Enhanced tumor retention of NTSR1-targeted agents by employing a hydrophilic cysteine cathepsin inhibitor

Development of Improved Tumor-Residualizing, GRPR-Targeted Agents: Preclinical Comparison of an Endolysosomal Trapping Approach in Agonistic and Antagonistic Constructs

“…1-4). Experiments involving distribution coefficient studies, peptide metabolic stability studies, inhibition of cathepsin B (CatB) activity, and determination of the inhibition constant (K i ) of the conjugates to CatB and cellular trafficking studies were performed according to published methods (13,14).…”

“…The conjugates (E-AG and E-AN) were labeled with nat EuCl 3 as described before (Supplemental Fig. 5) (14). For confocal trafficking studies, briefly, in a 4-well Lab-Tek chambered coverglass disk (Thermo-Fisher), PC-3 cells (3,000/well) were incubated with the nat Eu-labeled conjugate (5 mM) in the presence of LysoTracker Green (100 nM; Molecular Probes, Inc.) at 37°C for 4 h. Cells were subsequently washed with fresh medium and imaged at the desired time points.…”

“…To examine in vitro adduct formation, the radioconjugates (4.5 pmol, 0.7 MBq) were incubated with CatB (human liver) (3 nM, 10 mL) (Millipore Sigma) or with PC-3 (1 • 10 6 ) cells. The detailed protocols for the workup and the autoradiographic sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and centrifugal filtration analyses were as previously outlined (13,14). Blocking studies were performed by incubation with the CA-074 inhibitor (Apex-Bio) (10 mM, 10 mL) or an excess of unlabeled E-AG or E-AN (40 mM).…”

“…The mice were sacrificed at 24 h after injection, and the tumor, pancreas, liver, and kidney were excised, homogenized, and centrifuged to remove the pellet. An aliquot (18 mL) of the supernatant was examined by autoradiographic SDS-PAGE as described before (14). An additional aliquot (80 mL) of the supernatant was analyzed by centrifugal filtration (molecular weight cutoff, 10 kDa) to separate macromolecule-associated radioactivity from low-molecular-weight radioactivity.…”

“…To overcome this limitation, chemical approaches have been explored to enhance the tumor retention (i.e., residualization) of low-molecular-weight, receptor-targeted agents (13)(14)(15)(16). One of the approaches our laboratory has examined involves cysteine cathepsins (CCs), a family of endolysosomal proteases that are expressed predominately in endolysosomal vesicles and are associated with protein turnover within the cell (17,18).…”