iGEM 2023 — Team shanghai-medx


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{% block title %}Experiment Engineering Success{% endblock %}
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![](https://static.igem.wiki/teams/4889/wiki/team-bg.jpg)

#### Abstract：

Dysregulation of the Hippo pathway leads to abnormal development of organisms and related diseases such as tumors. Finding suitable targets is necessary for small-molecule high-throughput screening of anticancer medicines connected with the hippo signaling system. By reviewing the paper and taking human practice, we found that striatin 3 (STRN3) is upregulated in gastric cancer (one of the Hippo pathway-associated tumors) and dephosphorylated by interacting with MST1/2, leading to YAP activation downstream of the pathway and cancer development. Based on this, we developed AlphaScreen, a high-throughput screening system with two magnetic beads loaded with MST2 and STRN3 from engineered E. coli, to screen for drugs that disrupt the STRN3-MST2 interaction and reactivate the Hippo tumor suppressor. To validate the reliability of the AlphaScreen system, we selected a hit compound, CX6258, from the screening results and performed cell and animal experiments, which showed that the compound had a significant inhibitory effect on tumor cells and demonstrated the accuracy of this high-throughput screening system. 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.

##### 1. MST2 1-308 expression and purification

MST2, the key kinase upstream of the Hippo signaling pathway, contains two domains, kinase domain and SARAH. The kinase domain has kinase activity and its interaction with the CC domain of STRN3 has been demonstrated1. Therefore, we cloned MST2 1-308 (kinase domain, 921bp) into the pET28a-GST vector and expressed in fusion with the GST tag, which facilitated subsequent protein purification.

![](https://static.igem.wiki/teams/4889/wiki/engineeringsuccess/engineeringsuccess-1.png)

![](https://static.igem.wiki/teams/4889/wiki/engineeringsuccess/engineeringsuccess-2.png)

Figure 1. Construction of pET28a-GST-MST2

The plasmid was transformed into E. coli and bacterial PCR were used to measure the size of MST2 1-308. Originally 921 bp in size, MST2 was around 1500 bp due to the size of both ends. The outcomes demonstrated that the plasmid construction was effective.

![](https://static.igem.wiki/teams/4889/wiki/engineeringsuccess/engineeringsuccess-3.png)

Figure 2. Colony PCR of pET28a-GST-MST2 strain

The GST-MST2 1-308 protein is our target protein, and the "total" sample is a mixture of various proteins within the bacteria. To isolate our target proteins, we first processed the samples through steps such as centrifugation, which produced a "supernatant" and a "precipitate". These initial steps helped us separate soluble proteins (in the supernatant) from insoluble proteins (in the precipitate), allowing us to focus on the proteins we were interested in.

The "flow" step plays a critical role in removing unwanted contaminants. We use a column to pass the sample through a stationary phase that has a high affinity for GST-MST2 1-308. This step effectively captures our protein of interest while removing other molecules. After "Flow", we performed "Wash 1" and "Wash 2". The purpose of these steps is to remove any remaining impurities and ensure that the GST-MST2 1-308 protein is as pure as possible. "Wash 1" and "Wash 2" help to reduce background noise during the final elution.

Finally, we disrupted the binding between GST-MST2 1-308 and the stationary phase using reduced glutathione salt buffer (Elution) to obtain purified GST-MST2 1-308.

![](https://static.igem.wiki/teams/4889/wiki/engineeringsuccess/engineeringsuccess-4.png)

Figure 3. GST-MST2 protein purification

##### 2. STRN3 64-145 expression and purification

STRN is an evolutionarily conserved molecular, which has been implicated in the Hippo signaling pathway2. STRN3 contains two domains, the CC domain and the WD40 domain. The CC domain is involved in the interaction with MST2. Therefore, we cloned STRN3 64-145 (CC domain, 246bp) into the pET28a-HST vector and expressed in fusion with the HST tag, which facilitated subsequent protein purification.

