Lackner, A.; Leonidas, L.; Macapagal, A.; Lee, H.; Xu, H.; Qiu, Y.; Campos, M.; Cabral, J. E.; Onyuru, J.; Kulkarni, S.; Albrecht, L.; Hoffman, H. M.; McNulty, R.

The NLRP3 inflammasome plays a central role in innate immunity and is activated in response to mitochondrial dysfunction and oxidized DNA. Here, we demonstrate that repurposed small-molecule inhibitors originally developed for DNA glycosylases, TH5487 and SU0268, potently inhibit NLRP3 activation ex vivo in human Peripheral Blood Mononuclear Cells (PBMCs) with IC50 of 1.62 M and 3.24 M, respectively. We show that these inhibitors prevent mitochondrial localization of NLRP3 and directly block inflammasome assembly. They also reshape the immune landscape decreasing IL-1{beta} while increasing IFN-{beta}. Structural and biophysical analyses reveal a two-site DNA binding model in which NLRP3 engages oxidized DNA with a KD1 of 0.268 nM and KD2 3.02 nM. Importantly, these inhibitors block IL-1[beta] secretion in L353P Familial Cold Autoinflammatory Syndrome (FCAS) patient PBMCs where MCC950 fails, demonstrating the therapeutic potential for inflammasome-driven diseases. Together, our findings reveal a novel druggable mechanism of inflammasome inhibition through interference with oxidized DNA sensing and localization, offering new opportunities for treatment of chronic inflammatory disorders.