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

Our research focuses on screening active small molecules from natural products in traditional Chinese medicine. We have explored how to identify small molecular inhibitors of deubiquitinating enzymes from Salvia miltiorrhiza, utilize the props and the bioinformatics technology. Props for characterizing the activity of deubiquitinating enzymes include Ub-TAMRA, Ub-RHO, Ub-VME, Ub-PA, and others.

For the first time, we have identified Danshensu as a small molecular inhibitor of the deubiquitinating enzyme UCHL3.

[Narrator] To begin, on a computer, open the TCMSP database. Input the herb name Danshen. Then, press Search and click Radik Salvae from the result list. Click on each item and download its two dimensional structure one by one. Save the two dimensional structure in the .mol2 format. Next, open the PDB database and input UCHL3. Then press Search. Click on Homo sapiens then press Search in the Refinements. Select structure 1XD3 with small molecule protein co-crystallization, then click Download Files, and select PDB Format. Open the folder danshen UCHL3 docking, containing saved compounds of Salvia miltiorrhiza Bge and UCHL3 structures. Next, open Maestro software. Click File, then select Change Working Directory. Click Desktop, double click on the danshen_UCHL3_docking folder and click Choose Options. Now, click File and select Import Structures. Navigate to the Desktop, open the docking folder, then select the Salvia miltiorrhiza Bge structure file. And click Open to import the small molecules. Next, click Tasks and select the LigPrep option. In the LigPrep window, select Project Table on the use structure from option. Then, check the Determine chiralities from three dimensional structure option under the Computation. Leave all other settings as default. Change the job name to danshen_ligprep1 and click Run to execute the small molecule processing. To import protein structure, click File and select Import Structures. Then navigate to the Desktop and double click on the Danshen UCHL3 docking folder. Click on the UCHL3 protein structure file and select Open to import the protein structure file. For GLY 75, select the two covalent bonds connecting UCHL3 and the small molecule, and delete them using the Delete button. Now, select the incomplete protein residue and click the Build button, followed by the C button. Similarly, for CYS 95, select the two covalent bonds connecting UCHL3 and the small molecule, and delete them using the Delete button. Then, select the Build button, followed by other edits, and choose Mutate Residue and CYS. Next, 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. Next, click on Tasks. Choose Receptor Grid Generation and select Pick to identify the ligand molecule. Then, select the small molecule in the workspace where a pink docking box centered on the small molecule coordinates will appear. Keep the default settings, name the job 1XD3_danshen_glide_grid, and click Run to start the process. To perform molecular docking, click on Tasks and select the Ligand Docking option. Then, select the receptor grid, click from File, and click on Browse. Select the 1XD3_danshen_glide_grid zip file and click Open. Next, click on Use Ligands from his files, then click Browse. Click danshen_ligrep1 file. Then, select danshen_ligprep1-outmaegz file and click Open. Click Settings and select Precision as the SP option. Change the job name to danshen_UCHL3_glidedock_SP and click Run to execute the docking. To view docking results, click File and select Import Structures. Navigate to the danshen_UCHL3 docking folder on the Desktop. Double click on the danshen_UCHL3_glidedock_SP file. Then, select the danshen_UCHL3_glidedock_SP_pvmaegz file and click Open. Click on the Table option and view the score under the Docking Score column. Next, double click on the danshen_UCHL3_glidedock_SP_pvmaegz file and open it in Maestro. In the entry list, hold Shift, and simultaneously select Danshensu and the protein. Right click and select Merge to create a new structure. Select the new structure, right click, choose Export, click Structures. name the file danshensu_UCHL3, and export it in PDB format. Select the Merge Structure in the panel. Click Tasks and select 2D Sketcher to obtain a two dimensional structure image showing the interaction between danshensu and UCL3. Finally, import the danshensu_UCHL3 PDB file into PyMOL, and visualize it based on the two dimensional structure obtained from Maestro. To begin, centrifuge the UCHL3 protein at 100 G for three minutes. Determine the protein concentration using the BCA method. Dilute the protein to a concentration of 40 nanomoles using buffer one. Designate buffer one as the control group and UCHL3 as the experimental group. Add 200 microliters of each sample to separate wells in a 96 well plate group. Just before measurement, quickly add 250 nanomoles of Ub-AMC to each well. Measure optical density values at an excitation wavelength of 380 and a mission wavelength of 460 nanometers at 37 degrees Celsius, taking readings every 30 seconds for up to 10 minutes. Dilute the UCHL3 protein to a concentration of 10 micrograms per milliliter using buffer one. Weigh Danshensu and dilute it in a gradient using high purity water to concentrations of 5 millimoles, 1 millimole, 100 micromoles, 10 micromoles, and 1 micromole. After setting up the experimental groups, add 9 microliters of UCHL3 and 1 microliter of Danshensu at the corresponding concentrations to each drug treated group. Then add 1 microliter use PR 619 to the positive control group. For the negative control group, add high purity water and buffer one. Mix thoroughly by vortexing and incubate at 37 degrees Celsius for 30 minutes. Place the reaction samples on ice and add HA-UbVS to a final concentration of 1 micromolar. Mix thoroughly by vortexing again and incubate at 37 degrees Celsius for another 30 minutes. Add an appropriate amount of 5X sample buffer to each sample and heat the samples in a 100 degrees Celsius metal bath for 10 minutes. Allow the samples to cool to room temperature for five minutes and load them onto the gel. The purified UCH3 protein exhibited enzymatic activity based on the linear increase in fluorescence within two minutes during the Ub-AMC hydrolysis assay. Danshensu inhibited UCHL3 activity by reducing its ability to bind HA-UbVS with the IC50 value calculated at 12.91 micromolar.