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 identified in humans^7,8. They typically function as an isopeptidase responsible for cleaving the isopeptide bond between the C-terminus of ubiquitin and a lysine residue in a substrate or another ubiquitin molecule^9,10. Currently, they are primarily categorized into seven major families, namely: ubiquitin-specific peptidases (USPs), ovarian tumor proteases (OTUs), Jab1/Mov34/Mpr1 Pad 1 N-terminal + domain proteases (JAMMs), motif interacting with ubiquitin-containing novel DUB family proteases (MINDYs), ubiquitin C-terminal hydroxylases (UCHs), Machado-Josephin domain proteases (MJDs), and zinc finger-containing ubiquitin peptidase 1 (ZUP1)⁹. In addition to JAMMs, which belong to the zinc metalloprotease family¹¹, the other DUBs are cysteine proteases characterized by a catalytic triad consisting of catalytic cysteine, histidine, and a third acidic residue^12,13. This specificity opens up avenues for developing small-molecule inhibitors targeting the enzyme's active site or nearby allosteric pockets.

In the field of deubiquitinating enzyme research, there exists a significant challenge in characterizing their activity^14,15. The characterization based on active probes serves as a crucial approach for studying DUB inhibitors¹⁶. By performing competitive assays with activity-based probes and inhibitors in cell lysates or recombinant proteins, the activity of DUBs can be characterized, facilitating the development of small-molecule inhibitors targeting these enzymes. Ub-AMC is an early probe used to detect DUB activity, which has a fluorescent group attached to the C-terminal end of the ubiquitin^17,18. When DUBs exert their catalytic activity AMC is released in large quantities, and the fluorescent intensity of its detected light is enhanced accordingly. This probe has been widely used in high-throughput screening of DUB inhibitors^19,20. The HA-Ub-VS probe is also used to measure DUB activity²¹. It has a vinyl sulfone group at ubiquitin's C-terminus, making it a suicide substrate for DUBs. After sodium dodecyl sulfate-polyacrylamide Gel electrophoresis (SDS-PAGE) separation, active DUBs can be detected using western blotting^22,23,24.

Traditional Chinese medicine (TCM) has been using medicinal plants for more than 2000 years. Developing new drugs from natural products is of great medical significance, with a key focus on identifying active ingredients and elucidating their mechanisms²⁵. Salvia miltiorrhiza Bge is an herb widely used in the treatment of a variety of diseases, including cancer, cardiovascular, hepatic, and neurological²⁶. Currently, it contains known small molecules such as tanshinone²⁷, Danshensu²⁸, tanshinic acid²⁹, etc. These compounds exhibit diverse biological activities such as anti-thrombotic, antioxidant, and anti-tumor effects, making them highly valuable for research²⁶. Recent studies have identified Danshensu as a covalent inhibitor of the 3-chymotrypsin-like protease (3CLpro) of SARS-CoV-2³⁰. It has been shown to form a covalent bond with the active site residue C145 of 3CLpro, indicating the presence of potential small-molecule protease inhibitors in Salvia miltiorrhiza Bge.

Ubiquitin C-Terminal Hydrolase L3 (UCHL3) belongs to the cysteine proteases within the UCH family of DUBs. It relies on conserved residues like cysteine95, histidine169, and aspartic acid184 to catalyze its functions effectively^31,32. It plays crucial roles in multiple molecular pathways, including the cell cycle, homologous recombination, and repair of protein-linked DNA breaks³³. Additionally, it is up-regulated in various cancers such as ovarian, prostate, pancreatic, colorectal, and non-small cell lung cancers³⁴. Based on these studies, UCHL3 appears to be a promising target for treating diseases. Several small molecule inhibitors of UCHL3 have been identified and are progressing towards clinical use^35,36.

In this study, molecular docking was performed to investigate interactions between small molecules from Salvia miltiorrhiza Bge and UCHL3. Subsequently, an in vitro experiment using DUB-specific probes Ub-AMC and HA-Ub-VS identified Danshensu as a small molecule inhibitor of UCHL3. Molecular docking also predicted potential binding sites for Danshensu, suggesting its mechanism of action.

