UC San Diego

Cathepsin B (CatB) is a cysteine protease that is abundant in the lysosome of cells and is responsible for degradation of proteins to maintain cellular homeostasis. However, in certain pathogenic conditions such as cancer, neurodegenerative brain disorders, autoinflammatory, and pathogen-induced pyroptosis, CatB can be found outside the lysosome. Most lysosomal enzymes are optimally active at the acidic pH of lysosomes and become inactivated at neutral pH outside the lysosome. However, CatB retains good stability and catalytic activity at extra-lysosomal pH conditions such as the cytosol where it has been shown to participate in cellular apoptotic and inflammatory pathways. This has led to the hypothesis that translocation of CatB from the lysosome to either the cytosol, nucleus, or extracellular environment contributes to the pathogenesis of various diseases. To investigate such hypothesis, we sought to develop inhibitor probes of CatB that are selective for this protease at either neutral pH or acidic pH. In parallel, we aimed to develop inhibitor probes that are specific to CatB over related cathepsins. We found that established inhibitors such as E64c target CatB but also potently inhibit other cysteine cathepsins. CA-074 is an established selective inhibitor for CatB but we discovered that it is over 100-fold more potent at pH 4.6 than pH 7.2. We then proposed that it is possible to develop an inhibitor that is the converse of CA-074—being more potent at pH 7.2 than pH 4.6. This is based on the hypothesis that CatB may have distinct substrate and inhibitor interactions at different pH conditions. To evaluate this hypothesis, we profiled the cleavage specificity of CatB at pH 4.6 and pH 7.2, along with other related cathepsins, and developed substrates that are cleaved by CatB better at neutral pH than at acidic pH and not cleaved by other related cathepsins. We then modified the substrate with a cysteine reactive warhead to successfully develop a novel inhibitor that potently and selectively inhibits CatB at neutral pH to be used as a molecular probe for the investigation of extra-lysosomal CatB pathogenic role in various cell-death and inflammatory pathways of human diseases.