VU 0357121

2 years -80 in solvent

33 nM [1], 33 nM [1], 33 nM [1], 33 nM [1], 33 nM [1], 33 nM [1]

Radioligand Binding Assay, The allosteric antagonist MPEP analogue [3H]methoxyPEPy is used to evaluate the ability of test compounds to interact with the MPEP site on mGlu5. Membranes are prepared from rat mGlu5 HEK293 cells. VU0357121 is diluted in assay buffer (50 mM Tris/0.9% NaCl, pH 7.4) to a 5x stock, and 100 uL of the test compound is added to each well of a 96 deep-well assay plate. Three hundred microliter aliquots of membranes diluted in assay buffer (20 ug/well) are added to each well. One hundred microliters of [3H]methoxyPEPy (2 nM final concentration) is added, and the reaction is incubated at room temperature for 1 h with shaking. After the incubation period, the membrane-bound ligand is separated from free ligand by filtration through glass-fiber 96-well filter plates. The contents of each well are transferred simultaneously to the filter plate and washed 3 4 times with assay buffer using a cell harvester. Forty microliters of scintillation fluid is added to each well and the membrane-bound radioactivity determine by scintillation counting. Nonspecific binding is estimated using 5 uM MPEP. Concentration response curves are generated using a four parameter logistical equation in GraphPad Prism.

VU0357121 is a novel positive allosteric modulator (PAM) of mGlu5 with EC50 of 33 nM, is inactive or very weakly antagonizing at other mGlu receptor subtypes.