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In our project, we want to create and invent a highly targeted drug to overcome the shortcomings of current IPF treatment drugs, such as poor efficacy and strong side effects, in order to precisely target pulmonary fibrosis tissue, reverse the fibrosis process, and thus make IPF patients return to health.
To do this, we need a comprehensive understanding of the pathogenesis and important signaling pathways of IPF. Through literature review and brainstorming, we first clarified that TGF-related signaling pathways play a vital role in the development of fibrosis, and are very likely to be a breakthrough point in the treatment of IPF. We made our goal clear. We need to block the cell signaling pathway that induces pulmonary fibrosis to avoid the increase of fibroblasts and thereby inhibit the further deepening of pulmonary fibrosis.
We understand that fusion proteins are very suitable for this type of treatment. With reference to existing research on the treatment of fibrotic diseases, we have designed a variety of fusion proteins targeting lung fibrosis regions to block fibrosis signaling pathways. These proteins are made up of three main parts:
(1) The head is the fibrosis treatment-related domain. Here we consider using IPF truncated receptors to capture and inactivate TGF-β;
(2) The tail is the domain which can recognise and target nidus to enhance the targeting of drugs;
(3) The rigid linker stabilizes the protein structure and separate two functional domains.
There are many choices for the peptide sequence of the head and tail. We need to read the literature and do experiments to determine the optimal protein combination. Through literature review, we finally determined four candidates for the head TGF-β truncated receptor: the domains are M7824, P144, TMED10 and an unnamed peptide; We used HL5 as a rigid linker due to its good stability. At the same time, after extensively collecting and comparing the collagen types of various fibrotic diseases (including scars, liver fibrosis, pulmonary fibrosis) and the corresponding targeting methods, we selected six targeted probes. The picture below shows all possible combinations：
In dry lab, we use the Z-DOCK model to obtain the parameters of four truncated receptors and perform Z-score normalization on these parameters to compare receptors’ bind ability to TGF-β. The following result shows M7824 and Unnamed have strong binding ability to TGF-β. Considering that M7824 is already a relatively mature clinical targeted probe, we finally determined its choice as the head.
For probe targeting type I collagen, we used Z-DOCK and I-TASSER for molecular docking and stability experiments and select three types of collagen-binding targeting probes with KD values of 0.01, 0.1, 10 as the candidates. These probes need to be verified in wet experiments.
In wet lab, we realize the microcellular expression and prokaryotic expression of four fusion proteins (M7824-CILY, M7824-SILY, M7824-Type4, M7824-Type4(reversed)). Firstly, we need a quick and easy way to rapidly produce a certain amount of proteins for preliminary molecular verification test and cell verification test. We use cell-free protein synthesis which is used firstly in iGEM competition. We designed special primers for template reconstruction and make experiment to recover the glue with correct size of strip and then concentrate. The SDS-PAGE result is shown below to verify their successful expression:
Going further, we used E.Coli (BL21) with DH5-a and E.Coli (BL21) with pET28a to amplificate the plasmids inserted with our sequence. The direct SDS-PAGE and SDS-PAGE after purification results are shown below to verify their successful expression:
Then, we need to build a cell model to simulate the lesion tissue of IPF. We successfully establish a pulmonary fibrosis model with HFL-1 (treated by TGF-β1). After that, we
We successfully establish a pulmonary fibrosis model with HFL-1 (treated by TGF-β1) then set nine groups
and apply RT-q PCR to determine whether their epithelial-mesenchymal transition (EMT) process was blocked with the level of collagen I.
It can be obviously refered from the cell morphology that, the HEL-1 cells differentiated into myofibroblasts in the TGE-beta-treated group while this process was depressed in the drug-treated group.
To get more evidence to prove our cell model and drug treatment success, we used RT-qPCR to get more evidence in gene expression degree.
We can see that it occurs expression degree of collagen I differences between three groups though the difference is not that ovious. But we are making more drug concentration comparisons to detect the differences more obviously, and we believe that these experiments will provide guidance for later SPR experiments and animal experiments in the future.
Here are the five steps from research to testing. Design2&3 are the next steps for learning and improvement.
Collagen degeneration in fibrosis focus
Taking into account that the above treatment module can only delay the condition of IPF, but cannot reverse the lesion area. We considered designing a plan to restore normal function of the tissue in the lesion area. To this end, we focused on myofibroblasts. We hope to design drugs to properly degrade collagen to improve tissue compliance and restore normal tissue function. We knew that plasmin has been used in the treatment of thrombosis and ss a proteolytic enzyme, plasmin is also promising in applications in terminal pulmonary fibrotic diseases. In consideration of controlling side effects, we did not choose plasminogen activating peptides with enzymatic activity, but chose peptides with carboxyl terminal lysines. Among the peptides with carboxyl terminal lysines, the α2-antiplasmin C-terminal 26 peptide was selected. We chose a relatively mild activating peptide with dual effects and a highly selective collagen targeting peptide to control the scope of effect by enriching the drug in the lesion. We hope to reverse the lesion tissue of IPF patients by designing the above-mentioned fusion protein for administration.
Drug Delivery System Design
Through investigations in the hospital, we learned about the huge side effects of traditional IPF treatment drugs (such as nintedanib and pirfenidone), and we realized that the side effects of our fusion protein drugs are also a potential hidden danger. Although the targeted nature of the drug can limit side effects to a large extent, the harm to the body when the drug is metabolized in the body is still inevitable. In this regard, doctors suggest that we should consider to explore the inhibitory effects of different administration methods on side effects, and use liposomes to make sustained-release drug formulations to increase the utilization of drugs and reduce side effects. In response, we designed a pharmacokinetic experiment. By establishing a PBPK model, we can theoretically simulate the metabolism of drugs in the body.
We analyzed the metabolism of peptide drugs in animal models and humans in the two treatment modules, and used the physiological pharmacokinetic (PBPK) model prediction software GastroPlusTM to predict all peak concentration-time (Cp-time) curves. We focus on the metabolism of peptide drugs through intravenous administration and pulmonary administration, as well as the influence of the presence or absence of liposomes on drug metabolism. We use the second treatment module--the activation peptide (AP26 and SAK) involved in the degradation of collagen in fibrotic lesions as the research object.
We first established a PBPK model for intravenous administration, and the results are as follows:
Furthermore, we established a PBPK model for pulmonary administration as a comparison. The PK curve is as follows:
Our results prove that after lung administration, the deposition of the drug in the lungs is significantly increased and the bioavailability is higher, suggesting that lung administration is a better choice than intravenous administration.
In order to predict the effect of liposome delivery methods on drug metabolism, we established a release curve model of sustained-release preparations based on transdermal administration and predicted the PK curve. The simulation results show that the exposure of AP26 and SAK sustained-release preparations administered percutaneously in rats and humans is significantly increased, and their bioavailability basically reaches 100%.
The simulation results of pharmacokinetics gave us a lot of encouragement, and the results characterized the advantages of the drug delivery methods in our design, helped us understand the metabolism of drugs in the body and helped us to screen better drugs. After discussing with the Professor Zhenlei Wang, we knew that we can design in vitro experiments and animal experiments to optimize the parameter settings of the pharmacokinetic model, so that we can understand the metabolism of the drug in the body more accurately.