About Ubiquitin-Proteasome Pathway (UPP)
As we all know, cells produce new proteins every moment, and those “junk proteins” that have been used or expressed incorrectly will be degraded. Ubiquitin-Proteasome Pathway (UPP) is the main way to rapidly degrade “junk protein” in cells. UPP consists of ubiquitin molecule (Ubiquitin, Ub), 26S proteasome, ubiquitin activating enzyme E1, ubiquitin transferase E2 and ubiquitin ligase E3. In eukaryotic cells, there are 1 E1 and about 50 E2, but there are more than 600 E3 with different structures. E3 is mainly involved in the recognition process of target proteins, and different E3 can recognize different target proteins. The UPP process is shown in Figure: First, ubiquitin activating enzyme E1, which is responsible for activating the ubiquitin molecule and binding it to ubiquitin transferase E2; then ubiquitin ligase E3 binds to the target protein to be degraded; then, ubiquitin transferase the Ub attached to E2 is transferred to the target protein, and the target protein is labeled with ubiquitination; finally, the labeled target protein will be rapidly degraded into amino acid fragments by the proteasome.
Figure: UPP Process
Based on the above mechanism of ubiquitination degradation of target protein, can it be applied to disease treatment? For example, degradation of disease-related proteins can achieve therapeutic purposes. The idea is perfect, but the biggest difficulty is how to make E3 specifically recognize those disease-related proteins.
In order to solve the above difficulties, proteolysis targeting chimera (PROTAC) merged. It is a small molecule composed of target protein recognition ligand, Linker and E3 recognition ligand. The magic of PROTAC is that it can be used as a medium to connect ubiquitin ligase E3 and the target protein, which makes it possible to use the UPP mechanism to specifically degrade disease-related proteins. Different PROTAC can recognize different target proteins, so PROTAC, as a small molecule compound that specifically recognizes and induces degradation of the target protein, has invaluable clinical application value.
About Smad3
After understanding the background of UPP and PROTAC, let’s take a look at Smad3 protein. This protein is a protein in the cell that is closely related to tissue fibrosis and cancer. Tissue fibrosis is the main cause of death in many diseases at present. The main pathological manifestation is the increase of fibrous connective tissue in organ tissues and the decrease of parenchymal cells, which in turn can cause organ damage and The function is reduced. The study found that the splenocytes of Smad3-deficient mice will not undergo a proliferation response after specific stimulation, so reducing Smad3 protein levels has become one of the methods for treating tissue fibrosis and cancer.
Scheme design
Based on the potential application value of Smad3, the cooperative unit intends to use this protein as a research object and expects to obtain PROTAC with specific degradation of Smad3. After in-depth communication, researchers designed a specific research plan. First, use homology modeling and protein-protein docking methods to determine the binding site of Smad3 and PROTAC; then use molecular docking methods to conduct virtual screening of more than 1.3 million molecules in the Enamine compound library to obtain 100 compounds; obtained by artificial mesh screening Linker’s 13 target protein recognition ligands can be linked; the cooperative unit is verified by SPR to obtain compound 8 with the highest affinity to Smad3, and finally a PROTAC is obtained for biological activity testing.
Confirmation of the binding site of Smad3 and ligand
The key step of molecular docking is to determine the binding site of the target protein and ligand. After literature research, it was found that Smad3 had only its inhibitor binding site. Conventionally, is it not the best to use protein inhibitors as PROTAC’s target protein recognition ligands, on the one hand, it can exert an inhibitory function, and on the other hand, it can promote protein degradation. But Smad3 is very special, it is unscientific to design PROTAC based on the above mechanism. Because Smad3 can only be degraded after being phosphorylated, and the binding of the inhibitor will hinder its phosphorylation, therefore, the binding site of the inhibitor cannot be used as the site where PROTAC interacts with Smad3. Researchers need to find a new one on Smad3 Position to combine PROTAC.
We further investigated the literature and found that the co-repressor c-Ski can also bind to Smad3, and c-Ski can also accelerate the ubiquitination process of Smad3, but its specific binding site with Smad3 has not been reported. Therefore, we adopt the binding position of Smad3 and c-Ski as the binding site for designing PROTAC, and the specific steps to confirm the binding site are as follows:
- Use the homology modeling module of MolDesigner molecular simulation platform v1.2 to construct the three-dimensional structure of c-Ski and evaluate its structural rationality;
- Use the protein-protein docking module to search for the most ideal binding conformation of the two. By analyzing the overall structure, electrostatic potential and hydrophobic properties of the binding conformation, and referring to Fukuchi’s research, the final selection The Phe247 and His248 regions on Smad3 were designated as their binding sites with PROTAC.
Virtual screening
Virtual Screening can greatly shorten the drug development cycle, is a common technology for drug discovery. This study uses a virtual screening technology based on molecular docking. The basic idea is to predict the biological activity of candidate compounds by calculating the affinity of small molecules in the compound library with the target protein.
After determining the Smad3 binding site, researchers used a virtual screen combined with an artificial mesh screen to screen more than 1.3 million compounds in the Enamine library to obtain 13 candidate compounds with potential affinity for Smad3. The screening process mainly includes:
- Use the “Five Rules of Drug-Like” for preliminary screening;
- Screening by fast, standard and fine docking mode in turn, and take the top 10% of the score respectively, a total of 328 molecules;
- Use the MM/GBSA (Molecular mechanics combined with generalized Born and surface-area solvation) method to calculate the combined free energy of the compound obtained in the previous step, and take the top 100 compounds with the lowest energy;
- Based on the binding mode (mainly considering hydrogen bonding, hydrophobic interaction and electrostatic interaction) and PROTAC synthesizability (mainly considering whether it is easy to undergo acylation or esterification reaction with Linker, molecular polarity and flexibility of terminal amino or hydroxyl groups, etc.) 13 candidate compounds were obtained.
Verification of biological activity
The cooperative unit used SPR to test the affinity of 13 compounds with Smad3. The results showed that 11 compounds were active, accounting for 85%. Among them, compound 8 has the strongest affinity with Smad3. The final PROTAC was constructed using compound 8 as the target protein recognition ligand. The results of cell experiments show that the PROTAC can degrade Smad3 by ubiquitination. After bold assumptions, careful verification, and careful design, the resulting PROTAC lived up to expectations. The research results laid the foundation for the development of new drugs for Smad3 related diseases.
Outlook
In this project, a virtual screening technology based on molecular docking is used to select small molecules with high affinity to Smad3 from more than 1.3 million compound libraries, and then link Linker and E3 recognition ligands to obtain a complete PROTAC. The PROTAC has been verified by cell experiments and has the function of degrading Smad3 by ubiquitination. This research idea can provide guidance for the ubiquitination degradation of other proteins related to the occurrence and development of diseases, such as BET (Bromodomain and extra-terminal) protein related to anti-inflammatory and anti-tumor, KRAS protein, Amyloid β-protein, estrogen receptor protein, APOBEC3 enzyme related to antiretrovirus, etc.