Inhibitors of IDE 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 mM). 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 mM). D,E, Quantitative analysis of intracellular (D) and extracellular (E) FITC fluorescence from 5 replicate experiments. Data are mean 6 SEM. Note that these experiments were conducted at 22uC. 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 37uC in the presence of Ii1 (10 mM), ML3-XF (10 mM) or vehicle (DMSO). Data are mean 6 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 37uC in the presence of IDE inhibitors (10 mM) or vehicle then washed to remove insulin catabolites. Data are mean 6 SEM of 4 experiments expressed as a percentage of 125I present in control cells maintained at 4uC. doi:10.1371/journal.pone.0010504.g005 reported to be present in endosomes, where it may degrade insulin at neutral pH [27]. The development of Ii1—which potently inhibits IDE, but not cathepsin D (Fig. 2C)—enabled us for the first time to address cleanly this longstanding controversy. To that end, we conducted live-cell imaging of CHO-IR cells loaded with fluorescent insulin labeled exclusively at the N- terminus of the B chain with fluorescein isothiocyanate (FITC- ins), a modification that has been shown not to interfere with binding to the IR [28]. FITC-ins-loaded cells were washed then monitored for changes in fluorescence in the presence of Ii1 or vehicle (Fig. 5C). In vehicle-treated cells, intracellular fluores- cence decreased (Fig. 5D) and extracellular fluorescence increased (Fig. 5E) monotonically with time. By contrast, both intra- and extracellular fluorescence remained essentially constant in the presence of Ii1 (Figs. 5D,E). Consistent with previous studies of insulin catabolism[29], the fluorescent species secreted by vehicle-treated cells were confirmed to be proteolytic fragments of FITC-ins (Fig. S7). These results strongly suggest that the catabolism of internalized insulin is primarily, if not exclusively, carried out by IDE. Unanticipated effects of IDE inhibitors on insulin signaling Given the historic interest in IDE inhibition as a potential antidiabetic treatment, we assessed the effects of our novel inhibitors on insulin signaling. To simplify interpretation, we focused on the most upstream event in the insulin signaling cascade: IR autophosphorylation. To that end, CHO-IR cells were loaded with insulin at 4uC and washed extensively (in the absence of inhibitors), then warmed to 37uC for 5 min in the presence of IDE inhibitors (10 mM) or vehicle alone. The degree of insulin-stimulated (but not basal) IR autophosphorylation was consistently potentiated in the presence of Ii1 or the retro-inverso IDE inhibitor, ML3-XF (Fig. 5F; Table S4). Though surprisingly brief, the time-scale of this effect is consistent with classic studies of insulin degradation showing that the prebound pool of insulin is degraded very rapidly [29]. To verify this in our experimental system, we directly quantified the degree of degradation of 125I- insulin prebound to CHO-IR cells. In good agreement with previous studies [29], prebound 125I-insulin decreased ,30% within 5 min at 37uC in vehicle-treated cells, an effect that was inhibited completely by Ii1 and ML3-XF (Fig. 5G). Taken together, these results suggest that IDE normally regulates insulin signaling by virtue of its ability to rapidly degrade internalized pools of insulin; correspondingly, insulin signaling can be potentiated significantly by inhibiting IDE proteolytic activity. In addition to illustrating the utility of our novel compounds as experimental probes, these findings lend fresh support to the long- standing prediction that IDE inhibitors could hold therapeutic potential as primary or adjunct treatments for diabetes. Discussion Here we describe the rational design, synthesis, enzymologic characterization, and co-crystallographic analysis of potent and selective peptide hydroxamate inhibitors of IDE. In addition, we use these compounds to show that IDE regulates fundamental aspects of insulin catabolism and signaling in a manner that implies that IDE inhibitors could have anti-diabetic properties. Although the inhibitors described in this study are unlikely to have immediate value as therapeutic agents due to their peptidic nature, their development and the chemical biology they make possible are significant in several important respects. PLoS ONE | www.plosone.org 6 May 2010 | Volume 5 | Issue 5 | e10504