PACE4-Based Molecular Targeting of Prostate Cancer Using an Engineered 64Cu-Radiolabeled Peptide Inhibitor

The potential of PACE4 as a pharmacological target in prostate cancer has been demonstrated as this proprotein convertase is strongly overexpressed in human prostate cancer tissues and its inhibition, using molecular or pharmacological approaches, results in reduced cell proliferation and tumor progression in mouse tumor xenograft models. We developed a PACE4 high-affinity peptide inhibitor, namely, the multi-leucine (ML), and sought to determine whether this peptide could be exploited for the targeting of prostate cancer for diagnostic or molecular imaging purposes. We conjugated a bifunctional chelator 1,4,7-triazacyclononane-1,4,7- triacetic acid (NOTA) to the ML peptide for copper-64 (⁶⁴Cu) labeling and positron emission tomography (PET)– based prostate cancer detection. Enzyme kinetic assays against recombinant PACE4 showed that the NOTA-modified ML peptide displays identical inhibitory properties compared to the unmodified peptide. In vivo biodistribution of the ⁶⁴Cu/NOTA-ML peptide evaluated in athymic nude mice bearing xenografts of two human prostate carcinoma cell lines showed a rapid and high uptake in PACE4-expressing LNCaP tumor at an early time point and in PACE4-rich organs. Co-injection of unlabeled peptide confirmed that tumor uptake was target-specific. PACE4-negative tumors displayed no tracer uptake 15 minutes after injection, while the kidneys, demonstrated high uptake due to rapid renal clearance of the peptide. The present study supports the feasibility of using a ⁶⁴Cu/NOTA-ML peptide for PACE4-targeted prostate cancer detection and PACE4 status determination by PET imaging but also provides evidence that ML inhibitor–based drugs would readily reach tumor sites under in vivo conditions for pharmacological intervention or targeted radiation therapy.Abbreviations: ACN, acetonitrile; Ac, acetyl; ⁶⁴Cu, copper-64; DCM, dichloromethane; ¹⁸F]-FDG, ¹⁸F]-fluoro-2-deoxy-d-glucose; HR-MS, high-resolution mass spectrometry; K_i, inhibitory constant; ML, multi-leucine; DMF, N,N′-dimethylformamide; NOTA, 1,4,7-triazacyclononane-1,4,7-triacetic acid; PC, proprotein convertase; PET, positron emission tomography