We aim to produce two types of engineered proteins to target Non-Small Cell Lung Cancer (NSCLC) and check for any combination effects of those peptides with cisplatin, a common chemotherapy drug. Four peptides from Cordyceps militaris, a well‐known dietary therapy in anticancer treatment, were selected to test their effectiveness to lung cancer. Also, we modified those peptides by adding linker and cell-penetrating peptide.
Furthermore, we used RFdiffusion and ProteinMPNN-AI to generate peptide sequences by targeting PDEδ, which regulates KRAS signaling, a common gene mutation in cancerous cells. Then, those peptides were expressed by BL21. Finally, A549, a lung cancer cell line, was used to test the effectiveness of those peptides and check for whether two groups of peptides have any combination effects with cisplatin. If there are combination effects, lower concentration of cisplatin can be used, so that it can reduce the side effects of it.
According to the wet lab’s results, ACP5 is found to be the most promising ACP. It can cause a 70% inhibition in viability of 2D cancer cells and 20% inhibition in viability of 3D Multi-Cellular Tumor Spherord (MCTS). Hence, ACP5 is named KAPI ( KRAS and PDEδ Inhibitor ). In a preliminary study, with the supplement of KAPI a reduced concentration of cisplatin can achieve the same percentage of inhibition on cancer cell viability as that at its IC50
Cancer, especially lung cancer, imposes severe challenges to both the public health and the economy. According to the World Health Organisation, lung cancer is the most prevalent cancer type and accounts for the highest cancer mortality rates in the globe.[1] In general, about 80% to 85% of all lung cancers are Non-Small Cell Lung Cancer (NSCLC). [2] Furthermore, it is predicted that the global economic costs of lung cancer between 2020 and 2050 will amount to $3.9 trillion.[3] In Hong Kong, according to the Hong Kong Cancer Registry, lung cancer accounted for the highest cancer incidence and mortality rate (15.5% and 26.7%). [4] To treat cancer, chemotherapy drugs are commonly adopted.[5]
Cisplatin is one of the most commonly used chemotherapy drugs. It is estimated that 10% to 20% of all cancer patients use cisplatin and other similar platinum-based drugs.[6] Cisplatin is a highly effective anticancer drug used to treat various cancers. However, its clinical use is limited due to severe side effects, ranging from nephrotoxicity, neurotoxicity anototoxicty. These effects are always cumulative and dose-dependent. Between 20% and 35% of patients experience acute kidney injury (AKI) following cisplatin treatment and there there is no clinically effective drug to prevent or treat cisplatin-induced nephrotoxicity.[7] Acute kidney injury with repeating episodes can result in impaired renal tubular function, acute renal failure, chronic kidney disease, uremia, and hypertensive nephropathy. [8] Therefore, there is a necessity to explore new ways to reduce the side effect of cisplatin.
By literature review[9], we found that there is a anticancer peptide (AC-P19) which has been proved that it has anticancer ability against A549 cell line. After the discussion with our science teachers, we descided to synthesize anticancer peptides targeting Non-Small Cell Lung Cancer by synthetic biology approach. Futhermore, we have gained valuable preliminary diection is to find out a ACP which has combination effect with cisplatin, it can lower side effect of cisplatin.
Next, we had interviews with three Chinese medicine practitioners (CMPs) and then we come up with an new idea - blending Traditional Chinese and Western Medicine to fight lung cancer. After literature review[10] and interview with Prof. Promate, we chose four Cordyceps militaris peptides (C-ori, C-rds, CTP-ori and CTP-rds) to have further investigation.
Beside, we have more interviews with different experts. After collecting their opinions and comments, we come up with a new idea- to generate a all-new peptides targetting KRAS pathway by AI. For more information, please visit our human practice part to know more about our human practice journey.
- To choose four Cordyceps militaris peptides (C-ori, C-rds, CTP-ori and CTP-rds) as the potential condidates of anticancer peptide against Non-Small Cell Lung Cancer.
Some scientific papers have shown the extracts of Cordyceps militaris have anticancer activity on Colorectal cancer and Lung cancer.[11] [12] However, it hasn't been proven exactly which kinds of peptides are effective against lung cancer cell A549. Among the anti-cancer peptides, we have chosen 4 of them to be included in our plasmid first to test their effect. They are CTP-ori, CTP-rds, C-ori and C-rds.[10] we decided to find out ACPs (anticancer peptides) which have combination effects of cisplatin, aiming to reduce toxic effects while enhancing the efficiency of cisplatin.
KRAS is one of the most common gene mutations in cancerous cells. It is
responsible for the uncontrollable cell division of cancer cells.
[13]However, the reduction of the expression of KRAs was not obvious in
clinical trials[14]
PDEδ sustains the spatial organization of KRAS by facilitating its diffusion
in the cytoplasm and regulating its localization to endomembranes. This
allows KRAS signaling to take place. [15]So, PDEδ regulating KRAS signaling
is crucial for developing effective treatments.[16]
We identify the 3D structure of PDEδ (1-150 amino acids). Besides, we find
the hotspot residues which are viable amino acids for our ACP to bind within
PDEδ. They are Y149, C56, R61. This facilitates small molecules to bind to
PDEδ, instead of KRAS-PDEδ binding.[17]
The next step is to obtain the structure of PDEδ. At Protein Data Bank, we
found the structure of human PDEδ the same as the protein structure in the
literature. Its code is 4JV6 and it has specific structural features that
make it a good target for protein design.[18]
Next, we used RFdiffusion to generate 16 protein backbones. Then,
ProteinMPNN-AI is used to generate the sequence of the 16 peptides. After
that, the sequences are screened by AlphaFold to find out the most probable
sequence bound to PDEδ. Finally, we sent it to the wet lab for further
analysis.
