Tiancheng Liu, Lisa Y. Wu, Marat Kazak, Clifford E. Berkman

[corrected] Prostate-specific membrane antigen (PSMA) remains an attractive target for imaging and therapeutic applications for prostate cancer. Recent efforts have been made to conjugate inhibitors of PSMA with imaging agents. Compared to antibodies, small-molecule inhibitors of PSMA possess apparent advantages for in vivo applications. To date, there are no reports on the cellular fate of such constructs once bound the extracellular domain of PSMA. The present study was focused on precisely defining the binding specificity, time-dependent internalization, cellular localization, and retention of inhibitor conjugates targeted to PSMA on LNCaP cells. A novel fluorescent inhibitor was prepared as a model to examine these processes. Fluorescence microscopy of LNCaP and PC-3 cell lines was used to monitor the specificity, time-dependent internalization, cellular localization, and retention of a fluorescent PSMA inhibitor. Fluorescent inhibitor 2 was found to be a potent inhibitor (IC50 = 0.35 nM) of purified PSMA. Its high affinity for PSMA on living cells was confirmed by antibody blocking and competitive binding experiments. Specificity for LNCaP cells was demonstrated as no labeling by 2 was observed for negative control PC-3 cells. Internalization of 2 by viable LNCaP cells was detected after 30 min incubation at 37 degrees C, followed by accumulation in the perinuclear endosomes. It was noted that internalized fluorescent inhibitor can be retained within endosomes for up to 150 min without loss of signal. Our results suggest that potent, small-molecule inhibitors of PSMA can be utilized as carriers for targeted delivery for prostate cancer for future imaging and therapeutic applications.