Pharmacological Inhibition of Small Extracellular Vesicle Secretion by ALK5i SD-208 via Lysosomal Rerouting of CD63+...

Pharmacological tools to selectively modulate extracellular vesicle (EV) secretion are scarce. Here, we identify the ALK5 (TGF-β receptor I) inhibitor SD-208 as a potent suppressor of small EV (sEV) secretion that acts independently of its canonical anti-fibrotic activity. SD-208 not only reversed myofibroblast activation but also markedly inhibited sEV secretion. Strikingly, this inhibitory effect persisted in non-activated cardiac fibroblasts and non-fibrotic HEK293 cells, demonstrating that SD-208 regulates EV secretion through mechanisms uncoupled from TGF-β/Smad signalling. Mechanistic analyses revealed that SD-208 disrupts vesicle trafficking rather than EV biogenesis. Reduced secretion of CD63⁺ EVs was accompanied by intracellular accumulation of CD63⁺ structures and their selective diversion into LAMP1⁺ lysosomes. Proteomic profiling of SD-208-treated and control HEK293 cells and cardiac fibroblasts revealed dysregulation of vesicle trafficking pathways, enrichment of ubiquitin ligase complexes, and enhanced endosome-to-lysosome transport. Together, these findings demonstrate that SD-208 diverts CD63⁺ multivesicular bodies (MVBs) from a secretory fate toward lysosomal degradation. This work identifies SD-208 as a small-molecule tool to interrogate the secretory-versus-degradative fate of MVBs and uncovers a new regulatory link between lysosomal pathways and EV trafficking. Beyond its established role as an anti-fibrotic agent, SD-208 provides mechanistic and therapeutic opportunities for the control of EV secretion in diseases such as fibrosis, cardiac remodelling, hypertrophic cardiomyopathy, and cancer.