Bookmark and Share
BioAssay: AID 2437

Modulation of the Metabotropic Glutamate Receptor mGluR4

Screening Center Name & PI: Vanderbilt Screening Center for GPCRs, Ion Channels and Transporters, C. David Weaver ..more
 Tested Compounds
 Tested Compounds
 Tested Substances
 Tested Substances
AID: 2437
Data Source: Vanderbilt Screening Center for GPCRs, Ion Channels and Transporters (mGluR4_PAM_sum)
BioAssay Type: Summary, Candidate Probes/Leads with Supporting Evidence
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2010-02-26
Modify Date: 2011-01-05

Data Table ( Complete ):           Active    All
Sequence: Glutamate receptor, metabotropic 4 [Homo sapiens]
Description ..   
Protein Family: 7 transmembrane sweet-taste receptor of 3 GCPR

Gene:GRM4     Related Protein 3D Structures     More BioActivity Data..
BioActive Compound: Chemical Probe: 1    Active: 1
Depositor Specified Assays
Show more
2181Modulation of the Metabotropic Glutamate Receptor mGluR4: Selectivity at mGluR8confirmatory
2182Modulation of the Metabotropic Glutamate Receptor mGluR4: Selectivity at mGluR7confirmatory
2188Modulation of the Metabotropic Glutamate Receptor mGluR4: Selectivity at mGluR2confirmatory
2191Modulation of the Metabotropic Glutamate Receptor mGluR4: Selectivity at mGluR6confirmatory
2190Modulation of the Metabotropic Glutamate Receptor mGluR4: Selectivity at mGluR3confirmatory
2193Modulation of the Metabotropic Glutamate Receptor mGluR4: Selectivity at mGluR1confirmatory
2199Modulation of the Metabotropic Glutamate Receptor mGluR4: Selectivity at mGluR5confirmatory
2179Modulation of Metabotropic Glutamate Receptor mGluR4: Rat PAM Fold-Shiftconfirmatory
2183Modulation of the Metabotropic Glutamate Receptor mGluR4: Selectivity at mGluR4confirmatory
2185Modulation of the Metabotropic Glutamate Receptor mGluR4: Rat PAM Potencyconfirmatory
2180Modulation of Metabotropic Glutmate Receptor mGluR4: Human PAM Fold-Shiftconfirmatory
2197Modulation of the Metabotropic Glutamate Receptor mGluR4: Potency at human mGluR4confirmatory
2807Modulation of the Metabotropic Glutamate Receptor mGluR4: Calcium Assayconfirmatory
488971Rat PAM Fold-shift Assay with mGluR4 GIRKconfirmatoryfold shift assay for positive allosteric modulation of rat mGlu4
Assigned Assay Grant Number: NS053536-01
Screening Center Name & PI: Vanderbilt Screening Center for GPCRs, Ion Channels and Transporters, C. David Weaver
Chemistry Center Name & PI: Vanderbilt Specialized Chemistry Center for Accelerated Probe Development, Craig W. Lindsley
Assay Submitter & Institution: Colleen M. Niswender, Vanderbilt University

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 (mGluRs), including the group III mGluRs (mGluR4, -7 and -8), play important roles in regulating transmission through the BG and could serve as targets for novel PD therapeutics (Conn et al., 2005). For instance, mGluR4 activation reduces overactive GABA release at a specific inhibitory BG synapse (Macinnes and Duty, 2008; Marino et al., 2003; Valenti et al., 2003) and reverses motor deficits in a variety of rodent PD models (Konieczny et al., 2007; MacInnes et al., 2004; Marino et al., 2003; Ossowska et al., 2007; Valenti et al., 2003).

