Chemical Optimization of Advanced mGlu4 Lead Candidates (human_mGlu4_potency)
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 ..
Depositor Specified Assays
Modulation of the Metabotropic Glutamate Receptor mGlu4
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 (mGlus), including the group III mGlus (mGlu4, -7 and -8), play important roles in regulating transmission through the BG and could serve as targets for novel PD therapeutics (3). For instance, mGlu4 activation reduces overactive GABA release at a specific inhibitory BG synapse (12,19,9) and reverses motor deficits in a variety of rodent PD models (12,19,10,8,16).
To more selectively activate mGlu4 and improve upon the pharmacokinetic liabilities of glutamate analogs, we and others have developed novel positive allosteric modulators (PAMs) which potentiate glutamate function at mGlu4 (11,12,14,15,4,21,7,6); several of these tool compounds exhibit antiparkinsonian and neuroprotective effects in multiple rodent PD models (12,1,14,7,6). Unfortunately, many available compounds have lacked the necessary pharmacokinetic properties required for study of mGlu4 function via systemic routes of administration. Future compounds developed should exhibit sufficient potency, efficacy, and pharmacokinetic properties, including brain penetration, to make useful probes to progress mGlu4 biology, which will undoubtedly allow the intense study of mGlu4 activation in multiple areas of neuroscience such as psychiatric disorders (18,17), cancer (5), and addiction (2).
Potency and efficacy of compounds will be determined by performing concentration-response curves (CRCs, 10 points, ranging from approximately 30 uM-1 nM at 0.3% final DMSO concentration) at human mGlu4 using a calcium assay in which the cells express the chimeric G protein Gqi5 to couple mGlu4 to calcium mobilization (13,14,7,6). PAMs with EC50 values less than 500 nM versus human mGlu4 will next be evaluated for potency versus rat mGlu4 in a thallium flux assay measuring coupling of rat mGlu4 to G Protein-coupled Inwardly Rectifying Potassium (GIRK) Channels (13,6). Following potency evaluation at human and rat mGlu4, PAMs with EC50 values less than 500 nM at rat mGlu4 will be evaluated for their ability to left-shift the CRC of glutamate for rat mGlu4 in a thallium flux assay (13,14,7,6). PAMs with a fold-shift of the glutamate CRC of > 5 will then be evaluated for Tier 1 DMPK assays including plasma protein binding (PPB), intrinsic clearance, and inhibition of cytochrome p450 enzymes (CYP) (4,6). Next, compounds demonstrating PPB > 1% free and moderate clearance will be evaluated for CNS exposure and Plasma:Brain levels using an in vivo snapshot PK paradigm and will be examined for their selectivity for mGlu4 relative to other mGlu subtypes evaluated (4,6). Novel mGlu4 PAMs showing a CNS exposure > PAM EC50, a Brain:Plasma ratio of >0.5, and at least a 50-fold or greater selectivity versus other mGlu subtypes will next be evaluated in a preclinical model of PD: haloperidol-induced catalepsy (HIC) (20). mGlu4 PAMs demonstrating activity in HIC at a dose of < 30 mg/kg will have their ancillary pharmacology fully evaluated and those compounds that show no significant off-target activity, CaCo permeability > 10 cm/s-6, and suitable solubility will be declared an MLPCN probe. The ultimate goal of this project from the PI's perspective is to generate compounds which should exhibit sufficient potency, efficacy, and pharmacokinetic properties, including brain penetration, to make useful probes to progress mGlu4 biology.
1. Battaglia, G., C. L. Busceti, et al. (2006). "Pharmacological activation of mGlu4 metabotropic glutamate receptors reduces nigrostriatal degeneration in mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine." J Neurosci 26(27): 7222-9.
2. Blednov, Y. A., D. Walker, et al. (2004). "Mice lacking metabotropic glutamate receptor 4 do not show the motor stimulatory effect of ethanol." Alcohol 34(2-3): 251-9.
3. Conn, P. J., G. Battaglia, et al. (2005). "Metabotropic glutamate receptors in the basal ganglia motor circuit." Nat Rev Neurosci 6(10): 787-98.
4. Engers, D. W., C. M. Niswender, et al. (2009). "Synthesis and evaluation of a series of heterobiarylamides that are centrally penetrant metabotropic glutamate receptor 4 (mGluR4) positive allosteric modulators (PAMs)." J Med Chem 52(14): 4115-8.
5. Iacovelli, L., A. Arcella, et al. (2006). "Pharmacological activation of mGlu4 metabotropic glutamate receptors inhibits the growth of medulloblastomas." J Neurosci 26(32): 8388-97.
6. Jones, C. K., M. Bubser, et al. (2012). "The metabotropic glutamate receptor 4-positive allosteric modulator VU0364770 produces efficacy alone and in combination with L-DOPA or an adenosine 2A antagonist in preclinical rodent models of Parkinson's disease." J Pharmacol Exp Ther 340(2): 404-21.
7. Jones, C. K., D. W. Engers, et al. (2011). "Discovery, synthesis, and structure-activity relationship development of a series of N-4-(2,5-dioxopyrrolidin-1-yl)phenylpicolinamides (VU0400195, ML182): characterization of a novel positive allosteric modulator of the metabotropic glutamate receptor 4 (mGlu(4)) with oral efficacy in an antiparkinsonian animal model." J Med Chem 54(21): 7639-47.
