title: A small molecule inhibitor of endoplasmic reticulum oxidation 1 (ERO1) with selectively reversible thiol reactivity.
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.
authors.affiliation: Kimmel Center for Biology and Medicine of the Skirball Institute and the Departments of Cell Biology and Medicine, New York University School of Medicine, New York, NY 10016, USA. jaime.blais@med.nyu.edu
authors.forename: Jaime D
authors.initials: JD
authors.lastname: Blais
authors.forename: King-Tung
authors.initials: KT
authors.lastname: Chin
authors.forename: Ester
authors.initials: E
authors.lastname: Zito
authors.forename: Yuhong
authors.initials: Y
authors.lastname: Zhang
authors.forename: Nimrod
authors.initials: N
authors.lastname: Heldman
authors.forename: Heather P
authors.initials: HP
authors.lastname: Harding
authors.forename: Deborah
authors.initials: D
authors.lastname: Fass
authors.forename: Colin
authors.initials: C
authors.lastname: Thorpe
authors.forename: David
authors.initials: D
authors.lastname: Ron
mesh_terms: D000818:Animals; D002470:Cell Survival; D005347:Fibroblasts / Q000166:cytology / Q000502:physiology*; D005453:Fluorescence; D005982:Glutathione Transferase / Q000235:genetics; D006023:Glycoproteins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D007700:Kinetics; D051379:Mice; D018345:Mice, Knockout; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D010088:Oxidoreductases; D010101:Oxygen Consumption; D011489:Protein Denaturation; D019704:Protein Disulfide-Isomerases / Q000378:metabolism; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D019892:eIF-2 Kinase / Q000172:deficiency
chemical_list: D005982:Glutathione Transferase; D006023:Glycoproteins; D010088:Oxidoreductases; D019704:Protein Disulfide-Isomerases; D011993:Recombinant Fusion Proteins; D019892:eIF-2 Kinase; C491864:Ero1l protein, mouse