The Calpain Protease Active Site: A Target for Inhibitor and Activity-Based Probe Design
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The calpain family of intracellular Ca2+-dependent cysteine proteases is involved in a number of intracellular signaling processes. Calpain hyperactivity has also been implicated in ischemic injury, neurodegenerative diseases and cataract formation. However, the specific function of calpains in these normal and diseased states remains unclear. Competitive inhibition of calpain is useful for studying their functions and can lead to pharmacological treatments, while monitoring their activity with activity-based probes (ABPs) can reveal how calpain is regulated and be applied to screen for inhibitors in vivo. But these strategies are complicated by the similarity of the calpain active-site when compared to other intracellular cysteine proteases. Therefore, there is a need to design inhibitors and ABPs that selectively target calpain. Using X-ray crystallography, the interactions between the calpain active-site and each of two reversible inhibitors was studied. This led to the discovery of novel non-covalent aromatic stacking and hydrogen bonding interactions between the primed-side adenine group of one inhibitor and indole ring of an active-site Trp residue in μ-calpain. A substrate-based competition assay later confirmed that these interactions provided this compound with an inhibitory advantage over the other, which lacked any primed-side interactions, thereby providing insight into the development of new, more specific reversible calpain inhibitors. Next, a fluorescent ABP, containing features borrowed from an irreversible and presumably calpain-specific inhibitor, was evaluated for its ability to detect calpain activitiy. Although this probe appropriately targeted the calpain active site in its Ca2+-activated form, it was unable to detect calpain activity in a cell extract. Nevertheless, the results of this study have yielded insights into ways of improving the calpain detecting ability of this ABP.