8. Konieczny, J., J. Wardas, et al. (2007). "The influence of group III metabotropic glutamate receptor stimulation by (1S,3R,4S)-1-aminocyclo-pentane-1,3,4-tricarboxylic acid on the parkinsonian-like akinesia and striatal proenkephalin and prodynorphin mRNA expression in rats." Neuroscience 145(2): 611-20.
9. Macinnes, N. and S. Duty (2008). "Group III metabotropic glutamate receptors act as hetero-receptors modulating evoked GABA release in the globus pallidus in vivo." Eur J Pharmacol 580(1-2): 95-9.
10. MacInnes, N., M. J. Messenger, et al. (2004). "Activation of group III metabotropic glutamate receptors in selected regions of the basal ganglia alleviates akinesia in the reserpine-treated rat." Br J Pharmacol 141(1): 15-22.
11. Maj, M., V. Bruno, et al. (2003). "(-)-PHCCC, a positive allosteric modulator of mGluR4: characterization, mechanism of action, and neuroprotection." Neuropharmacology 45(7): 895-906.
12. Marino, M., O. Valenti, et al. (2003). "Glutamate receptors and Parkinson's disease : opportunities for intervention." Drugs Aging 20(5): 377-97.
13. Niswender, C. M., K. A. Johnson, et al. (2008a). "A novel assay of Gi/o-linked G protein-coupled receptor coupling to potassium channels provides new insights into the pharmacology of the group III metabotropic glutamate receptors." Mol Pharmacol 73(4): 1213-24.
14. Niswender, C. M., K. A. Johnson, et al. (2008b). "Discovery, characterization, and antiparkinsonian effect of novel positive allosteric modulators of metabotropic glutamate receptor 4." Mol Pharmacol 74(5): 1345-58.
15. Niswender, C. M., E. P. Lebois, et al. (2008c). "Positive allosteric modulators of the metabotropic glutamate receptor subtype 4 (mGluR4): Part I. Discovery of pyrazolo[3,4-d]pyrimidines as novel mGluR4 positive allosteric modulators." Bioorg Med Chem Lett 18(20): 5626-30.
16. Ossowska, K., J. Konieczny, et al. (2007). "An influence of ligands of metabotropic glutamate receptor subtypes on parkinsonian-like symptoms and the striatopallidal pathway in rats." Amino Acids 32(2): 179-88.
17. Stachowicz, K., E. Chojnacka-Wojcik, et al. (2006). "Anxiolytic-like effects of group III mGlu receptor ligands in the hippocampus involve GABAA signaling." Pharmacol Rep 58(6): 820-6.
18. Stachowicz, K., K. Klak, et al. (2004). "Anxiolytic-like effects of PHCCC, an allosteric modulator of mGlu4 receptors, in rats." Eur J Pharmacol 498(1-3): 153-6.
19. Valenti, O., M. J. Marino, et al. (2003). "Group III metabotropic glutamate receptor-mediated modulation of the striatopallidal synapse." J Neurosci 23(18): 7218-26.
20. Varty, G. B., R. A. Hodgson, et al. (2008). "The effects of adenosine A2A receptor antagonists on haloperidol-induced movement disorders in primates." Psychopharmacology (Berl) 200(3): 393-401.
21. Williams, R., C. M. Niswender, et al. (2009). "Positive allosteric modulators of the metabotropic glutamate receptor subtype 4 (mGluR4). Part II: Challenges in hit-to-lead." Bioorg Med Chem Lett 19(3): 962-6.
Human mGlu4 Potency
Cell line creation and culture of the human mGlu4/ Gqi5/CHO line. Human mGlu4 (hmGlu4)/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 mGlu4-mediated calcium mobilization using the method described below. hmGlu4/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.
Cells 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 following day, plated cells had their medium exchanged to Assay Buffer (Hank's balanced salt solution, 20 mM HEPES and 2.5 mM Probenecid (Sigma-Aldrich, St. Louis, MO)) using an ELX405 microplate washer (BioTek), leaving 20 uL/well, followed by addition of with 20 uL of 4.5 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 for 45 minutes at 37 degrees C. The dye was then exchanged to Assay Buffer using an ELX405, leaving 20 uL/well and the plates were incubated at room temperature for 10 min prior to assay. For concentration-response curve experiments, compounds were serially diluted 1:3 into 10 point concentration response curves in DMSO, were transferred to daughter plates using an Echo acoustic plate reformatter (Labcyte, Sunnyvale, CA), and diluted into Assay Buffer to generate a 2x stock. Calcium flux was measured using the Functional Drug Screening System 6000 or 7000 (FDSS6000 or FDSS7000, Hamamatsu, Japan). Baseline readings were taken (10 images at 1 Hz, excitation, 470+/-20 nm, emission, 540+/-30 nm) and then 20 uL/well test compounds were added using the FDSS's integrated pipettor. Approximately 2.5 minutes later an EC20 concentration of glutamate (10 uL of a 5x final concentration) was added followed approximately 2.0 minutes later by an EC80 concentration of glutamate (12 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).
Compounds with average EC50s greater than or equal to 30uM were assigned as 'Outcome' equals 'Inactive'. For compounds with average EC50s less than 30uM, 'Outcome' equals 'Active.' The 'Score' for 'Active' compounds was as follows: EC50 <30 to >= 5uM equals '50' and EC50 <5uM equals '100'.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)