All ACP inserts have been incorporated into our pET plasmid and then was tranformed into BL21 for protein expression. Those peptides would be purified and collected for cytotoxicity test.
For more information, please visit our experiments and results parts.
1.World Health Organization. (n.d.). (2024, February 1)Global cancer burden growing, amidst mounting need for services. World Health Organization.https://www.who.int/news/item/01-02-2024-global-cancer-burden-growing--amidst-mounting-need-for-services
2.What is lung cancer?: Types of lung cancer. Types of Lung Cancer | American Cancer Society. (n.d.).https://www.cancer.org/cancer/types/lung-cancer/about/what-is.html
3.Simiao Chen, S. (2023, April 1). Estimates and projections of the global economic cost of 29 cancers from 2020 to 2050. JAMA Oncology.https://jamanetwork.com/journals/jamaoncology/fullarticle/2801798
4.Hong Kong Cancer Registry, Hospital Authority. (2023, October). Lung Cancer in 2021. https://www3.ha.org.hk/cancereg/pdf/factsheet/2021/lung_2021.pdf
5.American Cancer Society. (n.d.). What is chemotherapy?: Chemo treatment for cancer. What is Chemotherapy? | Chemo Treatment for Cancer | American Cancer Society. https://www.cancer.org/cancer/managing-cancer/treatment-types/chemotherapy.html
6.Cancer Institute. (2014, May 30). Discovery – cisplatin and the treatment of testicular and other cancers. Discovery – Cisplatin and The Treatment of Testicular and Other Cancers - NCI. https://www.cancer.gov/research/progress/discovery/cisplatin
7.Fang, C., Lou, D., Zhou, L., Wang, J., Yang, B., He, Q., Wang, J., & Weng, Q. (2021, March 9). Natural products: Potential treatments for cisplatin-induced nephrotoxicity. Nature News. https://www.nature.com/articles/s41401-021-00620-9
8.Kellum, J. A., Romagnani, P., Ashuntantang, G., Ronco, C., Zarbock, A., & Anders, H.-J. (2021, July 15). Acute kidney injury. Nature News. https://www.nature.com/articles/s41572-021-00284-z
9.Int J Mol Sci. 2022 Dec; 23(24): 15594. Published online 2022 Dec 9. doi: 10.3390/ijms232415594
10.Chantawannakul, J., Chatpattanasiri, P., Wattayagorn, V., Kongsema, M., Noikaew, T., & Chumnanpuen, P. (2021, September 23). Virtual screening for Biomimetic Anti-Cancer peptides from cordyceps militaris putative Pepsinized Peptidome and validation on Colon Cancer Cell Line. Molecules (Basel, Switzerland). https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8510206/
11.Lee, H. H., Lee, S., Lee, K., Shin, Y. S., Kang, H., & Cho, H. (2015, July 4). Anti-cancer effect of cordyceps militaris in human colorectal carcinoma RKO cells via cell cycle arrest and mitochondrial apoptosis. Daru : journal of Faculty of Pharmacy, Tehran University of Medical Sciences. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4491205/
12.Jo, E., Jang, H.-J., Shen, L., Yang, K. E., Jang, M. S., Huh, Y. H., Yoo, H.-S., Park, J., Jang, I. S., & Park, S. J. (2020, May 26). cordyceps militaris exerts anticancer effect on non-small cell lung cancer by inhibiting hedgehog signaling via suppression of TCTN3. Integrative cancer therapies. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7265736/
13.Jancík, S., Drábek, J., Radzioch, D., & Hajdúch, M. (2010, June 7). Clinical relevance of KRAS in human cancers. Journal of biomedicine & biotechnology. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2896632/
14.Huang, L., Guo, Z., Wang, F., & Fu, L. (2021, November 15). Kras mutation: From undruggable to druggable in cancer. Nature News. https://www.nature.com/articles/s41392-021-00780-4
15.Zimmermann G, Papke B, Ismail S, Vartak N, Chandra A, Hoffmann M, Hahn SA, Triola G, Wittinghofer A, Bastiaens PI, Waldmann H. Small molecule inhibition of the KRAS-PDEδ interaction impairs oncogenic KRAS signaling. Nature. 2013 May 30;497(7451):638-42. doi: 10.1038/nature12205. Epub 2013 May 22. PMID: 23698361.v https://pubmed.ncbi.nlm.nih.gov/23698361/
16.Klein CH, Truxius DC, Vogel HA, Harizanova J, Murarka S, Martín-Gago P, Bastiaens PIH. PDEδ inhibition impedes the proliferation and survival of human colorectal cancer cell lines harboring oncogenic KRas. Int J Cancer. 2019 Feb 15;144(4):767-776. doi: 10.1002/ijc.31859. Epub 2018 Dec 4. PMID: 30194764; PMCID: PMC6519276. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6519276/
17.Dharmaiah S, Bindu L, Tran TH, Gillette WK, Frank PH, Ghirlando R, Nissley DV, Esposito D, McCormick F, Stephen AG, Simanshu DK. Structural basis of recognition of farnesylated and methylated KRAS4b by PDEδ. Proc Natl Acad Sci U S A. 2016 Nov 1;113(44):E6766-E6775. doi: 10.1073/pnas.1615316113. Epub 2016 Oct 17. PMID: 27791178; PMCID: PMC5098621. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5098621/
18.Gunther, Z., Papke, B., Ismail, S., Vartak, N., Chandra, A., Hoffmann, M., Hahn, S., Triola, G., Wittinghofer, A., Bastiaens, P., Waldmann, H. (2013, May 22). 4JV6: The crystal structure of PDE6D in complex to inhibitor-1. RCSB PDB. https://www.rcsb.org/structure/4jv6