Pseudoirreversible Inhibition of Prostate-Specific Membrane Antigen by Phosphoramidate Peptidomimetics

Abstract

The mode of inhibition for phosphoramidate peptidomimetic inhibitors of prostate-specific membrane antigen was determined by inhibition reversibility experiments. The results revealed that these inhibitors can be classified into three types: pseudoirreversible (compounds 1-3), moderately reversible (compounds 4-9), and rapidly reversible inhibitors (compounds 10 and 11). Representative compounds from each class were further evaluated for their ability to induce cellular internalization of PSMA. Results from these experiments revealed that the pseudoirreversible inhibitor 1 induced the greatest PSMA internalization. The discovery of pseudoirreversible PSMA inhibitors is expected to provide a new avenue of investigation and therapeutic applications for prostate cancer and neurological disorders.

Preclinical Evaluation of 18F-PSMA-1007, a New Prostate-Specific Membrane Antigen Ligand for Prostate Cancer Imaging

“…The blockade experiment with 2-PMPA as a competing ligand led to a significant, but not complete, reduction in tumor uptake. This result might be attributable to the binding mechanism of 2-PMPA, as it has been reported to be a fast and reversible inhibitor (21). Given the high internalization ratio of 18 F-PSMA-1007, only a small fraction of the ligand needs to be bound to be internalized-even in the presence of the competitorand thus might result in significant uptake into the tumor.…”

A method to determine the mode of binding for GCPII inhibitors using bio-layer interferometry

“…In addition, the correlation between reversibility of inhibition and GCP II internalization in LNCaP cells has been determined; we found that pseudo-irreversible inhibitors induced internalization to a greater extent than slowly reversible or reversible inhibitors 9 . Furthermore, we recently demonstrated that reversibility of inhibition has an effect on internalization and percent uptake of GCP II-targeted SPECT agents with the irreversible-targeting agent, demonstrating superior uptake and internalization in GCP II-positive (GCP II+) cells 10 .…”

“…We previously reported a series of phosphoramidate peptidomimetic inhibitors of PMSA and classified their reversibility of inhibition by monitoring the recovery of enzyme activity following rapid dilution of the enzyme-inhibitor complex 9 . In addition, the correlation between reversibility of inhibition and GCP II internalization in LNCaP cells has been determined; we found that pseudo-irreversible inhibitors induced internalization to a greater extent than slowly reversible or reversible inhibitors 9 .…”

“…These results confirm the significance of pseudo-irreversible inhibitors as targeting molecules in the development of targeted imaging and therapeutic agents and provide rationale for establishing a convenient and rapid method for ascertaining mode of inhibition. The focus of the work described herein was aimed at developing an efficient method to determine the mode of inhibition for inhibitors of GCP II; our current benchmark method (a rapid dilution, HPLC-based assay) is tedious 9 . An attractive aspect of a bio-layer interferometry (BLI)-based assay is the small quantities of reagents required, which can also be reused for subsequent assays.…”

“…In prior studies, we reported the IC 50 values and reversibility of inhibition for CTT54 (1, IC 50 ¼ 14 nM) 11 and CTT54.2 (2, IC 50 ¼ 144 nM) 12 ( Figure 1), utilizing a commonly employed method which involves monitoring the recovery of enzyme activity following rapid dilution of the enzyme-inhibitor complex by HPLC 9 . The results from these rapid dilution assays were used to validate the real-time BLI assay employed in this study as a qualitative method to assess the mode of inhibition.…”