Inhibitors of IDE Figure 4. Structure of human IDE complexed to inhibitor Ii1. A, Overall structure of Ii1-bound IDE. Ribbon diagram of monomeric IDE colored as green, blue, yellow and red for domains 1, 2, 3 and 4, respectively (left). The connecting loop between IDE-N and IDE-C is shown in cyan. 2Fo–Fc simulated annealing omit map contoured at 1 s is shown in blue around Ii1 (right). In this view, the C-terminal domains of IDE (yellow, red) have been omitted to highlight the interactions of Ii1 with the catalytic zinc atom in the N-terminal domain of IDE. B, Stereodiagram showing the detailed interactions of Ii1 with residues and zinc ion in the catalytic site of IDE. Protein residues involved in catalysis and in contact with Ii1, as well as the inhibitor are drawn in stick representation. Residues from the C-terminal half of IDE are underlined and highlighted in yellow. Ii1 in stick representation is colored the same as in (A). Carbon nitrogen and oxygen atoms of Ii1 are colored orange and those of IDE grey; nitrogen and oxygen atoms are colored blue and red, respectively; zinc and water are represented by spheres colored magenta and red, respectively. Hydrogen bonds are shown as dashed lines. Figures generated using Pymol [50]. doi:10.1371/journal.pone.0010504.g004 CHO cells overexpressing the human insulin receptor (IR) (CHO-IR cells) (Fig. 5A) and HeLa cells (Fig. S5A), Ii1 inhibited the degradation of extracellular insulin dose-dependently, with IC50s comparable to that obtained in vitro (c.f., Fig. 3A). Notably, at mM concentrations, insulin catabolism was inhibited essen- tially completely (Fig. 5B; Fig. S5B), consistent with other evidence implicating IDE as the major extracellular insulin protease [5]. No toxicity was observed for Ii1 or ML3-XF up to 100 mM (Fig. S6). Because IDE is in part a secreted protease [24], its role in degrading extracellular insulin is not surprising. By contrast, the extent to which IDE participates in the degradation of internalized insulin remains controversial. The prevailing dogma suggests that, following binding to the IR, insulin is rapidly endocytosed and then degraded in acidic compartments of the endolysosomal system [25]. Consistent with this view, the aspartyl protease cathepsin D has been implicated in the degradation of internalized insulin [26]. On the other hand, IDE has been PLoS ONE | www.plosone.org 5 May 2010 | Volume 5 | Issue 5 | e10504