Modulation of Metabotropic Glutmate Receptor mGluR4: Human PAM 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 ..
BioActive Compounds: 7
Assay Provider: Colleen Niswender
Assay Provider Affiliation: Vanderbilt University
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 (mGluRs), including the group III mGluRs (mGluR4, -7 and -8), play important roles in regulating transmission through the BG and could serve as targets for novel PD therapeutics (Conn et al., 2005). For instance, mGluR4 activation reduces overactive GABA release at a specific inhibitory BG synapse (Macinnes and Duty, 2008; Marino et al., 2003; Valenti et al., 2003) and reverses motor deficits in a variety of rodent PD models (Konieczny et al., 2007; MacInnes et al., 2004; Marino et al., 2003; Ossowska et al., 2007; Valenti et al., 2003).
To more selectively activate mGluR4 and improve upon the pharmacokinetic liabilities of glutamate analogs, we and others have developed novel positive allosteric modulators (PAMs) which potentiate glutamate function at mGluR4 (Engers et al., 2009; Maj et al., 2003; Marino et al., 2003; Niswender et al., 2008a; Niswender et al., 2008b; Williams et al., 2008); several of these tool compounds exhibit antiparkinsonian and neuroprotective effects in multiple rodent PD models (Battaglia et al., 2006; Marino et al., 2003; Niswender et al., 2008a). Unfortunately, many available compounds have lacked pharmacokinetic properties to make them useful tools for study of mGluR4 function via systemic routes of administration. The probe compound developed here exhibits sufficient potency, efficacy, and pharmacokinetic properties, including brain penetration, to make it a useful compound to progress mGluR4 biology, which will undoubtedly allow the intense study of mGluR4 activation in multiple areas of neuroscience such as psychiatric disorders (Stachowicz et al., 2006; Stachowicz et al., 2004), cancer (Iacovelli et al., 2006), and addiction (Blednov et al., 2004).
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Cell line creation and culture of the human mGluR4/ Gqi5/CHO line. Human mGluR4 (hmGluR4)/CHO cells were stably transfected with the chimeric G protein Gqi5 (Conklin et al., 1993) in pIRESneo3 (Invitrogen, Carlsbad, CA) and single neomycin-resistant clones were isolated and screened for mGluR4-mediated calcium mobilization using the method described below. hmGluR4/CHO cells were cultured in 90 percent Dulbecco's Modified Eagle Media (DMEM), 10 percent dialyzed fetal bovine serum (FBS), 100 units/ml penicillin/streptomycin, 20 mM HEPES (pH 7.3), 1 mM sodium pyruvate, 20 ug/ml proline, 2 mM glutamine, 400 ug/ml G418 sufate (Mediatech, Inc., Herndon, VA) and 5 nM methotrexate (Calbiochem, EMD Chemicals, Gibbstown, NJ). All cell culture reagents were purchased from Invitrogen Corp. (Carlsbad, CA) unless otherwise noted.
Fold shift determinations. Assays were performed within Vanderbilt University's High-Throughput Screening Center. Human mGluR4/CHO cells (30,000 cells/20 ul/well) were plated in black-walled, clear-bottomed, TC treated, 384 well plates (Greiner Bio-One, Monroe, North Carolina) in DMEM containing 10 percent dialyzed FBS, 20 mM HEPES, 100 units/ml penicillin/streptomycin, and 1 mM sodium pyruvate (Plating Medium). The cells were grown overnight at 37 degrees C in the presence of 5 percent CO2. The next day, the medium was removed and replaced with 20 uL of 1 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 percent (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 uL 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 20 uM stock. Ca2+ flux was measured using the Functional Drug Screening System 6000 (FDSS6000, Hamamatsu, Japan). Baseline readings were taken (10 images at 1 Hz, excitation, 47+/-20 nm, emission, 540+/-30 nm) and then 20 ul/well test compounds were added using the FDSS's integrated pipettor. Cells were incubated with test compounds (final concentration 30 uM) for 2.5 minutes and then increasing concentrations of glutamate were applied. FDSS data were analyzed using a custom analysis application.
For fold shift experiments, 20 ul of test compound (2X concentration, 30 uM final concentration) was applied approximately 2.5 minutes prior to increasing concentrations of glutamate (10 ul of a 5x final concentration). Curves were fitted using a four point logistical equation using Microsoft 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' and 'Score' was assigned as '1'. 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.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)