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Abstract

Cathepsin B is a lysosomal cysteine protease implicated in various pathological conditions
and therefore considered a potential therapeutic target. This study investigates the
selectivity and binding of a designed ankyrin repeat protein (DARPin) 4m3 towards mouse
cathepsin B (mCatB). It aims to explore the species-specific interaction of cathepsin B with
DARPin molecules to gain insights into the differences with human cathepsin B (hCatB),
including by comparing their cleavage site sequences with other human cathepsins using
clustering and a support vector machine (SVM) algorithm. The mCatB-DARPin 4m3
complex was confirmed by size exclusion chromatography (SEC), nano-differential
scanning fluorimetry (nano-DSF), and surface plasmon resonance (SPR) assay. The affinity
of 4m3 for mCatB was determined by SPR (KD = 65.7 nM), while the inhibition constant
(Ki = 26.7 nM) and mode of inhibition were determined by kinetic analysis, which showed
it to be hyperbolic mixed-type with a predominantly competitive inhibition. The crystal
structure of the complex of mouse cathepsin B with DARPin 4m3 was determined for the
first time at a resolution of 1.67 Å and compared with previously published structures of
hCatB with DARPin 81 and DARPin 8h6, providing insight into the interactions that may
be responsible for this selectivity. Structural analysis of the complexes revealed that
DARPin 4m3 binds to the structurally equivalent region on mCatB that corresponds to
the region targeted by DARPins 8h6 and 81 on hCatB. This region is less conserved among
cathepsins across different species, and candidate amino acid interactions that may
contribute to the selectivity of DARPin 4m3 were identified. DARPin 4m3 showed no
binding to hCatB, which was confirmed by SEC, nano-DSF, SPR and inhibition assays.
SVM-based analysis of cleavage-site sequences (P3–P4′) showed greater similarity between
mCatB and hCatB (AUC = 0.73) than between mCatB and human CatK, CatL, CatV,
CatS, or CatF (AUC = 0.94–0.97). The applied clustering and SVM-based comparison of
cleavage-site sequences provide a framework that could be extended to other proteases and
may help in the identification of selective substrates and protease-targeting tools. This
study supports the close functional relationship between mouse and human CatB while
demonstrating that species-specific differences can be exploited for selective targeting. The
findings highlight the potential of DARPins as species-selective CatB inhibitors and
provide structural and substrate-specificity insights relevant to the development of
protease-targeting tools.