Specific Aim

To identify small molecule positive allosteric modulators (PAMs) of mGluR8 and/or other group III mGluRs. Probe candidates will be highly selective versus the other seven mGluRs, and ideally be suitable for both in vitro and in vivo studies.

Significance

The metabotropic glutamate receptors (mGluRs) are members of the GPCR family C, characterized by a large extracellular amino-terminal binding domain (agonist binding site) along with a seven-transmembrane spanning (7TM) domain which is the binding site for most known mGluR allosteric modulators. (1–3) The eight cloned mGluRs have been assigned to three groups (group I: mGluRs 1 and 5, group II: mGluRs 2 and 3, and group III: mGluRs 4,6,7,8) based on their structural similarity, ligand specificity, and preferred coupling mechanisms. (4) Among the mGluRs, the group III receptors have thus far received less attention in terms of their therapeutic potential because of the paucity of selective ligands. However, recently there have been numerous reports detailing the potential benefits of mGluR4 activation in several disease models, most notably rodent models of Parkinson’s disease. (5,6) Parkinson’s disease (PD) is caused by the death of dopamine neurons in the basal ganglia and results in motor symptoms such as tremor and bradykinesia. Activation of metabotropic glutamate receptor 4 (mGluR4) has been shown to modulate neurotransmission in the basal ganglia, a mechanism that is expected to provide palliative benefit for the treatment of Parkinson’s disease (PD). (7) In addition, there have been recent reports detailing the neuroprotective effects of an mGluR4 PAM in cultured neurons and in vivo. (8,9) However, the leading mGluR4 probe compound is N-Phenyl-7-(hydroxyimino) cyclopropa[b]chromen-1a-carboxamide (PHCCC), a positive allosteric modulator (PAM) of mGluR4 that has been used to further validate the role of mGluR4 in PD; unfortunately, the compound suffers from a lack of selectivity, relatively low potency and poor solubility/PK (Figure 1). (8,10,11) Moreover, it is not systemically active and must be administered i.c.v. to show efficacy in PD models. Clearly, there is room for significant improvement. (10,11) Our mGluR8 screen employed mGluR4 as a representative group III mGluR counter-screen, which led to the discovery of a highly optimized mGluR4 in vitro and in vivo probe.

Figure 1

(-)-PHCCC, the prototypical mGluR4 PAM - weak, non-selective, poor physiochemical properties and no CNS penetration.

Rationale

In the present study, we developed and implemented a fluorescence-based calcium assay for high-throughput screening (HTS) of chemical libraries for novel modulators of mGluR8 function, and developed the appropriate counter screens within the mGluR family to identify other selective mGluR ligands as well. (12)

PubChem Bioassay Name

A direct assay for HTS of G_i/G_o-linked GPCRs: mGluR8 as the prototype

List of PubChem bioassay identifiers generated for this screening project (AIDs)

AID-2199, AID-2197, AID-2193, AID-2191, AID-2190, AID-2188, AID-2185, AID-2183, AID-2182, AID-2181, AID-2180, AID-2179, AID-2437

PubChem Primary Assay Description

A thallium flux assay was performed with human mGluR8 cells. These cell lines were grown in growth media containing 45% DMEM, 45% Ham’s F-12, 10% FBS, 20 mM HEPES, 2 mM L-glutamine, antibiotic/antimycotic, nonessential amino acids, 700 mg/mL G418, and 0.6 μg/mL puromycin at 37 °C in the presence of 5% CO2. In brief, mGluR8 GIRK cells were plated into 384-well, black-walled, clear-bottomed, poly(D-lysine)-coated plates at a density of 15 x 10³ cells/20 μL/well in plating medium and incubated overnight at 37 °C in the presence of 5% CO₂. The following day, the medium from the cells and 20 μL/well of 1.7 μM concentration of the indicator dye BTC-AM (Invitrogen) in assay buffer was added. Cells were incubated for 1 h at room temperature and the dye was replaced with 20 μL/well of assay buffer. For these assays, compounds were added at two times the final concentration, and then 2.5 min later, either an EC₂₀ or EC₈₀ concentration of glutamate (mGluR4 or 8) was added using the FDSS 6000. Agonists were diluted in thallium buffer (125 mM sodium bicarbonate, 1 mM magnesium sulfate, 1.8 mM calcium sulfate, 5 mM glucose, 12 mM thallium sulfate, and 10 mM HEPES) at five times the final concentration to be assayed. Five frames of data were collected (excitation, 470 + 20 nm; emission, 540 + 30 nm) at 0.5 Hz before compound addition. Data collection continued at 0.5 Hz until 10 s before agonist addition, when the rate was increased to 1 Hz for 2 min after agonist addition. Human mGluR4 (hmGluR4)/CHO cells were stably transfected with the chimeric G protein G_qi5 in pIRESneo3 (Invitrogen, Carlsbad, CA), and single neomycin-resistant clones were isolated and screened for mGluR4-mediated calcium mobilization using the method described below. hmGluR4/CHO cells were cultured in 90% Dulbecco’s modified Eagle’s medium (DMEM), 10% dialyzed fetal bovine serum (FBS), 100 U/mL penicillin/streptomycin, 20 mM HEPES, pH 7.3, 1 mM sodium pyruvate, 2 mM glutamine, 400 μg/mL G418 sulfate, 20 μM proline (Mediatech, Inc., Herndon, VA), and 5 nM methotrexate (Calbiochem, EMD Chemicals, Gibbstown, NJ). Culture of human embryonic kidney (HEK) 293 cell lines co-expressing rat mGluR4 and the G protein-regulated inwardly rectifying K⁺ channel (GIRK) have been described. All cell culture reagents were purchased from Invitrogen unless otherwise noted.

