Site-Specific Chemical Modulation of a Flexible Azaproline Transporter to Enhance Epirubicin Accumulation in Drug-Resistant Human Glioblastoma Cells and Blood–Brain Barrier Penetration in Adult Zebrafish

Cell-penetrating peptides (CPPs) have emerged as nonviral biological carriers for the delivery of macromolecular therapeutics into cells. Despite their lower immunogenicity and cellular toxicity, CPPs often lack target specificity and proteolytic stability. Various modified CPPs have been reported to improve such selective targeting and pharmacokinetic properties <i>in vivo</i>. On this frontier, we have previously reported the synthesis and <i>in vitro</i> activity of a protease-stable non-natural δ-azaproline (δ-azp) containing CPP, named <b>Flexible Azaproline Transporter-1</b> (<b>FAT-1</b>). In this study, we report the chemical synthesis of three modified <b>FAT</b> analogs (<b>FAT-2</b>, <b>FAT-3</b>, and <b>FAT-4</b>) and compare their biological efficacy <i>in vitro</i>. These analogs were designed by incorporating a β-alanine spacer between the two adjacent azaproline monomers, with structural variations (branched or linear) and terminal δ-<i>N</i> functionalization. Comparative biological efficacy studies demonstrated that <b>FAT-2</b> exhibited the highest potency among the series, with enhanced cellular uptake and efficient endosomal escape in CHO cells. For the functional evaluation of <b>FAT-2</b>, the scaffold was conjugated to the antineoplastic drug, Epirubicin. The conjugate (<b>Epi-FAT2</b>) showed efficient induction of apoptosis in drug-resistant human glioblastoma (LN-229) cells, inhibited cell migration, and reduced ABCG2/P-glycoprotein-mediated drug efflux. The intraperitoneal (IP) administration of <b>Epi-FAT2</b> in Wild Indian Karyotype (WIK) adult zebrafish revealed its superior blood-brain barrier (BBB) penetration capability with greater/diverse tissue-dependent accumulation. The promising results of <b>FAT-2</b> in both <i>in vitro</i> and <i>in vivo</i> studies highlight its potential for the delivery of CNS therapeutics and exemplify the importance of suitable scaffold modification in CPPs for future studies.