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.
2. Download the protein file.
   1. Open the PDB database (https://www.rcsb.org/), input UCHL3, then press search. Click Homo sapiens, then press search in refinements.
   2. Select structure 1XD3 with small molecule-protein co-crystallization, then click download files and select PDB format.

2. Molecular docking

1. Save the file.
   1. Create a new folder named danshen_UCHL3 docking on the desktop and save the compounds of the Salvia miltiorrhiza Bge and UCHL3 structures in this folder. Name the folder in English; otherwise, the file will fail to be imported.
2. Set the path.
   1. Open Maestro software, click File, select Change Working Directory, click Desktop, double-click to select the danshen_CUHL3 docking folder, and click Choose Options.
3. Small molecule processing
   1. Import small molecule structures.
      1. Click File and Import Structures options. Click Desktop, double-click on the docking folder, and click the Salvia miltiorrhiza Bge structure file. Then, click Open to import all of the small molecules.
   2. Prepare the small molecule.
      1. Click Tasks and select the LigPrep option. In the displayed LigPrep window, click the project table on the Use structure from option, check the Determine chiralities from 3D structure option under computation, and leave all other software settings as default.
      2. Change the job name to danshen_ligprep1, and then click Run to execute small molecule processing.
4. Protein structure processing
   1. Import the protein structure.
      1. Click the File and Import Structures options, click Desktop and double-click on the danshen_CUHL3 docking folder.
      2. Click on the UCHL3 protein structure file, and then click Open to import the protein structure file.
   2. Prepare the protein.
      1. Select the two covalent bonds connecting UCHL3 and the small molecule, and delete them using the Delete button. Then, select the two protein residues that are incomplete after the deletion. Click the Build button, select Other edits, and then for Gly75 click C button, for Cys95 choose Mutate Residue and CYS.
      2. Click Tasks and select the Protein Preparation Workflow option. Leave all other software settings as default. Change the job name to UCHL3_protein pre, then click Run to perform protein processing.
   3. Set up the docking box.
      1. Click on Tasks, choose Receptor Grid Generation, and select Pick to identify the ligand molecule. Select the small molecule in the workspac e, and a pink docking box centered on the small molecule coordinates will appear.
      2. Keep the default settings, name the job as 1XD3_danshen_glide_grid, and then click Run.
         NOTE: In 1XD3, the small molecule forms a covalent bond with the protein. It is necessary to remove this covalent bond to separate the small molecule from the protein. Otherwise, during the receptor grid setup process, the small molecule cannot be selected.
5. Perform molecular docking.
   1. Click Tasks and select the Ligand Docking option. Select the Receptor Grid, click From file, and then click on Browse. Select 1XD3_danshen_glide_grid.zip file, and then click Open.
   2. Click on Use Ligands from as files, then click Browse. Click danshen_ligprep1 file, select danshen_ligprep1-out. maegz file, and then click Open.
   3. Click Settings and select Precision as SP option, change the job name to danshen_UCHL3_ glidedock _SP, and click Run.
6. View the docking results.
   1. Click File and Import Structures options. Click Desktop and double-click on the danshen_CUHL3 docking folder.
   2. Double-click on the danshen_UCHL3_ glidedock _SP file, click the danshen_UCHL3_ glidedock _SP_pv.maegz file, and then click Open.
   3. Click the Table option, and then view the score under docking score.
7. Visualize danshensu and UCHL3 interactions.
   1. Double-click the danshen_UCHL3_glidedock _SP_pv.maegz file and open it in Maestro. In the entry list, hold Shift and simultaneously select danshensu and the protein.
   2. Right-click and select Merge to create a new structure. Select the new structure, right-click, choose Export, then click Structures, name the file danshensu_UCHL3, and export it in .pdb format.
   3. Select the merge structure in the panel, click Tasks, select 2D sketcher, and obtain a 2D structure picture of the interaction between danshensu and UCHL3.
   4. Import the danshensu_UCHL3.pdb file into the pymol software and visualize based on the 2D structure obtained from Maestro.

