Considerations on the Rational Design of Covalently Conjugated Cell-Penetrating Peptides (CPPs) for Intracellular Delivery of Proteins: A Guide to CPP Selection Using Glucarpidase as the Model Cargo Molecule

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.

Decoding the proteome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) for cell-penetrating peptides involved in pathogenesis or applicable as drug delivery vectors

“…The stability of SCV2-CPPs was estimated by analyzing their instability index values. Proteins and peptides with instability index values lower than 40 are expected to be stable in test tubes ( Behzadipour and Hemmati, 2019 ). According to the calculated data, 47.8% of SCV2-CPPs would be stable in vitro.…”

Scale-up fermentation of Escherichia coli for the production of recombinant endoglucanase from Clostridium thermocellum

“…The binding affinities ( ΔG values ) of native and mutated codons were determined by Mfold web server 28 , 29 . The mutation GTA > GTT has shown a promoting impact on mRNA binding with the ribosomes 30 . The best yield of active enzyme was obtained in modified M9NG media under 10 mM lactose.…”

Introducing a delivery system for melanogenesis inhibition in melanoma B16F10 cells mediated by the conjugation of tyrosine ammonia‐lyase and a TAT‐penetrating peptide

“…Furthermore, the rigidity and flexibility of ligand binding site residues are usually associated with specificity and tightness of ligand binding and the entrance of ligands into the binding site, respectively. 15,37 Therefore, any changes in flexibility may interfere with the correct conformational alterations of the enzyme and, as a result, enzyme function. No changes were observed in the flexibility of the enzymebinding pocket after the addition of the TAT sequence to the N-terminus of RsTAL.…”

“…One of the relatively new approaches for intracellular delivery of protein-based biotherapeutics is their conjugation to cell-penetrating peptides (CPPs). 15 CPPs are a large and still expanding family consisting of natural and nonnatural peptides that are being utilized as a promising tool to deliver membrane-impermeable molecules such as proteins across the cellular membrane. 16,17 TAT peptide derived from the HIV trans-activator transduction domain is the most recognized CPP, which has been used successfully for the intracellular delivery of a wide range of biomolecules.…”