Development of an ex vivo receptor occupancy assay for the Class C GPCR mGlu₅

E. Sergeev, E. Howley, K. Bennett, M. Barnes. Heptares Therapeutics Ltd, Welwyn Garden City, United Kingdom.

Introduction: Receptor occupancy (RO) assays play an important role in drug development, as they confirm target engagement in the tissue of interest. We have recently developed HTL0014242, a negative allosteric modulator of mGlu₅, and demonstrated RO in rat hippocampus using autoradiography¹. However, such methodology requires specialised equipment and thus we have established an ex vivo RO assay using a tissue homogenate filtration-based format. Here, we report assay development and validation using [³H]M-MPEP and HTL0014242 in murine brain tissue.

Method: HTL0014242 was synthesized as established in¹. Forebrain was dissected from six adult male CD-1 mice. Membranes were prepared from three mice as described previously² and protein linearity was performed to assess specific binding of [³H]M-MPEP. Using forebrain membranes (30 μg/well), saturation and kinetic [³H]M-MPEP binding experiments were performed. Competition binding assays were used to determine the pK_iof HTL0014242. All were carried out as described in¹. In RO experiments tissue needs to be rapidly processed to avoid dosed compound dissociation, therefore tissue was prepared by crude homogenisation (Polytron Homogeniser; 7,000 rpm; 20 s) immediately prior to use in protein linearity assays. Using chosen conditions (1.4 mg homogenate/well), the preparation was employed to perform both steady-state saturation and kinetic association assays at 4°C to select conditions resulting in full mGlu₅ RO.

Results: Increasing protein concentration correlated with increased specific binding of [³H]M-MPEP and 30 μg forebrain membranes/well resulted in high specific binding without radioligand depletion (Figure 1). Saturation and kinetic binding assays allowed K_d and association/dissociation rates to be calculated, and competition binding assays established that HTL0014242 displays high affinity for the murine mGlu₅ (Table 1). By using high [³H]M-MPEP concentrations (20xK_d) but short incubation times, it was demonstrated that all mGlu₅ receptors in tissue homogenates were occupied by 10 minutes, as calculated by comparing specific [³H]M-MPEP binding to the B_max determined in steady-state saturation binding studies (Figure 2).

Table 1. mGlu₅ ligand binding in murine forebrain. K_d of [³H]M-MPEP was determined by saturation binding (SB) or kinetic binding (KB) assays. Data shown are mean ± SD and as aim was to establish methodology, an n of 2 was performed to limit animal use.

Conclusion: Expression of mGlu₅ was confirmed in murine forebrain and binding of [³H]M-MPEP and HTL0014242 was characterised. In tissue from dosed animals, performing a 10-minute association of 20xK_d [³H]M-MPEP at 4°C would allow the radioligand to occupy all unliganded mGlu₅ but should prevent dissociation of HTL0014242. This data will serve as the groundwork for the final HTL0014242 dosing study to confirm mGlu₅ occupancy in the murine brain.

References:

1. Christopher JA et al. (2015). J Med Chem. 58: 6653-64.

2. Bradley SJ et al. (2011) Mol Pharmacol. 79: 874-85.