Cysteine cathepsin proteases include some of the most powerful human collagenases and elastases and are implicated in cardiovascular disease, where collagen and elastin are integral in artery biomechanics andsignaling. Of particular interest are cathepsins K, L, and S, which share 60% sequence homology and redundancyin target substrate proteins with different catalytic activities toward different extracellular matrix substrates, whichhave been investigated in isolation. However, cells secrete many proteases simultaneously to remodel arterial wallduring disease progression.This has made them highly sought after pharmaceutical targets, but of the 16 well-designed inhibitors thathave advanced to phase II clinical trials, all have failed except one due to cross-reactivity and off-target sideeffects, although there was efficacy for slowing disease progression. Previously, it has been shown that onespecies of cathepsin will preferentially degrade another, while also in the presence of matrix proteins; which wetermed cathepsin cannibalism. This led to the hypothesis that off-target side effects of pharmaceutical cathepsininhibitors may be due to an incomplete understanding of cathepsin regulation of each other, which could impactdrug dosing. Here we use computational modeling to shed light on the proteolytic network of cathepsincannibalism between multiple species and substrates to probe this network that is experimentally difficult to studyto understand the kinetics of small molecule inhibitors for appropriate dosing for small molecule inhibition ofthese cathepsins. Furthermore, we made site-specific mutants to confirm cannibalism cleavage directionality.
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