| As a reader for histone acetylation,BRD4 plays an important role in the gene transcription regulation of tumor development and survival,and is a hot target for the development of cancer therapy drugs.The BRD4 protein obtained by conventional experimental methods is fixed in the crystal lattice to present a single static conformation,but the biological macromolecules are constantly moving in the process of functioning in vivo,so observing the dynamic conformation of BRD4 protein is very critical to interpret its functional characteristics,molecular dynamics(MD)simulation makes up for the shortcomings of traditional experimental techniques,and its value in deciphering the functional mechanism of biological macromolecules such as proteins and revealing the structural basis of diseases has been confirmed.MD simulation has great advantages in the development of anti-tumor targeted drugs.In this paper,molecular dynamics simulation is used to explore the mechanism of protein inhibitor interaction in the BRD4 complex system.The research results can provide valuable clues for subsequent experimental research on BRD4 protein and the development of small molecule drugs.Firstly,the setup of the orthosteric system(we call the traditional active site of biological macromolecules the orthosteric site).Experimental research has been conducted to obtain BRD4 protein inhibitor compound 4 with a small molecular weight by retaining the core of inhibitor BI-2536,exploring the side chain,and replacing the group with compound 4 as the starting point to obtain transition compound 9.Compound 9 has been further optimized to obtain a potential pharmaceutical compound19 with excellent effects.Based on this,we constructed orthosteric systems: four relatively stable complex systems: Free-BRD4,BRD4-Cpd4,BRD4-Cpd9,and BRD4-Cpd19.Subsequently,we conducted conventional molecular dynamics simulations for the orthosteric system,and conducted two repeated experiments.Analysis of the simulated trajectory shows that:In four orthosteric systems,root mean square fluctuation analysis revealed structural changes in 80-100 residues.Secondary structure analysis based on root mean square fluctuation found that Compound 19 had a more significant impact on the secondary structure of BRD4 protein compared to other inhibitors,and 81-89 residues generated a more stable helix structure from the loop.It was speculated that the combination of Compound 19 and BRD4 could reduce its flexibility,Maintaining the ordered structure of BRD4 allows Compound 19 to bind more tightly to enzyme active sites,Subsequent temperature factor and binding free energy analysis can prove that Compound 19 binds to BRD4 protein more stably,which may be the reason why Compound 19 has a strong inhibitory effect on BRD4 protein.Subsequent experimental studies found a completely new site on the BRD4 protein that is different from the traditional acetylation binding site.The allosteric site is located at α Spiral between B and C,with classic KAc pockets back to back.The experiment selected the traditional orthosteric site inhibitor MS436 and allosteric site inhibitor ZL0590 to compare and observe the binding of allosteric sites.Exploring the mechanism of allosteric sites is helpful for the subsequent development of small molecule allosteric inhibitors.According to experimental guidance,we set up allosteric systems: Free-BRD4,BRD4-MS436,BRD4-ZL0590,BRD4-MS436-ZL0590 four individual structure complex systems.A conventional molecular dynamics simulation was performed on the system,and the trajectory was analyzed.The results showed that:We conducted stability analysis and active pocket display for the allosteric system,and clearly observed that the system undergoes allosteric inhibition.After the allosteric inhibitor ZL0590 binds to the active center of the BRD4 protein,it has an impact on the spatial structure of the active pocket of the BRD4 protein,preventing the binding of positive sites.Based on electrostatic potential analysis,we speculate that there is a gap between ZA loop and BC loop,and that the binding of a positive inhibitor to the active pocket causes the pocket to close,enabling stable binding of the ligand.However,after the addition of allosteric ligands,both sides of the active pocket are not fully closed,making it difficult for orthosteric inhibitors to penetrate.The simulation results show that the addition of allosteric ligands may affect the opening and closing state of the active pocket by affecting the charge distribution of the ZA loop.The results of this study have certain guiding significance for the study of the interaction between inhibitors and BRD4 protein,providing theoretical basis for revealing the reason why the orthosteric inhibitor Compound 19 has a strong inhibitory effect on BRD4 protein,and exploring the mechanism of BRD4 protein allosteric inhibitors,providing a theoretical basis for the future development of high-performance drugs targeting BRD4 protein. |
