Inhibitors of IDE 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 Ab (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. doi:10.1371/journal.pone.0010504.g003 account for the long-standing elusiveness of small-molecule IDE inhibitors, while at the same time auguring well for the eventual development of highly selective compounds (see Discussion). Unexpected potency of derivatized IDE inhibitors Because retro-inverso peptide hydroxamates could be generated with facility by solid-phase peptide synthesis, we explored the development of affinity labeled versions containing benzoylphe- nylalanine at either the P2’ or P3’ position together with a fluorescent EDANS label, and in some cases, a biotin group attached via a polyethylene glycol linker. The biotinylated versions avidly labeled recombinant IDE, but showed rather broad cross- reactivity in cell lysates under the conditions tested (not shown), probably due in part to their very large size (,1600 Da). Surprisingly, however, the addition of these extraneous function- alities resulted in considerable increases in potency, with Ki values in the range of 0.3 to 4 nM (Table S4), an effect that we speculate may relate to a reduced off rate due to entrapment within IDE’s internal chamber. Importantly, this effort resulted in the generation of 4-amino acid retro-inverso compounds that, relative to Ii1, were not only comparable in size and potency but also contained divergent residues at all positions but P1’ (e.g., ML3-XF; Table S4). Moreover, the inverted peptide backbone in these compounds is predicted to render them more resistant to degradation than conventional peptidic inhibitors, and they proved useful in downstream cellular assays. Effects on catabolism of extracellular and prebound insulin To assess the utility of these novel IDE inhibitors as chemical probes, we examined their effects on a range of endpoints relevant to insulin catabolism and signaling in cultured cells. In PLoS ONE | www.plosone.org 4 May 2010 | Volume 5 | Issue 5 | e10504