Search for Inhibitors against Inflammatory Responses of Microglia in a Meroterpenoid Library Constructed by Synthetic–Biological Manipulation of Biosynthetic Pathways

Microglia are the resident immune cells of the central nervous system. Activation-induced proinflammatory responses of microglia are associated with various neurodegenerative diseases, and therefore, inhibitors against microglial inflammatory responses are promising candidates for the treatment of those diseases as well as efficient tools to investigate the involvement of microglia in the pathologies. Here, we focused on meroterpenoids (MPs), which are hybrid compounds of a terpenoid and a nonterpenoid moiety such as a polyketide, phenol, alkaloid, or amino acid. We constructed a structurally diverse library of MPs by expressing combinations of enzymes catalyzing various biosynthetic steps, including polyketide synthesis, prenyl transfer, prenyl chain epoxidation, terpene cyclization, and postcyclization tailoring reactions, in Aspergillus oryzae as a heterologous host. The compounds obtained were purified, and the resulting library was screened for inhibitory activity toward activated microglia. Initial screening with immortalized mouse-derived BV2 microglial cells identified three hits with potential anti-inflammatory activity, namely, andrastin-type MPs 4c, 8c, and 9c. Follow-up study with primary cultures of rat microglia enabled us to calculate the half-maximal inhibitory concentrations (IC50) and the difference between the concentrations exhibiting efficacy and cytotoxicity. The IC50 values of 4c, 8c, and 9c for the release of interleukin-1β (IL1β) and interleukin-6 (IL6) were smaller than those of minocycline, but the safety margins of 4c, 8c, and 9c were narrower due to the cytotoxicity. However, their effects were more reproducible than that of minocycline at the concentrations tested. Compounds 4c, 8c, and 9c appear to be potential lead compounds for the development of inhibitors against proinflammatory responses of microglia, which warrant further optimization and in vivo pharmacology studies.