Examination of Charge Modifications of an Endolysosomal Trapping Inhibitor in an Antagonistic NTSR1-Targeted Construct for Colon Cancer.

Many low-molecular weight targeted radiotherapeutics (TRTs) are capable of rapidly achieving exceptional tumor to non-target ratios shortly after administration. However, the low tumor residence time of many TRTs limits therapeutic dose delivery and has become the Achilles heel to their clinical translation. To combat the tumor efflux of these otherwise promising agents, we have previously presented a strategy of equipping low-molecular weight TRTs with irreversible cysteine cathepsin inhibitors (e.g., E-64 analogues). These inhibitors are capable of forming irreversible adducts with cysteine proteases within the endolysosomal compartments of cells. Using these endolysosomal trapping agents (ETs), the receptor-targeted constructs are able to increase tumor retention and, thus, deliverable therapeutic doses. In this study, we examine this approach in the development of agents targeting the neurotensin receptor subtype 1 (NTSR1), a receptor overexpressed in numerous cancers. Using an antagonistic NTSR1-targeting vector, we explore the impact of charge modification of the ETs on the in vitro and in vivo biological performance of the constructs using HT-29 colon cancer models. Four ETs (based on the epoxysuccinyl peptide E-64) with various charge states were synthesized and incorporated into the structures of the NTSR1-targeted antagonist. These four