These findings lend fresh support to the longstanding prediction that IDE inhibitors could hold therapeutic potential as primary or adjunct treatments for diabetes. Here we describe the rational design, synthesis, enzymologic characterization, and co-crystallographic analysis of potent and Fulvestrant 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. First and foremost, these compounds constitute the first potent and selective inhibitors of IDE or, indeed, of any member of the inverzincin superfamily of zinc-metalloproteases. Given the longstanding interest in IDE in general, and the predicted therapeutic value of IDE inhibitors in particular, why has their development proved so elusive for so long? The answer can be traced to the distinctive structure of IDE��s active site, which in turn reflects the separate evolutionary origins of this protease superfamily. As documented by earlier studies and the present work, IDE��s active site is bipartite, consisting of two distinct domains contained within the C- and N-terminal halves of the protease. The active site becomes fully formed only when the protease is in the closed conformation, and it is disrupted completely upon transition to the open conformation. These very large conformational changes occurring during the catalytic cycle of IDE essentially render its active site a “moving GSI-IX target,” one that cannot easily be stably occupied by small molecules, even those containing a strong zinc-binding moiety. As our co-crystal structure reveals, the potency of Ii1 can be traced to its unique ability to interact simultaneously with both the N- and C-terminal portions of the active site. In so doing, Ii1 appears to “lock” the protease in the closed, inactive conformation-a feature that is likely to be indispensable for effective IDE inhibitors. Second, these IDE inhibitors grant several new insights into the enzymology of this poorly understood protease. A particularly puzzling property is the substrate-dependence of Ki values for inhibition of IDE by Ii1, wherein smaller substrates show lower Ki values than larger substrates. These two categories of substrate have in fact been shown to exhibit strikingly different behaviors in multiple contexts. For example, the hydrolysis of short substrates-but not longer ones-can be profoundly activated by ATP and other nucleotide polyphosphates, inorganic triphosphate, as well as by structurally unrelated drug-like molecules. In terms of the differences in Ki values, we speculate that larger substrates may be more capable than smaller ones of effecting the transition between the closed and open configurations, resulting in an increased off rate for the inhibitor. It may also be that the inhibitor can be trapped inside the internal chamber only in the case of smaller substrates. Alternatively, given that 2 residues within Ii1 protrude into the internal chamber, it may be that larger substrates sterically block a subset of binding modes of the inhibitor. In this context, it is relevant to note that larger substrates are known to interact with an exosite present inside the catalytic chamber but opposite to the active site.