diate-sized peptides, including atrial natriuric peptide, glucagon, and the amyloid b-protein (Ab) [11]; however, unlike insulin, most other IDE substrates are known to be hydrolyzed by multiple proteases. Diabetes melittus is a life-threatening and highly prevalent group of endocrinological disorders that, fundamentally, are characterized by impaired insulin signaling. Correspondingly, it is the common goal of most anti-diabetic therapies to enhance insulin signaling, either by direct injection of insulin, by stimulating the production or secretion of endogenous insulin, or by activating downstream targets of the insulin receptor (IR) signaling cascade [12]. In principle, it should be possible to enhance insulin signaling by inhibiting IDE-mediated insulin catabolism [13]. Pharmacological inhibitors of IDE in fact attracted considerable attention in the decades following the discovery of IDE in 1949 [14]. Quite significantly, a purified inhibitor of IDE (of undetermined identity) was found to potentiate the hypoglycemic action of insulin in vivo as early as 1955 [15]. Despite more than 60 years of research on IDE and its involvement in insulin catabolism, the development of small- molecule inhibitors of IDE has proved to be a surprisingly elusive goal [16]. We describe herein the design, synthesis, enzymologic characterization, and enzyme-bound crystal struc- ture of the first potent and selective inhibitors of IDE. In addition, we show that inhibition of IDE can potentiate insulin signaling within cells, by reducing the catabolism of internalized insulin. These novel IDE inhibitors represent important new pharmacological tools for the experimental manipulation of IDE and, by extension, insulin signaling. Furthermore, our results lend new support to the old idea that pharmacological inhibition of IDE may represent an attractive approach to the treatment of diabetes mellitus. Results Compound screening fails to identify effective IDE inhibitors The few IDE inhibitors currently in use are generally non- selective and/or highly toxic, and all suffer from extremely low potency, requiring mM concentrations to achieve complete inhibition (Table S1). To develop improved inhibitors of IDE, we initially conducted high-throughput screening on ,115,000 compounds, using a previously developed Ab degradation assay [17]. Although several inhibitors were discovered, most of these were either toxic, thiol-alkylating compounds [18] (Fig. S1A) or were compounds acting through other mechanisms that proved difficult to improve despite extensive medicinal chemistry optimization efforts (Fig. S1B). Among the remaining inhibitors, just a single compound, known as nullscript [19] (Fig. S1C), exhibited submicromolar IC50 values. Nullscript is predicted to target the active site of IDE because it contains the potent hydroxamic acid zinc-binding moiety, but it proved ineffective in cell-based assays (not shown). Consequently, we tested a range of peptidic and nonpeptidic hydroxamate inhibitors of conventional metalloproteases for possible effects on IDE. Surprisingly, IDE was unaffected by any of the nonpeptidic hydroxamates tested, some of which were potent inhibitors of a broad range of zinc-metalloproteases, and was only weakly inhibited by peptidic versions (Table S2). This result provides a striking confirmation that IDE’s active site—though obviously optimized to accommodate peptides—is indeed substantially different from the active site within canonical zinc-metallopro- teases, raising the possibility that highly selective zinc-binding inhibitors might be developed. Inhibitors of IDE Potent IDE inhibitors developed using a classic design strategy These initial results prompted us to revisit the classic design strategy originally used to develop zinc-metalloprotease inhib- itors [20]. The general approach is to attach a zinc-binding moiety to short peptides that correspond to the cleavage-site specificity of the protease of interest [21]. Because IDE lacks a clear cleavage-site specificity deducible from known substrates, we determined this de novo through proteomic analysis of the cleavage sites within combinatorial mixtures of short peptides (see Methods) [22]. This analysis revealed that IDE has a strong preference for Tyr<Arg . Phe at the P1’ position and for Arg at the P2’ position, while showing relatively little specificity at the remaining sites (Fig. 1A). Based on this analysis and considerations of synthetic tractability, we initially generated inhibitors based on the peptide Phe-Arg-Trp-Glu. A hydroxamic acid moiety was attached to the N-terminus, positioned appropriately by the inclusion of an a carbonyl moiety [21]. We generated pure diastereomers containing either all L-amino acids or a D-amino acid at the P1’ position, yielding Ki values of 0.11 (Fig. 1B) and 4.1 mM, respectively. While these compounds are far more potent than previously described IDE inhibitors, their potency is poorer than is typically achieved with peptide hydroxamates, and they proved non selective when tested against other metalloproteases (not shown). We postulated that these defi- ciencies were related to the incorporation of Phe at the P1’ position, which was not as strongly or uniquely preferred as Tyr or Arg (Fig. 1A). To optimize the P1’ position, we generated a focused library of peptide hydroxamates based on the sequence Xaa-Arg-Tyr, where Xaa represented a variety of natural and unnatural amino acids (Fig. 2A). These peptides were generated by solid-phase peptide synthesis in the ‘‘retro-inverso’’ config- uration, wherein the peptide backbone is reversed with respect to conventional peptides (see Fig. S2). The retro-inverso configuration required the use of all D-amino acids, and the need to incorporate an additional a carbon between the hydroxamic acid and the peptide sequence required the use of b-amino acids at the P1’ position. A ,100-fold gain in potency was realized when Phe was substituted with 2-naphthylalanine (2-Nap; Fig. 2B). We then generated a conventional peptide hydroxamate incorporating 2-Nap at the P1’ position, yielding an inhibitor, Ii1 (IDE inhibitor 1; Fig. 1C), with a Ki value of ,1.7 nM, or .105 more potent than IDE inhibitors currently in use (c.f., Table S1). Novel enzymological properties and unanticipated selectivity of Ii1 Enzymatic analysis of inhibitor Ii1 (Fig. 3) revealed several unexpected properties. First, the Ki values computed for Ii1 were found to be substrate dependent, with shorter substrates yielding lower values, and longer substrates—such as insulin and Ab— yielding values consistently ,10-fold higher (Fig. 3A). It is notable that a similar pattern of substrate dependence has consistently been observed for pharmacological activators of IDE [23] (see Discussion). In agreement with this result, Ii1 exhibited a purely competitive mode of inhibition of the hydrolysis of insulin (Fig. 3B) and Ab (Fig. S3). Second, the Hill slopes for inhibition of all substrates tested were ,1, suggesting some degree of negative cooperativity. Finally, and rather unexpectedly for this type of inhibitor, Ii1 proved to be remarkably selective for IDE vis-a`-vis a wide range of conventional zinc-metalloproteases and representa- tive members of other protease classes (Fig. 3C). PLoS ONE | www.plosone.org 2 May 2010 | Volume 5 | Issue 5 | e10504