Spacer length effects on in vitro imaging and surface accessibility of fluorescent inhibitors of prostate specific membrane antigen

Prostate-specific membrane antigen (PSMA), a type II transmembrane protein, has been becoming an active target for imaging and therapeutic applications for prostate cancer. Recently, the development of its various chemical inhibitor scaffolds has been explored to serve as carriers for therapeutic or diagnostic payloads targeted to PSMA-positive tumor cells. However, there have been few efforts to definitively determine the optimal length of linker between PSMA inhibitor cores and their payload molecules with regard to the affinity to PSMA and in vitro performance. In our present model study, three spacer-length varied fluorescent inhibitors (FAM-CTT-54, FAM-X-CTT-54 and FAM-PEG8-CTT-54) were synthesized, and further enzymatic inhibition studies displayed linker length-dependent changes in: inhibitory potency (IC50 = 0.41 nM, 0.35 nM, 1.93 nM), modes of binding (reversible, slowly reversible, irreversible), respectively. Furthermore, cell-labeling imaging revealed the spacer length-related change of fluorescence intensity (FAM-X-CTT-54 > FAM-PEG8-CTT-54 > FAM-CTT-54). These results suggest that selection of linkers and their lengths will be important considerations in the development of next-generation prostate tumor-targeted imaging probes and therapeutic agents that specifically home to PSMA on tumor cells.

A series of spacer-varied imaging probes were synthesized and evaluated, which exhibited spacer length-dependent effects on inhibitory potency, mode of inhibition, and fluorescence imaging of prostate cancer cells.Figure optionsDownload as PowerPoint slide