Spirodalesol analog 8A inhibits NLRP3 inflammasome activation and attenuates inflammatory disease by directly targeting adaptor protein ASC

Pharmacological inhibition of the Nod-like receptor family protein 3 (NLRP3) inflammasome contributes to the treatment of numerous inflammation-related diseases, making it a desirable drug target. Spirodalesol, derived from the ascomycete fungus Daldinia eschscholzii, has been reported to inhibit NLRP3 inflammasome activation. Based on the structure of spirodalesol, we synthesized and screened a series of analogs to find a more potent inhibitor. Analog compound 8A was identified as the most potent selective inhibitor for NLRP3 inflammasome assembly, but 8A did not inhibit the priming phase of the inflammasome. Specifically, while 8A did not reduce NLRP3 oligomerization, we found that it inhibited the oligomerization of adaptor protein apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC), as ASC speck formation was significantly reduced. Also, 8A interrupted the assembly of the NLRP3 inflammasome complex and inhibited the activation of caspase-1. Subsequently, we used a cellular thermal shift assay and microscale thermophoresis assay to demonstrate that 8A interacts directly with ASC, both in vitro and ex vivo. Further, 8A alleviated lipopolysaccharide-induced endotoxemia, as well as monosodium urate-induced peritonitis and gouty arthritis in mice by suppressing NLRP3 inflammasome activation. Thus, 8A was identified as a promising ASC inhibitor to treat inflammasome-driven diseases.