Lysosome-Directed Erlotinib Conjugates Reveal Dual Compartment Access and Targeted EGFR Suppression

The intracellular trafficking and localization of kinase inhibitors critically determine their ability to engage cytoplasmic targets. Here, we report the synthesis and bioimaging of two fluorescent erlotinib conjugates based on a naphthalimide core functionalized with either a pyridine or a morpholine moiety. While pyridine directs broad cytoplasmic distribution, morpholine, classically associated with lysosomal targeting, surprisingly retains the compound’s capacity to modulate EGFR activity. Live-cell imaging reveals that the morpholine-conjugated probe accumulates in lysosomes but also maintains a diffuse cytosolic presence, suggesting a lysosome-cytosol exchange or partial leakage. These findings challenge the prevailing notion that lysosomotropic delivery sequesters kinase inhibitors away from their cytoplasmic targets. Our study highlights the potential of rational probe design to manipulate subcellular localization while preserving functional engagement with oncogenic kinases, opening new avenues for targeted bioimaging and therapeutic delivery.