To more selectively activate mGluR4 and improve upon the pharmacokinetic liabilities of glutamate analogs, we and others have developed novel positive allosteric modulators (PAMs) which potentiate glutamate function at mGluR4 (Engers et al., 2009; Maj et al., 2003; Marino et al., 2003; Niswender et al., 2008a; Niswender et al., 2008b; Williams et al., 2008); several of these tool compounds exhibit antiparkinsonian and neuroprotective effects in multiple rodent PD models (Battaglia et al., 2006; Marino et al., 2003; Niswender et al., 2008a). Unfortunately, many available compounds have lacked pharmacokinetic properties to make them useful tools for study of mGluR4 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 mGluR4 biology, which will undoubtedly allow the intense study of mGluR4 activation in multiple areas of neuroscience such as psychiatric disorders (Stachowicz et al., 2006; Stachowicz et al., 2004), cancer (Iacovelli et al., 2006), and addiction (Blednov et al., 2004).

In the present study, we identified an mGluR4 PAM via an internal program (lead compound VU0153064) and optimized the compound to improve potency, efficacy, and pharmacokinetic properties; primary in vitro assays for mGluR4 have been previously described (Niswender et al., 2008a).

Chemical Probe Lead Optimization: Optimization efforts focused on the mGluR4 PAM lead (SID 85240633), which was comparable to (-)-PHCCC in terms of potency (EC50~ 5 uM, 135% Glu Max, 5-fold shift), but with a molecular weight of only 250 and ligand efficiency (LE) of 0.33. SID 85240633 represented a very attractive starting point for lead optimization in that small libraries could be rapidly prepared to survey the Western amide moiety and the Eastern aniline moiety employing simple amide coupling or acylation chemistry with a diverse array of commercially available building blocks. Our goal was to develop a best-in-class mGluR4 PAM (EC50 < 500 nM, fold-shift >10) with selectivity versus the other 7 mGluRs and ideally centrally penetrant to enable the study of selective mGluR4 activation in vivo and advance the field.

Our first library maintained the Eastern 3,4-dichloroaniline moiety and surveyed alternative amide moieties (heteroaryl, aryl and cycloalkyl). Like many allosteric ligands, SAR was shallow, with few actives from this effort. A 5-bromofuran (SID 85240634) showed slight improvement in potency, but the corresponding 5-phenyl congener was inactive. The majority of other groups explored (aryl, heteroaryl and cycloalkly) possessed EC50s >10 uM. Two exceptions were a 2-pyridyl amide, SID 85240642 (EC50 = 1.4 uM, 80% Glu Max) and a pyrimidine amide SID 85240643 (EC50 = 2.8 uM, 83% Glu Max). Based on the potency and physiochemical properties imparted by the 2-pyridyl amide, we next held this moiety constant, and surveyed alterative anilines and heterocyclic amines with diverse substiutents (AID 2197, AID 2185, AID 2180, AID 2179) (Engers et al., 2009).

This second generation library exploring alternatives for the Western 2-furyl amide was more productive. Multiple halo- and or alkoxy-substituted benzamides afforded potent mGluR4 PAMs with submicromolar EC50s, Glu Max values over 200% and fold-shifts of >30 for both human and rat. Multiple analogs represented a significant improvement over (-)-PHCCC. The most potent analogs, SID 85240643, SID 85240647 and SID 85240644 were selective for mGluR4 (>30 uM versus mGluRs 1,2,3,5,7,8). SID 85240643 and SID 85240647 met MLPCN probe criteria with mGluR4 PAM EC50s of 240 nM/110 nM (human/rat) and 340 nM/80 nM (human/rat), respectively. Both provided high efficacy (>100% Glu Max for rat and >200% Glu Max for human) and fold-shifts in the 20 to 40-fold range. Both are orders of magnitude better than the gold standard mGluR4 probe, (-)-PHCCC. SID 85240643 is in fact a positive allosteric modulator, having no effect on mGluR4 activation alone, but in the presence of an EC20 concentration of glutamate affords a dose-dependent increase in mGluR4 activation. The left-ward fold-shift of a fixed 30 uM concentration of SID 85240643 is ~33-fold (Engers et al., 2009).

