Targeting Rare Conformational States to Achieve Selective Caspase Protease Inhibition

Abstract

Caspases are cysteine proteasesthat are major players in key cellular processes, including apoptosis and inflammation. Amongst the human caspases, caspase-6 has been implicated in playing a unique and critical role in the neurodegenerative pathways of Alzheimer's disease. Unfortunately, structural similarities between caspase-6 and other caspases have hampered precise targeting of caspase-6 uniquely. All caspases can exist in a canonical conformation, in which the substrate binds atop a beta-strand platform in the 130's region. This caspase-6 region can also adopt a helical conformation that has not been seen in any other caspases. We have shown that caspase-6 is inherently and dramatically more conformationally dynamic than closely related caspase-7. In contrast to caspase-7, which rests constitutively in the strand conformation before and after substrate binding, hydrogen/deuterium exchange data for the L2’ and 130's regions suggested that prior to substrate binding, caspase-6 exists in a dynamic equilibrium between the helix and strand conformations. Caspase-6 transitions exclusively to the canonical strand conformation only upon substrate binding. Glu-135, which showed noticeably different calculated pKas in the helix and strand conformations, appears to play a key role in the interconversion between the helix and strand conformations. We have also mapped the local changes in the conformational flexibility of procaspase-6 at the discrete states that reflect series of cleavage events that ultimately lead to the fully active, substrate-bound state. The prodomain region was found to be intrinsically disordered, independent of the activation step of caspase-6; however, its complete removal resulted in the protection of the adjacent 26-32 region, suggesting a regulatory role. The molecular details of caspase-6 dynamics in solution provide a comprehensive scaffold for strategic design of therapeutics for neurodegenerative disorders. We have used this information to make the most potent caspase-6 inhibitor to date.