Chemical Optimization of Advanced mGlu4 Lead Candidates (rat_mGlu4_Fold_Shift)
The primary pathophysiological change giving rise to the symptoms of Parkinson's disease (PD) is a loss of the dopaminergic neurons in the substantia nigra pars compacta (SNc) that are involved in modulating the function of basal ganglia (BG) nuclei. Unfortunately, traditional therapies for treatment of PD based on dopamine replacement strategies eventually fail in most patients and are more ..
Modulation of the Metabotropic Glutamate Receptor mGlu4: Fold shift at rat mGlu4
The primary pathophysiological change giving rise to the symptoms of Parkinson's disease (PD) is a loss of the dopaminergic neurons in the substantia nigra pars compacta (SNc) that are involved in modulating the function of basal ganglia (BG) nuclei. Unfortunately, traditional therapies for treatment of PD based on dopamine replacement strategies eventually fail in most patients and are associated with numerous side effects. A great deal of effort has been focused on developing a detailed understanding of the circuitry and function of the BG to develop novel, nondopaminergic, approaches for restoring normal BG function in PD patients. Exciting advances suggest that metabotropic glutamate receptors (mGlus), including the group III mGlus (mGlu4, -7 and -8), play important roles in regulating transmission through the BG and could serve as targets for novel PD therapeutics (3). For instance, mGlu4 activation reduces overactive GABA release at a specific inhibitory BG synapse (12,19,9) and reverses motor deficits in a variety of rodent PD models (12,19,10,8,16).
To more selectively activate mGlu4 and improve upon the pharmacokinetic liabilities of glutamate analogs, we and others have developed novel positive allosteric modulators (PAMs) which potentiate glutamate function at mGlu4 (11,12,14,15,4,21,7,6); several of these tool compounds exhibit antiparkinsonian and neuroprotective effects in multiple rodent PD models (12,1,14,7,6). Unfortunately, many available compounds have lacked the necessary pharmacokinetic properties required for study of mGlu4 function via systemic routes of administration. Future compounds developed should exhibit sufficient potency, efficacy, and pharmacokinetic properties, including brain penetration, to make useful probes to progress mGlu4 biology, which will undoubtedly allow the intense study of mGlu4 activation in multiple areas of neuroscience such as psychiatric disorders (18,17), cancer (5), and addiction (2).
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 mGlu4 using a calcium assay in which the cells express the chimeric G protein Gqi5 to couple mGlu4 to calcium mobilization (13,14,7,6). PAMs with EC50 values less than 500 nM versus human mGlu4 will next be evaluated for potency versus rat mGlu4 in a thallium flux assay measuring coupling of rat mGlu4 to G Protein-coupled Inwardly Rectifying Potassium (GIRK) Channels (13,6). Following potency evaluation at human and rat mGlu4, PAMs with EC50 values less than 500 nM at rat mGlu4 will be evaluated for their ability to left-shift the CRC of glutamate for rat mGlu4 in a thallium flux assay (13,14,7,6). PAMs with a fold-shift of the glutamate CRC of > 5 will then be evaluated for Tier 1 DMPK assays including plasma protein binding (PPB), intrinsic clearance, and inhibition of cytochrome p450 enzymes (CYP) (4,6). Next, compounds demonstrating PPB > 1% free and moderate clearance will be evaluated for CNS exposure and Plasma:Brain levels using an in vivo snapshot PK paradigm and will be examined for their selectivity for mGlu4 relative to other mGlu subtypes evaluated (4,6). Novel mGlu4 PAMs showing a CNS exposure > PAM EC50, a Brain:Plasma ratio of >0.5, and at least a 50-fold or greater selectivity versus other mGlu subtypes will next be evaluated in a preclinical model of PD: haloperidol-induced catalepsy (HIC) (20). mGlu4 PAMs demonstrating activity in HIC at a dose of < 30 mg/kg will have their ancillary pharmacology fully evaluated and those compounds that show no significant off-target activity, CaCo permeability > 10 cm/s-6, 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 mGlu4 biology.
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2. Blednov, Y. A., D. Walker, et al. (2004). "Mice lacking metabotropic glutamate receptor 4 do not show the motor stimulatory effect of ethanol." Alcohol 34(2-3): 251-9.
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6. Jones, C. K., M. Bubser, et al. (2012). "The metabotropic glutamate receptor 4-positive allosteric modulator VU0364770 produces efficacy alone and in combination with L-DOPA or an adenosine 2A antagonist in preclinical rodent models of Parkinson's disease." J Pharmacol Exp Ther 340(2): 404-21.
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13. Niswender, C. M., K. A. Johnson, et al. (2008a). "A novel assay of Gi/o-linked G protein-coupled receptor coupling to potassium channels provides new insights into the pharmacology of the group III metabotropic glutamate receptors." Mol Pharmacol 73(4): 1213-24.