Summary of Screen

The initial mGluR screens were performed during the pilot phase, the MLSCN, when the MLSMR compound collection at Vanderbilt only contained ~110,000 compounds. From the primary mGluR8 screen of 110,814 compounds in 384 well plates, few hits were identified, and the average Z’ score was 0.75. The confirmation screen (singles at 10 μM) produced no active compounds, but the counter-screen versus mGluR4 identified one mGluR4 positive allosteric modulator (PAM) hit, CID 85240633 (SID-308065) that was devoid of activity at mGluR8.

Chemical Probe Lead Optimization

Optimization efforts focused on our one mGluR4 PAM lead (CID 308065), which was comparable to (-)-PHCCC in terms of potency (EC₅₀~ 5 μM, 135% Glu Max, 5-fold shift), but with a molecular weight of only 250 and ligand efficiency (LE) of 0.33 (Figure 2). CID 308065 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.

Figure 2

Strucutre of mGluR4 PAM hit CID 308065 and SAR plan.

Our first library (Table 1) 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 (CID 836051) 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 μM. Two exceptions were a 2-pyridyl amide, CID 4644726 (EC₅₀ = 1.4 μM, 80% Glu Max) and a pyrimidine amide CID 44191096 (EC₅₀ = 2.8 μM, 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 (Table 2). (13)

Table 1

SAR of the Western Amide Moiety.

Table 2

SAR of the Eastern aniline.

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, CID 44191096, CID 44189740 and CID 42644786 were selective for mGluR4 (>30 μM versus mGluRs 1,2,3,5,7,8). CID 44191096 and CID 44189740 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. Figure 3A demonstrates that CID 44191096 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 (Figure 3B) of a fixed 30 μM concentration of CID 44191096 is ~33-fold. (13)

Figure 3

Potency and efficacy of the novel mGluR4 PAM, CID 44191096. (a) CID 44191096 was added in progressively higher concentrations to cell co-expressing human mGluR4 and the chimeric G protein Gqi5 (white boxes). After a 2.5 minute incubation period, an EC20 (more...)

While CID 44191096 will advance the field in terms of both in vitro molecular pharmacology and electrophysiology studies, we were compelled to go deeper and determine if CID 44191096 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 CID 44191096 to determine a broader ancillary pharmacology profile for this MLPCN probe. In addition to selectivity versus the mGluR family, CID 44191096 possessed clean ancillary pharmacology, displaying no significant activity (no inhibition >50% at 10 μM for any GPCR, ion cannel or transporter targets in the 68 target Lead Profiling Screen). (13)

In vitro and in vivo DMPK

Based on these data, we evaluated CID 44191096 and several other potent analogs in a panel of in vitro Drug Metabolism assays such as microsomal stability and plasma protein binding (Table 3). 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. CID 44191096 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, CID 44191096 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 CID 44191096 were determined. CID 44191096 had reasonable PK, but excitingly, was centrally penetrant. The AUC_brain/AUC_plasma ratio was found to be 4.1, making CID 44191096 the first mGluR4 PAM with central levels after systemic dosing.(13)

Table 3

In vitro Drug Metabolism evaluation.

In vivo efficacy in a preclinical model of Parkinson’s disease (PD)

Based on the potency, efficacy and PK of CID 44191096, we evaluated CID 44191096 in a preclinical model of PD – haloperidol-induced catalepsy. (14) In this model, rats are injected with haloperidol, a dopamine antagonist to induce catalepsy, and then our mGluR4 PAM (CID 44191096) is administered. Latency to withdrawal is then evaluated at four doses (3, 10, 30 and 56.6 mg/kg) of CID 85240643 and two time points (90 and 120 minutes) relative to an Adenosine A2A receptor antagonist positive control (Figure 4). (14) At 56.6 mg/kg, CID 44191096 was as effective as a gold standard A2A antagonist at the same dose. Thus, CID 44191096 is the first mGluR4 PAM to show efficacy in a preclinical PD model upon systemic dosing. CID 44191096 would therefore be a useful in vivo tool to the scientific community to study the role of selective mGluR4 activation in vivo.

Figure 4

Effect of CID 44191096 on haloperidol(0.75 mg/kg)-induced catalepsy relative to A2A receptor antagonist positive control.

Solubility

~ 100 μM in DMSO. Homogeneous/microsuspension at 10 mg/mL in : 10% Tween-80, and 40% PEG-400.

Synthetic procedure and Spectral data for CID 44191096

MLS#s

0022608909, 002608910, 002608911, 002608912, 002608913, 002608914 (probe, 500 mg)

Solubility

Solubility in PBS (at pH = 7.4) for ML128 was 6.38 μM.

Stability

Stability (at room temperature = 23 °C) for ML128 in PBS (no antioxidants or other protectorants and DMSO concentration below 0.1%) is shown in the table below. After 48 hours, the percent of parent compound remaining was 109%, but the assay variability over the course of the experiment ranged from a low of 85% (at 15 minutes) to a high of 130% (at 1 hour).

Reactivity

As assessed through a glutathione (GSH) trapping experiment in phosphate buffered saline (with a substrate concentration of typically 5–50 μM and a GSH concentration of 5 mM, at t = 60 minutes) ML128 was found to not form any detectable GSH adducts.^*