Prostate-Specific Membrane Antigen Binding Assay

The PSMA binding affinities of the dye–urea conjugates for PSMA were measured using the N-acetylated-α-linked acidic dipeptidase (NAALADase) assay, as previously described.^{30 (link),31 (link)} PSMA is known also as glutamate carboxypeptidase II (GCPII), or NAALADase.^{32 (link),33 (link)} A reaction mixture (total volume of 50 µL) containing [³H]N-acetyl-aspartylglutamate ([³H]NAAG, 30 nM, 1850 GBq/mmol) and human recombinant GCPII (40 pM final) in Tris-HCl (pH 7.4, 40 mM) containing 1 mM CoCl₂ was used. The reaction was carried out at 37 °C for 15 min and stopped with ice-cold sodium phosphate buffer (pH 7.4, 0.1 M, 50 µL). Blanks were obtained by incubating the reaction mixture in the presence of 2-phosphonomethyl pentanedioic acid (2-PMPA, 1 µM final), a selective and potent inhibitor of PSMA.^{32 (link)} Dye-urea conjugates and controls were tested at log-unit final concentrations that ranged from 100 µM to < fM. A 90 µL aliquot from each terminated reaction was transferred to a well in a 96-well spin column containing AG1 × 8 ion-exchange resin. The plate was centrifuged at 1500 rpm for 5 min using a Beckman GS-6R centrifuge (Beckman Coulter, Inc., Brea, CA) equipped with a PTS-2000 rotor. [³H]NAAG bound to the resin and [³H]-glutamate eluted in the flowthrough. Columns were then washed twice with formate (1 M, 90 µL) to ensure complete elution of [³H]glutamate. The flowthrough and the washes were collected in a deep 96-well block. From each well with a total volume of 270 µL, a 200 µL aliquot was transferred to its respective well in a solid scintillator-coated 96-well plate (Packard, Meriden, CT) and dried to completion. The radioactivity corresponding to [³H]glutamate was determined with a scintillation counter (Topcount NXT, Packard, counting efficiency 80%). IC₅₀ curves were generated from CPM results, by use of both Microsoft Office Excel 2007 and GraphPad Prism 5 programs, with K_i values derived from the IC₅₀ values.^{34 (link)}