Development of Phosphoramidate Inhibitors for Cell Surface Proteases in Metastatic Cancers
Mendes, Desiree Ellene
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The development of highly tuned diagnostic agents are critical for the early detection of cancer and are commonly designed to bind cell surface receptors. A small library of first-generation phosphoramidate inhibitors leading to sub-micromolar potency as determined by a FRET-based spectrophotometric assay were generated as peptide mimics with a scaffold designed to interact with the S1, S1` register of the catalytic domain of the cell surface receptor matrix metalloproteinase (MMP-14). Candidate residues were selected for the scaffold using in silico modeling, and synthesized using organophosphate and peptide coupling methodologies. A phosphoramidate inhibitor and fluorescent conjugate were shown to specifically target prostate specific membrane antigen (PSMA) in human cancer cell lines was applied to a canine model of which the expression and enzymatic activity of PSMA were previously unknown. The expression of PSMA was determined by RT-PCR and western blot and found to be in lower abundance when compared to human prostate cancer PSMA expression. The kinetics of PSMA were established and inhibitory potency was determined by an end-point HPLC analysis, and was similar to that of human PSMA enzymatic activity. In addition, a fluorescent-conjugate was successfully used to label canine cancer cells in blood as detected using flow cytometry. Previous studies revealed that both PSMA and androgen receptor (AR) decreased following an extended period of androgen deprivation. When the biomarker expression of c-Met and calpain 2 are upregulated, it indicated a switch to a more aggressive prostate cancer phenotype. The expression of these biomarkers in prostate cancer cell lines subjected to prolonged androgen deprivation in an established in vitro model were investigated. After 10 passages of prolonged androgen deprivation, both c-Met and calpain 2 were found to be upregulated with the concomitant decrease in expression of PSMA and AR. In addition to switching signaling pathways, prostate cancer cells secrete exosomes as long-range cell-to-cell communication vehicles. The exosomes were found to be highly enriched with functionally active PSMA. Insights into inhibitor binding, signaling pathway alterations, and biomarker expression in extracellular vesicles could enable new trajectories for treatment and further the advancement toward powerful development of personalized cancer therapy.