Context-dependent pharmacology exhibited by negative allosteric modulators of metabotropic glutamate receptor 7

Abstract

Phenotypic studies of mice lacking metabotropic glutamate receptor subtype 7 (mGluR7) suggest that antagonists of this receptor may be promising for the treatment of central nervous system disorders such as anxiety and depression. Suzuki et al. (J Pharmacol Exp Ther 323:147-156, 2007) recently reported the in vitro characterization of a novel mGluR7 antagonist called 6-(4-methoxyphenyl)-5-methyl-3-(4-pyridinyl)-isoxazolo[ 4,5-c]pyridin-4(5H)-one (MMPIP), which noncompetitively inhibited the activity of orthosteric and allosteric agonists at mGluR7. We describe that MMPIP acts as a noncompetitive antagonist in calcium mobilization assays in cells coexpressing mGluR7 and the promiscuous G protein G alpha(15). Assessment of the activity of a small library of MMPIP-derived compounds using this assay reveals that, despite similar potencies, compounds exhibit differences in negative cooperativity for agonist-mediated calcium mobilization. Examination of the inhibitory activity of MMPIP and analogs using endogenous G(i/o)-coupled assay readouts indicates that the pharmacology of these ligands seems to be context-dependent, and MMPIP exhibits differences in negative cooperativity in certain cellular backgrounds. Electrophysiological studies reveal that, in contrast to the orthosteric antagonist (2S)-2-amino-2-[(1S,2S)-2-carboxyclycloprop-1-yl]-3-(xanth-9-yl) propanoic acid (LY341495), MMPIP is unable to block agonist-mediated responses at the Schaffer collateral-CA1 synapse, a location at which neurotransmission has been shown to be modulated by mGluR7 activity. Thus, MMPIP and related compounds differentially inhibit coupling of mGluR7 in different cellular backgrounds and may not antagonize the coupling of this receptor to native G(i/o) signaling pathways in all cellular contexts. The pharmacology of this compound represents a striking example of the potential for context-dependent blockade of receptor responses by negative allosteric modulators.

Fig. 1.

MMPIP antagonizes responses in mGluR7/BHK/Gα₁₅ cells in a noncompetitive fashion. A, cells coexpressing rat mGluR7a and the promiscuous G protein Gα₁₅ were incubated with increasing concentrations of MMPIP before the addition of an EC₈₀ concentration of the group III mGluR agonist l-AP4, and calcium mobilization was measured. EC₅₀ = 70.3 ± 20.4 nM. B, l-AP4 concentration-response curves were performed in the presence of increasing concentrations of MMPIP. Values represent mean ± S.E.M. of three independent experiments performed in quadruplicate.

Fig. 2.

MMPIP analogs exhibit a range of efficacies for inhibition of mGluR7-mediated calcium mobilization. A, compounds were preapplied to mGluR7a/BHK/Gα₁₅ cells before the addition of an EC₈₀ concentration of l-AP4, and calcium mobilization was measured. B, selected compounds were assessed in full concentration-response format for the inhibition of mGluR7-mediate calcium mobilization. MMPIP data are replotted from Fig. 1B for comparison. Potencies were as follows: VUSC001, 79.5 ± 11.8 nM; VUSC027, 49.2 ± 21.0 nM. Maximal inhibition values for each compound (calculated the lowest point of the curve fit as determined using a four site logistical equation in GraphPad Prism) were as follows: MMPIP, 100.7 ± 1.1%, VUSC001, 83.1 ± 4.1%; and VUSC027, 62.3 ± 7.0%. Values represent the mean ± S.E.M. of at least three experiments performed in quadruplicate.

Fig. 3.

