other proteases was assessed by monitoring the hydrolysis of OmniMMPTM Fluorogenic Substrate (Enzo Life Sciences International). Cell-based extracellular insulin degradation assays Recombinant human insulin (Sigma) was applied to CHO-IR cells or HeLa cells grown to near-confluency on 96-well plates under normal cell culture conditions, and its disappearance over time in the presence of different concentrations of IDE inhibitors or vehicle was quantified using a HTRF-based insulin assay (CIS- Bio). CHO-IR cells were a generous gift from Dr. Michel Bernier (National Instutute on Aging) and HeLa cells were purchased from American Type Culture Collection. Live-cell imaging of intracellular insulin catabolism CHO-IR cells were cultured on poly-D-lysine glass bottom culture dishes (MatTek Corp.), loaded with FITC-ins [28] (2 mM; Invitrogen) for 2 h at 37uC, washed with Hank’s balanced salt solution (HBSS), then imaged consecutively at fixed intervals by fluorescence microscopy at 22uC. Intracellular and extracellular fluorescence in the resulting images was quantified using MetaMorph software according to manufacturer’s recommenda- tions (Molecular Devices). IR autophosphorylation assays CHO-IR cells were grown to near confluency in 6-well plates, washed, then starved for 6 h in insulin-free medium (DMEM/ 0.1% BSA). Cells were then cold-loaded with human insulin essentially as described [49]. Briefly, after cooling the cells to 4uC, the medium was replaced with ice-cold HBSS/1% BSA supplemented with insulin (10 nM), incubated at 4uC for 1 h, washed 3 times with ice-cold HBSS/1% BSA. After addition of ice-cold HBSS/1% BSA supplemented with IDE inhibitors (10 mM) or DMSO, the cells were incubated at 37uC for 5 min, then rapidly cooled on ice. Cell lysates were harvested using manufacturer-provided cell-lysis buffer (Cell Signaling Technolo- gy) supplemented with additional phosphatase inhibitors (Milli- pore). IR autophosphorylation (Tyr 1146) and total IR levels were quantified by ELISAs (Cell Signaling Technology), and confirmed by western blotting with the same antibodies used for ELISAs or GAPDH as a loading control. Quantification of prebound insulin catabolism Confluent monolayers of CHO-IR cells grown in 6-well culture dishes were cold loaded with 240 pM 125I-insulin for 1 h and washed 3 times with ice-cold HBSS/1% BSA. Following addition of ice-cold HBSS/1% BSA supplemented with IDE inhibitors (10 mM) or DMSO, the cells were incubated at 37uC for 5 min (or as a control, maintained at 4uC), then rapidly cooled on ice and washed two times with ice-cold HBSS/1% BSA supplemented with Complete protease inhibitor cocktail (Roche) and Ii1 (10 mM). Following harvesting of cells by scraping, 125I levels were quantified by gamma counter (Beckman). Supporting Information Table S1 Examples of known IDE inhibitors. Found at: doi:10.1371/journal.pone.0010504.s001 (0.03 DOC) MB Table S2 IDE inhibition by commercially available hydroxamic acids. Found at: doi:10.1371/journal.pone.0010504.s002 (0.05 MB DOC) Inhibitors of IDE Table S3 Crystallographic statistics. Found at: doi:10.1371/journal.pone.0010504.s003 (0.07 DOC) MB Table S4 Potency of derivatized retro-inverso peptide hydroxamates. Found at: doi:10.1371/journal.pone.0010504.s004 (0.03 MB DOC) Figure S1 IDE inhibitors identified through high- throughput screening of ,115,000 compounds. A, Exam- ples of thiol-alkylating compounds, which made up the majority of identified inhibitors. B, Compound showing modest inhibition of IDE whose potency was not improved despite extensive medicinal chemistry efforts. C, Nullscript, a small-molecule hydroxamic acid, was the only inhibitor apart from thiol-alkylating compounds to show submicromolar potency. Found at: doi:10.1371/journal.pone.0010504.s005 (0.06 MB TIF) Figure S2 Structural comparison of conventional (A) and retro-inverso (B) peptide hydroxamates. Note that, in the retro-inverso compounds, the a-carbon adjacent to the hydroxamic acid moiety requires the use of b-amino acids, and that D-isomers must be used at all positions to mimic the relative orientation of residues in conventional peptides. Found at: doi:10.1371/journal.pone.0010504.s006 (0.06 MB TIF) Figure S3 Kinetic of Ii1-mediated inhibition of Ab degradation. A, Lineweaver-Burk plot of IDE-mediated Ab degradation in the absence or presence of Ii1 (30 nM). B, Quantitative kinetic data derived from A. Note pure competitive mode of inhibition. n = 4 replications. Found at: doi:10.1371/journal.pone.0010504.s007 (0.10 MB TIF) Figure S4 Surface representation of IDE showing the interior of the catalytic chamber defined by the N- and C-terminal domains. IDE-N and IDE-C are rotated by 290 degree (left) and +90 degree (right) with respect to IDE represented in the ribbon diagram above. Surfaces are colored by electrostatic potentials less than 6kT (red) or greater than +6kT (blue). The catalytic chamber of IDE-Ii1 contained extra electron density in the region previously shown to accommodate the N-terminus of substrates, which interact with the exosite of IDE. This extra electron density was fitted by a tri-alanine peptide. Surface potentials are displayed with Pymol. The monomeric IDE in the ribbon diagram is colored as green, blue, yellow and red for domains 1, 2, 3 and 4, respectively. The molecular surface of IDE is color coded by electrostatic potential, as calculated by APBS2. Ii1 and tri-alanine peptide are drawn in stick representation. Carbon, nitrogen, and oxygen atoms of Ii1 and the main chains of peptide at the exosite are colored orange, blue, and red, respectively. Figure generated using Pymol. Found at: doi:10.1371/journal.pone.0010504.s008 (1.58 MB TIF) Figure S5 Effects of Ii1 on catabolism of extracellular insulin in HeLa cells.- A, Dose-response curve of Ii1-mediated inhibition of insulin degradation by HeLa 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 HeLa cells in the absence or presence of Ii1 (10 mM). Note that insulin catabolism is completely inhibited by Ii1. Found at: doi:10.1371/journal.pone.0010504.s009 (0.05 MB TIF) PLoS ONE | www.plosone.org 9 May 2010 | Volume 5 | Issue 5 | e10504