While SID 85240643 will advance the field in terms of both in vitro molecular pharmacology and electrophysiology studies, we were compelled to go deeper and determine if SID 85240643 would be suitable for in vivo studies. At this point, the Lead Profiling Screen (68 GPCRs, ion channels and transporters) from MDS Pharma was performed on SID 85240643 to determine a broader ancillary pharmacology profile for this MLPCN probe. In addition to selectivity versus the mGluR family, SID 85240643 possessed clean ancillary pharmacology, displaying no significant activity (no inhibition >50% at 10 uM for any GPCR, ion cannel or transporter targets in the 68 target Lead Profiling Screen) (Engers et al., 2009).

In vitro and in vivo DMPK: Based on these data, we evaluated SID 85240643 and several other potent analogs in a panel of in vitro Drug Metabolism assays such as microsomal stability and plasma protein binding (Engers et al., 2009). In both human and rat liver microsomes (modeling PhI oxidative metabolism), the compounds displayed poor to moderate stability --not unexpected due to the amide moiety in the face of microsomal amidases. SID 85240643 in particular displayed poor metabolic stability in both human and rat liver microsomes (5.3% and 1.8%, respectively, after 30 minute incubation). Despite the poor in vitro stability, SID 85240643 was then dosed i.p. at 10 mg/kg as a microsuspension in 10% Tween-80 to male rats. Plasma and brain samples were taken at 0.5, 1 and 8 hours after administration and levels of SID 85240643 were determined. SID 85240643 had reasonable PK, but excitingly, was centrally penetrant. The AUCbrain/AUCplasma ratio was found to be 4.1, making SID 85240643 the first mGluR4 PAM with central levels after systemic dosing (Engers et al., 2009).

Based on the potency, efficacy and PK of SID 85240643, we evaluated SID 85240643 in a preclinical model of PD- haloperidol-induced catalepsy (Varty et al., 2008). In this model, rats are injected with haloperidol, a dopamine antagonist to induce catalepsy, and then our mGluR4 PAM (SID 85240643) is administered. Latency to withdrawal is then evaluated at four doses (3, 10, 30 and 56.6 mg/kg) of SID 85240643 and two time points (90 and 120 minutes) relative to an Adenosine A2A receptor antagonist positive control (Varty et al., 2008). At 56.6 mg/kg, SID 85240643 was as effective as a gold standard A2A antagonist at the same dose. Thus, SID 85240643 is the first mGluR4 PAM to show efficacy in a preclinical PD model upon ssystemic dosing. SID 85240643 would therefore be a useful in vivo tool to the scientific community to study the role of selective mGluR4 activation in vivo.


