Name: Screening Traditional Chinese Medicine Compounds for Inhibiting UCHL3 Activity Based on Molecular Docking and Deubiquitinating Enzyme Probe Technology (Video) | JoVE Journal
Uploaded: 2024-11-22T14:40:00.000+00:00
Duration: 10 min 25 s
Description: Discover molecular docking methods to screen traditional Chinese medicine compounds for UCHL3 inhibition using Danshensu from Salvia miltiorrhiza. This computational approach validates small molecule-protein interactions for therapeutic development.

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This work was supported by the National Natural Science Foundation of Beijing [grant number 7244498].

Virtual Screening and Validation of UCHL3 Inhibitors Using Molecular Docking

The authors declare no conflicts of interest.

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 burden38. TCM has a history of thousands of years of clinical use and a solid theoretical foundation in Chinese medicine, and its natural products play an irreplaceable role in the prevention and treatment of human diseases, as well as in drug opening and design25.
In this article, we report a method that combines molecular docking with ubiquitin-specific protease probe technology to identify natural products from traditional Chinese medicine that inhibit specific DUB targets. The results showed that Danshensu had high docking activity against UCHL3 in the small molecule library of the traditional Chinese medicine Salvia miltiorrhiza Bge. In vitro experiments demonstrated that Danshensu could competitively inhibit the activity of UCHL3 with HA-Ub-VS. The present method, which combines bioinformatics and molecular experiments, can be applied to the prediction of target inhibitors or agonists.
In the experiment, several key points require special attention. Firstly, before conducting molecular docking, it is crucial to thoroughly understand the structural features of the catalytic active site of the target protein. Prioritize selecting structures featuring co-crystallization of proteins with small molecules that have been reported to inhibit the activity of this protein. Secondly, during protein purification, it is essential to preserve its critical enzyme activity regions. If the protein is too large and difficult to express or purify in sufficient quantities, one option is to truncate its sequence to preserve its active regions. During protein use, efforts should be made to avoid repeated freeze-thaw cycles to prevent activity loss. Thirdly, for competitive inhibition experiments between small molecules and HA-Ub-VS, ensure the experiment is conducted at 37 &#176;C to facilitate sufficient interaction between the small molecule and DUB. Additionally, ensure the solvent of the small molecule does not cause protein inactivation to avoid generating false-positive results.
However, during the drug screening process, this technique may encounter false-negative results due to insufficient probe sensitivity and excessive protein concentrations. To address these potential issues, we propose several solutions. We aim to increase the concentration of the probes to ensure adequate interaction with the proteins while maintaining sub-saturation levels of the proteins. This approach helps prevent false negatives caused by competitive binding between the drug and the probe. In practical applications, we have observed that the probe effectively captures significant differences in protein activity induced by drugs. However, it remains less effective at detecting subtle differences. Therefore, during drug screening, we will incorporate more refined techniques, such as Surface Plasmon Resonance (SPR)39 and Cellular Thermal Shift Assay (CETSA)40, for comprehensive evaluation. Nevertheless, as an initial tool for screening DUB-active compounds, the probe technology still demonstrates a strong cost-performance ratio.
Molecular docking and ubiquitin probe technologies are both important methods in the study of DUB inhibitors. Molecular docking relies on target structure-based virtual predictions, utilizing specific algorithms to simulate interactions between small molecules and proteins41. Small molecules reported in TCM are ranked based on their docking scores, guiding subsequent in vitro activity validation experiments and serving as an initial screening method for natural products in Chinese medicine42,43. The application of ubiquitination activity probes is an important tool in the field of ubiquitination research, and the development of technology for DUB activity probes is continuously iterated and updated23,24. Ub-AMC is an early probe used to detect DUB activity. It is widely used in high-throughput screening of DUB inhibitors because of its convenient detection methods17. However, similar to other probes like Ub-TAMRA, its application range is limited by the restricted excitation wavelength detection range44. Whereas in recent years, Ub-Rho assay has emerged with a broader excitation wavelength range, expanding its application in drug discovery, compound kinetics, and molecular experiments45,46.
In addition to the application of probes for detecting the activity of purified proteins in vitro, DUBs can also be utilized in various other contexts. HA-Ub-VS, a probe where a nucleophilic head is linked to the C-terminus of ubiquitin, such as other similar designs like Ub-VME and Ub-PA, not only detects DUB activity but also forms a covalent bond with DUB through the nucleophilic attack of the head23. Under conditions with endogenous antibodies, DUB activity can be characterized using Western blotting. When Ub is attached to a tag or biotin-labeled, the DUB that undergoes covalent binding can be enriched and characterized by mass spectrometry. This approach allows simultaneous detection of multiple DUB activities, significantly enhancing detection efficiency46. DUBs are ubiquitin chain-specific, and the limitation of the above probes is that the chain-specificity factor is ignored7. But in recent years, chain-specific probes have also been successfully applied in several studies47,48. The application of selective DUB probes will be the key to the development of DUB inhibitors. Currently, it is challenging to target metalloprotease DUBs with ubiquitination probes. This area presents opportunities for further research in developing DUB inhibitors23,24.
In summary, molecular docking and ubiquitin probe technologies play a crucial role in researching DUB inhibitors, providing important methods and tools for the development of natural product drugs from traditional Chinese medicine.

