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P11013
Discovery of Novel Allosteric Modulators of Metabotropic Glutamate Receptor 8 (mGluR8) using GIRK-mediated Thallium Flux Modulation in High-Throughput Screening
Presenter Emily Days, Vanderbilt University, USA
Additional Authors: Michelle Lewis, Dehui Mi, Qingwei Luo, Daniel Dorset, Stephanie Bailey, Christopher Farmer, P.Jeffery Conn, Colleen Niswender, David Weaver

The metabotropic glutamate receptor mGluR8 has been implicated as a target in a number of psychiatric and neurological disorders. Tools to explore the function of this receptor are lacking and we sought to identify novel modulators of mGluR8 via high-throughput screening (HTS).  Challenges for accurate pharmacological measurements in vitro specific to mGluR8 have lead to the development of a new experimental design (Niswender et al., 2008) that we implemented for discovery of novel modulators of this target within Vanderbilt Center for GPCRS, Ion Channels and Transporters, part of the Molecular Libraries Screening Network (MLSCN).   
The previous demonstration of potassium channels such as G-protein regulated Inwardly Rectifying K+ (GIRK) channels to conduct the ion thallium (Weaver et al., 2004) set a platform for processing a screen to select a group of modulators for further investigation.  The screen was conducted to define either enhancement or inhibition of G[beta][gamma] mediated response to glutamate activation of mGluR8, governed by a per plate Z’ of acceptable control measurements (Zhang et al., 1999). Using 384 well plates containing stably transfected mGluR8 /GIRK HEK cells, we discovered approximately 1% of our 155,000 compounds as outliers or hits.  This diverse library of small molecules was tested in singlicate and analyzed using criteria of 3 standard deviations above or below the mean slope during thallium stimulation measured as fluorescent signal ratio over defined baseline fluorescence on the Hamamatsu FDSS6000.  These hit compounds were then reordered and retested in duplicate in the same assay format for mGluR8 and including a counter screen with a parental cell line containing GIRK channels and muscarinic M4, stimulating with either glutamate or acetylcholine, respectively.  Selection was made with repeating hits to further specify activity to mGluR8 demonstrating concentration dependant response that was not active for muscarinic receptor M4 nor a highly related group III mGluR, mGluR4. The activity of these compounds present an exciting breakthrough that will certainly yield a group of new leads to further characterize the structure activity relationship necessary for modulation of mGluR8 activity. 
Niswender CM, Johnson KA, Luo Q, Ayala JE, Kim C, Conn PJ, Weaver CD (2008) A novel assay of Gi/o-linked G protein coupled receptor coupling to potassium channels provides new insights into the pharmacology of the groupIII metabotropic glutamate receptors. Mol Pharmacol. 2008 Jan2; (in press) PMID 18171729
Weaver CD, Harden D, Dworetzky SI, Robertson B and Knox RJ (2004) A Thallium-sensitive, fluorescence-based assay for detecting and characterizing potassium channel modulators in mammalian cells.   J Biomol Screen 9(8)671-677.
Zhang JH, Chung TD and Oldenberg KR (1999) A Simple Statistical Parameter for use in evaluation and Validation of High-throughput Screening Assays.  J Biomol Screen 4(2)67-73