1. Battaglia G, Busceti CL, Molinaro G, Biagioni F, Traficante A, Nicoletti F and Bruno V (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):7222-7229.
2. Blednov YA, Walker D, Osterndorf-Kahanek E and Harris RA (2004) Mice lacking metabotropic glutamate receptor 4 do not show the motor stimulatory effect of ethanol. Alcohol 34(2-3):251-259.
3. Conn PJ, Battaglia G, Marino MJ and Nicoletti F (2005) Metabotropic glutamate receptors in the basal ganglia motor circuit. Nat Rev Neurosci 6(10):787-798.
4. Engers DW, Niswender CM, Weaver CD, Jadhav S, Menon UN, Zamorano R, Conn PJ, Lindsley CW and Hopkins CR (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):4115-4118.
5. Iacovelli L, Arcella A, Battaglia G, Pazzaglia S, Aronica E, Spinsanti P, Caruso A, De Smaele E, Saran A, Gulino A, D'Onofrio M, Giangaspero F and Nicoletti F (2006) Pharmacological activation of mGlu4 metabotropic glutamate receptors inhibits the growth of medulloblastomas. J Neurosci 26(32):8388-8397.
6. Konieczny J, Wardas J, Kuter K, Pilc A and Ossowska K (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):611-620.
7. Macinnes N and Duty S (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):95-99.
8. MacInnes N, Messenger MJ and Duty S (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.
9. Maj M, Bruno V, Dragic Z, Yamamoto R, Battaglia G, Inderbitzin W, Stoehr N, Stein T, Gasparini F, Vranesic I, Kuhn R, Nicoletti F and Flor PJ (2003) (-)-PHCCC, a positive allosteric modulator of mGluR4: characterization, mechanism of action, and neuroprotection. Neuropharmacology 45(7):895-906.
10. Marino MJ, Williams DL, Jr., O'Brien JA, Valenti O, McDonald TP, Clements MK, Wang R, DiLella AG, Hess JF, Kinney GG and Conn PJ (2003) Allosteric modulation of group III metabotropic glutamate receptor 4: a potential approach to Parkinson's disease treatment. Proc Natl Acad Sci U S A 100(23):13668-13673.
11. Niswender CM, Johnson KA, Weaver CD, Jones CK, Xiang Z, Luo Q, Rodriguez AL, Marlo JE, de Paulis T, Thompson AD, Days EL, Nalywajko T, Austin CA, Williams MB, Ayala JE, Williams R, Lindsley CW and Conn PJ (2008a) Discovery, characterization, and antiparkinsonian effect of novel positive allosteric modulators of metabotropic glutamate receptor 4. Mol Pharmacol 74(5):1345-1358.
12. Niswender CM, Lebois EP, Luo Q, Kim K, Muchalski H, Yin H, Conn PJ and Lindsley CW (2008b) 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):5626-5630.
13. Ossowska K, Konieczny J, Wardas J, Pietraszek M, Kuter K, Wolfarth S and Pilc A (2007) An influence of ligands of metabotropic glutamate receptor subtypes on parkinsonian-like symptoms and the striatopallidal pathway in rats. Amino Acids 32(2):179-188.
14. Stachowicz K, Chojnacka-Wojcik E, Klak K and Pilc A (2006) Anxiolytic-like effects of group III mGlu receptor ligands in the hippocampus involve GABAA signaling. Pharmacol Rep 58(6):820-826.
15. Stachowicz K, Klak K, Klodzinska A, Chojnacka-Wojcik E and Pilc A (2004) Anxiolytic-like effects of PHCCC, an allosteric modulator of mGlu4 receptors, in rats. Eur J Pharmacol 498(1-3):153-156.
16. Valenti O, Marino MJ, Wittmann M, Lis E, DiLella AG, Kinney GG and Conn PJ (2003) Group III metabotropic glutamate receptor-mediated modulation of the striatopallidal synapse. J Neurosci 23(18):7218-7226.
17. Williams R, Niswender CM, Luo Q, Le U, Conn PJ and Lindsley CW (2008) Positive allosteric modulators of the metabotropic glutamate receptor subtype 4 (mGluR4). Part II: Challenges in hit-to-lead. Bioorg Med Chem Lett.
18. Engers, D.W.; Niswender, C.M.; Weaver, C.D.; Jadhav, S.; Menon, U.; Conn, P.J.; Lindsley, C.W.; Hopkins, C.R. J. Synthesis and evaluation of a series of heterobiarylamides that are centrally penetrant metabotropic glutamate receptor 4 (mGluR4) positive allosteric modulators (PAMs). Med. Chem. 2009, 52, 4115-4118.
19. Varty, G.B.; Hodgson, R.A.; Pond, A.J.; Grzelak, M.E.; Parker, E.M.; Hunter, J.C. The effects of adenosine A2A receptor antagonists on haloperidol-induced movement disorders in primates. Psychopharmacology 2008, 200(3), 393-401.
Result Definitions
OutcomeThe BioAssay activity outcomeOutcome
Additional Information
Grant Number: NS053536

Data Table (Concise)