Assay for HTS of Gi/Go-linked GPCRs using mGluR8: Summary
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 Compound: 1
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).
The primary screen will be performed in singlicate at a 10 uM final concentration (generating a 0.125% final DMSO concentration which should be well tolerated in the assay). Compounds will be added and data then collected for 5 min. Following this 5 min period, 10 mul of either an EC20 (for example, columns 1, 3-23) or an ECmax concentration of agonist (e.g. columns 2, 24) will be added in the presence of thallium. Data will be collected for an additional 3 min and exported to a network server and analyzed by taking the slope of the fluorescence rise from 10-20 seconds after agonist addition. Preset criteria will be established for "hits" and will be identified using z score and b score determinations. Allosteric potentiator "hits" will be identified as those compounds that increase the slope by three standard deviations or more above the control EC20 population.
Compounds identified in the primary HTS will be characterized by a number of follow up assays to eliminate pharmacophores that have a non-selective mechanism of action and to identify the compounds that have the greatest potency and selectivity at mGluR8. Identified hits will be verified by running the GIRK assay in triplicate to confirm activity against cells expressing mGluR8 versus HEK/GIRK cells expressing the M4 receptor. In the M4 receptor experiment, compounds will be added as in the primary screen, but an EC20 concentration of acetylcholine or carbachol will be used as the agonist. If compounds are acting by a non-selective mechanism to induce GIRK activation, they may induce or potentiate GIRK activity in cells lacking mGluR8 or in cells expressing an unrelated Gi/o-linked GPCR. For this reason, compounds that give a response that is statistically different from the negative control population in these experiments will be eliminated at this stage.
Our next step will involve performing concentration-response curves (CRCs, 10 points, ranging from approximately 30 microM-1 nM) to determine the potency and efficacy of identified potentiators. For compounds passing the above steps, we will pursue follow-up studies to determine their selectivity for mGluR8 relative to other mGluR subtypes. Our goal for this project would be to generate an mGluR8-selective compound, with a potency under 10 microM, and with reasonable solubility in a solvent generally useful for in vitro experimentation (i.e., DMSO). If we do not obtain a suitable probe from the primary screen, initial chemistry would be performed within the MLPCN program to try to generate a useful compound. The ultimate goal of this project from the PI's perspective is to generate compounds that would be useful proof-of-concept molecules for in vivo testing in animal models of Parkinson's disease.
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).
Categorized Comment - additional comments and annotations
From MLP Probe Report:
Probe count: 1
MLP Probe ML# for probe 1: ML297
PubChem Substance ID (SID) for probe 1: 134418959
PubChem Compound ID (CID) for probe 1: 56642816
Probe type for probe 1: Activator
IC50/EC50 (nM) for probe 1: 160
Target for probe 1: GIRK 1/2
Disease relevance for probe 1: Adiction, Epilepsy
Anti-target for probe 1: GIRK 2/3
Fold selectivity for probe 1: >200
NCBI Book chapter link for probe 1: http://www.ncbi.nlm.nih.gov/books/NBK143539/ (ID: 3036062)
Grant number for probe 1: MH076398-01
NCBI Book chapter title for probe 1: Discovery and Characterization of a Selective Activator of the G-Protein Activated Inward-Rectifying Potassium (GIRK) Channel
Data Table (Concise)