A Successful Modification Technique for Creating Multifunctional Peptides Based on a Kunitz Family Trypsin Inhibitory Peptide

Jenifer Malia^*
*Correspondence: Jenifer Malia, Department of Pharmacy, Queen’s University Belfast, Belfast BT9 7BL, Northern Ireland, UK, Email:

Keywords

Peptides • Mutations • Protease inhibition

Introduction

Peptides are short chains of amino acids that play crucial roles in various biological processes. Among these, multifunctional peptides have garnered significant attention due to their ability to exhibit multiple biological activities. The Kunitz Family Trypsin Inhibitory peptides (KFTIs) are one such group known for their strong inhibitory action against trypsin and other serine proteases. This property makes them valuable in regulating proteolytic activities in biological systems. This paper presents a successful modification technique for creating multifunctional peptides based on a Kunitz family trypsin inhibitory peptide, detailing the methodology, results, and potential applications of these modified peptides [1].

Literature Review

The Kunitz family of protease inhibitors is characterized by a conserved structure, typically involving a compact domain stabilized by disulfide bonds. This structural stability makes them ideal candidates for engineering multifunctional peptides. Traditional applications of KFTIs include therapeutic uses such as anticoagulants, anti-inflammatory agents, and antitumor agents. However, the potential for multifunctionality—where a single peptide can perform multiple biological roles—opens new avenues for biomedical applications. The first step involves selecting a Kunitz peptide with a well-characterized structure. Typically, a peptide like the Bovine Pancreatic Trypsin Inhibitor (BPTI) is chosen due to its extensive study and known inhibitory profile. Sequence analysis tools and structural databases (e.g., PDB) are used to identify potential sites for modification without disrupting the core inhibitory function. Site-directed mutagenesis is employed to introduce specific amino acid changes. These changes are designed to impart new functionalities, such as binding to different protein targets, enhancing cellular uptake, or adding antimicrobial properties. Mutations are strategically placed in regions that do not interfere with the peptide's inhibitory activity. Techniques such as PCR-based mutagenesis are used for precise modifications [2,3].

The modified peptides are synthesized using Solid Phase Peptide Synthesis (SPPS), a method that allows for the sequential addition of amino acids. After synthesis, the peptides are purified using High-Performance Liquid Chromatography (HPLC). Correct folding and the formation of disulfide bonds are critical for maintaining the structural integrity of the peptide. This is typically achieved through oxidative folding protocols that mimic physiological conditions. The multifunctionality of the modified peptides is assessed through a series of assays. Protease inhibition assays to confirm that the modified peptide retains its inhibitory activity against trypsin and other proteases. Binding assays to test for new binding interactions introduced by the mutations [4].

Discussion

The modified peptides were tested for their ability to inhibit trypsin and other serine proteases. The results showed that the core inhibitory function was preserved, indicating that the structural integrity of the Kunitz domain was maintained despite the introduced mutations. In addition to protease inhibition, the modified peptides exhibited new functionalities. Antimicrobial activity some modified peptides showed significant antibacterial effects against Gram-positive and Gram-negative bacteria, likely due to the introduction of cationic residues that enhance membrane disruption. Certain mutations resulted in peptides that could inhibit pro-inflammatory cytokine production in vitro, suggesting potential applications in inflammatory diseases. Enhanced cellular uptake modifications led to improved cellular uptake, possibly due to the addition of cell-penetrating sequences, enhancing the peptides' potential as drug delivery vectors [5].

The successful creation of multifunctional peptides based on the Kunitz family trypsin inhibitory peptide highlights the versatility of this modification technique. By preserving the core inhibitory function and strategically introducing mutations, it is possible to design peptides with a broad range of biological activities. Maintaining the structural stability of the Kunitz domain was crucial. The introduction of mutations in flexible regions or loop areas minimized the risk of disrupting the overall fold. The use of bioinformatics tools for sequence and structure analysis was instrumental in identifying optimal mutation sites [6].

Conclusion

This study demonstrates a successful modification technique for creating multifunctional peptides from a Kunitz family trypsin inhibitory peptide. By carefully selecting mutation sites and employing rigorous synthesis and testing protocols, it is possible to endow these peptides with a range of new functionalities while retaining their core inhibitory activity. This approach not only expands the potential applications of KFTIs but also provides a blueprint for developing multifunctional peptides from other protein scaffolds. Further research will focus on optimizing the multifunctional properties of these peptides and exploring their in vivo efficacy and safety. Additionally, expanding the modification techniques to other peptide families could yield a new generation of multifunctional therapeutic agents.

Acknowledgement

None.

Conflict of Interest

None.

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