Screening Traditional Chinese Medicine Compounds for Inhibiting UCHL3 Activity Based on Molecular Docking and Deubiquitinating Enzyme Probe Technology

Summary

The experiment used here shows a method of molecular docking combined with probe technologies to predict and validate the interaction between small molecules of traditional Chinese medicine and protein targets.

Abstract

Deubiquitinating enzymes (DUBs) play a pivotal role in modulating ubiquitination homeostasis, with UCHL3 being an archetypal cysteine DUB intricately involved in a myriad of physiological and pathological processes. Therefore, developing small molecule inhibitors targeting Ubiquitin C-Terminal Hydrolase L3 (UCHL3) is of great significance. This protocol aims to establish a process for virtual screening and in vitro validation of small molecule inhibitors of cysteine DUB represented by UCHL3. Firstly, potential inhibitors of UCHL3 are virtually screened using molecular docking technology, and the interaction between drugs and protein targets is visualized. Subsequently, the effectiveness of the screened drug, Danshensu, is verified through in vitro activity inhibition assays. Ubiquitin-7-amino-4-methylcoumarin (Ub-AMC) and hemagglutinin-ubiquitin-vinyl sulfone (HA-Ub-VS) are used as probes for in vitro activity testing, as they can competitively bind to DUB with small molecule inhibitors to assess the activity of UCHL3. The results indicate that Danshensu has a good binding affinity with UCHL3 in molecular docking, and it can competitively inhibit the activity of UCHL3 with HA-Ub-VS. These findings provide important references for further research and development of therapeutic drugs targeting UCHL3.

Introduction

Ubiquitination is a post-translational modification of proteins, a process by which E1 ubiquitin-activating enzymes, E2 ubiquitin-conjugating enzymes, and E3 ubiquitin ligases attach ubiquitin to the target protein, and the entire process of ubiquitination can be reversed by deubiquitinating enzymes (DUBs)^1,2,3,4. Due to their important physiological and pathological role, DUBs are considered important targets for drug discovery^5,6.

Over 100 DUBs have been....

Protocol

1. Downloading the structures of small molecules of Salvia miltiorrhiza Bge and the UCHL3

1. Download the small molecule file.
   1. Open the TCMSP database (https://old.tcmsp-e.com), input danshen (herb name), then press search and click the Radix Salviae in the result list.
   2. Click on download one by one of the items and save the 2D structure in the .mol2 format.<.......

Discussion

DUBs play a crucial role in regulating the homeostasis of the entire ubiquitin system by removing ubiquitin from substrates or polyubiquitin chains³⁷. In recent years, these enzymes have also attracted much attention as targets for drug development¹³. However, there are challenges in the process of small-molecule drug development. For instance, high-throughput screening involving tens of thousands of small molecule libraries results in high costs and a significant workload .......

Materials

Name	Company	Catalog Number	Comments
30% Acrylamide	Beijing Lablead Biotech Co., Ltd	A3291
Ammonium persulfate	China National Medicines Corporation Ltd	10002616
Anti-rabbit IgG, HRP-linked Antibody #7074	Cell Signaling Technology	7074P2
BeyoECL Plus	Beyotime	P0018S
Bradford Protein Assay Kit	Beyotime	P0006
ClonExpress Ultra One Step Cloning Kit	Vazyme	C115-01
Danshensu	Shanghai yuanye Bio-Technology Co., Ltd	B20254
DMSO	Ameresco, Inc.	21K2356571
Electrophoresis System	Liuyi Biotechnology	112-0630
HEPES	Sigma	H3375
His-tagged protein purification kit (NTA-Ni agarose magnetic beads)	Beyotime	P2247S
Immun-Blot PVDF Membrane, Roll, 26 cm x 3.3 m	Bio-Rad Laboratories (Shanghai) Co., Ltd	1620177
Isopropyl alcohol	Macklin	I811925
M5 Prestained Protein Ladder	Mei5 Biotechnology Co.Ltd	MF-212-01
Maestro	Schrödinger’s	https://www.schrodinger.com/platform/products/maestro/
Methyl alcohol	China National Medicines Corporation Ltd	10014108
MF-Millipore	Millipore	HAWP04700
MyFug mini centrifuge	Sigma	Z764183
Pierce Dilution-Free Rapid Gold BCA Protein Assay	Thermo Scientific	A55860
PR-619	Cell Signaling Technology	26065S
Primary Antibody Dilution Buffer for Western Blot	Macklin	P917820
Recombinant Human HA-Ubiquitin Vinyl Sulfone Protein, CF	R&D Systems	U-212-025
Recombinant Human Ubiquitin AMC Protein, CF	R&D Systems	U-550-050
Skim Milk	Becton,Dickinson and Company	232100
Sodium Dodecyl Sulfate (SDS)	Ameresco, Inc.	205-788-1
TEMED	Ameresco, Inc.	2545C134
Tween 20	Beijing Lablead Biotech Co., Ltd	0777-1
UCHL3 (D25E6) Rabbit mAb	Cell Signaling Technology	8141T