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).
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).
Figure 3.
In vitro enzymatic analysis of IDE inhibition by Ii1.
A, Representative dose-response curves for a variety of IDE substrates and Ki values and Hill slopes computed from 4 to 6 replications per substrate. Note that [S]/KM was kept constant for dose-response studies, permitting visual comparison of relative Ki values. B, Lineweaver-Burk plot of IDE-mediated insulin degradation in the absence or presence of Ii1 (30 nM) and kinetic parameters calculated from 4 independent experiments. These data were obtained using recombinant human insulin and 125I-insulin at a fixed ratio (1000∶1), as described [48]. Note that the mode of inhibition is purely competitive, and was also observed using Aβ (Fig. S3). C, Dose-response curves showing the selectivity of Ii1 for IDE as compared to several other zinc-metalloproteases—neprilysin (NEP), endothelin-converting enzyme-1 (ECE1), angiotensin-converting enzyme-1 (ACE1) and matrix-metalloprotease-1 (MMP1)—and representative members of other protease classes, including cysteine (cathepsin B), aspartate (cathepsin D), serine (trypsin and plasmin) and threonine (20S proteasome). Data are mean of 2 to 3 experiments per condition.
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 σ 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].
Figure 5.
Effects of IDE inhibitors on insulin catabolism and signaling in cells.
A, Dose-response curve of Ii1-mediated inhibition of insulin degradation by CHO-IR cells. Note that the potency of Ii1 in this context is comparable to that obtained in vitro (c.f., Fig. 3A). B, Progress curve of insulin degradation by CHO-IR cells in the absence or presence of Ii1 (10 µM). Note that insulin catabolism is completely inhibited by Ii1. C–F, Effects of Ii1 on insulin catabolism in live cells. C, Representative images of live CHO-IR cells pre-loaded with FITC-ins and imaged at various time points in the presence or absence of Ii1 (10 µM). D,E, Quantitative analysis of intracellular (D) and extracellular (E) FITC fluorescence from 5 replicate experiments. Data are mean ± SEM. Note that these experiments were conducted at 22°C. F, Effects of Ii1 on insulin signaling in CHO-IR cells cold-loaded with insulin. Graph shows IR autophosphorylation (phospho-IR) determined by ELISA in response to 5-min incubation at 37°C in the presence of Ii1 (10 µM), ML3-XF (10 µM) or vehicle (DMSO). Data are mean ± SEM of 6 independent experiments, where data are normalized to IR autophosphorylation obtained for insulin and vehicle alone. *p<0.01. Western analysis of a representative experiment (lower panels) providing biochemical confirmation of the results obtained by ELISA. G, Preloaded insulin is rapidly catabolized by CHO-IR cells and blocked by IDE inhibitors. Graph shows amounts of 125I present in CHO-IR cells cold-loaded with 125I-insulin then incubated for 5 min at 37°C in the presence of IDE inhibitors (10 µM) or vehicle then washed to remove insulin catabolites. Data are mean ± SEM of 4 experiments expressed as a percentage of 125I present in control cells maintained at 4°C.