This study investigated the interaction between MST2-STRN3, a crucial element in the Hippo signaling pathway. In our project, we engineered E. coli to produce two key components (MST2 and STRN3) of the human hippo pathway, the interaction of which leads to dysfunctional hippo pathway signaling and cancer development. After obtaining the purified protein by affinity chromatography, we used it to develop a high-throughput drug screening system, AlphaScreen, which detects changes in fluorescence intensity to indicate changes in the proximity of MST2-STRN3 binding and is used to screen drug candidates that can restore normal Hippo signaling. This drug screening system enables high-throughput, large-scale screening of existing drug libraries, accelerating the process of bringing new drugs to market, and is expected to provide more precise, lower-risk therapies for cancer patients in the future.
The Hippo pathway includes a group of conserved kinases that serves as a signaling pathway, inhibiting cellular proliferation. Recent research has demonstrated that the Hippo signaling pathway is a crucial regulator of organ size in Drosophila and mammals, and its malfunctions cause multiple cancers, encompassing liver, gastric, colon, and lung cancers. Dephosphorylation of MST1/2, which is a crucial element of this pathway because of STRN3, results in irregular cell proliferation and is accountable for certain types of cancer. To re-activate the Hippo signaling pathway, we have created a high-throughput drug screening method that concentrates on breaking up the MST2-STRN3 interaction to reinstate the phosphorylated state of MST2. MST2 1-308 is the kinase domain of the MST2 protein, which has kinase activity and can interact with the CC domain (64-145) of STRN3. This drug screening system requires that we first express and purify the structural domains of the two proteins that interact with each other separately.
We chose the pET28a expression vectors pET28a-GST-MST2 1-308 and pET28a-HST-STRN3 64-145 as well as the corresponding expression host E. coli BL21(DE3) to achieve high expression capability. pET28a vectors contain the T7 promoter for transcription initiation and a Shine–Dalgarno (SD) sequence to mediate translation initiation. The two vectors separately include affinity tags glutathione S-transferase (GST) and histidine (HST) which enable the purification of target proteins. Specifically, we cloned the MST2 1-308 cDNA into the pET28a-GST plasmid to obtain the recombinant GST-MST 1-308 fusion protein which could be further affinity-purified by the GST antibody-conjugated beads. In addition, the STRN3 64-145 cDNA was cloned into the pET28a-HST plasmid to express the STRN3-recombinant protein which could be purified using the beads conjugated with the anti-HST antibody.
Affinity chromatography is characterized by high selectivity, high resolution, and high capacity in most protein purification schemes. It has the advantage of exploiting the biological structure or function of the protein for purification, often simplifying time-consuming and complex purification processes. Affinity chromatography is based on the specific and reversible binding of proteins to matrix-bound ligands. The ligand can bind either directly to the protein of interest or to a tag covalently attached to the protein. This specific affinity interaction captures the target while removing impurities or other molecules from the solution.
AlphaScreen contains both donor and acceptor magnetic beads. In our experiments, both beads were paired with MST2 1-308 and STRN3 64-145. The donor beads contain a photosensitizer (phthalocyanine) that excites ambient oxygen to a singlet state when irradiated at 680 nm. Each donor bead produces approximately 60,000 monomorphic oxygen per second, and as the interaction of MST2 with STRN3 brings the two beads closer together, it causes the acceptor beads to receive the energy of the monomorphic oxygen and emit light. We performed a high-throughput screen by adding various small molecules to ten plates containing 24 lengthwise wells and 16 widthwise wells and measuring the fluorescence intensity of the magnetic receptor beads to determine how tightly MST2 and STRN3 bind. The first two columns and the last two columns of the plates are negative and positive controls, respectively. For the negative control, we add only magnetic beads that contain no proteins and therefore produce very little light. The positive control produces high-intensity light. The drug candidate libraries include the TargetMol-Natural Compound Library, TargetMol-Approved Drug Screening Library, and TargetMol-Inhibitor Library, with a total of 3106 compounds. Calculation of MST2 and STRN3 recovery rates:
1. Set the number except the first and last two columns equal to x
2. Average the negative and positive controls respectively and label them as AN and AP
3. Put data into the equation: [1- (x-AN) /AP]×100 (a.u.)
GST-MST2 1-308 binds to donor magnetic beads via a biotin-streptavidin bond, while HST-STRN3 64-145 binds to acceptor magnetic beads via a Ni-HST bond. Biotin-streptavidin and its homologs are widely used in molecular research because of their selective and stable interactions with biotin. In addition, polyhistidine tags are widely used for the purification of recombinant target proteins by immobilized metal affinity chromatography. These beads enable rapid separation and purification of HST-tagged fusion proteins on a small scale by manual or automated methods.
