Abstract
Access of proteins to their intracellular targets is limited by a hydrophobic barrier called the cellular membrane. Conjugation with cell-penetrating peptides (CPPs) has been shown to improve protein transduction into the cells. This conjugation can be either covalent or non-covalent, each with its unique pros and cons. The CPP-protein covalent conjugation may result in undesirable structural and functional alterations in the target protein. Therefore, we propose a systematic approach to evaluate different CPPs for covalent conjugations. This guide is presented using the carboxypeptidase G2 (CPG2) enzyme as the target protein. Seventy CPPs —out of 1155— with the highest probability of uptake efficiency were selected. These peptides were then conjugated to the N- or C-terminus of CPG2. Translational efficacy of the conjugates, robustness and thermodynamic properties of the chimera, aggregation possibility, folding rate, backbone flexibility, and aspects of in vivo administration such as protease susceptibility were predicted. The effect of the position of conjugation was evaluated using unpaired t-test (p < 0.05). It was concluded that N-terminal conjugation resulted in higher quality constructs. Seventeen CPP-CPG2/CPG2-CPP constructs were identified as the most promising. Based on this study, the bioinformatics workflow that is presented may be universally applied to any CPP-protein conjugate design.
Highlights
Biotherapeutics such as protein-based therapeutics are a fast-growing group of pharmaceuticals [1]. the advantage of biotherapeutics is their target specificity, one of the major challenges in the way of further development of protein-based therapeutics is their intracellular delivery
cell-penetrating peptides (CPPs) with the highest uptake efficiency are preferred for conjugation
This study presented a bioinformatics workflow applicable as a comprehensive approach useful to select CPP-cargo constructs for any therapeutic application
Summary
Biotherapeutics such as protein-based therapeutics are a fast-growing group of pharmaceuticals [1]. the advantage of biotherapeutics is their target specificity, one of the major challenges in the way of further development of protein-based therapeutics is their intracellular delivery. A huge hydrophilic proteinaceous macromolecule cannot cross the hydrophobic lipid bilayer membrane surrounding a cell [2] This obstacle, can be overcome with the assistance of a cell-penetrating peptide (CPP). CPPs are short relatively non-toxic peptide sequences, usually less than 30 amino acids, which have the ability to cross cellular membranes, and can co-transport a variety of biologically active molecules (cargoes) inside the cells [1,2]. Since their discovery in 1988, a wide range of different CPPs has been identified and subjected to clinical trial studies [3,4]. CPPs are Molecules 2019, 24, 4318; doi:10.3390/molecules24234318 www.mdpi.com/journal/molecules
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
