Design and Optimization of an α-Helical Bundle Dimer Cell-Penetrating Peptide for In Vivo Drug Delivery

Abstract

To deliver membrane-impermeable drugs into eukaryotic cells, a lot of cell-penetrating peptides (CPPs) were discovered. Previously we designed an amphipathic α-helical peptide which dimerizes itself via its two C-residues. This bis-disulfide-linked dimeric bundle, LK-3, has remarkable cell-penetrating ability at nanomolar concentration, which is an essential prerequisite for CPP. In an effort to optimize the sequence of LK-3, we adjusted its length and evaluated changes in the dimerization rate. We found that a 10-amino-acid monomer has the fastest dimerization rate and subsequently modified its hydrophobic and hydrophilic residues to construct a small peptide library. The evaluation of cell permeability of these derivatives showed that their cell-penetrating ability is comparable to that of the LK-3, except V- or H-containing ones. In this library, diLR10 was found to display fast nanomolar cell membrane penetration, low toxicity, and ease of production. The methotrexate (MTX) conjugate of diLR10, MTX-diLR10, has a 19-fold increased efficacy over MTX in MDA-MB-231 cells and efficiently deflates lesions in a rheumatoid arthritis (RA) in vivo mouse model.

Proline-Hinged α-Helical Peptides Sensitize Gram-Positive Antibiotics, Expanding Their Physicochemical Properties to Be Used as Gram-Negative Antibiotics

“…The cell viability assay was performed as described previously . HK2 cells were seeded in a 96-well plate with 7 × 10 3 cells/well in complete RPMI medium.…”

“…The cell viability assay was performed as described previously. 65 HK2 cells were seeded in a 96-well plate with 7 × 10 3 cells/well in complete RPMI medium. The cells were incubated for 24 h and media were removed by aspiration.…”

Cell Penetrating Peptides: Classification, Mechanisms, Methods of Study, and Applications

“…Synthetic designed S5-S5 stapled peptides, alpha helical [127] TatRI qpprrrqrrkkrg Designed to deliver a iCal36 CFTR [a] stabilizing peptide Retro-inverso [128] diLR10 LCKLLKKLCK Synthetic designed Alpha helical, self-assembling in a dimeric form [129] [a] Cystic fibrosis transmembrane conductance regulator.…”

Cyclohexylalanine-Containing α-Helical Amphipathic Peptide Targets Cardiolipin, Rescuing Mitochondrial Dysfunction in Kidney Injury

“…First, we demonstrated that 10-aa long monomer peptides in this family rapidly form oxidative dimeric bundles. Second, we found that substitution(s) of hydrophobic or hydrophilic residue(s) for Leu and Lys in the original monomer peptides did not alter oxidative dimeric bundle formation or cell penetration ability . We identified a cell-penetrating bundle dimer peptide based on the LR10 monomer, which rapidly forms oxidative dimeric bundles and readily enters cells at nanomolar concentrations.…”

“…Second, we found that substitution(s) of hydrophobic or hydrophilic residue(s) for Leu and Lys in the original monomer peptides did not alter oxidative dimeric bundle formation or cell penetration ability. 14 We identified a cell-penetrating bundle dimer peptide based on the LR10 monomer, which rapidly forms oxidative dimeric bundles and readily enters cells at nanomolar concentrations. This peptide may target mitochondria or CL owing to its four positively charged residues, capable of strong binding to multiple CLs, and its hydrophobic moieties that interact with the fatty acidic region of CL.…”