biostudies-literatureZaric MThe Slovenian Research and Innovation Agency11910https://www.ebi.ac.uk/biostudies/studies/S-EPMC12732864biostudies-literatureUnknown26(24)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 (K_D = 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 rInternational journal of molecular sciencesStructural and Proteomic Analysis of the Mouse Cathepsin B-DARPin 4m3 Complex Reveals Species-Specific Binding Determinants.PMC12732864P1-0140; P1-0048Novak MTurk BGevaert KUsenik ATurk DKramer LVasiljeva OImpens FTusar LZaric MfalseStructural 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 (K_D = 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 r2025-01-01T00:00:00Z2025 Dec2026-05-28T03:21:15.918Z2026-05-28T03:11:44.65ZS-EPMC127328644146533610.3390/ijms262411910