Novel indole-based derivatives as promising NLRP3 inflammasome inhibitors: Design, synthesis, biological evaluation,...

Seeking to identify a new chemotype for the development of NLRP3 inhibitors, inspired by the structure of the tool compound MCC950 (9), we designed and synthesized new derivatives by choosing the indole heterocycle as spacer between the furanyl moiety and the chemically handy sulfonamide chain core. Four compounds (19c, 19e, 19g, and 19j) selectively reduced NLRP3-dependent IL-1β levels with micromolar inhibitory activity in THP-1 cells, and were safe at the same concentrations. Moreover, they inhibited lactate dehydrogenase release, caspase-1 enzymatic activity, and ASC speck formation with a dose-response effect. Indoles 19c and 19g confirmed their biological activity in primary human macrophages, with IC₅₀ of 19 and 15 µM, respectively. Furthermore, no off-target effects were observed as the compounds did not inhibit LPS-induced TNF release. In silico studies helped us rationalize the binding mode and showed that these derivatives can accommodate the NACHT domain and make several interactions with crucial key residues of the protein. Remarkably, the target engagement assay displayed that 19c and 19g can displace MCC950, thus confirming their direct binding to the NACHT domain. These preliminary results suggest the potential for future development of this new class of indoles as NLRP3 inhibitors.