Inhibitors of IDE Figure 1. Cleavage-site specificity of IDE and first-generation inhibitors derived therefrom. A, Heat map showing amino acids favored (red) or disfavored (green) by IDE at different positions relative to the cleavage site, as determined from proteomic analysis of peptide mixtures. B, Conventional peptide hydroxamate synthesized on the basis of the analysis in (A). C, Structure of inhibitor Ii1, incorporating optimized P1’ moiety deduced from analysis of a focused library of retro-inverso peptide hydroxamates (see Figure 2). doi:10.1371/journal.pone.0010504.g001 Crystal structure of IDE-Ii1 complex reveals a novel mode of inhibition To elucidate the mechanistic basis for these intriguing properties, we solved the co-crystal structure of Ii1 complexed to catalytically inactive, cysteine-free human IDE at 2.6- A˚ resolution (Fig. 4; Fig. S4; Table S3). The mode of binding of Ii1 is remarkable in several key respects, which have attendant implications for IDE’s mechanism of catalysis and for future drug design. First, the IDE-inhibitor interaction is restricted to the catalytic zinc and subsites S1’ and S2’ (Fig. 4A). The P3’ and P4’ residues of Ii1 face the center of the catalytic chamber of IDE and Figure 2. IDE inhibition by a focused library of retro-inverso peptide hydroxamates containing modifications at the P1’ position. A, Structure of compound library. Note that compounds were generated in the retro-inverso configuration (see Fig. S2). B, Relative Ki values of inhibitors containing different R groups (see A). doi:10.1371/journal.pone.0010504.g002 do not contact IDE directly; consequently, only the main chain of these two residues could be modeled (Fig. 4B). This is consistent with IDE’s strong cleavage-site preferences at P1’ and P2’ and weak preferences at P3’ and P4’ (Fig. 1A). Second, the active site of IDE is configured such that the P1’ and P2’ residues of Ii1 both point away from IDE’s internal chamber (Fig. 4A,B), thereby inducing a partial turn within the amide backbone of Ii1. This partial turn, which is also present within several full-length IDE substrates [9], appears to be a structural prerequisite permitting extended peptides to fit within IDE’s internal chamber. This configuration would appear to disfavor hydrolysis of substrates that extend from the inside to the outside of the protease, a feature that likely accounts for IDE’s inability to process larger protein substrates and its lack of exopeptidase activity. Finally, and quite significantly, Ii1’s hydroxamate moiety, amide backbone, and P2’ residue all make substantial interactions not only with residues in the N-terminal portions of IDE involved in zinc-binding and catalysis, but also with multiple residues within distal C-terminal portions of the protease (Fig. 4B; Fig. S4). This finding highlights the fact that IDE’s active site is not completely formed unless and until the N- and C-terminal portions of the protease are juxtaposed, which occurs only in the closed conformation and only during a portion of IDE’s catalytic cycle [9,18]. Consequently, Ii1 appears to inhibit IDE not only by active-site blockade but also by the novel mechanism of locking IDE in the closed, inactive conformation. Together, these atypical attributes of IDE’s active site and its catalytic mechanism may PLoS ONE | www.plosone.org 3 May 2010 | Volume 5 | Issue 5 | e10504