1. Academic Validation
  2. High-Throughput Screening for Linear Ubiquitin Chain Assembly Complex (LUBAC) Selective Inhibitors Using Homogenous Time-Resolved Fluorescence (HTRF)-Based Assay System

High-Throughput Screening for Linear Ubiquitin Chain Assembly Complex (LUBAC) Selective Inhibitors Using Homogenous Time-Resolved Fluorescence (HTRF)-Based Assay System

  • SLAS Discov. 2018 Dec;23(10):1018-1029. doi: 10.1177/2472555218793066.
Ken Katsuya 1 Yuji Hori 1 Daisuke Oikawa 2 Tomohisa Yamamoto 1 Kayo Umetani 1 Toshiki Urashima 1 Tomomi Kinoshita 1 Kumiko Ayukawa 1 Fuminori Tokunaga 2 Masahiro Tamaru 1
Affiliations

Affiliations

  • 1 1 Biological Research Laboratories, Central Pharmaceutical Research Institute, Japan Tobacco Inc., Takatsuki, Osaka, Japan.
  • 2 2 Department of Pathobiochemistry, Graduate School of Medicine, Osaka City University, Abeno-ku, Osaka, Japan.
Abstract

The nuclear factor κB (NF-κB) pathway is critical for regulating immune and inflammatory responses, and uncontrolled NF-κB activation is closely associated with various inflammatory diseases and malignant tumors. The Met1-linked linear ubiquitin chain, which is generated by linear ubiquitin chain assembly complex (LUBAC), is important for regulating NF-κB activation. This process occurs through the linear ubiquitination of NF-κB essential modulator, a regulatory subunit of the canonical inhibitor of the NF-κB kinase complex. In this study, we have established a robust and efficient high-throughput screening (HTS) platform to explore LUBAC inhibitors, which may be used as tool compounds to elucidate the pathophysiological role of LUBAC. The HTS platform consisted of both cell-free and cell-based assays: (1) cell-free LUBAC-mediated linear ubiquitination assay using homogenous time-resolved fluorescence technology and (2) cell-based LUBAC assay using the NF-κB luciferase reporter gene assay. By using the HTS platform, we performed a high-throughput chemical library screen and identified several hit compounds with selectivity against a counterassay. Liquid chromatography-mass spectrometry analysis revealed that these compounds contain a chemically reactive lactone structure, which is transformed to give reactive α,β-unsaturated carbonyl compounds. Further investigation revealed that the reactive group of these compounds is essential for the inhibition of LUBAC activity.

Keywords

HTRF; HTS; LUBAC; inhibitor.

Figures
Products
  • Cat. No.
    Product Name
    Description
    Target
    Research Area
  • HY-122881
    LUBAC Inhibitor
    IKK