A Small Molecule Inhibitor of Endoplasmic Reticulum Oxidation 1 (ERO1) with Selectively Reversible Thiol Reactivity

Jaime D Blais,King-Tung Chin,Ester Zito,Yuhong Zhang,Nimrod Heldman,Heather P Harding,Deborah Fass,Colin Thorpe,David Ron

doi:10.1074/jbc.m110.126599

Abstract

Endoplasmic reticulum oxidation 1 (ERO1) is a conserved eukaryotic flavin adenine nucleotide-containing enzyme that promotes disulfide bond formation by accepting electrons from reduced protein disulfide isomerase (PDI) and passing them on to molecular oxygen. Although disulfide bond formation is an essential process, recent experiments suggest a surprisingly broad tolerance to genetic manipulations that attenuate the rate of disulfide bond formation and that a hyperoxidizing ER may place stressed cells at a disadvantage. In this study, we report on the development of a high throughput in vitro assay for mammalian ERO1alpha activity and its application to identify small molecule inhibitors. The inhibitor EN460 (IC(50), 1.9 mum) interacts selectively with the reduced, active form of ERO1alpha and prevents its reoxidation. Despite rapid and promiscuous reactivity with thiolates, EN460 exhibits selectivity for ERO1. This selectivity is explained by the rapid reversibility of the reaction of EN460 with unstructured thiols, in contrast to the formation of a stable bond with ERO1alpha followed by displacement of bound flavin adenine dinucleotide from the active site of the enzyme. Modest concentrations of EN460 and a functionally related inhibitor, QM295, promote signaling in the unfolded protein response and precondition cells against severe ER stress. Together, these observations point to the feasibility of targeting the enzymatic activity of ERO1alpha with small molecule inhibitors.

Highlights

Disulfide bonds stabilize the tertiary structures of secreted proteins [1], and disulfide formation is essential in all known life forms
ERO1 genes are under transcriptional control, and their mRNA levels increase with the load of unfolded reduced proteins in the ER, a phenomenon mediated by the unfolded protein response [9, 10]
ERO1 is an essential enzyme in yeast, and its dysfunction leads to a rapid decline in oxidative protein folding, strong activation of the unfolded protein response, and marked loss of viability [11, 12]

Summary

Introduction

Disulfide bonds stabilize the tertiary structures of secreted proteins [1], and disulfide formation is essential in all known life forms. Exposure to TrxAred lead to reduction of the regulatory disulfides, and a new steady state was attained whereby the enzyme was reduced, activated, and AMS-reactive, as demonstrated by its lower mobility on SDS-PAGE (Fig. 2E, compare lanes 9 and 10).

Results

Conclusion