Assay for HTS of Gi/Go-linked GPCRs using mGluR8: Primary Screening
Parkinson's disease (PD) is a common neurodegenerative disorder characterized by disabling motor impairment due to the loss of substantia nigra dopaminergic neurons involved in modulating function of the basal ganglia (BG). The treatment of PD has traditionally relied on strategies for replacing lost dopamine; Levodopa (L-DOPA), the immediate precursor of dopamine, remains the most effective PD more ..
BioActive Compounds: 2166
Parkinson's disease (PD) is a common neurodegenerative disorder characterized by disabling motor impairment due to the loss of substantia nigra dopaminergic neurons involved in modulating function of the basal ganglia (BG). The treatment of PD has traditionally relied on strategies for replacing lost dopamine; Levodopa (L-DOPA), the immediate precursor of dopamine, remains the most effective PD drug (1). As the disease progresses, however, L-DOPA efficacy becomes unpredictable and many patients experience debilitating dyskinesias and behavioral disturbances. Thus, novel approaches for symptomatic treatment of PD are desperately needed. New therapeutic agents that bypass the dopamine system have the potential of providing more sustained efficacy and fewer side effects or could allow for co-administration with lower doses of L-DOPA to delay the development of adverse effects.
In recent years, G-protein-linked glutamate receptors, termed metabotropic glutamate receptors (mGluRs) have emerged as exciting new targets for PD treatment (2), particularly mGluR8. mGluR8 does not regulate transmission through the BG in normal animals and the mGluR8 agonist (S)-3,4-DCPG (DCPG) does not have antiparkinsonian activity in acute models of dopamine depletion or dopamine receptor blockade. However, DCPG has robust antiparkinsonian effects in rodent models in which dopamine is depleted or receptors are blocked for more prolonged periods as well as in animals with chronic 6-OHDA-induced lesions of dopamine neurons. Interestingly, DCPG has more robust antiparkinsonian activity than do mGluR4 PAMs or mGluR5 antagonists. Furthermore, mGluR8 is not widely expressed in normal animals (3) and the fact that mGluR8 does not regulate BG or motor function except in animals with chronic parkinsonian motor impairments could provide an ideal profile for a novel antiparkinsonian agent that may be selectively active in PD patients. We and others have found that activities of other mGluR subtypes in the basal ganglia are dramatically regulated by loss of dopamine (4-7).
1. Poewe, W. and Granata, R., In: Movement Disorders: Neurological principles and practice (Watts RL, ed.), 201 (1997).
2. Conn, P. J., Battaglia, G., Marino, M. J., and Nicoletti, F., Nat Rev Neurosci 6 (10), 787 (2005). PMID: 16276355
3. Corti, C. et al., Eur J Neurosci 10 (12), 3629 (1998); Saugstad, J. A. et al., Mol Pharmacol 51 (1), 119 (1997).
4. Poisik, O. V. et al., J Neurosci 23 (1), 122 (2003).
5. Marino, M. J., Awad-Granko, H., Ciombor, K. J., and Conn, P. J., Neuropharmacology 43 (2), 147 (2002).
6. Wittmann, M., Marino, M. J., and Conn, P. J., J Pharmacol Exp Ther 302 (2), 433 (2002).
7. Picconi, B. et al., Brain 125 (Pt 12), 2635 (2002).
Using Human Embryonic Kidney Cells (HEKs) containing rat mGluR8, Poly-D-Lysine coated black/clear bottom Greiner 384 well plates, were seeded at 15,000 cells/well and grown overnight at 37 degrees C in 5 percent CO2. The next day, FluoZin2 was added to each well and the plate incubated for 50 minutes at RT. The cells were washed with HBSS + 20mM HEPES, pH 7.4 leaving 20uL residual volume. The cell plate was loaded in to the Hamamatsu FDSS and 5 frames of background were collected before addition of compound at 10uM final concentration. Data collection continued at 2 sec intervals until the addition of EC20 glutamate/thallium stimulus, then the sampling interval changed to 1 sec intervals for a total collection time of 4 min. EC20 and ECMax of glutamate controls were used to calculate the Z prime value for each window as a measure of assay robustness.
The data were processed using an automated data analysis protocol generated with Pipeline Pilot (Accelrys, San Diego, CA) and R statistics package (www.r-project.org). Data were normalized by dividing each value in each trace by the initial value for that trace. For each well, 3 windows were calculated as follows: 1)window 1 was the ratio value 5 seconds post-addition of compound; 2)window 2 was the ratio value 5 seconds pre-addition of EC20/thallium and 3)window 3 was the slope value between 165 and 175 seconds. Thallium flux modulators or "hits" were compounds where Z score > 3 or Z < -3 in Window 3. Windows 1 and 2 were used to evaluate fluorescent and environmentally sensitive compounds.
Compounds denoted as 'hits' were given the score of '100' and outcome of 'active'. All other compounds were denoted as 'inactive' and given a score of '0'.
Data Table (Concise)