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 discovery5,6.
Over 100 DUBs have been identified in humans7,8. 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 molecule9,10. 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 residue12,13. This specificity opens up avenues for developing small-molecule inhibitors targeting the enzyme&#39;s active site or nearby allosteric pockets.
In the field of deubiquitinating enzyme research, there exists a significant challenge in characterizing their activity14,15. 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 ubiquitin17,18. 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 inhibitors19,20. The HA-Ub-VS probe is also used to measure DUB activity21. It has a vinyl sulfone group at ubiquitin&#39;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 blotting22,23,24.
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 effectively31,32. 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 use35,36.
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.

<table><tbody><tr><td>30% Acrylamide</td><td>Beijing Lablead Biotech Co., Ltd</td><td>A3291</td><td></td></tr><tr><td>Ammonium persulfate</td><td>China National Medicines Corporation Ltd</td><td>10002616</td><td></td></tr><tr><td>Anti-rabbit IgG, HRP-linked Antibody&amp;nbsp;#7074</td><td>Cell Signaling Technology</td><td>7074P2</td><td></td></tr><tr><td>BeyoECL Plus</td><td>Beyotime</td><td>P0018S</td><td></td></tr><tr><td>Bradford Protein Assay Kit</td><td>Beyotime</td><td>P0006</td><td></td></tr><tr><td>ClonExpress Ultra One Step Cloning Kit</td><td>Vazyme</td><td>C115-01</td><td></td></tr><tr><td>Danshensu</td><td>Shanghai yuanye Bio-Technology Co., Ltd</td><td>B20254</td><td></td></tr><tr><td>DMSO</td><td>Ameresco, Inc.</td><td>21K2356571</td><td></td></tr><tr><td>Electrophoresis System</td><td>Liuyi Biotechnology</td><td>112-0630</td><td></td></tr><tr><td>HEPES</td><td>Sigma</td><td>H3375</td><td></td></tr><tr><td>His-tagged protein purification kit (NTA-Ni agarose magnetic beads)</td><td>Beyotime</td><td>P2247S</td><td></td></tr><tr><td>Immun-Blot PVDF Membrane, Roll, 26 cm x 3.3 m</td><td>Bio-Rad Laboratories (Shanghai) Co., Ltd</td><td>1620177</td><td></td></tr><tr><td>Isopropyl alcohol</td><td>Macklin</td><td>I811925</td><td></td></tr><tr><td>M5 Prestained Protein Ladder</td><td>Mei5 Biotechnology Co.Ltd</td><td>MF-212-01</td><td></td></tr><tr><td>Maestro</td><td>Schr&amp;ouml;dinger&amp;rsquo;s</td><td>https://www.schrodinger.com/platform/products/maestro/</td><td></td></tr><tr><td>Methyl alcohol</td><td>China National Medicines Corporation Ltd</td><td>10014108</td><td></td></tr><tr><td>MF-Millipore</td><td>Millipore</td><td>HAWP04700</td><td></td></tr><tr><td>MyFug&amp;nbsp;mini centrifuge</td><td>Sigma</td><td>Z764183</td><td></td></tr><tr><td>Pierce Dilution-Free Rapid Gold BCA Protein Assay</td><td>Thermo Scientific</td><td>A55860</td><td></td></tr><tr><td>PR-619</td><td>Cell Signaling Technology</td><td>26065S</td><td></td></tr><tr><td>Primary Antibody Dilution Buffer for Western Blot</td><td>Macklin</td><td>P917820</td><td></td></tr><tr><td>Recombinant Human HA-Ubiquitin Vinyl Sulfone Protein, CF</td><td>R&amp;amp;D Systems</td><td>U-212-025</td><td></td></tr><tr><td>Recombinant Human Ubiquitin AMC Protein, CF</td><td>R&amp;amp;D Systems</td><td>U-550-050</td><td></td></tr><tr><td>Skim Milk</td><td>Becton,Dickinson and Company</td><td>232100</td><td></td></tr><tr><td>Sodium Dodecyl Sulfate (SDS)</td><td>Ameresco, Inc.</td><td>205-788-1</td><td></td></tr><tr><td>TEMED</td><td>Ameresco, Inc.</td><td>2545C134</td><td></td></tr><tr><td>Tween 20</td><td>Beijing Lablead Biotech Co., Ltd</td><td>0777-1</td><td></td></tr><tr><td>UCHL3 (D25E6) Rabbit mAb</td><td>Cell Signaling Technology</td><td>8141T</td><td></td></tr></tbody></table>

screening traditional chinese medicine compounds for inhibiting uchl3 activity based on molecular docking and deubiquitinating enzyme probe technology

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

Deubiquitinating enzymes&#160;(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.

