The Screening Of Small Molecular Inhibitors On TRIM24 Bromodomain

Bromodomain?BRD?,with unique sequence and structure,is a protein-protein interaction module that was first identified and named in the fruit fly genome in the1980s.At present,61 bromodomain have been found in 46 proteins,which are divided into 8 families according to their similarities and differences in sequence and structure.TRIM24?Tripartite motif-containing 24?proteins belong to the fifth family,TRIM24 protein also belongs to the TRIM family,and the most significant structural feature of TRIM family is the highly conservative sequence of structural domains of RBCC.Even if family members lack a structural domain,other structural domains are still conservative in order and space.TRIM24 was originally named TIF1??transcriptional regulator 1??,is a co-regulator of retinoic acid signaling pathways.It is involved in apoptosis,cycle regulation and other important life processes,and can be used as a co-regulator to regulate the transcriptional activity of nuclear factors.Studies have shown that abnormal expression of TRIM24 protein can result in multiple dysfunction mechanisms,thereby promoting tumorigenesis and development.However,due to the difficulty in the study of TRIM24 inhibitors as small molecules to be prepared into drugs,there are few inhibitors in the market,few experimental methods for screening activity.So the screening of small molecule inhibitors with TRIM24 bromodomain was studied,it has important research value and practical significance.In this study,TRIM24 protein was used as a target,Screening the activity of small molecule inhibitors,Small molecule inhibitors with relatively high activity were screened out.First of all,the expression of proteins is carried out by using the expression system of E.coli,High purity TRIM24 target protein was obtained by affinity chromatography and gel filtration chromatography.Then the activity was screened by AlphaScreen assay,16 small molecules with high activity were screened from three small molecule inhibitor libraries in the laboratory,the activity of F8,L6,L7 and L8 is relatively high,The IC₅₀ values are 5.91,4.73,5.53 and 4.77?M,respectively.On the basis of the structure of these four small molecule inhibitors,structural optimization was carried out,16 small molecules were synthesized,Compounds F15,L19,L27 and L34 had higher activity.At the AlphaScreen level,the IC₅₀ value is about 1?M.In subsequent TSA experiments,the activity of?T_m reached about 2°C,which was relatively good.The experimental data of AlphaScreen and TSA were synthesized,4 small molecule inhibitors with relatively high activity and potential for optimization were obtained.After the above experiments and data analysis,the eutectic experiment was carried out by selecting small molecular inhibitors with better activity,to study the real binding mode of TRIM24 protein to small molecule inhibitors.The purified protein was recrystallized about the aim is to improve the success rate of crystallization.The crystal was cultured by suspension drop method,and then the crystal was optimized to achieve the diffraction condition.The crystal and structure of the complex of small molecule inhibitor L19 and TRIM24were obtained through the above experiments.The crystal structure has important reference significance for the study of the binding mode of compounds to proteins and the structural design of small molecule inhibitors,it provides theoretical basis and further research ideas for the subsequent discovery and design of TRIM24 about small molecule inhibitors.In this study,the screening of small molecular inhibitors of TRIM24bromodomain was studied.Through a comprehensive reading of the literature,a series of experimental designs and explorations,the relatively high activity of small molecule inhibitors F15,L19,L27,L34 were finally screened.The eutectic experiments were carried out to obtain the crystals of the protein complex of small molecule inhibitor L19 and TRIM24,and the real binding mode was analyzed.This research provides theoretical and material basis for the research and development of TRIM24 related biological structure and small molecule inhibitors.