Discovery of Small Molecule Antagonists of the USP5 Zinc Finger Ubiquitin-Binding Domain.

Mandeep K Mann,Renato Ferreira De Freitas,Wolfram Tempel,Masoud Vedadi,Cheryl H Arrowsmith,Leanna Smith,Scott Houliston,Rachel J Harding,Ivan Franzoni,Matthieu Schapira

doi:10.1021/acs.jmedchem.9b00988

Abstract

USP5 disassembles unanchored polyubiquitin chains to recycle free monoubiquitin, and is one of the 12 ubiquitin specific proteases featuring a zinc finger ubiquitin-binding domain (ZnF-UBD). This distinct structural module has been associated with substrate positioning or allosteric modulation of catalytic activity, but its cellular function remains unclear. We screened a chemical library focused on the ZnF-UBD of USP5, crystallized hits in complex with the protein, and generated a preliminary structure-activity relationship, which enables the development of more potent and selective compounds. This work serves as a framework for the discovery of a chemical probe to delineate the function of USP5 ZnF-UBD in proteasomal degradation and other ubiquitin signaling pathways in health and disease.

Highlights

Ubiquitination is a reversible, post-translational modification involving the conjugation of a conserved 76 amino acid protein, ubiquitin (Ub), to substrate proteins for proteasomal targeting or the regulation of cell signaling[1,2,3]
The library was docked using ICM-Pro[37] (Molsoft, CA) and Glide[38] (Schrodinger, NY) to the X-ray crystal structure conformation of USP5 zinc finger ubiquitin-binding domain (ZnF-UBD) (PDB: 2G45) and an alternate conformational state modelled after the structure of HDAC6 ZnF-UBD in complex with inhibitors
Since one of the two tryptophan side-chains of USP5 ZnF-UBD is located at the targeted ubiquitin C-terminal binding pocket (Figure 1a), we decided to use a 19F nuclear magnetic resonance (NMR) spectroscopy assay as a primary, qualitative screening method

Summary

Introduction

Ubiquitination is a reversible, post-translational modification involving the conjugation of a conserved 76 amino acid protein, ubiquitin (Ub), to substrate proteins for proteasomal targeting or the regulation of cell signaling[1,2,3]. The ZnF-UBD recognizes the C-terminal di-glycine motif of ubiquitin[11], and has been associated with allosteric modulation of USP5 activity[8,11,14], and alternatively, with substrate recognition and positioning[12]. USP5 plays a role in regulation of ubiquitin levels in DNA damage aCC-BY 4.0 International license.

Results

Conclusion