Fusion Proteins with Cleavable Dithiocyclopeptide Spacers
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Solution Overview
Problem
Current methods lack an effective approach for the oral delivery of protein drugs due to their large size, charge, and sensitivity to digestive enzymes, making it difficult to achieve oral administration and resulting in inconvenient and ineffective treatments for chronic diseases.
Innovation Solution
Development of recombinant fusion proteins with a dithiocyclopeptide spacer containing a protease cleavage site, allowing for the separation of protein domains inside the body, enabling efficient oral delivery and biological activity of each component.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of operation
If protein drugs are administered orally, then patient convenience and compliance improve, but the drugs cannot be effectively delivered due to their large size, charge, and sensitivity to digestive enzymes
Solution Approach 1:
The fusion protein is segmented into multiple functional domains (transferrin domain for receptor binding, dithiocyclopeptide spacer for cleavage, and therapeutic protein domain for biological activity). This segmentation allows the protein to be delivered orally as a fusion construct while releasing the active therapeutic domain at the target site, resolving the contradiction between oral administration convenience and delivery effectiveness.
Solution Approach 2:
The dithiocyclopeptide spacer acts as an intermediary between the transferrin domain and the therapeutic protein domain. It provides a cleavable linkage that allows the fusion protein to remain intact during oral delivery and transit to the target tissue, then releases the active therapeutic domain at the destination, enabling both oral administration and effective delivery.
2Reliability
If fusion proteins with cleavable spacers are used, then oral bioavailability and therapeutic efficacy improve, but the device complexity increases due to the need for specific protease cleavage sites and disulfide bond reduction
Solution Approach 1:
The fusion protein is designed to self-cleave at the dithiocyclopeptide spacer through protease recognition and self-reduce through disulfide bond formation/breakage. The protease cleavage site and disulfide bonds enable the protein to automatically separate its domains at the target site without external intervention, improving oral bioavailability while managing structural complexity through autonomous functionality.
3Stability of the object's composition
If the dithiocyclopeptide spacer is cyclized by disulfide bond, then the stability of the fusion protein improves, but the manufacturing precision becomes more difficult due to the need for proper disulfide bond formation
Solution Approach 1:
The dithiocyclopeptide spacer is pre-designed with specific cysteine residues positioned to form disulfide bonds under physiological conditions. The amino acid sequence and spatial arrangement are predetermined to facilitate spontaneous cyclization and disulfide bond formation during or after translation, ensuring proper folding and stability while simplifying manufacturing by relying on self-organization rather than complex post-translational modification control.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The fusion proteins effectively transport and release active protein domains, such as G-CSF and Tf, across epithelial barriers, enhancing oral bioavailability and therapeutic efficacy while minimizing side effects.
Implementation Method 1
The disulfide bond in the dithiocyclopeptide is reduced during the transport or within the cell, thereby separating the first protein domain from the second protein domain
Implementation Method 2
the dithiocyclopeptide is cleaved by the protease at the protease cleavage site
Data Source
AI summary
A polypeptide comprising a first protein domain, a second protein domain, and a dithiocyclopeptide spacer containing at least one protease cleavage site, wherein the dithiocyclopeptide is exogenous relative to the first or second protein domain, and wherein the first and second protein domains are operably linked by the dithiocyclopeptide. Also disclosed are methods of producing the polypeptide and delivering the protein domains into a cell.