A novel small molecule enhances the release of extracellular vesicles with immunostimulatory potency

Extracellular vesicles (EVs) transfer antigens and immunomodulatory molecules in immunologic synapses as a part of intracellular communication, and EVs equipped with immunostimulatory functions have been utilized for vaccine formulation. Hence, University of California San Diego researchers sought small-molecule compounds that increase immunostimulatory EVs released by antigen-presenting dendritic cells (DCs) for enhancement of vaccine immunogenicity. The researchers previously performed high-throughput screening on a 28K compound library using three THP-1 reporter cell lines with CD63 Turbo-Luciferase, NF-κB, and interferon-sensitive response element (ISRE) reporter constructs, respectively. Because intracellular Ca2+ elevation enhances EV release, they screened 80 hit compounds and identified ethyl 2-(benzo[c][1,2,5]thiadiazole-4-sulfonamido)-4,5-dimethylthiophene-3-carboxylate (hereafter designated as 634) as a Ca2+ influx inducer. 634 enhanced EV release in murine bone marrow-derived dendritic cells (mBMDCs) and increased costimulatory molecule expression on the surface of EVs and the parent cells. EVs isolated from 634-treated mBMDCs induced T cell proliferation in the presence of antigenic peptides. To assess the roles of intracellular Ca2+ elevation in immunostimulatory EV release, the researchers performed structure-activity relationship (SAR) studies of 634. The analogues that retained the ability to induce Ca2+ influx induced more EVs with immunostimulatory properties from mBMDCs than did those that lacked the ability to induce Ca2+ influx. The levels of Ca2+ induction of synthesized analogues correlated with the numbers of EVs released and costimulatory molecule expression on the parent cells. Collectively, this study presents that a small molecule, 634, enhances the release of EVs with immunostimulatory potency via induction of Ca2+ influx. This agent is a novel tool for EV-based immune studies and vaccine development.

Sako Y, Sato-Kaneko F, Shukla NM, Yao S, Belsuzarri MM, Chan M, Saito T, Lao FS, Kong H, Puffer M, Messer K, Pu M, Cottam HB, Carson DA, Hayashi T. (2023) Identification of a Novel Small Molecule That Enhances the Release of Extracellular Vesicles with Immunostimulatory Potency via Induction of Calcium Influx. ACS Chem Biol [Epub ahead of print]. [article]

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