A series of hit compounds can be obtained based on the AlphaScreen high-throughput screening system. To verify the reliability of this high-throughput screening system, we selected one of the candidates for the following in vitro pull-down and point mutation experiments to further confirm the accuracy of the screening results.
After finding small molecule drug candidates by AlphaScreen, it is necessary to further verify the reliability of high-throughput screening results by in vitro interaction experimental methods, such as pull-down. GST pull-down is a method to study protein interactions in vitro, and the basic principle is as follows: assuming that proteins A and B may have interactions, we will fuse one of the proteins such as protein A with a GST tag. Then, GST-A and B and Sephrose4B beads, which can specifically bind GST, are incubated for a certain period of time, the unbound proteins are washed sufficiently, the beads are boiled for SDS-PAGE electrophoresis, and finally radiolabeling is performed, and the corresponding bands of GST-A and B are seen, indicating that the GST-A and B are pulled down by GST-A due to the interaction. If there is no interaction, only one band corresponds to GST-A. By this method, the experimental group and the blank control group were set up at the same time when GST-MST2 1-308, HST-STRN3 64-145, and Sephrose4B beads were incubated with the candidate compounds, and the results could be observed to see whether the candidate small molecule drugs could break the interaction between the two types of proteins.
The binding sites between the medicine molecule and target component are determined through the virtual simulation of the interaction established between the medicine molecule and the target component (MST2), and the interaction is predicted through an approximation of the Gibbs free energy of the binding pair, calculated from the imported data (of shape and structure) of each of the pair through the summation of the virtual prediction of interactive factors including London-dispersion, electrostatic, and hydrogen bond energies combined with consideration to the effect of entropy; the binding sites of the ligand component will be predicted in reference to interaction through built-in algorithms.
We will use PCR to mutate the bases in the plasmid that may encode the site of interaction of the MST2 protein with small molecule drug candidates. The final ring-opening plasmid with the mutated sequence is produced by designing primers for base substitutions that bind to the template DNA. Point mutations were used to confirm the correct site of interaction of MST2 with the drug candidate. It was also tested whether the drug molecules obtained using AlphaScreen could effectively disrupt the interaction between MST2 and STRN3, thereby restoring the Hippo pathway.
GST pulldown was able to test whether MST2 mutants could dissociate from STRN3 in the presence of small molecule drugs. Assuming that small molecule drugs are therefore unable to interfere with the interaction of the MST2 mutant with the STRN3 protein, this validates the site of interaction of small molecule drugs with the MST2 protein and provides theoretical support for restoring the Hippo pathway.
In short, our experimental design can be divided into four parts:
1. Expression and purification of GST-MST2 1-308 and HST-STRN3 64-145 proteins in the microbial strain E. coli BL21 through genetic engineering.
2. Formation of donor magnetic beads (biotin-Streptavidin linked with GST-MST2 1-308) and acceptor magnetic beads (Ni-HST linked with HST-STRN3 64-145), followed by utilizing the AlphaScreen high-throughput screening system to screen for small molecule drugs.
3. Target identification of the interaction between MST2 and candidate small molecules through structural simulations involving site-directed mutagenesis of MST2. Validation of their interaction sites will be conducted using pull-down assays.
4. Assessment of the potential inhibitory effects of candidate compounds on tumor cells in AGS cell lines or a Drosophila colorectal cancer model.
This study focuses on the interaction between critical components of the Hippo signaling pathway, MST2-STRN3, as a target. By integrating the AlphaScreen high-throughput screening system, we screened a library of FDA-approved small molecule compounds to identify those capable of disrupting the MST2-STRN3 interaction. These compounds are being considered candidate drugs, and their therapeutic effects will be tested in both tumor cell lines and a Drosophila colorectal cancer model2, 3. This achievement aims to provide a high-throughput screening method for identifying drugs targeting tumors related to the Hippo pathway. Moreover, the compound identified through this screening has demonstrated remarkable tumor-inhibiting effects. In the future, this could broaden the options for tumor treatment, offering more precise and low side-effect therapies for cancer patients.
1.Eglen, Richard M, et al. “The Use of AlphaScreen Technology in HTS: Current Status.” Current Chemical Genomics, vol. 1, 25 Feb. 2008, pp. 2–10,
2.Messina B, Lo Sardo F, Scalera S, et al. Hippo pathway dysregulation in gastric cancer: from Helicobacter pylori infection to tumor promotion and progression. Cell Death Dis. 2023;14(1):21.
3.Liang K, Zhou G, Zhang Q, Li J, Zhang C. Expression of hippo pathway in colorectal cancer. Saudi J Gastroenterol. 2014;20(3):188-194.