The first significant achievement corresponds to Aim 3: Inflammasome inhibition of the proposal (SA3-2: PROTACS and molecular glues targeting NLRP3) where we report the first successful development of PROTACs targeting the NLRP3 inflammasome (1). As chemical scaffold, we adapt a thiophene-based analogue of the known inhibitor MCC950 and link it to ligands for different E3 ubiquitin ligases (VHL, CRBN, and IAP). Together with my colleague Michael Gütschow from the Pharmaceutical Institute of the University of Bonn, we synthesized a series of 17 PROTAC molecules using click chemistry and demonstrated through thermal shift assays that these compounds bind both NLRP3 and their respective E3 ligases, enabling ternary complex formation and NLRP3 degradation. A second major achievement was on the mechanisms of NLRP3 activation and inhibition as elucidated by functional analyses of disease-associated variants (2). Here we systematically investigate the functional consequences of 534 disease-associated NLRP3 variants using a high-throughput cell-based assay measuring ASC speck formation as a proxy for inflammasome activation. This measured parameter was used to generate a quantitative ‘ASC50’ score to assess variant activity. By reclassifying a significant fraction of variants in clinical databases and correlating functional activity with disease severity, this work provides a robust framework for improving cryopyrin-associated periodic syndromes (CAPS) diagnosis, understanding inflammasome activation mechanisms, and guiding patient stratification for targeted therapies. Another achievement focussing on Aim 2: NLRP3 characterization was the demonstration that NLRP3 inflammasome activation is driven not by a specific subcellular location, but by scaffold-induced clustering of NLRP3, which serves as a unifying mechanism across diverse stimuli (3). Using engineered NLRP3 variants targeted to multiple organelles (e.g. ER, Golgi, mitochondria, lysosomes, and plasma membrane), we were able to show that inflammasome activation, including IL-1β release, caspase-1 activation, and ASC speck formation, occurs robustly regardless of localization.
Two other studies summarize our efforts to identify and develop new inhibitors of the NLRP3 inflammasome that would be able to pass the blood-brain barrier. In the first study, we report the discovery and characterization of BAL-0028 and its derivative BAL-0598 as potent, selective small-molecule inhibitors of the human NLRP3 inflammasome with a novel mechanism of action (4). Using a DNA-encoded library screen, we identified BAL-0028, which inhibits NLRP3-driven IL-1β release, caspase-1 activation, ASC speck formation, and pyroptosis across multiple human cell types without affecting other inflammasomes (e.g. AIM2, NLRC4, NLRP1) or upstream LPS signalling. In a follow-up study we describe the discovery and structural characterization of BAL-1516, a novel indazole-based small-molecule inhibitor of the NLRP3 inflammasome. Biophysical assays such as surface plasmon resonance (SPR) show that BAL-1516 binds NLRP3 with nanomolar affinity (~14 nM), exceeding the potency of MCC950, while thermal stability experiments confirm that the compound stabilizes the NLRP3 protein without disrupting its oligomeric decamer structure. Importantly, we determined the cryo-EM structure of human NLRP3 bound to BAL-1516, showing that the compound occupies a previously unrecognized binding groove on the NACHT domain which is distinct from the MCC950 site. Notably, it forms a characteristic network of three hydrogen bonds with the β-sheet of the nucleotide-binding domain, alongside extensive hydrophobic interactions.
