Fluorescence-based cell-based primary high throughput screening assay to identify positive allosteric modulators (PAMs) of the human cholinergic receptor, muscarinic 4 (CHRM4)
Name: Fluorescence-based cell-based primary high throughput screening assay to identify positive allosteric modulators (PAMs) of the human cholinergic receptor, muscarinic 4 (CHRM4). ..more
BioActive Compounds: 1450
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC)
Affiliation: The Scripps Research Institute, TSRI
Assay Provider: Colleen Niswender, Vanderbilt University School of Medicine
Network: Molecular Library Probe Production Centers Network (MLPCN)
Grant Proposal Number: MH077606-01
Grant Proposal PI: Colleen Niswender, Vanderbilt University School of Medicine
External Assay ID: CHRM4_PAM_FLUO8_1536_1X%ACT PRUN
Name: Fluorescence-based cell-based primary high throughput screening assay to identify positive allosteric modulators (PAMs) of the human cholinergic receptor, muscarinic 4 (CHRM4).
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 (1-5). 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 (6, 7). 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 (8). Numerous pharmacological studies have provided evidence for a diversity of mAChR subtypes in brain and other tissues (4, 5, 9). These molecularly distinct receptors have important differences, including preferential coupling to various effector systems (see (4, 5) for reviews). In general, it is thought that coupling to Gq and phospholipase C is the predominant effector system coupled to M1, M3, and M5, whereas M2 and M4 most often couple to Gi/Go and inhibition of adenylyl cyclase or ion channels.
Clinical trials with xanomeline, a M1/M4-preferring orthosteric agonist, which also has sub-micromolar activity toward M5, 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. In addition, xanomeline has been associated with unwanted gastrointestinal side effects and syncope that resulted in patient non-compliance during the trials (10). 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. Further, in vivo active tool compounds for the M5 receptor are also unavailable. These tools will prove invaluable for verifying that novel M1 or M4 compounds do not have off target effects on M5 in vivo and assist with determining the role of M5 in the CNS.
1. Bonner, T.I., N.J. Buckley, A.C. Young, and M.R. Brann, Identification of a family of muscarinic acetylcholine receptor genes. Science, 1987. 237(4814): p. 527-32.
2. Bonner, T.I., A.C. Young, M.R. Brann, and N.J. Buckley, Cloning and expression of the human and rat m5 muscarinic acetylcholine receptor genes. Neuron, 1988. 1(5): p. 403-10.
3. Peralta, E.G., A. Ashkenazi, J.W. Winslow, D.H. Smith, J. Ramachandran, and D.J. Capon, Distinct primary structures, ligand-binding properties and tissue-specific expression of four human muscarinic acetylcholine receptors. Embo J, 1987. 6(13): p. 3923-9.
4. Caulfield, M.P., Muscarinic receptors--characterization, coupling and function. Pharmacol Ther, 1993. 58(3): p. 319-79.
5. Hulme, E.C., N.J. Birdsall, and N.J. Buckley, Muscarinic receptor subtypes. Annu Rev Pharmacol Toxicol, 1990. 30: p. 633-73.
6. Levey, A.I., S.M. Edmunds, V. Koliatsos, R.G. Wiley, and C.J. Heilman, Expression of m1-m4 muscarinic acetylcholine receptor proteins in rat hippocampus and regulation by cholinergic innervation. J Neurosci, 1995. 15(5 Pt 2): p. 4077-92.
7. Jakubik, J., P. Michal, E. Machova, and V. Dolezal, Importance and prospects for design of selective muscarinic agonists. Physiol Res, 2008. 57 Suppl 3: p. S39-47.
8. Conn, P.J., C.K. Jones, and C.W. Lindsley, Subtype-selective allosteric modulators of muscarinic receptors for the treatment of CNS disorders. Trends Pharmacol Sci, 2009. 30(3): p. 148-55.
9. Waelbroeck, M., M. Tastenoy, J. Camus, and J. Christophe, Binding of selective antagonists to four muscarinic receptors (M1 to M4) in rat forebrain. Mol Pharmacol, 1990. 38(2): p. 267-73.
10. Bodick, N.C., W.W. Offen, A.I. Levey, N.R. Cutler, S.G. Gauthier, A. Satlin, H.E. Shannon, G.D. Tollefson, K. Rasmussen, F.P. Bymaster, D.J. Hurley, W.Z. Potter, and S.M. Paul, Effects of xanomeline, a selective muscarinic receptor agonist, on cognitive function and behavioral symptoms in Alzheimer disease. Arch Neurol, 1997. 54(4): p. 465-73.
PAM, CHRM4, HM4, M4R, M4, mAChR, primary, singlicate, cell-based, CHO, GPCR, receptor, acetylcholine, acetylcholine receptor, muscarinic, allosteric, positive, agonist, calcium, dye, Fluo8, FLINT, FLIPR, fluorescence, fluor, activate, activator, activation, CNS, brain, central nervous system, mood, psychosis, dementia, kinetic, Parkinson's, Lewy body dementia, rat, vascular dementia, schizophrenia, HTS, high throughput screen, 1536, Scripps, Scripps Florida, MLSMR, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
The purpose of this assay is to identify compounds that act as positive allosteric modulators (PAMs) and increase activity of the human M4 muscarinic receptor (CHRM4; M4) in cells pre-treated with a known agonist. In this assay, CHO-K1 cells stably expressing human M4 are loaded intracellularly with the calcium indicator dye, Fluo-8, followed by addition of test compounds and subsequent treatment with the activator acetylcholine at a concentration that results in 20% activation (EC20). As designed, compounds that act as CHRM4 PAMs will increase calcium mobilization, increased intracellular calcium and relative fluorescence of the indicator dye beyond that of the EC20 of acetylcholine. Compounds are tested in singlicate at a final nominal concentration of 3 uM.
