Allosteric modulation of group III metabotropic glutamate receptor 4: A potential approach to Parkinson’s disease treatment Michael J. Marino*†, David L. Williams, Jr.*, Julie A. O’Brien‡, Ornella Valenti‡, Terrence P. McDonald, Michelle K. Clements, Ruiping Wang, Anthony G. DiLella, J. Fred Hess, Gene G. Kinney, and P. Jeffrey Conn§ Department of Neuroscience, Merck Research Laboratories, West Point, PA 19486 Communicated by Edward M. Scolnick, Merck Research Laboratories, West Point, PA, September 5, 2003 (received for review May 13, 2003) Parkinson’s disease (PD) is a debilitating movement disorder that nergic tone leads to an imbalance between direct inhibition and afflicts >1 million people in North America. Current treatments indirect excitation, such that an excessive basal ganglia output focused on dopamine-replacement strategies ultimately fail in disrupts motor behavior. As indicated on Fig. 1, surgical inter- most patients because of loss of efficacy and severe adverse effects ventions bypass the dopamine system and produce a decrease in that worsen as the disease progresses. The recent success of basal ganglia outflow that results in palliative benefit. Therefore, surgical approaches suggests that a pharmacological intervention a pharmacological intervention that mimics these surgical meth- that bypasses the dopamine system and restores balance in the ods could provide palliative benefit to a larger number of basal ganglia motor circuit may provide an effective treatment patients without the need for invasive surgery. Furthermore, by strategy. We previously identified the metabotropic glutamate bypassing the dopamine system, such a treatment should pro- receptor 4 (mGluR4) as a potential drug target and predicted that duce fewer side effects and may actually slow the disease process selective activation of mGluR4 could provide palliative benefit in by normalizing overactive glutamatergic input to midbrain PD. We now report that N-phenyl-7-(hydroxylimino)cyclopropa[b]- dopamine-containing neurons. chromen-1a-carboxamide (PHCCC) is a selective allosteric potenti- We have been interested in the role of metabotropic glutamate ator of mGluR4. This compound selectively potentiated agonist- receptors (mGluRs) in the function of the basal ganglia and the induced mGluR4 activity in cultured cells expressing this receptor potential that these receptors may provide useful targets for the and did not itself act as an agonist. Furthermore, PHCCC potenti- treatment of PD (for review, see ref. 8). Recent studies indicate -ated the effect of L-(؉)-2-amino-4-phosphonobutyric acid in inhib- that mGluR4 decreases GABAergic transmission at the inhibi iting transmission at the striatopallidal synapse. Modulation of the tory striatopallidal synapse within the basal ganglia (9). In PD, striatopallidal synapse has been proposed as a potential therapeutic increased GABAergic inhibition at this synapse is thought to target for PD, in that it may restore balance in the basal ganglia motor play an important role in motor dysfunction (Fig. 1). Based on circuit. Consistent with this, PHCCC produced a marked reversal of this concept, reduction of ␥-aminobutyric acid (GABA) release reserpine-induced akinesia in rats. The closely related analogue at the striatopallidal synapse by a selective agonist of mGluR4 7-(hydroxylimino)cyclopropachromen-1a-carboxamide ethyl ester, has been suggested as an approach for the treatment of PD. which does not potentiate mGluR4, had no effect in this model. Recently, allosteric modulators of several family C G protein- These results are evidence for in vivo behavioral effects of an coupled receptors (including two of the three groups of mGluRs) allosteric potentiator of mGluRs and suggest that potentiation of have been described, which suggested to us that mGluR4 might mGluR4 may be a useful therapeutic approach to the treatment be amenable to allosteric modulation. We have found that of PD. the group I antagonist N-phenyl-7-(hydroxylimino)cyclopropa- [b]chromen-1a-carboxamide (PHCCC) (10) is a potentiator of arkinson’s disease (PD) is a debilitating neurodegenerative human and rat mGluR4. PHCCC did not itself exhibit mGluR4 Pdisorder that afflicts Ϸ1% of people older than 55 years. The agonist activity, and the closely related analogue 7-(hydroxyl- primary pathology underlying PD is a degeneration of neurons imino)cyclopropa[b]chromen-1a-carboxamide ethyl ester (CPC- in the substantia nigra pars compacta (1). The finding that these COEt) (10) had no mGluR4 potentiator activity, suggesting it neurons are dopaminergic cells that provide a dense innervation might be useful as a control for in vivo studies. Characterization of the striatum (2) led to the development of dopamine- of PHCCC revealed that it