Novel mGluR4 Radioligands Reveal Distinct Sites for Positive Allosteric Modulators

Two novel positive allosteric modulators (PAMs) of the human mGluR4 receptor were optimized by medicinal chemistry efforts to improve the potency of compounds initially identified by high- throughput screening. These compounds potentiate the activity of the mGluR4 agonist L-AP4, with a potency of 34 nM for the phthalimide, PAM1, and 160 nM for the sulfonamide, PAM2, and an efficacy, relative to the agonist L-AP4, of 150% and 115%, respectively. There is no appreciable agonist activity with the identified compounds. The activity of the two novel PAMs was determined using FLIPR, in a CHO cell line overexpressing the human mGluR4 receptor and containing the promiscuous G-protein Gqi5. The site of action of PAM 1 and PAM2 was mapped using a set of chimeric receptors, which revealed that both compounds mediate their effect by interacting with the seven-transmembrane spanning domain of the mGluR4 receptor. PAM1 and PAM2 were radiolabeled with tritium and evaluated for the ability to bind to the human mGluR4 receptor over-expressed in CHO cells. Saturation binding analysis yields a Kd of 11 nM for 3H-PAM1 and a Kd of 26 nM for 3H-PAM2. These radiotracers are suitable for use in competition binding analysis; PAM1 displaces 3H-PAM2 with an IC50 of 8 nM; and PAM2 displaces 3H-PAM1 with an IC50 of 50 nM. Interestingly, the binding of both radiolabeled PAMs is dependent upon the presence of agonist. Finally, the mGluR4 PAM VU0003423 enhances, rather than displaces, binding of radiolabeled PAM1; providing direct evidence for two distinct positive allosteric modulator sites. Taken together, these data provide novel insight into the mechanism of PAM action on the mGluR4 receptor.