lifelong, pancellular deletion of IDE present in these animals, drugs that only partially or transiently block IDE activity and/or target only a subset of IDE molecules would not be expected to give rise to these compensatory changes, but would instead be expected to improve glycemic control [38]. IDE knockout mice also show elevated levels of cerebral Ab, raising concern that chronic inhibition of IDE could increase the risk for AD [5]. However, pharmacological inhibitors of IDE could easily be engineered to be blood-brain barrier impermeant, thereby obviating this concern. Moreover, in light of the recent finding that intranasal insulin improves cognition in early AD patients [39], and given insulin’s beneficial effects on learning and memory [1], it may be overly simplistic to assume that IDE’s role in AD pathogenesis is limited to its predicted effects on Ab alone. Finally, the inhibitors we have developed constitute important new tools for the experimental manipulation of IDE, tools that are long overdue [16]. In this connection, it is significant to note that EDTA does not inhibit IDE except after prolonged incubation [18,40]. The use of IDE inhibitors might therefore be critical for many routine experimental as well as clinical applications involving short peptides. Thus, despite their peptidic nature, the IDE inhibitors described herein should be of immediate use for addressing a number of outstanding questions concerning the chemical biology of this important protease, as well as for accurately quantifying its substrates. Moreover, the insights derived from our co-crystal structure, the first IDE-inhibitor complex, provide a key starting point for the development of more drug-like IDE inhibitors. Given that IDE resides predominantly in the cytosol and other intracellular compartments [41], where its function remains ill-defined, the development of cell-permeant IDE inhibitors will be a particularly important goal. Furthermore, the development of IDE inhibitors that are stable and non-toxic in vivo will permit a more thorough evaluation of the physiological and pathophys- iological roles of IDE proteolytic activity in animal models of disease, which in turn could lead to new treatments for diabetes and other disorders. Methods Analysis of IDE’s cleavage-site specificity To determine the amino acids preferred by IDE at positions C- terminal to the scissile bond, IDE was allowed to partially hydrolyze a mixture of N-terminally acetylated 12-mer peptides (Ac-XXXXXXXXXXXX-amide, where ‘‘X’’ refers to any amino acid except cysteine). The resulting C-terminal peptide fragments, containing unblocked N-termini, were analyzed by successive rounds of Edman degradation as described [22]. To determine amino acids preferred at positions N-terminal to the scissile bond, the results of the above analysis were used to generate a second peptide mixture (MGXXXXYKPEDK [K-biotin]-amide) de- signed to promote hydrolysis N-terminal to the sequence YKPE. Following partial hydrolysis with IDE, biotinylated species, comprising intact peptides and C-terminal fragments, were removed with an avidin column, and the remaining peptides, consisting of freshly cleaved N-terminal fragments, were analyzed by Edman degradation. Synthesis of conventional peptide hydroxamic acids The synthesis of the conventional peptide hydroxamic acids is described in Fig. S8. Synthesis of distereomerically pure intermediates used to confirm the stereochemical assignments of the conventional peptide hydroxamic acids is also described in Figs. S9, S10. Inhibitors of IDE Synthesis of retro-inverso peptide hydroxamic acids 9-fluorenylmethyleneoxy-carbonyl (Fmoc)-based solid-phase peptide peptide synthesis was carried out manually using a 2- chlorotritylhydroxyl amine resin (Sigma-Aldrich). Following de- protection of the resin with 20% piperidine in dichloromethane (DCM), Fmoc-protected b-D-amino acids (5 eq.) were coupled in a 16-hr reaction using HATU (5 eq.), HOAt (5 eq.) and DIEA (10 eq.) in DCM, and subsequent amino acids (4 eq.) were coupled using HBTU (4 eq.), HOBt (4 eq.) and DIEA (4 eq.) in dimethylformamide (DMF). Cleavage from the resin and removal of protecting groups was effected by a mixture of trifluoroacetic acid (95%), triisopropylsilane (2.5%) and water (2.5%).Peptides were purified by reverse-phase liquid chromatography using a linear acetonitrile:water gradient and masses were verified by matrix-assisted laser desorption/ionization time-of-flight (MALDI- TOF) mass spectrometry. Crystallization and structural refinement of IDE-Ii1 complex IDE-CF-E111Q, a catalytically inactive IDE mutant free of cysteines (C110L, C171S, C178A, C257V, C414L, C573N, C590S, C789S, C812A, C819A, C904S, C966N, and C974A) was constructed using the QuickChange Multi Site-Directed Mutagenesis Kit according to manufacturer’s recommendations (Stratagene). IDE-CF has catalytic activity comparable to wild- type IDE, and IDE-CF-E111Q readily forms diffracting crystals. Expression and purification of IDE-CF-E111Q were performed as previously described [42]. The complex of CF-IDE-E111Q and the peptide hydroxamate Ii1 was formed by mixing protein and Ii1 in a 1:1 molar ratio and isolated by a superdex-200 column. Crystals of IDE-Ii1 were grown by hanging drop and cryopro- tected as described [42]. Data from a crystal that diffracted to 2.6 A˚ resolution was collected at 100 K at the Advanced Photon Source Structural Biology Center ID19 (Argonne, IL) and processed with the programs HKL2000/SCALEPACK [43]. Using substrate-free IDE-Y831F (PDB code 2JG4; Ref [31]) as the model in PHASER [44], a satisfactory molecular replacement solution of IDE-Ii1 was obtained. Refinement of the model was performed using anisotropic scaling, bulk solvent correction, rigid body refinement, coordinate and individual restraint B factor refinement, and simulated annealing using PHENIX [45]. Building of IDE structure was performed using Coot [46]. Simulated annealing 2Fo–Fc composite omit map was then calculated using CNS [47] and used in modelling Ii1 at the active site. The final model of IDE-Ii1 (PDB code 3E4A) had Rwork and Rfree of 16.7% and 22.5%, respectively. The statistics of diffracting data and refinement are summarized in Table S3. In vitro enzymatic assays IDE activity was quantified by monitoring hydrolysis of FRET1 [31], SP1 [23], FAbB [17] or 125I-insulin [48] as described previously, using recombinant human IDE purified from bacteria [33]. Degradation of unmodified recombinant human insulin (Sigma-Aldrich) and FITC-ins [28] (Invitrogen) was assessed using a homogeneous time-resolved fluorescence (HTRF)-based assay (CIS-Bio). Quantitative kinetic data were derived by hyperbolic regression analysis using the computer program HYPER.EXE created by John S. Easterby (University of Liverpool). Activity of cathepsin B was assessed by monitoring hydrolysis of Z-LR-AMC (Enzo Life Sciences International), that of cathepsin D using the Cathepsin D Assay Kit (Sigma-Aldrich), and that of the 20S proteasome using the 20S Proteasome Assay Kit for Drug Discovery (Enzo Life Sciences International). The activity of all PLoS ONE | www.plosone.org 8 May 2010 | Volume 5 | Issue 5 | e10504