does not potentiate or activate any replacement therapies for the treatment of this disease. Drugs other mGluR subtype but acts as an antagonist of some of the such as the dopamine precursor L-dopa and dopamine receptor mGluRs. In brain-slice electrophysiological studies of the rat agonists provide dramatic amelioration of the motor signs of PD striatopallidal synapse, PHCCC was found to potentiate the effect at early stages of the disease. However, prolonged treatment of the mGluR4 agonist L-(ϩ)-2-amino-4-phosphonobutyric acid with these drugs leads to a loss of reliable efficacy and a variety (L-AP4) in inhibiting transmission. Finally, PHCCC was found of motor and cognitive side effects (3). In addition, disagreement still exists as to whether or not L-dopa therapy may actually speed disease progression through increased oxidative damage (for Abbreviations: CPCCOEt, 7-(hydroxylimino)cyclopropa[b]chromen-1a-carboxamide ethyl review, see refs. 4 and 5). Therefore, interest has been renewed ester; GPe, external globus pallidus; L-AP4, L-(ϩ)-2-amino-4-phosphonobutyric acid; PD, Parkinson’s disease; PHCCC, N-phenyl-7-(hydroxylimino)cyclopropa[b]chromen-1a- in the design of therapeutic methods that bypass the dopamine carboxamide; FLIPR, fluorometric imaging plate reader; MPEP, 2-methyl-6-(phenylethy- system. nyl)-pyridine; mGluR, metabotropic glutamate receptor; GABA, ␥-aminobutyric acid. One such method has been suggested by the recent resurgence *M.J.M. and D.L.W. contributed equally to this work. and advances in surgical interventions such as pallidotomy or †To whom correspondence should be addressed at: Department of Neuroscience, Merck deep-brain stimulation. These approaches have led to both Research Laboratories, Merck & Co., Inc., WP46-200, 770 Sumneytown Pike, West Point, PA dramatic palliative benefits for PD patients and an unprece- 19486. E-mail: michael࿝[email protected]. dented refinement of the model of basal ganglia dysfunction ‡J.A.O. and O.V. contributed equally to this work. associated with PD (for review, see refs. 6 and 7). In brief, this §Present address: Department of Pharmacology, Vanderbilt University Medical Center, model suggests that two pathways exist between the striatum and 23rd Avenue South at Pierce, 452-B Preston Research Building, Nashville, TN 37232-6600. the basal ganglia output nuclei. In PD, loss of striatal dopami- © 2003 by The National Academy of Sciences of the USA 13668–13673 ͉ PNAS ͉ November 11, 2003 ͉ vol. 100 ͉ no. 23 www.pnas.org͞cgi͞doi͞10.1073͞pnas.1835724100 Downloaded by guest on October 2, 2021 care and use of animals. Animals were group-housed with access to food and water ad libitum. Brain-Slice Preparation. All experiments were performed on brain slices from 26- to 30-day-old Sprague–Dawley rats (Taconic Farms). Animals were killed by decapitation, and their brains were rapidly removed and submerged in an ice-cold solution containing the following (in millimolar): choline chloride, 126; KCl, 2.5; NaH2PO4, 1.2; MgCl2, 1.3; MgSO4, 8; glucose, 10; and ͞ NaHCO3, 26 equilibrated with 95% O2 5% CO2 (13). The brain was glued to the chuck of a vibrating blade microtome (Leica Microsystems, Nussloch, Germany) and parasagittal slices (300- ␮m-thick) were obtained. Slices were immediately transferred to a 500-ml holding chamber containing artificial cerebrospinal fluid (in millimolar): NaCl, 124; KCl, 2.5; MgSO4, 1.3; NaH2PO4, 1.0; CaCl2, 2; glucose, 20; and NaHCO3, 26; equilibrated with ͞ 95% O2 5% CO2 that was maintained at 32°C. After 20 min at 32°C, the holding chamber was allowed to gradually decrease to room temperature. In all experiments, 5 ␮M glutathione, 500 ␮M pyruvate, and 250 ␮M kynurenic acid were included in the choline chloride buffer and in the holding chamber artificial cerebrospinal fluid. Electrophysiology. Whole-cell patch-clamp recordings were ob- tained as described (9). During recording, brain slices were Fig. 1. Model of the basal ganglia motor circuit. (A) In the normal state, the maintained fully submerged on the stage of a 1-ml brain-slice direct and indirect projections from the striatum to the output nuclei are chamber at 32°C and perfused continuously with equilibrated balanced by striatal dopaminergic tone. (B) In PD, the loss of striatal dopamine artificial cerebrospinal fluid (2–3ml͞min). Neurons were visu- produces an imbalance in the direct and indirect pathways such that an alized by using a differential interference contrast microscope excessive excitation of the output nuclei leads to a large increase in basal ganglia outflow. The sites of action for dopamine-replacement therapy and and an infrared video system. Patch electrodes were pulled from borosilicate glass on a two-stage puller and had resistances in the for surgical interventions