![](https://static.igem.wiki/teams/4889/wiki/engineeringsuccess/engineeringsuccess-5.png)

![](https://static.igem.wiki/teams/4889/wiki/engineeringsuccess/engineeringsuccess-6.png)

Figure 4. Construction of pET28a-HST-STRN3

The plasmid was transformed into E. coli and bacterial PCR were used to measure the size of STRN3 64-145. The result showed that STRN3 64-145 was around 250 bp and demonstrated that the plasmid construction was effective.

![](https://static.igem.wiki/teams/4889/wiki/engineeringsuccess/engineeringsuccess-7.png)

Figure 5. Colony PCR of pET28a-HST-STRN3 strain

Purification of proteins is a necessary step before performing any protein-related experiments. The testification of the HST-STRN3 64-145 protein determined the composition of the sample after each step of purification according to their stripe. The graph shows the protein composition of the solution at different stages of the purification process. The most important category to check here is lane 7, the composition of the final eluted protein. This is because it directly symbolizes whether or not the production and purification of the protein was successful - in this case, it was as there is only one stripe here representing HST-STRN3 64-145. lanes 5~6 are similar to the solution after washing the beads, and none of them have clear stripes. Other details are the same as for MST2 1-308.

![](https://static.igem.wiki/teams/4889/wiki/engineeringsuccess/engineeringsuccess-8.png)

Figure 6. HST-STRN3 protein purification

##### 3. High-throughput screening of drugs capable of breaking the MST2-STRN3 interaction using AlphaScreen

We use AlphaScreen technology in this part. We bind “Donor” and “Acceptor” beads with MST2 and STRN3 in our experiment, separately. Donor beads contain a photosensitizing agent (phthalocyanine) that, when irradiated at 680 nm, excites ambient oxygen to a singlet state. Excitation of each Donor bead generates approximately 60,000 oxygen singlets per second, resulting in a glowing of receptor beads. We used the public platform of the School of Life Sciences to perform the high throughput screening. The library of small molecules screened included TargetMol's Natural Compound Library, Approved Drug Screening Library, and Inhibitor Library, totaling 3106 molecules. MST2 and STRN3 binding tightness were determined by simultaneously measuring the fluorescence intensity of each well in a 384-well plate. The first two columns and the last two columns of the plate are negative and positive controls, respectively. For the negative control, we added only magnetic beads without protein, so the light intensity produced will be very low. And the light intensity produced by the positive control will be very high. Therefore, we can use this method to calculate the separation rate of MST2 and STRN3:

![](https://static.igem.wiki/teams/4889/wiki/engineeringsuccess/engineering-19.png)

Figure 7. Schematic and results of AlphaScreen. A. 384-well plate layout and partial presentation of results. B. Schematic of AlphaScreen. C. Separation rates of some representative candidate small molecules. Higher separation rates mean more effective drugs and the calculations are as follows.

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.)

After the calculation, our group found CX6258 (A potent, kinase-selective inhibitor of pan-Pim kinase that has passed the preclinical studies and clinical trials, and their biological activities and safety have been verified) which has the highest separation rate for doing the rest of the experiment.

![](https://static.igem.wiki/teams/4889/wiki/engineeringsuccess/engineering-20.png)

Figure 8. CX-6258 Chemical Structure (CAS No. : 1202916-90-2)

Functional validation of small molecule drug candidates using pull-down experiments. Through the interview with Dr. Song during human practice, we learned that although the AlphaScreen technology has the advantages of a wide range of applications, including small molecules to large complexes, high speed, and high sensitivity, its main limitations should not be ignored. These include the sensitivity of the reaction system to bright light or prolonged room light; the trapping of singlet oxygen molecules by some compounds can reduce the optical signal; and the bleaching effect of donor beads makes single-pass signal detection preferable. To verify the screening results in the previous step, we performed pull-down experiments with CX6258 to clarify the separation of MST2-STRN3.

GST pull-down is a method to study protein interactions in vitro, and the basic principle is as follows: assuming that MST2 and STRN3 may have interactions, we will fuse MST2 with a GST tag. Then, GST-MST2, HST-STRN3, 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-MST2 and HST-STRN3 are seen, indicating that the two proteins are pulled down due to the interaction. If there is no interaction, only one band corresponds to GST-MST2.

