9s60

Publication Abstract from PubMed

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 A 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.

Structural and Proteomic Analysis of the Mouse Cathepsin B-DARPin 4m3 Complex Reveals Species-Specific Binding Determinants.,Zaric M, Tusar L, Kramer L, Vasiljeva O, Novak M, Impens F, Usenik A, Gevaert K, Turk D, Turk B Int J Mol Sci. 2025 Dec 10;26(24):11910. doi: 10.3390/ijms262411910. PMID:41465336^[2]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.