Human M2 PAM Extended Characterization CounterScreen (CRC)
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. 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. In contrast, the adverse effects of more ..
BioActive Compound: 1
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. 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. In contrast, the adverse effects of cholinergic agents are thought to be primarily due to activation of peripheral M2 and M3 mAChRs. 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 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.
Clinical trials with xanomeline, a M1/M4-preferring orthosteric agonist, demonstrated efficacy as both a cognition-enhancing agent and an antipsychotic agent. In follow-up studies in rats, xanomeline displayed an antipsychotic-like profile comparable to clozapine. However, a long standing question concerned 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 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. 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. These data, coupled with findings that schizophrenic patients have altered hippocampal M4 but not M1 receptor expression, suggest that selective activators of M4 may provide a novel treatment strategy for schizophrenia patients.
However, multiple studies suggest that M1 may also play an important role in the antipsychotic effects of mAChR agonists and that the relative contributions of M1 and M4 to the antipsychotic efficacy of xanomeline or antipsychotic-like effects of this compound in animal models are not known. Highly selective centrally penetrant activators of either M1 or M4 have not been available, making it impossible to determine the in vivo effects of selective activation of these receptors.
1. Conklin BR, Farfel Z, Lustig KD, Julius D, Bourne HR. Substitution of three amino acids switches receptor specificity of Gq alpha to that of Gi alpha. Nature. 1993 May 20;363(6426):274-6.
hM2 Muscarinic Receptor CounterScreen
Cell line creation and culture of the human M2/Gqi5/CHO line.
Human M2 (hM2) CHO cells were a gift from the laboratory of Allan Levey (Emory University). hM2/CHO cells were stably transfected with the chimeric G protein Gqi5 (1) in pIREShygro (Invitrogen, Carlsbad, CA) and single hygromycin- and neomycin-resistant clones were isolated and screened for M2-mediated calcium mobilization using the method described below.
hM2/CHO-Gqi5 cells were cultured in Ham's F-12; 10% FBS, 20mM HEPES, 50mug/mL G418 (Mediatech, Inc., Herndon, VA), 500 microg/ml Hygromycin. All cell culture reagents were purchased from Invitrogen Corp. (Carlsbad, CA) unless otherwise noted.
Assays were performed within the Vanderbilt Center for Neuroscience Drug Discovery's Screening Center. Human M2/Gqi5/CHO cells (15,000 cells/20 mul/well) were plated 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 next day, the medium was removed and replaced with 20 microL of 2.3 microM 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 (Hank's balanced salt solution, 20 mM HEPES and 2.5 mM Probenecid (Sigma-Aldrich, St. Louis, MO)) for 45 minutes at 37 degrees C. Dye was removed and replaced with 20 microL of Assay Buffer. 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 EC20 and 80 were prepared at a 5X stock solution in assay buffer prior to addition to assay plates. Ca2+ mobilization was measured at 37 degrees C using a Functional Drug Screening System 6000 (FDSS6000, 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, acetylcholine (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 acetylcholine. Microsoft XLfit (IDBS, Bridgewater, NJ) was utilized for curves 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".
Compound concentrations (log [M]):
* Activity Concentration. ** Test Concentration.
Data Table (Concise)