Identification of a Novel Small Molecule That Enhances the Release of Extracellular Vesicles with Immunostimulatory Potency via Induction of Calcium Influx

Abstract

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, we sought small-molecule compounds that increase immunostimulatory EVs released by antigen-presenting dendritic cells (DCs) for enhancement of vaccine immunogenicity. We 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, we screened 80 hit compounds and identified compound 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, we 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, our 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.

Structure–Activity Relationship Studies in Benzothiadiazoles as Novel Vaccine Adjuvants

“…A focused SAR study was published involving a small set of analogues of compound 1 (structures shown in Figure 1B). 15 This study showed that all of the compounds bearing the aliphatic esters retained the ability to enhance CD63 reporter and Ca 2+ influx activities, except the smaller alkyl ester analogue 2G179A and the aryl ester 2G179G. Additionally, losing the ester functional group as in the carboxylic acid analogue 2H013 or the amide analogue 2E241 or modifying the sulfonamide group by alkylating the nitrogen as in 2F186 led to complete loss of activity.…”

“…The previous focused SAR studies 15 pointed to the necessity of preserving a small alkyl ester functional group for potency in CD63 reporter and Ca 2+ influx assays. Thus, we continued to explore the ester site and other sites on the scaffold to identify potent compounds as well as sites on the scaffold that could retain activity upon modification and allow us to introduce functional groups such as an amino group as potential handles for further derivatization.…”

“…We have shown that EVs secreted by these compounds induce T cell receptor-dependent CD4 + T cell proliferation in vitro and thus wanted to compare adjuvant potencies in vivo. 15 FDA-approved TLR4 agonistic adjuvant MPLA was used as a positive control and a comparator. Immunization experiments in mice (4 mice/group) were performed with selected lead compounds (200 nmol/injection) or MPLA (1 μg/injection) added as adjuvants to chicken egg ovalbumin (OVA, 5 μg/injection) as a model antigen.…”

“…Syntheses of compounds shown in Figure 1 have been previously published. 15 Syntheses of intermediates 10m, 11, 12h, 12m, 15, 16, and 19 are given in the Supporting Information, Schemes S1−S2.…”

“…13,14 Collectively, these studies led to the identification of a benzothiadiazole class of compounds (compound 634, Figure 1A). 15 In this report, compound 634 served as the initial lead for further structure−activity relationship (SAR) studies and is referred to as compound 1. Previously, compound 1 was characterized to induce Ca 2+ influx, increase costimulatory molecule expression on murine bone-marrow-derived dendritic cells (mBMDCs), and enhance EV release with increased immunostimulatory surface ligands on these activated APCs.…”