Cationic Oligopeptides Research Articles

DNA/RNA heteroduplex technology with cationic oligopeptide reduces class-related adverse effects of nucleic acid drugs

Antisense oligonucleotides (ASOs) are a therapeutic modality for incurable diseases. However, systemic injection of gapmer-type ASOs causes class-related toxicities, including prolongation of activated partial thromboplastin time (aPTT) and thrombocytopenia. We previously reported that cholesterol-conjugated DNA/RNA heteroduplex oligonucleotides (Chol-HDOs) exhibit significantly enhanced gene silencing effects compared to ASOs, even in the central nervous system, through crossing the blood-brain barrier. In the present study, we initially evaluated the effect of the HDO structure on class-related toxicities. The HDO structure ameliorated the class-related toxicities associated with ASOs, but they remained to some extent. As a further antidote, we have developed artificial cationic oligopeptides, L-2,4-diaminobutanoic acid oligomers (DabOs), which bind to the phosphates in the major groove of A-type double helical structure of HDOs. DabO/Chol-HDO complex showed significantly improved aPTT prolongation and thrombocytopenia in mice while maintaining gene silencing efficacy. Moreover, the conjugation with DabOs effectively prevented cerebral infarction, a condition frequently observed in mice intravenously injected with high-dose Chol-HDO. These approaches, combining HDO technology with DabOs, offer distinct advantages over conventional strategies in reducing toxicities. Consequently, the DabO/HDO complex represents a promising platform for overcoming the class-related toxicities associated with therapeutic ASOs.

A Fluorous Peptide Amphiphile with Potent Antimicrobial Activity for the Treatment of MRSA-induced Sepsis and Chronic Wound Infection.

The rising prevalence of global antibiotic resistance evokes the urgent need for novel antimicrobial candidates. Cationic lipopeptides have attracted much attention due to their strong antimicrobial activity, broad-spectrum and low resistance tendency. Herein, a library of fluoro-lipopeptide amphiphiles was synthesized by tagging a series of cationic oligopeptides with a fluoroalkyl tail via a disulfide spacer. Among the lipopeptide candidates, R6F bearing six arginine moieties and a fluorous tag shows the highest antibacterial activity, and it exhibits an interesting fluorine effect as compared to the non-fluorinated lipopeptides. The high antibacterial activity of R6F is attributed to its excellent bacterial membrane permeability, which further disrupts the respiratory chain redox stress and cell wall biosynthesis of the bacteria. By co-assembling with lipid nanoparticles, R6F showed high therapeutic efficacy and minimal adverse effects in the treatment of MRSA-induced sepsis and chronic wound infection. This work provides a novel strategy to design highly potent antibacterial peptide amphiphiles for the treatment of drug-resistant bacterial infections.

A Fluorous Peptide Amphiphile with Potent Antimicrobial Activity for the Treatment of MRSA‐induced Sepsis and Chronic Wound Infection

The rising prevalence of global antibiotic resistance evokes the urgent need for novel antimicrobial candidates. Cationic lipopeptides have attracted much attention due to their strong antimicrobial activity, broad‐spectrum and low resistance tendency. Herein, a library of fluoro‐lipopeptide amphiphiles was synthesized by tagging a series of cationic oligopeptides with a fluoroalkyl tail via a disulfide spacer. Among the lipopeptide candidates, R6F bearing six arginine moieties and a fluorous tag shows the highest antibacterial activity, and it exhibits an interesting fluorine effect as compared to the non‐fluorinated lipopeptides. The high antibacterial activity of R6F is attributed to its excellent bacterial membrane permeability, which further disrupts the respiratory chain redox stress and cell wall biosynthesis of the bacteria. By co‐assembling with lipid nanoparticles, R6F showed high therapeutic efficacy and minimal adverse effects in the treatment of MRSA‐induced sepsis and chronic wound infection. This work provides a novel strategy to design highly potent antibacterial peptide amphiphiles for the treatment of drug‐resistant bacterial infections.

Self-Assembled Oligopeptoplex-Loaded Dissolving Microneedles for Adipocyte-Targeted Anti-Obesity Gene Therapy.

