Modulation of the Metabotropic Glutamate Receptor mGluR4
Screening Center Name & PI: Vanderbilt Screening Center for GPCRs, Ion Channels and Transporters, C. David Weaver ..more
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.
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Categorized Comment - additional comments and annotations
From MLP Probe Report:
Probe count: 3
MLP Probe ML# for probe 1: ML128
PubChem Substance ID (SID) for probe 1: 85240643
PubChem Compound ID (CID) for probe 1: 44191096
Probe type for probe 1: Modulator
IC50/EC50 (nM) for probe 1: 240 (human); 110 (rat)
Target for probe 1: mGluR4 (gi: 120660252)
Disease relevance for probe 1: Parkinson's disease
Anti-target for probe 1: mGluRs 1, 2, 3, 5, 7, 8
Fold selectivity for probe 1: >50
NCBI Book chapter link for probe 1: http://www.ncbi.nlm.nih.gov/books/NBK50684/ (ID: 2376216)
Grant number for probe 1: NS053536-01
MLP Probe ML# for probe 2: ML182
PubChem Substance ID (SID) for probe 2: 99309089
PubChem Compound ID (CID) for probe 2: 46869947
Probe type for probe 2: Modulator-Allosteric
IC50/EC50 (nM) for probe 2: <376
Target for probe 2: rmGlu4; hmGlu4 (gi: 120660252)
Disease relevance for probe 2: Parkinson's disease
Anti-target for probe 2: mGlu1,mGlu2, mGlu3, mGlu5, mGlu7, mGlu8, PanLabs (Ricerca)
Fold selectivity for probe 2: >50
NCBI Book chapter link for probe 2: http://www.ncbi.nlm.nih.gov/books/NBK143194/ (ID: 3034104)
Grant number for probe 2: NS053536-01
MLP Probe ML# for probe 3: ML228
PubChem Substance ID (SID) for probe 3: 111978103
PubChem Compound ID (CID) for probe 3: 46742353
Probe type for probe 3: Activator
IC50/EC50 (nM) for probe 3: <1600
Target for probe 3: HIF-1a (gi: 120660252)
Anti-target for probe 3: Proteasome Inhibition
NCBI Book chapter link for probe 3: http://www.ncbi.nlm.nih.gov/books/NBK143197/ (ID: 3034068)
Grant number for probe 3: MH082355-01A2
PubMed Publication ID (PMID) for probe 1: 19469556
PubMed Publication ID (PMID) for probe 2: 21966889
PubMed Publication ID (PMID) for probe 3: 22172704
NCBI Book chapter title for probe 1: Discovery of a potent, selective and in vivo active mGluR4 positive allosteric modulator
NCBI Book chapter title for probe 2: Discovery of a potent, selective and orally active in vivo mGlu4 positive allosteric modulator
NCBI Book chapter title for probe 3: Discovery of a Small Molecule Activator of the Hypoxia Inducible Factor Pathway
Data Table (Concise)