Selective Neutral pH Inhibitor of Cathepsin B Designed Based on Cleavage Preferences at Cytosolic and Lysosomal pH Conditions

Cathepsin B is a
cysteine protease that normally functions within
acidic lysosomes for protein degradation, but in numerous human diseases,
cathepsin B translocates to the cytosol having neutral pH where the
enzyme activates inflammation and cell death. Cathepsin B is active
at both the neutral pH 7.2 of the cytosol and the acidic pH 4.6 within
lysosomes. We evaluated the hypothesis that cathepsin B may possess
pH-dependent cleavage preferences that can be utilized for design
of a selective neutral pH inhibitor by (1) analysis of differential
cathepsin B cleavage profiles at neutral pH compared to acidic pH
using multiplex substrate profiling by mass spectrometry (MSP-MS),
(2) design of pH-selective peptide–7-amino-4-methylcoumarin
(AMC) substrates, and (3) design and validation of Z-Arg-Lys-acyloxymethyl
ketone (AOMK) as a selective neutral pH inhibitor. Cathepsin B displayed
preferences for cleaving peptides with Arg in the P2 position at pH
7.2 and Glu in the P2 position at pH 4.6, represented by its primary
dipeptidyl carboxypeptidase and modest endopeptidase activity. These
properties led to design of the substrate Z-Arg-Lys–AMC having
neutral pH selectivity, and its modification with the AOMK warhead
to result in the inhibitor Z-Arg-Lys–AOMK. This irreversible
inhibitor displays nanomolar potency with 100-fold selectivity for
inhibition of cathepsin B at pH 7.2 compared to pH 4.6, shows specificity
for cathepsin B over other cysteine cathepsins, and is cell permeable
and inhibits intracellular cathepsin B. These findings demonstrate
that cathepsin B possesses pH-dependent cleavage properties that can
lead to development of a potent, neutral pH inhibitor of this enzyme.