Chemical optimization of in vitro pharmacology and DMPK properties of the highly selective mAChR 4 (M4) Positive Allosteric Modulator (PAM) Series with Greatly Improved Human Receptor Activity (hM4 Calcium Potency)
To date, five muscarinic acetylcholine receptor (mAChR) subtypes have been identified (M1-M5) and play important roles in mediating the actions of ACh in the peripheral and central nervous systems (Langmead et al. 2008). Of these, M1 and M4 are the most heavily expressed in the CNS and represent attractive therapeutic targets for cognition, Alzheimer's disease, and schizophrenia (Felder et al. more ..
To date, five muscarinic acetylcholine receptor (mAChR) subtypes have been identified (M1-M5) and play important roles in mediating the actions of ACh in the peripheral and central nervous systems (Langmead et al. 2008). Of these, M1 and M4 are the most heavily expressed in the CNS and represent attractive therapeutic targets for cognition, Alzheimer's disease, and schizophrenia (Felder et al. 2000; Clader and Wang 2005; Wess et al. 2007; Langmead et al. 2008). For example, clinical trials with xanomeline, a M1/M4-preferring orthosteric agonist, demonstrated efficacy as both a cognition-enhancing agent and an antipsychotic agent (Bodick et al. 1997a; Bodick et al. 1997b; Shekhar et al. 2008). However, a long standing question concerns whether or not the antipsychotic efficacy or antipsychotic-like activity in animal models is mediated by activation of M1, M4, or a combination of both receptors. Data from mAChR knockout (KO) mice led to the suggestion that a selective M1 agonist would be beneficial for cognition, whereas an M4 agonist would provide antipsychotic activity for the treatment of schizophrenia (Gomeza et al. 1999; Gomeza et al. 2001; Anagnostaras et al. 2003; Tzavara et al. 2004). This proposal is further supported by recent studies demonstrating that M4 receptors modulate the dynamics of cholinergic and dopaminergic neurotransmission and that loss of M4 function results in a state of dopamine hyperfunction (Tzavara et al. 2004). These data, coupled with findings that schizophrenic patients have altered hippocampal M4 but not M1 receptor expression (Raedler et al. 2007), suggest that selective activators of M4 may provide a novel treatment strategy for schizophrenia patients.
Unfortunately, xanomeline lacks true M1/M4 specificity and also has significant affinity and efficacy at M2 and M3 mAChRs. Thus xanomeline, like many other cholinergic agents including acetylcholinesterase (AChE) inhibitors, displays significant adverse effects including bradycardia, GI distress, excessive salivation, and sweating, which are thought to be primarily due to activation of peripheral M2 and M3 mAChRs (Bymaster et al. 2003; Wess et al. 2007; Langmead et al. 2008). Due to the high sequence homology and conservation of the orthosteric ACh binding site among the mAChR subtypes, development of chemical agents that are selective for a single mAChR subtype has been largely unsuccessful, and in the absence of highly selective activators of M4, it has been impossible to test the role of selective M4 activation. However, in recent years, we have been highly successful in the identification of multiple highly selective allosteric activators for M1 (Bridges et al. 2008; Jones et al. 2008; Ma et al. 2009; Marlo et al. 2009; Shirey et al. 2009; Bridges et al. 2010) and M4 (Brady et al. 2008; Shirey et al. 2008; Kennedy et al. 2009). Though we are encouraged by these results, unfortunately, highly selective centrally penetrant activators of either M1 or M4 remain unavailable, making it impossible to determine the in vivo effects of selective activation of these receptors. Future compounds developed should exhibit sufficient potency, efficacy, and pharmacokinetic properties, including brain penetration, to make useful probes to progress M4 biology, which will undoubtedly allow the intense study of M4 activation in multiple areas of neuroscience.
