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Molecular Features of CA-074 pH-Dependent Inhibition of Cathepsin B
Michael C Yoon1,2, Mitchell P Christy3, Von V Phan1,2
1Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, California 92093-0021, United States.
Abstract:
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.
Insights
CA-074 effectively inhibits cathepsin B at acidic pH, showing over 100-fold greater potency than at neutral pH. This pH-dependent inhibition is crucial for understanding cathepsin B
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Cathepsin B, a lysosomal cysteine protease, plays a role in cellular functions and disease mechanisms.
- Cathepsin B translocates between acidic lysosomes (pH 4.6) and neutral cellular compartments (pH 7.2).
- Understanding cathepsin B inhibition by CA-074 across different pH environments is critical.
Purpose of the Study:
- To evaluate the molecular features, potency, and selectivity of CA-074 for cathepsin B inhibition at acidic and neutral pH.
- To elucidate the pH-dependent inhibition mechanism of CA-074.
Main Methods:
- In vitro enzyme inhibition assays at pH 4.6 and pH 7.2.
- Assessment of CA-074 specificity against other cysteine cathepsins.
- Profiling mass spectrometry to analyze substrate cleavage.
- Molecular docking simulations to investigate enzyme-inhibitor interactions.
Main Results:
- CA-074 demonstrated significantly higher potency (nM range) in inhibiting cathepsin B at acidic pH 4.6 compared to neutral pH 7.2 (over 100-fold difference).
- The free C-terminal carboxyl group of CA-074 is essential for its pH-dependent inhibition.
- CA-074 maintained specificity for cathepsin B, displayed irreversible inhibition, and affected diverse substrate cleavages.
- Molecular docking revealed pH-dependent ionic interactions between CA-074 and cathepsin B's S2' subsite.
Conclusions:
- CA-074 exhibits potent and specific inhibition of cathepsin B, which is highly dependent on acidic pH.
- The findings highlight the importance of pH in modulating CA-074's efficacy and provide insights into its interaction mechanism.
- Adjusted concentrations of CA-074 or its prodrug CA-074Me could be explored for differential modulation of cathepsin B activity in various cellular compartments.
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