3. Purification of protein UCHL3

1. Construction of the pHUE-UCHL3 prokaryotic expression plasmid
   1. Obtain the coding sequence of the recombinant protein UCHL3 gene from NCBI (isform2). Integrate the gene fragment obtained into the pHUE-10HIS vector via homologous recombination and transform it into DH5α competent cells. Extract the plasmid DNA to obtain the desired construct.
2. Induction and purification of recombinant proteins
   1. Bacterial culture:
      1. Transform the recombinant plasmids into BL21 (DE3) competent cells and spread them on Luria-Bertani (LB) agar plates containing ampicillin. Incubate the plates at 37 °C overnight.
      2. Pick single colonies, inoculate them into 12 mL of liquid LB medium supplemented with 50 µg/mL ampicillin, and culture overnight at 37 °C.
   2. Transformation and induction
      1. Dilute the overnight bacterial culture to 2% using a fresh medium. When OD600 reached 0.4-0.6, add isopropyl-beta-D-thiogalactopyranoside (IPTG) to a final concentration of 0.4 mM for low-temperature induction at 16 °C for 12 h.
   3. Collection of bacterial culture
      1. Transfer the bacterial culture into sterile centrifuge tubes and centrifuge at 2200 x g for 10 min. Remove the supernatant and store the strains at -80 °C for preservation.
   4. Sonication
      1. Resuspend the strain in 25 mL (1/20 of the collected bacterial culture) of Buffer 1 (50 mM HEPES pH 7.5, 200 mM NaCl, 1 mM EDTA). Perform bacterial sonication under the following conditions: on for 4 s, off for 6 s, energy set at 60%, for 15 min. Add 1% Triton X-100 and lyse at 4 °C for 30-60 min.
   5. Separation of supernatant and pellet
      1. Centrifuge the sample at 9000 x g at 4 °C for 30 min. Collect the supernatant and filter it through a 0.45 µm membrane filter. Reserve 50 µL of the supernatant as the input.
      2. Resuspend the pellet in Buffer 1, add an appropriate amount of 5x sample buffer (25% 1M Tris-HCl [pH 6.8], 10 % sodium dodecyl sulfate, 0.5% bromophenol blue, 41.67% glycerol, and 10% DL-dithiothreitol), and boil at 100 °C for 10 min.
3. Protein purification
   1. Column equilibration
      1. Load 1 mL of NiNTA nickel resin into the column, allow it to settle for 10 min, then sequentially equilibrate the column with 5 column volumes of buffer 2 (50 mM HEPES pH 7.5, 50 mM NaCl) containing 10 mM, 500 mM, and 10 mM imidazole.
      2. Use a pipette to add 5 mL (approximately 5 column volumes) of the 10 mM imidazole solution along the wall of the column. Do this process without controlling the flow rate.
      3. Once the liquid just covers the packing material and is nearly drained, sequentially add 5 column volumes of the 500 mM imidazole solution and the 10 mM imidazole solution for equilibration. When the remaining 10 mM imidazole solution just covers the packing material, close the flow controller to stop the equilibration.
   2. Protein purification
      1. Pass the filtered protein supernatant through the column at a flow rate of 0.5 mL/min (<15 s per drop), collecting the flow-through fraction. Prepare a concentration gradient of 10 mM, 30 mM, 50 mM, and 100 mM imidazole using Buffer 2, along with 0.5 M NaCl.
      2. Elute sequentially from low to high concentrations of imidazole, using a pipette to add approximately 5 column volumes of solution along the wall of the column without controlling the flow rate. Collect the eluate in clean 1.5 mL microcentrifuge tubes, taking 1 mL flow-through per tube.
      3. After collecting five tubes, take 1 µL of flow-through from each tube to react with 1 µL of Bradford buffer. If the reaction remains blue, continue the steps; if it turns transparent, stop elution at that concentration and switch to the next one.
      4. After eluting with 100 mM imidazole, wash with 10 column volumes of 0.5 M NaCl without controlling the flow rate or collecting the eluate.
      5. Finally, elute with 500 mM imidazole (<15 s per drop). Using a pipette, add 1 mL of the solution to the column, maintaining the aforementioned slow elution rate. Collect 1 mL of the eluate using a 1.5 mL microcentrifuge tube, and repeat this process 10 times.
   3. Assess the purified protein using Coomassie Brilliant Blue staining.
      1. Quantify the previously obtained pellet, pre-purification supernatant, and the collected 10 protein tubes by taking 10 µL from each group and adding the appropriate 5x sample buffer, then heating at 100 °C for 5 min.
      2. Prepare the bovine serum albumin (BSA) standard solution at 1 mg/mL, 500 µg/mL, and 100 µg/mL. Then add the appropriate 5x sample buffer and heat at 100 °C for 5 min.
      3. Conduct SDS-PAGE gel electrophoresis on the samples, then remove the gel and incubate it at room temperature (RT) in Coomassie Brilliant Blue solution on a low-speed shaker overnight.
      4. The next day, transfer the gel to a decolorizing solution (40% ethanol, 10% acetic acid, 50% H₂O) for decolorization, applying gentle heat as needed. Once the gel is transparent, observe under a development instrument to quantify the purified protein based on BSA levels and assess purity by checking for a single band.
   4. Protein storage: Aliquot the protein into microcentrifuge tubes at 50 µL per tube, flash-freeze in liquid nitrogen, and store at -80 °C.

