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Review
. 2021 Apr 27;22(9):4546.
doi: 10.3390/ijms22094546.

Advances in the Development Ubiquitin-Specific Peptidase (USP) Inhibitors

Shiyao Chen  1 , Yunqi Liu  1 , Huchen Zhou  1
Affiliations
Review

Advances in the Development Ubiquitin-Specific Peptidase (USP) Inhibitors

Shiyao Chen et al. Int J Mol Sci. .

Abstract

Ubiquitylation and deubiquitylation are reversible protein post-translational modification (PTM) processes involving the regulation of protein degradation under physiological conditions. Loss of balance in this regulatory system can lead to a wide range of diseases, such as cancer and inflammation. As the main members of the deubiquitinases (DUBs) family, ubiquitin-specific peptidases (USPs) are closely related to biological processes through a variety of molecular signaling pathways, including DNA damage repair, p53 and transforming growth factor-β (TGF-β) pathways. Over the past decade, increasing attention has been drawn to USPs as potential targets for the development of therapeutics across diverse therapeutic areas. In this review, we summarize the crucial roles of USPs in different signaling pathways and focus on advances in the development of USP inhibitors, as well as the methods of screening and identifying USP inhibitors.

Keywords: USP inhibitors; drug screening; signaling pathways; ubiquitin-specific peptidases.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Key events in the ubiquitylation and deubiquitylation process [1,2,3]. E1: ubiquitin-activating enzyme; E2: ubiquitin-conjugating enzyme; E3: ubiquitin-protein ligase; Ub: ubiquitin. Really interesting new gene (RING) E3 ligases which represent the vast majority of E3 ligases are depicted here as an example.
Figure 2
Figure 2
Structure of USP7. (A) Schematic of the USP7 domain organization [27]. (B) Crystal structure of the dimer of the USP7CD-UBL4/5-ubiquitin complex (PDB ID: 5JTV). Ubiquitin: blue; USP7 catalytic domain: orange; UBL4/5: gray. In this structure, the USP7 N-terminal TRAF-like domain and ubiquitin-like (UBL) domains 1–3 were truncated. The catalytic triad of USP7 (Cys223-His464-Asp481) was shown as red sticks.
Figure 3
Figure 3
USPs inhibitor screening methods. (A) Activity-based probe (ABP) structures; (B) Ub-7-amino-4-methylcoumarin (AMC) structure; (C) Ub-phospholipase A2 (PLA2) assay; (D) Time-resolved fluorescence resonance energy transfer (TR-FRET) assay; (E) UBA52 structure and the SDS-PAGE-Coomassie assay; (F) Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) method.
Figure 4
Figure 4
Structures of reported USP inhibitors in clinical trials.
Figure 5
Figure 5
Structures of representative USP1 inhibitors.
Figure 6
Figure 6
Structures of representative USP2 inhibitors.
Figure 7
Figure 7
Cocrystal structure of the USP2-Ub-6TG complex and a close-up on the 6TG-binding site (PDB ID: 5XU8). Ubiquitin: magenta; USP2 catalytic domain: gray; 6TG: cyan sticks. In the close-up figure, hydrogen bonds made by 6TG are indicated by yellow dotted lines. Key residues involved in the interactions and the catalytic triad of USP2 (Cys276-His557-Asp574) are shown as sticks, with Cys276 in orange.
Figure 8
Figure 8
Structure of compound PR619.
Figure 9
Figure 9
Structure of compound RA-9. The exact chemical structures of AM146 and RA-14 were not reported.
Figure 10
Figure 10
Structures of reported USP2/7 inhibitors.
Figure 11
Figure 11
Structure of vialinin A.
Figure 12
Figure 12
Structure of curcusone D.
Figure 13
Figure 13
Structures of reported USP5/9X/14/24/UCHL5 inhibitors.
Figure 14
Figure 14
Structures of representative USP7 inhibitors.
Figure 15
Figure 15
Comparison of the structures of USP7-GNE-6776 (PDB ID: 5UQX) and USP7-ALM2 (PDB ID: 5N9R). A ubiquitin molecule is modeled into the complexes based on a superposition with a USP7CD-ubiquitin complex (PDB ID: 1NBF). Ubiquitin: magenta; USP7 catalytic domain: gray; GNE-6776: green sticks; ALM2: cyan sticks. Hydrogen bonds: yellow dotted lines. Key residues involved in binding: sticks. (A) Crystal structure of USP7-GNE-6776 and a close-up of the GNE-6776-binding site. In the close-up figure, the ubiquitin peptide AGKQLED is omitted for clarity. (B) Crystal structure of USP7-ALM2 and a close-up of the ALM2-binding site. In the close-up figure, the catalytic triad of USP7 (Cys223-His464-Asp481) is also shown as sticks, with Cys223 in orange, and the ubiquitin C-terminal peptide LRLRGG is omitted for clarity.
Figure 16
Figure 16
Structures of reported USP7/8 inhibitors.
Figure 17
Figure 17
Structures of reported USP7/10 inhibitors.
Figure 18
Figure 18
Structures of reported USP7/47 inhibitors.
Figure 19
Figure 19
Structure of Spautin-1.
Figure 20
Figure 20
Structure of mitoxantrone.
Figure 21
Figure 21
Crystal structure of the USP15-mitoxantrone complex and a close-up of the binding site (PDB ID: 6GH9). A di-ubiquitin molecule is modeled into the complex based on a superposition with a USP30 C77A Lys6-linked di-ubiquitin structure (PDB ID: 5OHP), the closest available USP structure in complex with a substrate. Ubiquitin: magenta; USP15 catalytic domain: gray; mitoxantrone: cyan sticks. In the close-up figure, key residues involved in binding and the catalytic triad (Cys269-His862-Asp879) are shown as sticks, with Cys269 marked orange, and the di-ubiquitin molecule is omitted for clarity.
Figure 22
Figure 22
Structures of representative USP14 inhibitors.
Figure 23
Figure 23
Cocrystal structure of USP14-IU1-248 and a close-up of the binding site (PDB ID: 6IIN). A ubiquitin molecule is modeled into the complex based on the superposition with a USP14CD-ubiquitin complex (PDB ID: 2AYO). Ubiquitin: magenta; USP14 catalytic domain: gray; IU1-248: cyan sticks. Hydrogen bonds: yellow dotted lines. Key residues involved in binding and the catalytic triad (Cys114-His435-Asp451) are shown as sticks, with Cys114 in orange. The ubiquitin C-terminal peptide RLRGG is omitted for clarity.
Figure 24
Figure 24
Structures of representative USP14/UCLH5 inhibitors.
Figure 25
Figure 25
Structure of compound AZ1.
Figure 26
Figure 26
Structures of reported USP30 inhibitors.
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