The results showed that lane 1 was used to exclude the possibility of the GST tag interacting with STRN3. Lanes 4-7, as the INPUT of each component, served as a control and to make the results more convincing. Lanes 2 and 3 were used to test whether CX6258 could disrupt the MST2-STRN3 interaction. Lane 2, due to the addition of CX6258 in the sample, showed only one band in the SDS-PAGE (only GST-MST2 was pulled down), while lane 3 showed two bands (indicating that GST-MST2 and HST-STRN3 were pulled down, respectively), indicating that CX6258 has the ability to disrupt the MST2-STRN3 interaction.

![](https://static.igem.wiki/teams/4889/wiki/engineeringsuccess/engineeringsuccess-10.png)

Figure 9. CX6258 Pull-down testing

##### 4. Confirmation of drug candidates that disrupt the MST2-STRN3 interaction and investigation of the mechanisms involved

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.

Virtual docking predicts sites of CX6258 interaction with MST2. 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).

![](https://static.igem.wiki/teams/4889/wiki/engineeringsuccess/engineering-new.png)

Figure 10. Virtual docking predicts sites of CX6258 interaction with MST2

The binding site predicted by virtual docking was mutagenized by PCR to determine the mode of action of CX6258. Based on the pET28a-GST-MST2 1-308 plasmid, we designed the primer sequences corresponding to the three amino acids to be mutated, i.e. GCA instead of CTT for L34A, GCA instead of GAA for E36A, and GCA instead of GTA for V42A, and then performed overlap PCR.

![](https://static.igem.wiki/teams/4889/wiki/engineeringsuccess/engineeringsuccess-16.png)

![](https://static.igem.wiki/teams/4889/wiki/engineeringsuccess/engineeringsuccess-17.png)

Figure 11. Construction of pET28a-GST-MST2 (mutant)

We performed protein purification of mutant MST2 (MST2 3 mutant). The experimental design was similar to the previous purification of MST2 and STRN3. The clear single band in lane 7 shows that the GST-MST2 3 mutant protein was successfully expressed and purified.

![](https://static.igem.wiki/teams/4889/wiki/engineeringsuccess/engineeringsuccess-18.png)

Figure 12. GST-MST2 (mutant) protein purification

After the MST2 3 mutant protein was purified, we tested the role of CX6258 between MST2-STRN3 and MST2 3 mutant-STRN3. The results of lanes 1-4 show that after the binding site of CX6258 to MST2 is mutated, CX6258 no longer affects the interaction between MST2 and STRN3 (two bands in both lanes 1 and 2); the unmutated MST2 protein can still be affected by CX6258 and separated from STRN3 (only one distinct band in lane 3). Lanes 5 and 6 were used to exclude the effect of non-specific binding of GST-MST2 to HST tag or GST tag to HST-STRN3 on the results. Lanes 7-11 were used as the INPUT of each component and were used as a control to make the results more convincing. Overall, this result validates that the sites where CX6258 interacts with MST2 are L34, E36, and V42, elucidating the mechanism by which this small molecule is able to disrupt the MST2-STRN3 interaction. In addition, the elucidation of this mechanism will increase the commercial and translational value of the project results.

![](https://static.igem.wiki/teams/4889/wiki/engineeringsuccess/engineeringsuccess-22.png)

Figure 13. GST-MST2 VS GST-MST2 (mutant) Pull-down testing

##### 5. CX6258 functional testing

We chose a cancer cell called AGS to explore the effect of CX3. We prepared three different concentrations of CX, to investigate which concentration is the most effective. The three concentrations are 0 nmol/L, 25 nmol/L, and 50 nmol/L. We add the CX into about 300 AGS cells and leave them in the cell chamber. After 24 hours, we used Crystal Violet Staining Solution to dye the cells and count the number of the cells. Crystal Violet Staining Solution is a dye often used in tissue or cell staining to stain the nucleus a deep purple color. When it dissolves, it can be ingested by living cells, and it can color DNA, proteins, and fats. Quantitative analysis can evaluate cell growth and reproduction.

