Selective targeting of lysosomal cysteine proteases with radiolabeled electrophilic substrate analogs

Background: The lysosomal cysteine proteases of the papain family are some of the best studied proteolytic enzymes. Small-molecule inhibitors and fluo rogenic substrate mimics have been used to probe the physiological roles of these proteases. A high degree of homology between family members and over lap in substrate specificity have made elucidating individual protease func tion, expression and activity difficult. Results: Using peptide vinyl sulfones and epoxide as templates, we have gen erated probes that can be tagged with radioactive iodine. The resulting com pounds covalently label various cathepsins and several unidentified polypep tides likely to be proteases, MB-074 was found to be a highly selective pro be of cathepsin B activity. Probes that labeled several cathepsins were use d to examine the specificity and cell permeability of the CA-074 family of inhibitors. Although CA-074 reportedly acts in vivo, we find it is unable t o penetrate cells, Esterifying CA-074 resulted in a cell-permeable inhibito r with dramatically reduced activity and specificity for cathepsin B. The p robes were also used to monitor protease activity in primary human tumor ti ssue and cells derived from human placenta. Conclusions: We have generated a highly selective cathepsin B probe and sev eral less specific reagents for the study of cathepsin biology. The reagent s have several advantages over commonly used fluorogenic substrates, allowi ng inhibitor targets to be identified in a pool of total cellular enzymes. We have used the probes to show that cathepsin activity is regulated in tum or tissues and during differentiation of placental-derived cytotrophoblasts to invasive cells required for establishing blood circulation in a develop ing embryo.