(-)-PHCCC, a positive allosteric modulator of mGluR4: mechanism of action, and neuroprotection.

Group-III metabotropic glutamate receptors (mGluR4, -6, -7, and -8) modulate neurotoxicity of excitatory amino acids and beta-amyloid-peptide (βAP) as well as epileptic convulsions, most likely via presynaptic inhibition of glutamatergic neurotransmission. Due to the lack of subtype-selective ligands for group-III receptors, we previously utilized knock-out mice to identify mGluR4 as the primary receptor mediating neuroprotection of unselective groupIII agonists such as L-AP4 or (+)-PPG, whereas mGluR7 is critical for anticonvulsive effects. In a recent effort to find group-III subtype-selective drugs we identified (+/-)-PHCCC as a positive allosteric modulator for mGluR4. The positive modulation of GTPγ[S] binding on hmGluR4a-tranfected stable cell lines, by 3 μM and 10 μM of (-)-PHCCC was completely blocked by the group-III mGluR-selective antagonists CPPG and MSOP. In order to determine half-maximally effective concentrations (EC50s) of (-)-PHCCC, we performed concentrationresponse curves in the absence and presence of constant L-AP4 concentrations. Without LAP4, (-)-PHCCC showed an EC50 of >30 μM; at 0.2 and 0.6 μM of L-AP4, the EC50 of (-)PHCCC was approximately 6 μM; and in the presence of 10 μM L-AP4, the EC50 of (-)PHCCC decreased to 3.8 μM. All the activity of (+/-)-PHCCC resides in the (-)-enantiomer, which is inactive at mGluR2, -3, -5a, -6, -7b and -8a, but shows partial antagonist activity at mGluR1b (30% maximum antagonist efficacy). Chimeric receptor studies showed that the binding site of (-)-PHCCC is localized in the transmembrane region. Finally, (-)-PHCCC showed neuroprotection against NMDAand βAP-toxicity in mixed cultures of mouse cortical neurons (NMDA, 100 μM = 100 + 6; NMDA + (-)-PHCCC, 30 μM = 71.4 + 2; βAP(25-35), 25 μM = 100 + 5; βAP(25-35) + (-)-PHCCC, 10 μM = 20.5 + 3.5). This neuroprotection was additive to that induced by the highly efficacious mGluR1 antagonist CPCCOEt and was blocked by MSOP, a group-III mGluR antagonist. Our data provide evidence for a novel pharmacological site on mGluR4, which may be used as a target-site for therapeutics.