4. UCHL3 activity assay (Ub-AMC assay)

1. Protein preparation
   1. Thaw the protein on ice, centrifuge the sample at 1000 x g at RT for 3 min, and determine protein concentration using the BCA method. Dilute the protein to a concentration of 40 nM using Buffer 1.
2. Setting up groups
   1. Designate Buffer 1 as the control group and UCHL3 as the experimental group. Add 200 µL of each sample per well in a 96-well plate, setting up 3 replicates per group.
3. Detection
   1. Just before measurement, quickly add Ub-AMC to each well and shake vigorously for 2-5 s. The concentration of Ub-AMC used is 250 nM. Measure OD values at 380 nm excitation wavelength and 460 nm emission wavelength under 37 °C conditions, taking readings every 30 s for up to 10 min.

5. Inhibition assay of UCHL3 activity by HA-Ub-VS (HA-Ub-VS assay)

1. Protein preparation
   1. Thaw the protein on ice, centrifuge the sample at 1000 x g at RT for 3 min, and determine protein concentration using the BCA method. Dilute the protein to a concentration of 10 µg/mL.
2. Preparation of small molecules
   1. Weigh Danshensu and dilute it in a gradient using high-purity water to 5 mM, 1 mM, 100 µM, 10 µM, and 1 µM.
3. Setting up groups
   1. Set up the pure protein group and the group without small molecule drugs as negative control groups. Use PR619 (DUB inhibitor) as the positive control group, and the other groups as the small molecule drug treatment groups.
4. Sample preparation
   1. According to the grouping, sequentially add 9 µL of UCHL3 and 1 µL of Dansensu at the corresponding concentrations to each drug-treated group. Add 1 µL of PR619 (50 µM in use) to the positive control group. Use high-purity water and Buffer 1 to make up for the negative control group.
   2. Mix thoroughly by vortexing, and incubate at 37 °C for 30 min. Place the reaction samples on ice, add HA-Ub-VS to a final concentration of 1 µM, mix thoroughly by vortexing, and incubate at 37 °C for 30 min.
   3. Add an appropriate amount of 5x sample buffer and heat in a 100 °C metal bath for 10 min. Put the samples at RT for 5 min and load onto the gel.

