5 research outputs found
Design and Optimization of an α‑Helical Bundle Dimer Cell-Penetrating Peptide for <i>In Vivo</i> Drug Delivery
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
Nonhemolytic Cell-Penetrating Peptides: Site Specific Introduction of Glutamine and Lysine Residues into the α‑Helical Peptide Causes Deletion of Its Direct Membrane Disrupting Ability but Retention of Its Cell Penetrating Ability
Cell-penetrating
peptides (CPPs) often have cationic and amphipathic
characteristics that are commonly associated with α-helical
peptides. These features give CPPs both membrane demolishing and penetrating
abilities. To make CPPs safe for biomedical applications, their toxicities
resulting from their membrane demolishing abilities must be removed
while their cell penetrating abilities must be retained. In this study,
we systematically constructed mutants of the amphipathic α-helical
model peptide (LKKÂLÂLÂKÂLÂLÂKÂKÂLÂLÂKÂLAG,
LK peptide). The hydrophobic amino acid leucine in the LK peptide
was replaced with hydrophilic amino acids to reduce hemolytic or cell
toxicity. Most of the mutants were found to have weakened membrane
disrupting abilities, but their cell penetrating abilities were also
weakened. However, the L8Q and L8K mutants were found to have low
micromolar range cell penetrating ability and almost no membrane disrupting
ability. These selected mutants utilize energy-dependent endocytosis
mechanisms instead of an energy-independent direct cell penetrating
mechanism to enter cells. In addition, the mutants can be used to
deliver the anticancer drug methotrexate (MTX) to cells, thereby overcoming
resistance to this drug. To determine if the effect of these mutations
on the membrane disrupting and cell penetrating abilities is general,
Q and K mutations of the natural amphipathic α-helical antimicrobial
peptide (AMP), LL37, were introduced. Specific positional Q and K
mutants of LL37 were found to have lower hemolytic toxicities and
preserved the ability to penetrate eukaryotic cells such as MDA-MB-231
cells. Taken together, observations made in this work suggest that
interrupting the global hydrophobicity of amphipathic α-helical
CPPs and AMPs, by replacing hydrophobic residues with mildly hydrophilic
amino acids such as Q and K, might be an ideal strategy for constructing
peptides that have strong cell penetrating abilities and weak cell
membrane disrupting abilities
Apoptosis Inducing, Conformationally Constrained, Dimeric Peptide Analogs of KLA with Submicromolar Cell Penetrating Abilities
The apoptosis inducing KLA peptide,
(KLAKLAK)<sub>2</sub>, possesses
an ability to disrupt mitochondrial membranes. However, this peptide
has a poor eukaryotic cell penetrating potential and, as a result,
it requires the assistance of other cell penetrating peptides for
effective translocation in micromolar concentrations. In an effort
to improve the cell penetrating potential of KLA, we have created
a library in which pairs of residues on its hydrophobic face are replaced
by Cys. The double Cys mutants were then transformed to bundle dimers
by oxidatively generating two intermolecular disulfide bonds. We envisioned
that once transported into cells, the disulfide bonds would undergo
reductive cleavage to generate the monomeric peptides. The results
of these studies showed that one of the mutant peptides, dimer B,
has a high cell penetrating ability that corresponds to 100% of fluorescence
positive cells at 250 nM. Even though dimer B induces disruption of
the mitochondrial potential and cytochrome c release followed by caspase
activation at submicromolar concentrations, it displays an LD<sub>50</sub> of 1.6 μM under serum conditions using HeLa cells.
Taken together, the results demonstrate that the strategy involving
formation of bundle dimeric peptides is viable for the design of apoptosis
inducing KLA peptide that translocate into cells at submicromolar
concentrations
Screening of Pre-miRNA-155 Binding Peptides for Apoptosis Inducing Activity Using Peptide Microarrays
MicroRNA-155, one of the most potent
miRNAs that suppress apoptosis
in human cancer, is overexpressed in numerous cancers, and it displays
oncogenic activity. Peptide microarrays, constructed by immobilizing
185 peptides containing the C-terminal hydrazide onto epoxide-derivatized
glass slides, were employed to evaluate peptide binding properties
of pre-miRNA-155 and to identify its binding peptides. Two peptides,
which were identified based on the results of peptide microarray and
in vitro Dicer inhibition studies, were found to inhibit generation
of mature miRNA-155 catalyzed by Dicer and to enhance expression of
miRNA-155 target genes in cells. In addition, the results of cell
experiments indicate that peptide inhibitors promote apoptotic cell
death via a caspase-dependent pathway. Finally, observations made
in NMR and molecular modeling studies suggest that a peptide inhibitor
preferentially binds to the upper bulge and apical stem-loop region
of pre-miRNA-155, thereby suppressing Dicer-mediated miRNA-155 processing
Photoswitching of Cell Penetration of Amphipathic Peptides by Control of α‑Helical Conformation
We
stapled an amphipathic peptide mainly consisting of leucine
(L) and lysine (K) by an azobenzene (Ab) linker for photocontrol of
the secondary structure. The <i>cis</i>–<i>trans</i> isomerization of the Ab moieties could stabilize and destabilize
the α-helical conformation of the LK peptide along with dramatic
change of associated peptide structures in a reversible manner by
UV–vis irradiation. The cell-penetrating activities of the
LK peptide can be readily regulated by the photocontrol, as the stabilized <i>cis</i>-Ab-LK peptide showed remarkable increase of cell penetration
compared to the destabilized <i>trans</i>-Ab-LK peptide.
The photoswitchable cell-penetrating peptides would provide important
structural information for cell permeability as well as an effective
targeting strategy for peptide-based pharmaceuticals with spatiotemporal
specificity