DS_DISC786509 – Supplemental material for Development and Characterization of Quantitative, High-Throughput-Compatible Assays for Proteolytic Degradation of Glucagon

<p>Supplemental material, DS_DISC786509 for Development and Characterization of Quantitative, High-Throughput-Compatible Assays for Proteolytic Degradation of Glucagon by Caitlin N. Suire, Shelley Lane and Malcolm A. Leissring in SLAS Discovery</p

Age-dependent changes in basal blood glucose and body weight.

<p><b><i>A</i></b>, Blood glucose in 2-, 4- and 6-mo-old wild-type (WT) and IDE-KO (KO) mice following overnight fasting. Note that 2-mo-old IDE-KO mice exhibit significantly lower basal glucose levels relative to controls. <b><i>B</i></b>, Body weight of fasted 2-, 4- and 6-mo-old WT and IDE-KO mice. Note that IDE-KO mice weigh significantly less than wild-type controls mice at 2 months yet significantly more at 6 months of age. Data are mean ± SEM of 10–12 mice per group. *P<0.05 IDE-KO <i>vs.</i> WT as determined by 2-tailed Student's t test.</p

Constitutive hyperinsulinemia in IDE-KO mice.

<p>Insulin levels in serum of 2-, 4- and 6-mo-old wild-type (WT) and IDE-KO (KO) mice following overnight fasting. Data are mean ± SEM of 10–12 mice per group. *P<0.05 determined by 2-tailed Student's t test.</p

IDE-KO mice show age-dependent reductions in IR levels and insulin-stimulated glucose uptake that correlate with the onset of the diabetic phenotype.

<p><b><i>A</i></b>–<b><i>C</i></b>, IR levels in muscle (<b><i>A</i></b>) adipose (<b><i>B</i></b>) and liver (<b><i>C</i></b>) tissue from wild-type (WT) and IDE-KO (KO) mice. Note that IR levels in IDE-KO mice are significantly decreased in all tissues at 6 months, but not at 2 months of age. Graphs show mean ± SEM of IR levels quantified by luminescent imaging (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020818#s4" target="_blank"><i>Materials and Methods</i></a>) and normalized to IR levels in 2-mo-old WT mice. *P<0.05 relative to 6-mo-old WT mice; ^#P<0.01 relative to 2-mo-old KO mice and P<0.05 relative to 6-mo-old WT and KO mice; ^⧫P<0.05 relative to 2-mo-old KO mice, all determined by 2-tailed Student's t tests. <b><i>D</i></b>, Insulin-stimulated glucose uptake is significantly impaired in primary adipocytes isolated from 6-mo-old IDE-KO mice. Quantification was performed by measuring cellular uptake of ¹⁴C-deoxyglucose (¹⁴C-DOG) various times after addition of insulin (16.7 nM). Data are mean of 2 independent experiments utilizing epididymal adipose tissue collected from 4 mice per condition for each replication.</p

Structure and activity of P12-3A.

<p><b><i>A</i></b>, Structure of cyclized <b>P3-12A</b>. <b><i>B</i></b>, Dose-response of <b>P12-3A</b> against insulin degradation by IDE. Data are mean ± SEM of 5 independent experiments.</p

Peptides derived by phage display.

<p><b><i>A</i></b>, Peptide sequences deduced from DNA sequencing of 20 clones from the Ph.D.™-C7C library. <b><i>B</i></b>, Consensus sequence derived from analysis of all data. <b><i>C</i></b>, Parent peptides selected for synthesis and testing. <b><i>C</i></b>,<b><i>D</i></b>, Peptide sequences deduced from DNA sequencing of 39 clones from the Ph.D.™-12 library, conducted as two independent runs (<b><i>D</i></b> and <b><i>E</i></b>). Note that Seq-12A-07 did not yield a decipherable sequence. <b><i>F</i></b>, Parent peptides selected for subsequent synthesis and testing based on prevalence.</p

Effects of P12-3A on cultured skin cells.

<p><b><i>A</i></b>, Insulin concentrations as a function of time in logarithmically growing primary murine skin fibroblasts in the absence or presence of <b>P12-3A</b> (100 μM). Note that insulin levels remain constant in the presence of <b>P12-3A</b>, reflecting both the effectiveness of the peptide inhibiting insulin degradation and also the stability of the peptide in biological milieu. Data are mean ± SEM of 4 independent replications. *<i>P<0</i>.<i>05</i>, <i>**P<0</i>.<i>01</i>. <b><i>B</i></b>, Proliferation of cells in the absence or presence of <b>P12-3A</b> (100 μM). Data are mean ± SEM of 6 independent replications. No significant differences were observed. <b><i>C</i></b>,<b><i>D</i></b>,<b><i>E</i></b>, <b>P12-3A</b> (100 μM) potentiates insulin-induced collagen production in skin fibroblasts. Collagen production was assessed by <i>COL1A1</i> mRNA levels (<b><i>C</i></b>), levels of hydroxyproline secreted into the medium (<b><i>D</i></b>), and cell-associated mature alpha-1 type I collagen levels detected by Western blotting (<b><i>E</i></b>). Data are mean ± SD of 4 independent replications. *<i>P<0</i>.<i>05</i>, <i>**P<0</i>.<i>01</i>. <b><i>F</i></b>, <b>P12-3A</b> (100 μM) potentiates the migration of keratinocytes in a scratch wound assay. Migration of HaCaT cells 48 h after induction of a scratch wound in the presence of the indicated quantities of insulin and/or <b>P12-3A</b> (100 μM). Data are mean ± SEM of 6 independent replications. *<i>P<0</i>.<i>05</i>, <i>**P<0</i>.<i>01</i>.</p

Vulnerability of peptides to degradation by IDE assessed by activity assays.

<p>IC₅₀ values obtained for selected peptides pre-incubated with IDE 0 or 4 h before testing with the FRET1 assay. Note that all peptides except <b>P12-3A</b> and <b>P12-3B</b> showed reductions in apparent potency after prolonged incubation with IDE, reflecting degradation. Data are the average of duplicate assays that did not differ by more than 5%.</p

Selective Targeting of Extracellular Insulin-Degrading Enzyme by Quasi-Irreversible Thiol-Modifying Inhibitors

Many therapeutically important enzymes are present in multiple cellular compartments, where they can carry out markedly different functions; thus, there is a need for pharmacological strategies to selectively manipulate distinct pools of target enzymes. Insulin-degrading enzyme (IDE) is a thiol-sensitive zinc-metallopeptidase that hydrolyzes diverse peptide substrates in both the cytosol and the extracellular space, but current genetic and pharmacological approaches are incapable of selectively inhibiting the protease in specific subcellular compartments. Here, we describe the discovery, characterization, and kinetics-based optimization of potent benzoisothiazolone-based inhibitors that, by virtue of a unique quasi-irreversible mode of inhibition, exclusively inhibit extracellular IDE. The mechanism of inhibition involves nucleophilic attack by a specific active-site thiol of the enzyme on the inhibitors, which bear an isothiazolone ring that undergoes irreversible ring opening with the formation of a disulfide bond. Notably, binding of the inhibitors is reversible under reducing conditions, thus restricting inhibition to IDE present in the extracellular space. The identified inhibitors are highly potent (IC₅₀^app = 63 nM), nontoxic at concentrations up to 100 μM, and appear to preferentially target a specific cysteine residue within IDE. These novel inhibitors represent powerful new tools for clarifying the physiological and pathophysiological roles of this poorly understood protease, and their unusual mechanism of action should be applicable to other therapeutic targets