Structure-Based Insights into Stefin-Mediated Targeting of Fowlerpain-1: Towards Novel Therapeutics for Naegleria fowleri Infections.

Background/Objectives: Naegleria fowleri is a free-living protozoan that causes primary amoebic meningoencephalitis, a rapidly progressing central nervous system infection with high mortality rates and limited treatment options. Targeting virulence-associated proteins is essential for effective drug development. Fowlerpain-1 (FWP1), a papain-like cysteine protease (CP) implicated in extracellular matrix degradation and host-cell cytotoxicity, has been investigated as a therapeutic target. This study aimed to evaluate the FWP1 pocket geometry and stefin binding using an integrated in silico structural biology approach. Methods: A computational pipeline was used, including AlphaFold2-Multimer modeling of FWP1-stefin complexes, 20-ns molecular dynamics simulations under NPT conditions for conformational sampling, and molecular mechanics Poisson-Boltzmann surface area free energy calculations. Three natural CP inhibitors (stefins) were investigated. Structural stability was assessed using root mean square deviations, and binding profiles were characterized using protein-protein interaction analysis. Results: Stable FWP1-stefin interaction interfaces were predicted, with human stefin A showing favorable binding free energy. Two conserved motifs (PG and QVVAG) were identified as critical mediators of active-site recognition. Druggability analysis revealed a concave pocket with both hydrophobic and polar characteristics, consistent with a high-affinity ligand-binding site. Conclusions: This computational study supports a structural hypothesis for selective FWP1 inhibition and identifies stefins as promising scaffolds for developing structure-guided protease-targeted therapeutics against N. fowleri.