In Silico Identification of Spironolactone as a Walker B Associated Candidate in the NLRP3 NACHT ATPase Domain

Abstract The NLRP3 inflammasome is a cytosolic complex whose activation depends on ATP binding and hydrolysis within the central NACHT domain. MCC950 (CRID3) targets this ATPase region and engages residues proximal to the Walker B motif, providing a structural benchmark for inhibitor discovery. Here, we performed molecular docking based virtual screening of 100 FDA-approved small molecules against the NLRP3 NACHT ATP-binding cavity using the cryo-EM structure of NLRP3 bound to CRID3 (PDB ID: 7PZC). Redocking of MCC950 yielded a best binding energy of −9.79 kcal/mol and reproduced localization within the Walker B–associated sub-pocket, including a predicted hydrogen bond with Asp274 and a buried surface area of 455.68 Ų. Top-ranked compounds exhibited predicted binding energies between −10.7 and −9.6 kcal/mol; however, spatial analysis showed that several localized outside the MCC950-associated site. Incorporation of centroid deviation, buried surface area, and proximity to Asp274 enabled prioritization of structurally aligned candidates. Spironolactone demonstrated the closest alignment to MCC950, with a docking score of −9.99 kcal/mol, centroid deviation of 1.35 Å, comparable burial (460.35 Ų), and predicted interaction with Asp274. These results identify Spironolactone as a structurally aligned NACHT-binding candidate warranting experimental validation.