Structure–Activity Relationship Studies in Substituted Oxadiazoles as Inducers of Extracellular Vesicles

We characterized small-molecule compounds discovered through a prior high-throughput screening campaign that enhanced the release of immunostimulatory extracellular vesicles (EVs) and identified compound #645 (compound 1) as an immunomodulatory compound. Here, we performed systematic structure–activity relationship (SAR) studies to probe the scaffold for analogs inducing tetraspanin (CD63) reporter activity, a marker of EV biogenesis and release, in human THP-1 cells. A potent analog 39 was identified, which also stimulated the expression of an immunostimulatory cytokine and costimulatory molecules by antigen-presenting cells and promoted antigen cross-presentation to CD8+ T cells. As its mechanism of action, compound 39 was found to interfere with tubulin polymerization, possibly binding to the nocodazole binding site on beta-tubulin as evident by molecular docking studies. Additionally, 39 preferentially impaired the viability in proliferating cells and may be a potential therapeutic agent for cancer and immunotherapy applications.