Receptor binding assay

PSMA inhibitory activities of 1-3 were determined using a fluorescence-based assay according to a previously reported procedure.¹⁷ Briefly, 25 μL lysates of PSMA-positive human prostate adenocarcinoma (LNCaP) cells were incubated with the inhibitor (12.5 μL) in the presence of 4 μM N-acetylaspartylglutamate (NAAG) (12.5 μL) for 120 min. The amount of the glutamate released from NAAG hydrolysis was measured by incubating with a working solution (50 μL) of the Amplex Red Glutamic Acid Kit (Molecular Probes Inc., Eugene, OR) for 60 min. Fluorescence was measured with a VICTOR3V multilabel plate reader (Perkin Elmer Inc., Waltham, MA) with excitation at 530 nm and emission at 560 nm. Inhibition curves were determined using semi-log plots and IC₅₀ values were determined at the concentration at which enzyme activity was inhibited by 50%. Assays were performed in triplicate. Enzyme inhibitory constants (K_i values) were generated using the Cheng-Prusoff conversion.³⁸ Data analysis was performed using GraphPad Prism version 4.00 for Windows (GraphPad Software, San Diego, California).