Cocrystal Structures of Primed Side-Extending α-Ketoamide Inhibitors Reveal Novel Calpain-Inhibitor Aromatic Interactions

Abstract

Calpains are intracellular cysteine proteases that catalyze the cleavage of target proteins in response to Ca 2+ signaling. When Ca 2+ homeostasis is disrupted, calpain over-activation causes unregulated proteolysis, which can contribute to diseases such as post-ischemic injury and cataract formation. Potent calpain inhibitors exist, but of these many cross-react with other cysteine proteases and will need modification to specifically target calpain. Here, we present crystal structures of rat calpain 1 protease core (μI-II) bound to two α-ketoamide-based calpain inhibitors containing adenyl and piperazyl primed-side extensions. An unexpected aromatic-stacking interaction is observed between the primed-side adenine moiety and the Trp298 side chain. This interaction increased the potency of the inhibitor towards μI-II and heterodimeric m-calpain. Moreover, stacking orients the adenine such that it can be used as a scaffold for designing novel primed-side address regions, which could be incorporated into future inhibitors to enhance their calpain specificity.In response to Ca 2+ signaling, the calpain family of intracellular cysteine proteases catalyzes the limited cleavage of target proteins, resulting in changes to processes such as gene expression, cytoskeleton remodeling and apoptosis. 1 Problems arise following ischemic or cerebral injury, when cells lose their ability to regulate Ca 2+ influx to the cytoplasm. The elevated Ca 2+ concentration leads to calpain hyperactivation, which causes uncontrolled proteolysis and irreversible cell damage. Since their overactivation has been linked to the development of pathological conditions such as stroke, Alzheimer disease, Duchenne muscular dystrophy and cataractogenesis, calpains represent an important class of targets for pharmacological inhibition. 2,3To date, all known calpain isoforms are multidomain enzymes, 4 with a catalytic cleft located at the interface between domains I and II. 5 These two domains, which encompass the enzyme's proteolytic core, must each bind one Ca 2+ ion to facilitate the rearrangement of the catalytic triad and substrate binding pocket into an active conformation. 6 Although the various other domains also contribute somewhat to calpain activation, the susceptibility of full-length calpain to autolysis, subunit dissociation and aggregation following Ca 2+ activation has complicated its study in the full-length form. 7 The protease core though, remains resistant to autolysis and maintains its Ca 2+ -dependent activity, albeit, at a significantly reduced level. 8 In addition, because of the relative ease with which they can be expressed in E. coli and crystallized, these *To whom correspondence should be addressed: Department of Biochemistry, Queen's University, Kingston, ON, CANADA K7L 3N6. Tel: 613-533-6821; Fax: 613-533-2497; E-mail: robert.campbell@queensu.ca. c Current address: Cytochroma Inc., 330 Cochrane Drive, Markham, ON, Canada L3R 8E4 PDB: coordinates and structure factors for the complexes of the protease core of μ-cal...

Calpains: Attractive Targets for the Development of Synthetic Inhibitors

“…This characteristic hydrogen bond pattern is clearly evident in the structures of I-II in complex with AK-295-D2 (shown in Fig. (6), [138,139,166]) and the other inhibitors depicted in Fig. (4) [162,163,166], and also in that of ZLLYCH 2 F (Fig.…”

“…(6), [138,139,166]) and the other inhibitors depicted in Fig. (4) [162,163,166], and also in that of ZLLYCH 2 F (Fig. 5) bound to I-II [164].…”

“…However, the X-ray structures of Ca 2+ -bound calpain 2 in complex with domain I [132] and domain IV [133] of calpastatin, respec-tively, have now been published. In addition, engineered constructs of the protease core of calpains 1, 2 and 9, so-called 'minicalpains', have been crystallized [159][160][161] and used to perform crystallographic studies on complexes with various inhibitors [162][163][164][165][166].…”

“…Crystal structures [162][163][164][165][166] of Ca 2+ -activated minicalpain 1 ( I-II) complexed with reversible ( Fig. (4), [168][169][170]) and irreversible (Fig.…”

“…However, despite these differences, all inhibitors shown in Figs. (4) and (5) form a pair of hydrogen bonds between their P 1 or P 1 ' CO group [162][163][164][165][166] and the residues of the oxyanion hole of I-II (side chain amide of Gln109 and backbone NH group of Cys115, see also Fig. (6)).…”