Introduction: NLRP3 is a cytosolic pattern recognition receptor that plays a crucial role in inflammatory responses to pathogens, danger signals, and other stimuli. Upon activation, NLRP3 forms an inflammasome containing the NLRP3 molecular sensor, the apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) and pro-caspase-1, resulting in caspase-1 activation, release of proinflammatory cytokines named IL-1β and IL-18 and eventually pyroptosis. The broad involvement of the NLRP3 inflammasome in various inflammatory diseases such as multiple sclerosis, type 2 diabetes, and cancer makes it a highly attractive drug target. A range of pharmacological inhibitors of NLRP3 inflammasome has been previously described while no specific inhibitor exists for the various components of inflammasome complexs such as pyd domains of NLRP3 and ASC.
Methods: In this study, the split luciferase complementation assay was used as an approach to investigate interactions between the NLRP3-pyd and ASC. Both proteins were expressed in E.coli and purified by Ni-NTA-sepharose. The effect of two chemical compounds QM380 and QM381 on these homotypic interactions was checked by luciferase activity assay. The Firefly luciferase enzyme was used in a split form in that the pyd domain of NLRP3 is attached to one fragment and ASC is attached to another fragment forming NLuc-NLRP3pyd and CLuc-ASC respectively. Also, we applied molecular docking via autodock vina to better understand interactions between chemical compounds and our proteins.
Results: Both proteins were expressed, purified, and checked out by SDS-PAGE. Bioinformatics analysis demonstrated that QM380 interacts with approximately the second interface of the pyd domain which is less important among the other interfaces and also, QM381 is involved in the first and second interfaces. In vitro results show the effects of the chemical compounds on the purified proteins that QM381 in 3mM concentration inhibits 50 percent of interactions while QM380 did not affect these homotypic interactions confirming our bioinformatics analysis.
Conclusion: NLRP3 inflammasome is the most studied of the inflammasome family and plays a crucial role in the development of inflammatory diseases. In the past, cytokine therapy and antibodies were used for targeting IL-1β and IL-18 which led to an increased risk of infection and various side effects. Secretion of IL-1β and IL-18 is regulated by NLRP3 inflammasome, which targeting NLRP3 is a highly attractive strategy in curing inflammatory disease. According to this study, these chemical compounds have shown promising effects on homotypic interactions of pyd domains or could be a potential lead for further study.