The CHO-hM4 cell line was routinely cultured in T-175 sq cm flasks at 37 C and 95% relative humidity (RH). The growth media consisted of Ham's F-12 Nutrient Media (F-12) supplemented with 10% v/v heat-inactivated qualified fetal bovine serum, 20 mM HEPES, 500 ug/mL Geneticin, 200 ug/mL Hygromycin and 1X antibiotic mix (penicillin and streptomycin).
The day before the assay 750 cells in 3 uL of growth media were seeded into each well of 1536 well microtiter plates and allowed to incubate at 37 C, 5% CO2, and 95 % RH for 17-24 hours. Next, 2 uL of the fluorogenic Fluo-8 intracellular calcium indicator mixture (prepared according to the manufacturer's protocol) was added to each well. Plates were then incubated for 60 minutes at 37 C, 5% CO2, and 95 % RH, followed by 30 minute incubation at room temperature. Then, 15 nL of test compound in DMSO were dispensed to appropriate wells.The assay was started by performing a basal read of plate fluorescence (470-495 nm excitation and 515-575 nm emission) for 5 seconds on the FLIPR Tetra (Molecular Devices) followed by a 140 second read after compound addition. Then a basal read of 5 seconds is started prior to all wells being treated with an EC20 concentration of acetylcholine. Then a real time fluorescence measurement was immediately performed for the remaining 140 seconds of the assay. This read is the measurement for the PAM mode. A ratio for each well was calculated to normalize assay data, according to the following mathematical expression:
Ratio = I_Max / I_Min
I_Max represents the maximum measured fluorescence emission intensity over the PAM mode 140 second read and;
I_Min represents the minimum (basal) measured fluorescence emission intensity before EC20 was added.
The percent activation was calculated from the median ratio as follows:
%_Activation = ( ( Ratio_Test_Compound - Median_Ratio_Low_Control ) / ( Median_Ratio_High_Control - Median_Ratio_Low_Control ) ) * 100
Test_Compound is defined as wells containing test compound.
Low_Control is defined as wells containing Acetylcholine at EC20 and DMSO.
High_Control is defined as wells containing Acetylcholine (EC100) and DMSO.
A mathematical algorithm was used to determine nominally activating compounds in the primary screen. Two values were calculated for each assay plate: (1) the average percent activation of test compound wells and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter for the entire run, i.e. any compound that exhibited greater % activation than the cutoff parameter determined for each plate (plate based cutoff) was declared active.
PubChem Activity Outcome and Score:
The reported PubChem Activity Score has been normalized to 100% observed primary activation. Negative % activation values are reported as activity score zero.
In this assay not all plates were run in the same batch. This resulted in batch-to-batch variation among the different batches of plates, thereby necessitating the use of a plate-based activity cutoff. For this reason the inactive and active scores overlap.
The PubChem Activity Score range for active compounds is 100-14, and for inactive compounds 42-0.
List of Reagents:
Cell line: Chinese Hamster Ovary (CHO) cells containing hM4 receptor and Gqi5; (Conn Lab)
Calcium sensitive dye: Fluo-8 No Wash Calcium Assay Kit; (AAT Bioquest, part 36316)
Growth media: Ham's F-12; 10% FBS, 20 mM HEPES, 500 ug/mL G418, 200 ug/mL Hygromycin
Assay media: Ham's F-12, 10% FBS, 20 mM HEPES
Assay plates: Corning black/clear 1536well FLIPR plate; (Corning, part 7338)
Probenecid: 250mM (pH 8.0); (Sigma P8761)
Agonist: Acetylcholine (50mM stock in water); Sigma A6625
Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, and compounds that modulate well fluorescence. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided by the MLSMR.
Categorized Comment - additional comments and annotations
From BioAssay Depositor:
BAO: assay design: redistribution reporter: second messenger: calcium
BAO: assay format: cell-based format
BAO: assay format detail: assay phase characteristic: homogeneous assay
BAO: bioassay specification: assay biosafety level: bsl1
BAO: bioassay specification: assay footprint: microplate: 1536 well plate
BAO: bioassay specification: assay measurement throughput quality: single concentration single measurement
BAO: bioassay specification: assay measurement type: kinetic assay
BAO: bioassay specification: assay readout content: assay readout method: regular screening
BAO: bioassay specification: assay readout content: content readout type: single readout
BAO: bioassay specification: assay stage: primary
BAO: bioassay specification: bioassay type: binding
BAO: detection technology: fluorescence: fluorescence intensity
BAO: detection technology detail: detection instrumentation: flipr tetra
BAO: detection technology detail: detection instrumentation manufacturer: molecular devices
BAO: endpoint detail: perturbagen concentration: concentration unit: micromolar
BAO: meta target detail: binding reporter specification: interaction: protein-small molecule
BAO: meta target: biological process target: regulation of molecular function
BAO: meta target: molecular target: protein target: receptor: transmembrane receptor: g protein coupled receptor
BAO: version: 1.4b1090
Assay Format: Cell-based
Assay Cell Type: CHO
** Test Concentration.
Data Table (Concise)