A Pre-Specified, Structure-Based Cascade Protocol for the Discovery of Exosite-Directed, Zinc-Sparing Inhibitors of Insulin-Degrading Enzyme

Abstract Background Zn 2+ -dependent metallopeptidase that regulates the clearance of insulin and several bioactive peptides. Therapeutic IDE inhibition is complicated by the conservation of catalytic zinc-binding features among metallopeptidases and by the potential consequences of central IDE inhibition. Objective To define a reproducible, prospectively specified computational framework for identifying exosite-directed, zinc-sparing IDE inhibitors while explicitly evaluating selectivity against neprilysin (NEP) and angiotensin-converting enzyme (ACE). Methods The workflow comprises structural verification and standardized preparation of IDE, NEP, and ACE; self-docking validation of crystallographic reference ligands; curation of non-chelating chemical libraries; parallel three-target virtual screening; quantitative selectivity filtering; pose-based exosite assessment; molecular dynamics and MM-GBSA rescoring; cross-target dynamic controls; and in silico ADMET triage, including exclusion of predicted blood–brain barrier penetration. Results Protocol verification identified a critical structural assignment error in the original target set: PDB 2G54 contains Zn 2+ -bound IDE complexed with insulin B-chain, whereas the IDE–Ii1 complex is PDB 3E4A. Accordingly, 2G54 is designated for substrate-engaged structural mapping and 3E4A for Ii1 redocking validation. The protocol also replaces the previously stated Ii1 potency value with the approximately 2 nM value reported in the primary study. No virtual screening, molecular dynamics, or ADMET calculations were performed; therefore, no candidate-level computational outcomes are claimed. The corrected protocol is intended to guide subsequent experimental laboratory validation. Conclusion This framework establishes explicit, falsifiable decision criteria for prospective IDE inhibitor discovery and is designed to reduce catalytic-site zinc chelation, NEP/ACE cross-reactivity, and central nervous system exposure before experimental testing.