20.500.12556/DiRROS-25322 Structural and proteomic analysis of the mouse cathepsin B-DARPin 4m3 complex reveals species-specific binding determinants Cathepsin B (CatB) is a lysosomal cysteine protease that plays a major role in various pathologies and is therefore considered a valuable therapeutic target. To address species-specific inhibitor challenges, we characterized the selective binding of designed ankyrin repeat protein (DARPin) 4m3 toward mouse cathepsin B (mCatB) over human CatB (hCatB). The mCatB–DARPin 4m3 complex was validated by size-exclusion chromatography (SEC), nano-differential scanning fluorimetry (nano-DSF), and surface plasmon resonance (SPR), revealing high affinity binding (KD = 65.7 nM) and potent inhibition (Ki = 26.7 nM; mixed competitive/noncompetitive). DARPin 4m3 showed no binding/inhibition toward hCatB. The 1.67 Å crystal structure of the complex—the first for mCatB—identified key interaction residues (e.g., I65/Q66 in mCatB vs. S65/M66 in hCatB) conferring selectivity. Proteomic analysis of endogenous substrates using a support vector machine (SVM) revealed greater similarity between mCatB and hCatB cleavages (Area Under the Curve (AUC) = 0.733) than between mCatB and other human cathepsins (AUC = 0.939–0.965). Clustering and SVM methods offer broadly applicable tools for protease specificity profiling in drug discovery. This study demonstrates the utility of DARPins for species-selective targeting and highlights the importance of integrated structural and proteomic approaches for dissecting protein–protein interactions. cathepsin B protein inhibitor proteomics katepsin B proteomika zaviralci true false true MDPI Švica Angleški jezik Slovenski jezik © 2025 by the authors. Neznano 2026-01-16 09:25:41 2026-01-16 09:25:42 2026-01-17 04:06:03 0000-00-00 00:00:00 2025 0 0 Nasl. z nasl. zaslona; Soavtorji iz Slovenije: Livija Tušar, Lovro Kramer, Olga Vasiljeva, Matej Novak, Aleksandra Usenik, Dušan Turk, Boris Turk; Opis vira z dne 15. 1. 2026; str. 1-37 iss. 24, [article no.] 11910 Vol. 26 Dec. 2025 0000-00-00 Zaloznikova Objavljeno NiDoloceno 2025-10-16 2025-12-04 2025-12-10 577 1422-0067 10.3390/ijms262411910 264865795 https://www.wwpdb.org/pdb?id=pdb_00009s60 1 f53bcfc5-f2b4-11f0-a15d-001a4af901a5 https://dirros.openscience.si/Dokument.php?lang=slv&id=38307 https://www.mdpi.com/1422-0067/26/24/11910 1 f424d8a4-f2b4-11f0-a15d-001a4af901a5 https://dirros.openscience.si/Dokument.php?lang=slv&id=38306 RAZ_Zaric_Miki_2025.pdf RAZ_Zaric_Miki_2025.pdf Povezava do raziskovalnih podatkov: https://doi.org/10.2210/pdb9S60/pdb. 1 D895CB9BBADEC9746BEEC00BA4606EC1 2b21818e00d96f7af87c687c3da522ab5a9205e4b2b8aa0aacbb7cfc15758db5 53cf6eb7-f2b5-11f0-a15d-001a4af901a5 https://dirros.openscience.si/Dokument.php?lang=slv&id=38309 Institut Jožef Stefan 0 0 0