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20. Varty, G. B., R. A. Hodgson, et al. (2008). "The effects of adenosine A2A receptor antagonists on haloperidol-induced movement disorders in primates." Psychopharmacology (Berl) 200(3): 393-401.
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Cell line creation and culture of the rat mGlu4 GIRK cell line.
HEK/GIRK cells stably expressing the M4 muscarinic receptor were grown in 45% Dulbecco's Modified Eagle Media (DMEM), 45% Ham's F12, 10% fetal bovine serum (FBS), 100 units/ml penicillin/streptomycin, 20 mM HEPES (pH 7.3), 1 mM sodium pyruvate, 2 mM glutamine, and 700 ug/ml G418. The rat mGlu4 cell line was prepared by PCR amplification of the entire coding sequence of each receptor and cloning into pIRES puro 3 (Invitrogen). Cloning sites were BamHI/Not I. HEK/GIRK/M4 cells were transfected with 24 ug of DNA and stable transfectants were selected with puromycin. And a monoclonal cell lines was established. Cells were grown in 45% Dulbecco's Modified Eagle Media (DMEM), 45% Ham's F12, 10% fetal bovine serum (FBS), 100 units/ml penicillin/streptomycin, 20 mM HEPES (pH 7.3), 1 mM sodium pyruvate, and 2 mM glutamine (Growth Media. mGlu/GIRK lines were supplemented with 600 ng/ml puromycin dihydrochloride (Sigma-Aldrich) and 700 ug/ml G418 (Mediatech, Inc., Herndon, VA). Cells for experiments were generally maintained for approximately 15-20 passages. All cell culture reagents were purchased from Invitrogen Corp. (Carlsbad, CA) unless otherwise noted.
Cells were plated into 384 well, black-walled, clear-bottom poly-D-lysine coated plates (Greiner) at a density of 15,000 cells/20 uL/well in DMEM containing 10% dialyzed FBS, 20 mM HEPES, and 100 units/ml penicillin/streptomycin (Assay Media). Plated cells were incubated overnight at 37 degrees C in the presence of 5% CO2. The following day, plated cells had their medium exchanged to Assay Buffer (Hanks Balanced Salt Solution (Invitrogen) containing 20 mM HEPES pH 7.3) using an ELX405 microplate washer (BioTek), leaving 20 uL/well, followed by addition of with 20 uL of 330 nM FluoZin-2 AM (Invitrogen, Carlsbad, CA) prepared as a 2.85 mM stock in DMSO and mixed in a 1:1 ratio with 10 percent (w/v) pluronic acid F-127 and diluted in Assay Buffer for 1 hour at room temperature. 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. Glutamate was diluted in Thallium Buffer (125 mM sodium bicarbonate (added fresh the morning of the experiment), 1 mM magnesium sulfate, 1.8 mM calcium sulfate, 5 mM glucose, 12 mM thallium sulfate, 10 mM HEPES, pH 7.3) at 5x the final concentration to be assayed. For fold shift experiments, compounds were diluted to a 60 uM (2x final) concentration in Assay Buffer. Thallium flux was measured using the Functional Drug Screening System 6000 or 7000 (FDSS6000 or FDSS7000, Hamamatsu, Japan). Baseline readings were taken (10 images at 1 Hz, excitation, 470+/-20 nm, emission, 540+/-30 nm) and then 20 uL/well test compounds were added using the FDSS's integrated pipettor. Approximately 2.5 minutes later 10 uL of Thallium Buffer +/- agonist was added. After the addition of agonist, data were collected for an approximately 3 additional min.
Thallium sulfate requires special handling and disposal precautions and investigators are cautioned to contact their Environmental Health and Safety Department to ensure proper procedures are followed.
Data were analyzed using Microsoft Excel. Raw data were opened in Excel and each data point in a given trace was divided by the first data point from that trace (static ratio). For experiments in which antagonists/potentiators were added, data were again normalized by dividing each point by the fluorescence value immediately before the agonist addition to correct for any subtle differences in the baseline traces after the compound incubation period. The slope of the fluorescence increase beginning five seconds after thallium/agonist addition and ending fifteen seconds after thallium/agonist addition was calculated. Curves were fitted using a four point logistical equation using XLfit (IDBS, Bridgewater, NJ). Subsequent confirmations of concentration-response parameters were performed using independent serial dilutions of source compounds and data from multiple days experiments were integrated and fit using a four point logistical equation in GraphPad Prism (GraphPad Software, Inc., La Jolla, CA).
For compounds that changed the EC50 of glutamate 3 fold or less, 'Outcome' was assigned as 'Inactive'. For compounds that changed the EC50 of glutamate greater than 3 fold, 'Outcome' was assigned as 'Active'. 'Score' was assigned as '50' for those with a fold-shift less than or equal to 10 or as '100' for those with a fold-shift greater than 10.
** Test Concentration.
Data Table (Concise)