6. Western blot

1. Preparation of SDS-PAGE gel
   1. Rinse a set of glass panels with ultrapure water. Place the plates into the clamping slot, then on a transparent board. Add ultrapure water and leak check for 10 min.
   2. Prepare the separation glue according to the glue dispensing table.
   3. Pour the high-purity water from the gel cassette and absorb any remaining water with filter paper. Quickly and evenly add the prepared 12% separating gel. Then add 1 mL of isopropanol and wait for approximately 30 min until the gel solidifies.
   4. Prepare the top layer of concentrated glue according to the glue dispensing table.
   5. Remove the isopropanol from the top of the separating gel, wash it 3 times with high-purity water, and blot dry with filter paper. Quickly add the prepared 3% stacking gel, insert a 1.0 mm 15-hole comb, and wait for approximately 30 min.
      NOTE: Keep the comb perpendicular to the glue surface, and ensure no air bubbles appear.
2. Electrophoresis
   1. Vertically remove the comb and pour a sufficient amount of running buffer into the inner chamber of the electrophoresis apparatus, ensuring the liquid covers the sample wells.
   2. Place the protein marker and the prepared experimental samples at RT, vortex thoroughly, and briefly centrifuge. Add 3 µL of protein marker into the first left-hand sample well, followed by 10 µL of experimental sample into each subsequent well.
   3. Add an appropriate amount of running buffer into the outer chamber of the electrophoresis tank, cover the tank with its lid, and connect the power supply. Ensure the power supply is connected correctly.
   4. Turn on the power supply and set the voltage to 80 V until the samples concentrate into lines, then increase the voltage to 120 V once separation begins in the separating gel.
      NOTE: During the process, ensure no bubbles form at the bottom. If bubbles appear, tilt the electrophoresis apparatus to one side to expel them.
3. Transfer to membrane
   1. Use a plastic knife to pry open the gel cassette. Cut off a corner at the top left where the sample loading started to mark the orientation. Soak the gel and required filter paper in the transfer buffer for 1-2 min.
   2. Measure and cut thepolyvinylidene difluoride (PVDF) membrane according to the size needed for the target protein. Activate the PVDF membrane by soaking it in methanol for 20 s to 1 min, then immerse it with the gel in the transfer buffer.
   3. Pour a small amount of transfer buffer into the transfer apparatus, and use a roller to wet the semi-dry transfer unit. Arrange the components in the following order from bottom to top: three layers of filter paper, gel, PVDF membrane, and three more layers of filter paper.
   4. After placing each layer, use the roller to remove any bubbles. Wet the lid with transfer buffer and then cover the apparatus.
      NOTE: Ensure an adequate amount of transfer buffer is present, adding a small amount after completing each layer. Prepare markings in advance, distinguishing between the left and right sides of the gel where samples were loaded, as well as identifying the front and back of the membrane. After each layer is placed, gently roll to remove any bubbles.
   5. Turn on the power supply, ensuring correct polarity. Adjust the current to 190 mA and set the time for 45 min.
4. Blocking
   1. Prepare a 5% non-fat dry milk solution in TBST (1% Tween20) as the blocking buffer. Place the PVDF membrane with the front side up into a hybridization box, pour in 10 mL of blocking buffer, and gently shake on a low-speed shaker at RT for 1 h to block.
5. Incubation of primary antibody
   1. Prepare the primary antibody with antibody solution1 at 1:1000 and add 10 mL of primary antibody to the new incubation box. Place the incubation box in a 4 °C cold room and keep it on a shaker overnight.
6. Incubation of secondary antibody
   1. Collect the primary antibody in the incubation box the next day, then add proper TBST solution and place it on a shaker to wash for 5 min, repeat 3 times.
   2. Prepare the secondary antibody with 5% milk solution at 1:5000. Drop 1 mL of secondary antibody onto the bottom of a moistened hybridization box. Place the PVDF membrane with the protein side facing down onto the secondary antibody, and then incubate at RT on a low-speed shaker for 1.5 h.
   3. Add proper TBST solution and place it on a shaker to wash for 5 min; repeat 3 times.
7. Exposing the strips
   1. Take equal volumes of chemiluminescent reagents A and B, shake, and mix well. Shield the solution from light.
   2. Apply the developing solution evenly onto the PVDF membrane, gently shaking it to ensure the membrane is uniformly coated with the solution.
   3. Place the membrane into the gel imaging system and set the exposure time, number of exposures, and other relevant parameters.
      NOTE: The developer must completely cover the strip, and the development should be in a dark environment.

Results

To screen out the small molecules in the Salvia miltiorrhiza Bge that can effectively inhibit UCHL3, we performed molecular docking between the small molecules obtained from the TCMSP website with UCHL3. The top 30 small molecules in the docking results and their scores are shown in Table 1. The docking results for all small molecules are presented in Supplementary Table 1. We selected Danshensu as a representative small molecule for the research. As shown in

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