13 research outputs found
Two helices in the third intracellular loop determine anoctamin 1 (TMEM16A) activation by calcium
Small-Molecule Inhibitors and Degraders Targeting KRAS-Driven Cancers
Drug resistance continues to be a major problem associated with cancer treatment. One of the primary causes of anticancer drug resistance is the frequently mutated RAS gene. In particular, considerable efforts have been made to treat KRAS-induced cancers by directly and indirectly controlling the activity of KRAS. However, the RAS protein is still one of the most prominent targets for drugs in cancer treatment. Recently, novel targeted protein degradation (TPD) strategies, such as proteolysis-targeting chimeras, have been developed to render āundruggableā targets druggable and overcome drug resistance and mutation problems. In this study, we discuss small-molecule inhibitors, TPD-based small-molecule chemicals for targeting RAS pathway proteins, and their potential applications for treating KRAS-mutant cancers. Novel TPD strategies are expected to serve as promising therapeutic methods for treating tumor patients with KRAS mutations
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An Amphiphilic Peptide Induces Apoptosis Through the miR29b-p53 Pathway in Cancer Cells
Peptides have been in the limelight, as therapeutic agents for cancer treatment through various applications due to their high target selectivity and exceptional ability to penetrate the cell membrane. Recent studies have revealed that synthesized peptides bind to hairpin structures of RNA that affect their activities such as changing the efficacy of microRNA maturation. MicroRNA-mediated p53 activation by the microRNA-29 (miR29) family is one of the most important regulatory pathways in cancer therapeutics. By targeting the suppressors of p53, a tumor suppressor protein, miR29 induces apoptosis of cancer cells through p53 stabilization. Here, we identify a novel synthesized amphiphilic peptide, LK-L1C/K6W/L8C, which enhances expression of miR29b and promotes p53 activity. In the presence of LK-L1C/K6W/L8C, pre-miR29b preferentially forms a complex with the Dicer protein through interaction of LK-L1C/K6W/L8C with the terminal loop region of pre-miR29b, leading to an increase in Dicer processing. Furthermore, LK-L1C/K6W/L8C stimulates apoptosis by improving p53 stability in miR29-inducible HeLa and MCF7 cells. Collectively, our study shows that a peptide can directly influence the miR29b-mediated p53 activation pathway in cancer cells. Therefore, our findings provide the basis for a new, potentially promising peptide-based drug for cancer therapy
Method development for 14C-labelingĀ of IgG antibodiesĀ in preparation for clinical trials
Abstract Objectives Carbon-14 (14C) labeling is a standard technology for tracing molecules and providing their pharmacokinetic profiles. However, its primary focus has been on small molecules, with limited application to biomacromolecules. Particularly in the development of new biological entities (NBE), the utilization of microdosing with a 14C-labeled biomacromolecule proves beneficial in the early stages of drug development, contributing to significant time and cost savings. This study investigates the 14C-labeling of antibody and explores the stability of 14C-labeled antibody under various storage conditions. Methods and results In this study, the utilization of 14C-formaldehyde for labeling target antibodies at various molar ratios revealed a direct correlation between labeling efficiency and the quantity of 14C-formaldehyde applied: 1.5Ā mol/mol for 14C-labeled antibody with the use of 10 equivalents of 14C-formaldehyde, 3.8Ā mol/mol for 14C-labeled antibody with the use of 10 equivalents of 14C-formaldehyde, and 10.5Ā mol/mol for 14C-labeled antibody with the use of 60 equivalents of 14C-formaldehyde. All the reaction conditions exhibited no antibody degradation, as evidenced by the absence of a significant change in HPLC purity compared to the unlabeled antibody. Stability tests revealed that all groups maintained their purities over a 4-week period at both āā75āĀ±ā10Ā Ā°C and 5āĀ±ā3Ā Ā°C. Given safety concerns related to internal radiation exposure in potential human subjects during microdosing, this study established optimal conditions for employing 14C-labeled antibodies. Therefore, it is optimized that 10 equivalents of 14C-formaldehyde can be used for 14C-antibody labeling through reductive amination, storing the antibodies at 5āĀ±ā3Ā Ā°C, and assigning a storage period of 4Ā weeks. Conclusion The findings from this study offer valuable insights into the effective application of 14C-labeling in microdosing studies, especially for larger molecules such as antibodies
An amphipathic cell penetrating peptide aids cell penetration of cyclosporin A and increases its therapeutic effect in an in vivo mouse model for dry eye disease
Cell penetrating peptide (CPP), LK-3, causes a ca. 10-fold increase in the cell penetration of cyclosporin A (CsA) at nanomolar concentrations. The results of an in vivo dry eye mouse model demonstrated that a 100-fold lower dose of the CsA/LK-3 complex than that of Restasis (R) is sufficient to cause the same therapeutic effect.N
One-bead-one-compound screening approach to the identification of cyclic peptoid inhibitors of cyclophilin D as neuroprotective agents from mitochondrial dysfunction
In an effort designed to discover superior inhibitors of cyclophilin D (CypD), we identified and screened members of a one-bead-one-compound (OBOC) library of cyclic peptoid analogues of cyclosporin A (CsA). The results show that the one member of this cyclic peptoid family, I11, inhibits mitochondrial membrane potential changes mediated by CypD.11Nsciescopu
Design and Optimization of an Ī±āHelical 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 Ī±-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 Ī±āHelical 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 Ī±-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 Ī±-helical
model peptide (LKKĀLĀLĀKĀLĀLĀKĀKĀLĀLĀKĀLAG,
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 Ī±-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 Ī±-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 Ī¼M 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