Author(s) name and affiliation:
Kui (Kathy) Chen1, Klaus Michelsen2 & Rob Wahl1
HTS & Molecular Pharmacology1 & Molecular Structure2
Amgen, Inc. Thousand Oaks, CA 91320
Insulin-degrading enzyme (IDE) is a member of the inverzincin metalloprotease family with the inverse common zinc-binding motif (HXXEH). In addition to insulin, IDE is capable of recognizing and hydrolyzing amyloid-ß peptide (Aß) in brain. Recent success of the co-crystallization of IDE with Aß and promising data of IDE over-expression in rodents have brought great interest to the pharmaceutical industry in searching for small molecule allosteric IDE activators for prevention and treatment of Alzheimer’s Disease.
To facilitate our internal HTS campaign and hit triage, we have developed and optimized four IDE assays in three different assay platforms (IDE FRET assay with Substrate V, IDE FRET assay with Substrate Aß, IDE TR-FRET assay with Substrate Aß & IDE direct binding assay in Biacore).
This poster presents the study of 9 compounds, including literature-reported activators, general MMP inhibitors and physiological substrates, in the above IDE assays. The literature-reported IDE activators (Dynorphin Bs and TNP-ATP) have demonstrated activation in the IDE FRET assay with Substrate V, but not in the IDE FRET assay with Substrate Aß nor in the IDE TR-FRET assay with Substrate Aß. In the IDE Biacore analysis, the binding stoichiometry of Dynorphin Bs to IDE has been confirmed to be 2:1 with Kd values of ~ 50 µM. However, the binding of TNP-ATP to IDE seems to be non-specific and non-stoichiometric. On the other hand, the IC50s of general MMP inhibitors (EDTA and 1,10-Phenanthroline) and physiological substrates (Insulin and Aß40) determined in our two IDE FRET assays and in the IDE TR-FRET assay are consistent with the literature-reported IC50s and Km values, respectively.
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