The goal of our team is to construct a drug screening system for Hippo pathway-related cancers that is capable of screening antitumor drugs in a high-throughput and large-scale manner. Based on the investigation of the Hippo pathway, we have learned that MST2-STRN3 is a drug-screening target worth trying. The interaction between the two leads to oncogene expression and tumorigenesis. So we genetically engineered E. coli by inserting DNA fragments encoding MST2 and STRN3 proteins into the pET28a plasmid to obtain pure MST2 and STRN3 proteins, respectively. Both were integrated into the AlphaScreen system, which is widely used to study protein interactions, and a fluorescence detection system was also loaded to screen various small molecule libraries, we obtained CX6258. In addition, we functionally validated CX6258 using a gastric cancer tumor cell line and Drosophila colon cancer, and the results showed that it has the ability to inhibit cancer cell growth and is expected to be a future new drug for the treatment of Hippo pathway-related cancers, bringing benefits to more patients.
We have reached the following achievements.
We constructed pET28a-GST-MST2 1-308, pET28a-HST-STRN3 64-145, pET28a-GST-MST2 1-308 (mutant), expressing GST-MST2 1-308, HST-STRN3 64-145 and GST-MST2 1-308 (mutant) under the T7 promoter proteins. These proteins were expressed in fusion with the corresponding tags, which will facilitate subsequent isolation and purification to obtain functional proteins. By loading two differently tagged proteins into the AlphaScreen system, the strength of protein interactions can be determined by observing changes in fluorescence intensity. Future iGEM teams can use these tagged plasmid backbones as well as our research ideas to explore the function of protein interactions and use the function to find application perspectives.
From further human practice on the issue of future development of the drug, we learned that the current application of our drug in the treatment of hippo-related cancers belongs to the stage of lead compounds, and the subsequent chemical modifications need to be continuously carried out to achieve better efficacy and lower side effects. These optimizations are based on the understanding of the binding site of CX6258 with the target protein MST2 and the crystal structure of the binding site. Therefore, we used virtual docking prototypes to predict and validate the binding sites of both.
Autodock Lab is a computer software widely used for molecular docking research of drug molecules. Its principle is based on the theories of molecular mechanics and computational chemistry, used to predict the binding ability and binding mode between compounds. By simulating intermolecular interactions, Autodock can provide important information about molecular binding and drug development for drug designers. We imported the protein sequence information of MST2 and the molecular structure information of CX6258 into the software, and obtained the following prediction result graph, showing that the binding sites are L34, E36, and V42 (steps for using Autodock virtual docking are shown in the protocol). The accuracy of the prediction system was proven by our subsequent expression of the MST2 protein after site mutation and in vitro pull-down validation. Future iGEM teams can use this software to analyze structure and binding site prediction of protein to protein, protein to small molecule, etc. (software details and instructions are available on the protocol page).
Protocol for Virtual docking