Potency and efficacy of compounds will be determined by performing concentration-response curves (CRCs, 10 points, ranging from approximately 30 uM-1 nM at 0.3% final DMSO concentration) at human M4 using a calcium assay in which the cells also express the chimeric G protein Gqi5 to couple M4 to calcium mobilization (Brady et al. 2008; Shirey et al. 2008; Kennedy et al. 2009). PAMs with EC50 values < 1 uM versus human M4 will next be evaluated for potency versus rat M4 in an analogous calcium assay utilizing Gqi5. Following potency evaluation at human and rat M4, PAMs with EC50 values less than 1 uM at rat M4 will be evaluated for their ability to left-shift the CRC of acetylcholine (ACh) for rat M4 in a calcium assay and will be examined for their selectivity for M4 relative to other mAChR subtypes (Brady et al. 2008; Shirey et al. 2008; Kennedy et al. 2009). PAMs with a fold-shift of the ACh CRC of > 5 will then be evaluated for Tier 1 DMPK assays including plasma protein binding (PPB), intrinsic clearance (Obach 1997; Obach 1999), and inhibition of cytochrome p450 enzymes (CYP) (Youdim et al. 2008; Engers et al. 2009; Lebois et al. 2011; Jones et al. 2012). Next, compounds demonstrating PPB > 0.01 fraction unbound (Fu), a clean CYP profile, and moderate clearance will be evaluated for CNS exposure and Plasma:Brain levels using an in vivo snapshot PK paradigm (Frick et al. 1998; Engers et al. 2009; Lebois et al. 2011; Jones et al. 2012). Novel M4 PAMs showing a CNS exposure > PAM EC50, a Brain:Plasma ratio of >0.5, and at least a 10-fold or greater selectivity versus other mAChR subtypes will next be evaluated in a preclinical model of antipsychotic drug action: amphetamine-induced hyperlocomotion (AHL) (Brady et al. 2008; Jones et al. 2008). M4 PAMs demonstrating activity in AHL will have their ancillary pharmacology fully evaluated and those compounds that show no significant off-target activity and suitable solubility will be declared an MLPCN probe. The ultimate goal of this project from the PI's perspective is to generate compounds which should exhibit sufficient potency, efficacy, and pharmacokinetic properties, including brain penetration, to make useful probes to progress M4 biology.
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hM4 Muscarinic Receptor Calcium Concentration Response
Cell line creation and culture of the human M4/Gqi5/CHO line
Human M4 (hM4) cDNA in pcDNA3.1(+) was purchased from www.cDNA.org and stably transfected into Chinese hamster ovary (CHO K1) cells purchased from the ATCC (www.atcc.org). To make a stable hM4 cell line for use in calcium mobilization assays, cells were cotransfected with the chimeric G protein Gqi5 (Conklin et al., 1993). All transfections utilized Lipofectamine 2000. hM4-Gqi5 cells were cultured in Ham's F-12; 10% heat-inactivated fetal bovine serum (FBS), 20mM HEPES, 500 ug/mL G418 (Mediatech, Inc., Herndon, VA), and 500 ug/mL hygromycin B. All cell culture reagents were purchased from Invitrogen Corp. (Carlsbad, CA) unless otherwise noted.
Conklin, B.R., Farfel, Z., et al. (1993). "Substitution of three amino acids switches receptor specificity of Gq alpha to that of Gi alpha." Nature 20;363(6426):274-6.
Assays were performed within the Vanderbilt Center for Neuroscience Drug Discovery's Screening Center. CHO cell lines expressing muscarinic acetylcholine receptors were plated (15,000 cells/20 uL/well) in black-walled, clear-bottomed, TC treated, 384 well plates (Greiner Bio-One, Monroe, North Carolina) in Ham's F-12, 10% FBS, 20 mM HEPES. The cells were grown overnight at 37 degrees C in the presence of 5% CO2. The following day, plated cells had their medium exchanged to Assay Buffer (Hank's balanced salt solution, 20 mM HEPES and 2.5 mM Probenecid (Sigma-Aldrich, St. Louis, MO)) using an ELX405 microplate washer (BioTek), leaving 20 uL/well, followed by addition of with 20 uL of 4.5 uM Fluo-4, AM (Invitrogen, Carlsbad, CA) prepared as a 2.3 mM stock in DMSO and mixed in a 1:1 ratio with 10% (w/v) pluronic acid F-127 and diluted in Assay Buffer for 45 minutes at 37 degrees C. The dye was then exchanged to Assay Buffer using an ELX405, leaving 20 uL/well and the plates were incubated at room temperature for 10 min prior to assay. Test compounds were transferred to daughter plates using an Echo acoustic plate reformatter (Labcyte, Sunnyvale, CA) and then diluted into Assay Buffer to generate a 2x stock in 0.6% DMSO (0.3% final). Acetylcholine (ACh) EC20 and EC80 were prepared at a 5X stock solution in assay buffer prior to addition to assay plates. Calcium mobilization was measured at 37 degrees C using a Functional Drug Screening System 6000 or 7000 (FDSS6000 or FDSS7000, Hamamatsu, Japan) kinetic plate reader according to the following protocol. Cells were preincubated with test compound (or vehicle) for 144 seconds prior to the addition of an EC20 concentration of the agonist, ACh. 86 seconds after this addition, an EC80 concentration of ACh was added. Control wells also received a maximal ACh concentration (1 mM) for eventual response normalization.
The signal amplitude was first normalized to baseline and then as a percentage of the maximal response to ACh. Microsoft XLfit (IDBS, Bridgewater, NJ) was utilized for curve fitting and EC50 value determination using a four point logistical equation.
Compounds showing dose-dependency were assigned 'Outcome' = 'Active', EC50='Value', and % ACh max='Value'.
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)