Structural and Proteomic Analysis of the Mouse Cathepsin B-DARPin 4m3 Complex Reveals Species-Specific Binding Determinants.

ABSTRACT: 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 r

SUBMITTER: Zaric M 

PROVIDER: S-EPMC12732864 | biostudies-literature | 2025 Dec

REPOSITORIES: biostudies-literature