MHz, d6 - DMSO) δ 181.99, 158.73, 158.33, 149.00, 143.83, 130.35, 128.88, 126.94, 121.32, 118.79, 117.66, 114.02, 112.36, 55.05, 42.45. LCMS (214 nm) 3.37 min (>98%); m/z 352.1 [M+H]. HRMS calcd for C17H13F3NO4 [M+H] 352.0797 found 352.0795.
002608917, 002608918, 002608916, 002608915 (Probe, 500 mg)
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For information on the MDS Pharma Lead Profiling Screen see: www
APPENDIX I. Solubility, Stability and Reactivity data as determined by Absorption Systems
Solubility in PBS (at pH = 7.4) for ML129 was 3.14 μM.
Stability (at room temperature = 23 °C) for ML129 in PBS (no antioxidants or other protectorants and DMSO concentration below 0.1%) is shown in the table below. After 48 hours, the percent of parent compound remaining was 79%, but the assay variability over the course of the experiment ranged from a low of 79% (at 1 hour and 48 hours) to a high of 112% (at 30 minutes).
As assessed through a glutathione (GSH) trapping experiment in phosphate buffered saline (with a substrate concentration of typically 5–50 μM and a GSH concentration of 2 μM, at t = 60 minutes), ML129 was found to form one detectable GSH adduct with an exact mass of 573.1652 (M+H). Relative to parent AUC (at t = 0), this conjugate was 13-fold more abundant at 60 minutes, but not knowing the respective extinction coefficients (for parent molecule and GSH conjugate) it is difficult to quantify the extent of GSH reactivity. Furthermore, given that the reported solubility for ML129 (in PBS at pH = 7.4 – determined at Absorption Systems*) is only 3.14 μM, this apparent large increase in the concentration of the GSH adduct might be a result of dramatically increased solubility for the GSH adduct over the relatively insoluble substrate, which would then inflate the relative abundance of the GSH conjugate. And, while it is possible to propose a structure for this mass which corresponds to displacement of the trifluoromethoxy group on the isatin by GSH, it is interesting to note that this sort of reactivity was not observed for the same isatin core in ML172.
APPENDIX II. Liquid Chromatography-Mass Spectrometry (LCMS) and Nuclear Magnetic Resonance (NMR) as prepared by Vanderbilt Specialized Chemistry Center
Solubility (PBS at pH = 7.4), Stability and Reactivity experiments were conducted at Absorption Systems. For additional information see: https://www
Thomas M Bridges, L Michelle Lewis, C David Weaver, and Craig W Lindsley1.
Received: December 18, 2009; Last Update: October 20, 2010.
National Center for Biotechnology Information (US), Bethesda (MD)
Bridges TM, Lewis LM, Weaver CD, et al. Discovery of the first mAChR 5 (M5) selective ligand, an M5 Positive Allosteric Modulator (PAM) 2009 Dec 18 [Updated 2010 Oct 20]. In: Probe Reports from the NIH Molecular Libraries Program [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2010-.