Mast cells (MCs) are known to regulate innate and adaptive immunity. MC activators have recently been described as safe and effective vaccine adjuvants. Many currently known MC activators are inadequate for in vivo applications, however, and research on identifying novel MC activators is limited. In this study, we identified novel MC activators by using high-throughput screening (HTS) assays using approximately 55,000 small molecules. Data sets obtained by the primary HTS assays were statistically evaluated using quality control rules and the B-score calculation, and compounds with B-scores of >3.0 were chosen as mast cell activators (hits). These hits were re-evaluated with secondary and tertiary HTS assays, followed by further statistical analysis. From these hits, we selected 15 compounds that caused degranulation in murine and human MCs, with potential for flexible chemical modification for further study. Among these 15 compounds, ST101036, ST029248, and ST026567 exhibited higher degranulation potency than other hit compounds in both human and mouse MCs. In addition, the 15 compounds identified promote de novo synthesis of cytokines and induce the release of eicosanoids from human and mouse MCs. HTS enabled us to identify small-molecule MC activators with unique properties that may be useful as vaccine adjuvants.

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Mast cells (MCs) are highly granulated tissue resident hematopoietic cells and because of their capacity to degranulate and release many proinflammatory mediators, they are major effectors of chronic inflammatory disorders including asthma and urticaria. As MCs have the unique capacity to reform their granules following degranulation in vitro, their potential to undergo multiple cycles of degranulation and regranulation in vivo has been linked to their pathogenesis. However, it is not known what factors regulate MC regranulation let alone if MC regranulation occurs in vivo. Here, we report that IgE-sensitized mice can undergo multiple bouts of regranulation, following repeated anaphylactic reactions. mTORC1, a critical nutrient sensor that activates protein and lipid synthesis, was found necessary for MC regranulation. mTORC1 activity in MCs was regulated by a glucose-6-phosphate transporter, Slc37a2, which was found to be necessary for increased glucose-6-phosphate and ATP levels during regranulation, two upstream signals of mTOR. Slc37a2 is highly expressed at the cell periphery early during regranulation where it appears to colocalize with mTORC1. Additionally, this transporter was found to concentrate extracellular metabolites within endosomes which are trafficked directly into nascent granules. Thus, the metabolic switch associated with MC regranulation is mediated by the interactions of a cellular metabolic sensor and a transporter of extracellular metabolites into MC granules.