Discovery of Selective Inhibitor Leads by Targeting an Allosteric Site in Insulin-Regulated Aminopeptidase.

Ioannis Temponeras,Efstratios Stratikos,Lykourgos Chiniadis,Athanasios Papakyriakou

doi:10.3390/ph14060584

Abstract

Insulin-Regulated aminopeptidase (IRAP) is a zinc-dependent aminopeptidase with several important biological functions and is an emerging pharmaceutical target for cognitive enhancement and immune system regulation. Aiming to discover lead-like IRAP inhibitors with enhanced selectivity versus homologous enzymes, we targeted an allosteric site at the C-terminal domain pocket of IRAP. We compiled a library of 2.5 million commercially available compounds from the ZINC database, and performed molecular docking at the target pocket of IRAP and the corresponding pocket of the homologous endoplasmic reticulum aminopeptidase 1 (ERAP1). Of the top compounds that showed high selectivity, 305 were further analyzed by molecular dynamics simulations and free energy calculations, leading to the selection of 33 compounds for in vitro evaluation. Two orthogonal functional assays were employed: one using a small fluorogenic substrate and one following the degradation of oxytocin, a natural peptidic substrate of IRAP. In vitro evaluation suggested that several of the compounds tested can inhibit IRAP, but the inhibition profile was dependent on substrate size, consistent with the allosteric nature of the targeted site. Overall, our results describe several novel leads as IRAP inhibitors and suggest that the C-terminal domain pocket of IRAP is a promising target for developing highly selective IRAP inhibitors.

Highlights

Targeting allosteric sites of enzymes in order to develop inhibitors can be challenging since binding of the compound does not directly compete with binding of the substrate at the catalytic site
The conserved nature of many enzymatic active sites, while it can empower rational design, can make achievement of high degrees of selectivity difficult. This has been reported previously for Insulin-Regulated aminopeptidase (IRAP) using phosphinic pseudopeptides that act as transition-state analogues and which are potent inhibitors, but often target homologous aminopeptidases such as endoplasmic reticulum aminopeptidase 1 (ERAP1) and ERAP2 [29]
We targeted an allosteric site in IRAP that has been shown to be able to bind small molecules in the homologous ERAP1 [15]

Summary

Introduction

IRAP is under investigation as a pharmaceutical target for cognitive disorders [5]. Several IRAP inhibitors have been developed [6,7,8,9] no clinical applications have yet been reported. Most efforts on developing IRAP inhibitors have either targeted the active site or utilized random library screening [10,11,12]. The IRAP active site is highly homologous to other aminopeptidases of the M1 family and is characterized by structural plasticity that could complicate the development of potent and selective inhibitors [13]. As a result, targeting other sites in IRAP could constitute a viable strategy that would allow the discovery of novel leads that could be developed to inhibitors with superior selectivity and pharmacodynamics. In a recent study, a spiro-oxindole dihydroquinazolinone derivative was demonstrated to be an un-competitive inhibitor of IRAP trimming a small dipeptide substrate, the exact mechanism of inhibition is yet to be determined [12]

Methods

Results

Conclusion