UC San Diego

MG-132 is a tripeptide-aldehyde (Cbz-leu-leu-leucinal) and is extensively used in cellular studies to understand the role of the proteasome in cells as it is a potent inhibitor of the human proteasome. Early articles of MG-132 revealed its ability to inhibit cysteine cathepsins and calpains (1) and advised the community that controls such as a cysteine protease inhibitor also be used distinguish between cellular function of the proteasome and that of cysteine proteases. Many papers in the last 25 years have made conclusions about the proteasome based on phenotypic changes that have occurred following treatment with MG-132 but without a control compound. We previously determined that MG-132 is not only a potent inhibitor of cathepsin B and cathepsin L, but also potently inhibits the aspartyl proteases, cathepsin D and E. We then assayed HeLa and A549 lysates for protease activity at pH 3.5 and 5.5 with MG-132 and observed complete inhibition with 10 µM of MG-132 while other proteasome inhibitors such as marizomib, bortezomib and carfilzomib did not significantly inhibit this enzyme activity. In dose response assays with A549 lysate, we determined that the IC50 value for MG-132 at pH 3.5 and pH 5.5 was 5.0 nM and 8.7 nM, respectively. In summary, MG-132 is a potent inhibitor of aspartyl protease and cysteine protease within the concentration range that is used in cellular studies. Therefore, the phenotypic and biochemical changes that occur upon treatment of cells with this inhibitor are unlikely to be solely due to inhibition of the proteasome.