Clickable, selective, and cell-permeable activity-based probe of human cathepsin B - Minimalistic approach for enhanced selectivity.

Human cathepsin B is a cysteine-dependent protease whose roles in both normal and diseased cellular states remain yet to be fully delineated. This is primarily due to overlapping substrate specificities and lack of unambiguously annotated physiological functions. In this work, a selective, cell-permeable, clickable and tagless small molecule cathepsin B probe, KDA-1, is developed and kinetically characterized. KDA-1 selectively targets active site Cys25 residue of cathepsin B for labeling and can detect active cellular cathepsin B in proteomes derived from live human MDA-MB-231 breast cancer cells and HEK293 cells. It is anticipated that KDA-1 probe will find suitable applications in functional proteomics involving human cathepsin B enzyme.

Cell penetrable, clickable and tagless activity-based probe of human cathepsin L.

Human cathepsin L is a ubiquitously expressed endopeptidase and is known to play critical roles in a wide variety of cellular signaling events. Its overexpression has been implicated in numerous human diseases, including highly invasive forms of cancer. Inhibition of cathepsin L is therefore considered a viable therapeutic strategy. Unfortunately, several redundant and even opposing roles of cathepsin L have recently emerged. Selective cathepsin L probes are therefore needed to dissect its function in context-specific manner before significant resources are directed into drug discovery efforts. Herein, the development of a clickable and tagless activity-based probe of cathepsin L is reported. The probe is highly efficient, active-site directed and activity-dependent, selective, cell penetrable, and non-toxic to human cells. Using zebrafish model, we demonstrate that the probe can inhibit cathepsin L function in vivo during the hatching process. It is anticipated that the probe will be a highly effective tool in dissecting cathepsin L biology at the proteome levels in both normal physiology and human diseases, thereby facilitating drug-discovery efforts targeting cathepsin L.

Development of a highly potent, selective, and cell-active inhibitor of cysteine cathepsin L-A hybrid design approach.

A hybrid-design approach is undertaken to develop a highly potent and selective inhibitor of human cathepsin L. Studies involving human breast carcinoma MDA-MB-231 cells establish that this inhibitor can successfully block intracellular cathepsin L activity, and retard the cell-migratory potential of these highly metastatic cells.

Development of cell-active non-peptidyl inhibitors of cysteine cathepsins.

Cysteine cathepsins are an important class of enzymes that coordinate a variety of important cellular processes, and are implicated in various types of human diseases. However, small molecule inhibitors that are cell-permeable and non-peptidyl in nature are scarcely available. Herein the synthesis and development of sulfonyloxiranes as covalent inhibitors of cysteine cathepsins are reported. From a library of compounds, compound 5 is identified as a selective inhibitor of cysteine cathepsins. Live cell imaging and immunocytochemistry of metastatic human breast carcinoma MDA-MB-231 cells document the efficacy of compound 5 in inhibiting cysteine cathepsin activity in living cells. A cell-motility assay demonstrates that compound 5 is effective in mitigating the cell-migratory potential of highly metastatic breast carcinoma MDA-MB-231 cells.

Stromal protein Ecm1 regulates ureteric bud patterning and branching.

The interactions between the nephrogenic mesenchyme and the ureteric bud during kidney development are well documented. While recent studies have shed some light on the importance of the stroma during renal development, many of the signals generated in the stroma, the genetic pathways and interaction networks involving the stroma are yet to be identified. Our previous studies demonstrate that retinoids are crucial for branching of the ureteric bud and for patterning of the cortical stroma. In the present study we demonstrate that autocrine retinoic acid (RA) signaling in stromal cells is critical for their survival and patterning, and show that Extracellular matrix 1, Ecm1, a gene that in humans causes irritable bowel syndrome and lipoid proteinosis, is a novel RA-regulated target in the developing kidney, which is secreted from the cortical stromal cells surrounding the cap mesenchyme and ureteric bud. Our studies suggest that Ecm1 is required in the ureteric bud for regulating the distribution of Ret which is normally restricted to the tips, as inhibition of Ecm1 results in an expanded domain of Ret expression and reduced numbers of branches. We propose a model in which retinoid signaling in the stroma activates expression of Ecm1, which in turn down-regulates Ret expression in the ureteric bud cleft, where bifurcation normally occurs and normal branching progresses.