Structure-based design of peptide inhibitors targeting the SPSB2–iNOS interaction: a computational approach to modulating nitric oxide signaling

Dysregulation of inducible nitric oxide synthase (iNOS) plays a central role in inflammatory diseases, cancer, and neurodegeneration due to excessive nitric oxide (NO) production. Suppressor of Cytokine Signaling Box Protein 2 (SPSB2) is a key negative regulator of iNOS through selective protein–protein interactions. Targeting the SPSB2–iNOS interface offers a novel strategy for therapeutic modulation of NO signaling. In this study, we employed a comprehensive in silico approach to design and evaluate peptide inhibitors derived from the native iNOS sequence. A series of mutant peptides was generated via residue scanning, followed by molecular docking, molecular dynamics (MDs) simulations, and MM/PBSA free-energy calculations. Among the designed variants, two peptides – D2Q and E8F emerged as lead candidates. The D2Q mutant demonstrated enhanced electrostatic interactions and maintained structural stability, while the E8F mutant exhibited the most compact conformational behavior and the lowest binding free energy (BFE) (−50.57 ± 3.74 kcal/mol). In contrast, the N5R and V7T mutations were associated with increased flexibility and reduced interaction stability. Principal component analysis (PCA) and free energy landscape (FEL) mapping further confirmed the restricted motion and high thermodynamic stability of D2Q and E8F complexes. These findings highlight D2Q and E8F as promising peptide inhibitors capable of modulating SPSB2–iNOS interactions. This work provides a strong foundation for future in vitro and in vivo studies aimed at developing targeted therapies for NO-related pathologies.