MMPIP analogs exhibit lower efficacies in thallium flux assays, and the potency of MMPIP is lower than in calcium mobilization studies. A, compounds were preapplied to mGluR7a/HEK/GIRK cells before the addition of an EC₈₀ concentration of l-AP4, and thallium flux through the GIRK channel was measured. B, MMPIP concentration-response in thallium flux assays using cells expressing mGluR4, mGluR7, mGluR8, and M4 muscarinic receptor. In each case, concentration-response curves were applied to cells before an appropriate EC₈₀ concentration of either l-AP4 (mGluR4, 7, and 8) or acetylcholine (M4). The potency of MMPIP in these experiments was 2.0 ± 0.2 μM. C, corrected concentration-response curve for MMPIP after normalizing for nonspecific effects of the compound on M4/GIRK cells. Methods for this correction are described under Materials and Methods. Revised potency of MMPIP, 717 ± 89 nM. Values represent mean ± S.E.M. and are an n of three experiments performed in quadruplicate.

Fig. 4.

The level of MMPIP blockade is not probe-dependent in the GIRK/thallium flux assay. A 1 μM final concentration of MMPIP was preapplied before the addition of increasing concentrations of l-AP4 (A) or glutamate (B), and thallium flux was measured. The response remaining was 71 ±3.8% for l-AP4 (A) and 67.7 ± 6.8%, glutamate. Data represent the mean ± S.E.M. of three independent experiments performed in quadruplicate.

Fig. 5.

MMPIP raises basal cAMP levels and fails to effectively block l-AP4-mediated inhibition of cAMP accumulation. A, MMPIP induced a concentration-dependent increase in cAMP accumulation in mGluR7/HEK cells (EC₅₀ = 690 ± 130 nM). B, in contrast to blockade obtained in the presence of 100 μM concentration of the orthosteric antagonist LY341495 (●), a 1 μM concentration of MMPIP (□) increased basal cAMP levels but did not effectively block l-AP4-mediated inhibition of cAMP accumulation (compared maximal blockade to curve of l-AP4 alone, ■). Data represent the mean ± S.E.M. of three independent experiments performed in triplicate.

Fig. 6.

l-AP4 and MMPIP induce opposing responses in mGluR7/HEK cells using Epic technology. mGluR7/HEK cells were plated into 384-well Epic plates as described under Materials and Methods. Increasing concentrations of l-AP4 (A) or MMPIP (B) were applied to cells, and Epic responses were measured.

Fig. 7.

MMPIP antagonizes l-AP4 responses at mGluR7 with distinct and saturable efficacies in mGluR7/HEK and mGluR7/BHK cells as assessed using Epic technology. Increasing concentrations of l-AP4 were added to mGluR7/BHK (A) or mGluR7/HEK (B) cells in the presence (□) and absence (■) of 1 μM MMPIP and the responses, measured as a change in refractive index indicative of cellular mass distribution, were determined. C. increasing concentrations of MMPIP were incubated with mGluR7/HEK (■) or mGluR4/HEK (□) cells in the absence of agonist. MMPIP induced decreases in refractive index in cells expressing mGluR7 but not cells expressing mGluR4. D, increasing concentrations of MMPIP were incubated with mGluR7/BHK (●) mGluR7/HEK (○) cells in the absence or presence of an EC₈₀ concentration of l-AP4. Individual responses in the presence of l-AP4 were subtracted by the response induced by MMPIP in the absence of l-AP4. The potency of MMPIP in mGluR7/BHK cells was 50 ± 15 nM and in mGluR7/HEK cells was 156 ± 28 nM. The percentage of maximal blockade was 75.2 ± 3.3% in mGluR7/BHK cells and 46.0 ± 1.8% in mGluR7/HEK cells Data for each panel represent mean ± S.E.M. of three independent experiments performed in quadruplicate.

Fig. 8.

MMPIP does not block l-AP4-mediated depression of synaptic transmission at the Schaffer collateral-CA1 synapse. fEPSPs were measured at the SC-CA1 synapse in adult rat hippocampus. A, the effect of a submaximal concentration of l-AP4 on fEPSP slope (400 μM) over time is shown (dark symbols). This response was significantly blocked by a 5-min preincubation with 100 μM LY341495 (open symbols). After normalizing for responses across slices, the maximal effect of LY341495 over a 5-min period was quantified as shown in Fig. 8C (*, p = 0.0065). B, a 5-min preincubation with 10 μM MMPIP before l-AP4 addition had no significant effect on fEPSP slope (quantified in D). Data are from four to five slices derived from two to three animals.