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 Figure 1, Danshensu and UCHL3 have hydrogen bonding with ARG-186, HIS-169, VAL-166, and ASN-93, respectively, and the docking scores between them are -4.75 kcal/mol with favorable binding activity.
To validate the inhibitory activity of Danshensu on UCHL3, we proceeded by purifying the recombinant protein of UCHL3 in vitro, as depicted in Figure 2A. Next, Ub-AMC hydrolysis assays were conducted to assess the enzymatic activity of the purified UCHL3 protein. As shown in Figure 2B, compared to the control group, the UCHL3 group's fluorescence value was higher under the condition of 380 nm excitation light and 460 nm emission light. In addition, within 2 min the fluorescence values of the UCHL3 group showed a linear relationship with the reaction time and reached a plateau after 2 min. It demonstrated that the purified UCHL3 retains significant enzymatic activity in vitro, meeting experimental requirements.
Here, we report another probe, HA-Ub-VS, which can covalently bind with UCHL3. The inhibitory effect of Danshensu on UCHL3 activity can be demonstrated through competitive binding with HA-Ub-VS. As shown in Figure 3A, when UCHL3 protein is added alone, it migrates at around 25 kDa. However, upon the addition of the probe with a molecular weight of approximately 10 kDa, the band shifts to around 35 kDa, indicating inhibited UCHL3. UCHL3's ability to bind HA-Ub-VS decreases gradually with increasing concentrations of Danshensu. As depicted in Figure 3B, the IC50 value of Danshensu for competitively inhibiting UCHL3 binding to HA-Ub-VS is 12.91 µM.
<img alt="Protein-ligand complex interaction, structural diagram, molecular analysis, binding site exploration." class="xfigimg" src="/files/ftp_upload/67432/67432fig01.jpg" data-altupdatedat="1788304762000" data-alt="Figure 1"> 
Figure 1: Interaction between Danshensu and UCHL3 (PDB: 1XD3) protein. <a href="https://www.jove.com/files/ftp_upload/67432/67432fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="SDS-PAGE gel and fluorescence chart for enzyme kinetics, protein analysis, and activity assay results." class="xfigimg" src="/files/ftp_upload/67432/67432fig02.jpg" data-altupdatedat="1788304762000" data-alt="Figure 2"> 
Figure 2: Ub-AMC hydrolysis assay catalyzed by UCHL3. (A) The SDS-PAGE image of the purified UCHL3 target protein in vitro. (B) The result of the Ub-AMC hydrolysis assay catalyzed by UCHL3, Data are represented as mean ± SD. <a href="https://www.jove.com/files/ftp_upload/67432/67432fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Western blot analysis of UCHL3 activity inhibition by Danshensu; IC50 determination graph." class="xfigimg" src="/files/ftp_upload/67432/67432fig03.jpg" data-altupdatedat="1788304762000" data-alt="Figure 3"> 
Figure 3: Using HA-Ub-VS to detect Danshensu's inhibition activity against UCHL3. (A) The inhibitory effect of Danshensu on UCHL3 at different concentrations. (B) The quantification curve of Danshensu inhibiting UCHL3 activity, with Danshensu competitively inhibiting HA-Ub-VS, yielded an IC50 value of 12.91 µM. Data are represented as mean ± SD. <a href="https://www.jove.com/files/ftp_upload/67432/67432fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: The names and scores of the top 30 small molecules in molecular docking. <a href="https://www.jove.com/files/ftp_upload/67432/Table%201.xlsx" target="_blank">Please click here to download this Table.</a>
Supplementary Table 1: The docking results of all small molecules from Salvia miltiorrhiza Bge with UCHL3.&gt; <a href="https://www.jove.com/files/ftp_upload/67432/Supplementary%20Table%201.zip" target="_blank">Please click here to download this File.</a>

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.

Deubiquitinating enzymes&#160;(DUBs) play a pivotal role in modulating ubiquitination homeostasis, with UCHL3 being an archetypal cysteine DUB intricately ...

screening-traditional-chinese-medicine-compounds-for-inhibiting-uchl3

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Discover molecular docking methods to screen traditional Chinese medicine compounds for UCHL3 inhibition using Danshensu from Salvia miltiorrhiza. This computational approach validates small molecule-protein interactions for therapeutic development.

Screening Traditional Chinese Medicine Compounds for Inhibiting UCHL3 Activity Based on Molecular Docking and Deubiquitinating Enzyme Probe Technology (Video) | JoVE Journal

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

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