Modulation of the Metabotropic Glutamate Receptor mGluR4: Selectivity at mGluR5
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 Compound: 1
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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1. Cells are plated at a density of 20,000 cells/20 ul well 1 day prior to assay in Assay Media (DMEM with 20mM HEPES, 10% dialyzed FBS, 1mM sodium pyruvate).
2. The following day, media is removed from the cells.
3. 20 ul/well of Fluo-4 AM dye (1 uM) in Assay Buffer (HBSS + 2.5mM probenecid) is added.
4. The plates are incubated for 45 min to one hour at RT.
5. Dye is removed from the cells.
6. 20 ul of assay buffer is added and removed. 20 ul of assay buffer is added.
8. Plates are loaded into a Hamamatsu FDSS.
9. Compounds are tested for selectivity at a 10 uM final concentration (prepared as a 2X stock, 20 ul added per well) and the agonist is added at a 5X concentration (10 ul of agonist added per well, 50 uL total volume). A baseline read is taken for 1.5 seconds, the first addition (compound or DMSO-matched vehicle) occurs at 1.5 seconds and the second addition (agonist) is added at 142 seconds. The read continues for 300 seconds.
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 max fluorescence increase beginning after the agonist addition was calculated. Curves were fitted using a four point logistical equation using Microsoft XLfit (IDBS, Bridgewater, NJ).
This compound was not active as a positive allosteric modulator of mGluR5 and was assigned an 'Outcome' of 'Inactive' and a 'Score' of '0'. Furthermore, the 3-fold decrease in glutamate EC50 in the presence of compound would suggest antagonistic modulation.
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* Activity Concentration. ** Test Concentration.
Data Table (Concise)