posted on 2022-02-04, 20:12authored byMichael
C. Yoon, Mitchell P. Christy, Von V. Phan, William H. Gerwick, Gregory Hook, Anthony J. O’Donoghue, Vivian Hook
CA-074 is a selective
inhibitor of cathepsin B, a lysosomal cysteine
protease. CA-074 has been utilized in numerous studies to demonstrate
the role of this protease in cellular and physiological functions.
Cathepsin B in numerous human disease mechanisms involves its translocation
from acidic lysosomes of pH 4.6 to neutral pH 7.2 of cellular locations,
including the cytosol and extracellular environment. To gain in-depth
knowledge of CA-074 inhibition under these different pH conditions,
this study evaluated the molecular features, potency, and selectivity
of CA-074 for cathepsin B inhibition under acidic and neutral pH conditions.
This study demonstrated that CA-074 is most effective at inhibiting
cathepsin B at an acidic pH of 4.6 with nM potency, which was more
than 100-fold more potent than its inhibition at a neutral pH of 7.2.
The pH-dependent inhibition of CA-074 was abolished by methylation
of its C-terminal proline, indicating the requirement for the free
C-terminal carboxyl group for pH-dependent inhibition. Under these
acidic and neutral pH conditions, CA-074 maintained its specificity
for cathepsin B over other cysteine cathepsins, displayed irreversible
inhibition, and inhibited diverse cleavages of peptide substrates
of cathepsin B assessed by profiling mass spectrometry. Molecular
docking suggested that pH-dependent ionic interactions of the C-terminal
carboxylate of CA-074 occur with His110 and His111 residues in the
S2′ subsite of the enzyme at pH 4.6, but these interactions
differ at pH 7.2. While high levels of CA-074 or CA-074Me (converted
by cellular esterases to CA-074) are used in biological studies to
inhibit cathepsin B at both acidic and neutral pH locations, it is
possible that adjusted levels of CA-074 or CA-074Me may be explored
to differentially affect cathepsin B activity at these different pH
values. Overall, the results of this study demonstrate the molecular,
kinetic, and protease specificity features of CA-074 pH-dependent
inhibition of cathepsin B.