As Figure 14 shows, the left one is 0 nmol/L, the middle one is 25 nmol/L and the right one is 50 nmol/L. It is obvious that there are more stained cells in the left one and in the right one there are fewer stained cells. In addition, we quantified the inhibitory effect of CX on AGS gastric cancer cell lines using histograms. Overall, the results showed that the higher concentration of CX in the concentration range of 0-50 nmol/L had a greater effect on reducing cancer cells, demonstrating that CX has the potential to inhibit tumor development.

![](https://static.igem.wiki/teams/4889/wiki/engineeringsuccess/engineeringsuccess-23.png)

Figure 14. The effect of CX6258 on AGS cell lines

Due to the short growth cycle and high fecundity of Drosophila and the high homology of the hippo pathway to humans, we have chosen the Drosophila model of colorectal cancer as the experimental animal for our project4. We will use two types of Drosophila, the experimental group Esgts>Yki/Esgts>GFP and the control group Esgts>ctrl/Esgts>GFP. Specific overexpression of Yki in Drosophila mesenteric stem cells promotes mesenteric stem cell proliferation, which thickens the colon and produces a colorectal tumor-like phenotype that can lead to Drosophila colorectal cancer. The Esg-GFP is specifically overexpressed in midgut stem cells. We fed 3-day-old adult Drosophila CX6258 for 7 days at 30°C. After dissection, the midgut was fixed and stained with p-H3 and Dapi to represent growing midgut stem cells and nuclei, respectively.

We found that the inhibition of midgut stem cell growth by CX6258 was significant in both experimental and control groups. In the experimental group, the proliferation of colon stem cells was abnormally active due to the overexpression of Yki (similar to colorectal carcinogenesis), and the application of CX6258 resulted in a nearly 3-fold decrease in the number of colon stem cells compared to the untreated group. These results suggest that CX6258 inhibits the proliferation of colon stem cells, and the inhibitory effect is even more pronounced in the colorectal cancer model.

![](https://static.igem.wiki/teams/4889/wiki/engineeringsuccess/engineeringsuccess-13.png)

![](https://static.igem.wiki/teams/4889/wiki/engineeringsuccess/engineeringsuccess-14.png)

Figure 15. The effect of CX6258 on growing midgut stem cells in Drosophila

Overall, the reliability of the AlphaScreen high-throughput drug screening system was confirmed by further experimental validation of the screening results, which is expected to provide us with more valuable hit compounds.

#### Summary：

In our project, we engineered E. coli to produce two key components of the human hippo signaling pathway (MST2 and STRN3), the interaction of which leads to dysregulation of hippo signaling. We used this to develop a drug screening system, AlphaScreen, to screen drug candidates that disrupt the MST2-STRN3 interaction and restore normal hippo signaling. As shown in the results above, we found that CX6258 has the ability to inhibit the growth of human gastric cancer cells and Drosophila colon cancer cells (both cancers have been shown to be associated with dysregulation of the Hippo signaling pathway3, 4) and confirmed the targeting of this small molecule. The validation of the mechanism and function of this small molecule further confirms the accuracy of the AlphaScreen high-throughput drug screening system. This will contribute to the development of drugs targeting the Hippo pathway, improve the precision of oncology drugs, and alleviate patient suffering.

#### Reference：

1.Tang Y, Fang G, Guo F, et al. Selective Inhibition of STRN3-Containing PP2A Phosphatase Restores Hippo Tumor-Suppressor Activity in Gastric Cancer. Cancer Cell. 2020;38(1):115-128.e9.

2.Tang Y, Chen M, Zhou L, et al. Architecture, substructures, and dynamic assembly of STRIPAK complexes in Hippo signaling. Cell Discov. 2019;5:3.

3.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.

4.Liang K, Zhou G, Zhang Q, Li J, Zhang C. Expression of hippo pathway in colorectal cancer. Saudi J Gastroenterol 2014;20:188-94.

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