Discovery of a Highly Selective in vitro and in vivo M4 Positive Allosteric Modulator(PAM) : Activity with human M4
Grant Title: Discovery of a Highly Selective in vitro and in vivo M4 Positive Allosteric Modulator(PAM) ..more
BioActive Compounds: 4
Assay Provider: Colleen Niswender
Assay Provider Affiliation: Vanderbilt University
Grant Title: Discovery of a Highly Selective in vitro and in vivo M4 Positive Allosteric Modulator(PAM)
Grant Number: MH077607-1
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. However, 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.
Data collected by Millipore GPCR Profiler Screening
Percentage activations were determined by comparing well data to Emax values upon addition of reference agonists after five minute incubation of vehicle or Vanderbilt compounds. These samples were run using a 'Single Addition' assay protocol for the first and second assay run.
Master stock solution
Unless specified otherwise, all sample compounds are diluted in 100% anhydrous DMSO including all serial dilutions. If sample compounds were provided in a different solvent all master stock dilutions are performed in the specified solvent. All control wells contain identical solvent final concentrations as sample compound wells.
Compound plate for assay
Sample compounds were transferred from a master stock solution into a daughter plate that was used in the assay. Each sample compound was diluted into assay buffer (1x HBSS with 20mM HEPES and 2.5mM Probenecid) at an appropriate concentration to obtain final concentrations specified by Vanderbilt University.
Calcium Flux Assay
Vanderbilt University compounds were added to the assay plate in order to achieve an eight-point, four-fold serial dose response with a top concentration of 30uM (concentrations described here reflect the final concentration during the addition of EC20 ACh). Reference agonist was handled as mentioned above serving as assay control. The reference agonist was handled as described above for Emax. Read for 180 seconds using the FLIPRTETRA. At the completion of the first 'Single Addition' assay run, assay plate is removed from the FLIPRTetra and placed at 25 degrees C for 7 minutes. Read for 180 seconds using the FLIPRTETRA (This assay added EC20 Acetylcholine to the respective wells then fluorescence measurements were collected to determine Vanderbilt compounds increasing the g-protein activation relative to EC20 ACh).
All plates were subjected to appropriate baseline corrections. Once baseline corrections were processed, maximum fluorescence values were exported and data manipulated to calculate percentage activation, percentage inhibition, Z', EC50, and IC50. Dose response curves were generated using GraphPad Prism. The curves were fit by utilizing Sigmodial dose response (variable slope)fitting with the bottom parameter fixed at '0.' Compounds with fits having R squared greater than 0.8 were considered 'Active' and assigned a 'Score' of '100.' 'Inactive' compounds were scored as '0.'
* Activity Concentration. ** Test Concentration.
Data Table (Concise)