Characterisation of the mechanism of action of positive allosteric modulators of the mGlu2 receptor

Andrew Weaver, Kirstie Bennett, Chris Langmead. Heptares Therapeutics, Welwyn Garden City, Herts., UK

The human metabotropic glutamate receptor subtype 2 (hmGlu2) has been implicated in many central nervous system disorders including schizophrenia and Alzheimer’s disease. The development of orthosteric drugs at hmGlu2 has largely failed due to lack of subtype selectivity, making allosteric modulators attractive alternatives as therapeutic ligands. Herein we study the action of three allosteric modulators, LY487379, BINA and JNJ-40068782 at the hmGlu2 receptor.

[3H]LY341495 saturation and competition binding experiments were performed on HEK293-hmGlu2 membranes (2.5 µg/well) using assay conditions previously described (Johnson et al., 1999). Three-way binding data was analysed using an extended allosteric ternary complex model which estimates the co-operativity of binding (α) between allosteric modulator and L-glutamate as well as the affinity of the allosteric modulator at the unoccupied receptor (Lazerano and Birdsall, 1995). For functional [35S]GTPγS assays, 5µg/well of hmGlu2 expressing Chem-1 cell membranes (Millipore, USA) were permeabilized by addition of saponin to an equal concentration by mass and mixed with buffer (20 mM HEPES, pH 7.4, 100 mM NaCl, 10 mM MgCl2, and 2 µM GDP) with a range of concentrations of L-glutamate and/or JNJ-40068782. After pre-incubation (15 minutes, 30 °C), 0.1 nM [35S]GTPγS was added before another incubation period (30 minutes, 30 °C). The binding reactions were then transferred to GF/B filters pre-wetted with water, washed three times with water, dried, and then counted. Data was analysed using an equation derived from the operational ternary complex model to determine the functional co-operativity between JNJ-40068782 and L-glutamate (discussed in Leach et al., 2007).

L-glutamate fully displaced [3H]LY341495 binding (pKI = 5.7 ± 0.2, n = 3), whilst BINA, LY487379, and JNJ-40068782 had no effect on equilibrium radioligand binding (neutral co-operativity; α = 1, n = 3). BINA, LY487379 and JNJ-40068782 all gave saturable increases in the affinity of L-glutamate for the mGlu2 receptor. Analysis according to an extended allosteric ternary complex model yielded estimates of affinity co-operativity between L-glutamate and each of the allosteric modulators (α = 4.8 ± 0.1, 2.3 ± 0.2 and 6.7 ± 0.4 for BINA, LY487379, and JNJ-40068782, respectively) as well as affinity estimates of the allosteric modulators for mGlu2 (pKI = 7.4 ± 0.1, 5.9 ± 0.2, and 5.8 ± 0.3, respectively n = 3). JNJ-40068782 was further studied and shown to be a positive allosteric modulator of LY354740 affinity (α = 4.4 ± 0.5). [35S]GTPγS binding assays showed that JNJ-40068782 acted as a partial agonist with respect to L-glutamate (pEC50 = 5.9 ± 0.1, Emax = 62% of L-glutamate Emax; n = 2). The potency of L-glutamate was also increased by the presence of JNJ-400687282. Using the operational ternary complex model the estimated functional co-operativity between L-glutamate and JNJ-40068782 was calculated (αβ = 42±1.6), suggesting that JNJ-40068782 exerts its effects primarily through changes in glutamate affinity.

Herein we confirm that BINA, LY487379 and JNJ-40068782 act as positive allosteric modulators of hmGlu2 receptor. Furthermore we were able to discern that increase in L-glutamate potency in the presence of JNJ-40068782 is mainly due to an increase in L-glutamate affinity rather than efficacy.

Johnson BG, Wright RA, Arnold MB, Wheeler WJ, Ornstein PL, and Schoepp DD (1999). Neuropharmacology 38:1519-1529.

Lazerano S, and Birdsall NJM (1995). Mol.Pharmacol. 48:362-378.

Leach K, Sexton PM, and Christopoulos A (2007). TiPS 28:382-389.