Cruzain, a cysteine protease in the cathepsin family, is pivotal to the life-cycle of Trypanosoma cruzi, the etiological agent in Chagas disease. Current inhibitors of cruzain suffer from drawbacks involving gastrointestinal and neurological side effects and as a result have spurred the search for alternative anti-trypanocidals. Through sequence alignment studies and intra-residue interaction analysis of the pro-protein of cruzain (pro-cruzain), we have identified a host of non-active site residues that are conserved among the cathepsins. We hypothesize that these conserved amino acids play a critical role in structure-stabilizing interactions among the cathepsins and are therefore crucial for eventually gaining protease activity. As predicted, mutation of selected conserved non-active site amino-acid candidates in cruzain resulted in a compromised structural stability and a corresponding loss in enzymatic activity relative to wild-type enzyme. By advancing the discovery of novel, non-active-site-based targets to arrest enzymatic activity our results potentially open the field of alternative inhibitor design. The advantages of defining such a non-active-site inhibitor design space is discussed.
Keywords: Auto-activation; Chagas disease; Circular dichroism; Cysteine protease; Expression; Pro-cruzain; Trypanosoma cruzi.