Advancements in gene delivery systems are pivotal for gene-based therapeutics in oncological, inflammatory, and infectious diseases. This study delineates the design of a self-assembled oligopeptoplex (SA-OP) optimized for shRNA delivery to adipocytes, targeting obesity and associated metabolic syndromes. Conventional systems face challenges, including instability due to electrostatic interactions between genetic materials and cationic oligopeptides. Additionally, repeated injections induce discomfort and compromise patient well-being. To circumvent these issues, a dissolvable hyaluronic acid-based, self-locking microneedle (LMN) patch is developed, with improved micro-dose efficiency, for precise SA-OP delivery. This platform offers pain-free administration and improved SA-OP storage stability. In vitro studies in 3T3-L1 cells demonstrated improvements in SA-OP preservation and gene silencing efficacy. In vivo evaluation in a mice model of diet-induced type 2 diabetes yielded significant gene silencing in adipose tissue and a 21.92 ± 2.51% reduction in body weight with minimum relapse risk at 6-weeks post-treatment, representing a superior therapeutic efficacy in a truncated timeframe relative to the GLP-1 analogues currently available on the market. Additionally, SA-OP (LMN) mitigated insulin resistance, inflammation, and hepatic steatosis. These findings establish SA-OP (LMN) as a robust, minimally invasive transdermal gene delivery platform with prolonged storage stability for treating obesity and its metabolic comorbidities.

Cationic Oligopeptides with Amino Groups as Synthetic Nucleolar Localization Signals for the Rational Design of Nucleolus-Staining Probes.

Cationic oligopeptides with amino groups were found to function as synthetic nucleolar localization signals for directing various fluorophores to the nucleolus with high selectivity in the cells with a view toward the development of nucleolus-staining probes.

A new triphenylphosphonium-conjugated amphipathic cationic peptide with improved cell-penetrating and ROS-targeting properties

We study for the first time whether triphenylphosphonium (TPP) moiety can improve cellular delivery and redox properties of amphipathic cationic peptides based on YRFK/YrFK cell-penetrating and cytoprotective motif. TPP moiety was found to increase reducing activity of both stereoisomeric peptides in solution and on electrode surface in association with TPP-mediated intramolecular interactions. Among TPP-conjugated peptides, newly synthesized TPP3-YrFK featured both increased antioxidant efficacy and proteolytic resistance. TPP-conjugated peptides preferably mitigated endogenic ROS in mitochondria and cytoplasm of model glioblastoma cells with increased oxidative status. This anti-ROS effect was accompanied by mild reversible decrease of reduced glutathione level in the cells with relatively weak change in glutathione redox forms ratio. Such low interference with cell redox status is in accordance with non-cytotoxic nature of the compounds. Intracellular concentrations of label-free peptides were analyzed by LC–MS/MS, which showed substantial TPP-promoted penetration of YrFK motif across cell plasma membrane. However, according to ΔΨm analysis, TPP moiety did not profoundly enhance peptide interaction with mitochondrial inner membrane. Our study clarifies the role of TPP moiety in cellular delivery of amphipathic cationic oligopeptides. The results suggest TPP moiety as a multi-functional modifier for the oligopeptides which is capable of improving cellular pharmacokinetics and antioxidant activity as well as targeting increased ROS levels. The results encourage further investigation of TPP3-YrFK as a peptide antioxidant with multiple benefits.

Acquisition of absorption-enhancing abilities of cationic oligopeptides with short chain arginine residues through conjugation to hyaluronic acid

Cell-penetrating peptides such as oligoarginines are one of promising tools that improve mucosal absorption of poorly membrane-permeable biologics. We have already demonstrated that conjugation of L-octaarginine to hyaluronic acid via a tetraglycine spacer resulted in a 3-fold enhancement of nasal absorption of somatropin (Mw: ca. 22.1 kDa) in mice when compared with the unmodified peptide. Here, we evaluated absorption-enhancing abilities and safety profiles of oligopeptides with short chain arginine residues conjugated to hyaluronic acid. Somatropin absorption was hardly ever enhanced by diglycine-L-tetraarginine. The peptide acquired the absorption-enhancing ability through the conjugation; however, it disappeared when arginine residues were halved. In vivo data were consistent to in vitro cellular uptake of somatropin. When somatropin was substituted with exendin-4 (Mw: ca. 4.2 kDa), cellular uptake was significantly enhanced by diglycine-L-diarginine conjugated to hyaluronic acid under comparison with the unmodified peptide. The conjugate also exhibited the enhancement ability in mice, as observed for hyaluronic acid derivatives with four and more arginine residues. Another cell studies revealed that oligoarginine-linked hyaluronic acid tended to be less toxic as arginine residues were reduced. Results indicated that diglycine-L-tetraarginine-linked hyaluronic acid was the most suitable candidate as an absorption enhancer whose Mw-independent enhancement ability and safety were well-balanced.

Controlling liquid–liquid phase separation of G-quadruplex-forming RNAs in a sequence-specific manner

We constructed a minimum liquid-liquid phase separation model system to form liquid droplets using only G-quadruplex-forming oligonucleotides and R- and G-rich oligopeptides. We found that the G-quadruplex structure is an essential component for RNA to form droplets with the peptide. Based on this model system and our findings, droplet redissolution via structure transition from a G-quadruplex to a duplex was achieved in a sequence-specific manner.

Adsorption of Recombinant Human β-Defensin 2 and Two Mutants on Mesoporous Silica Nanoparticles and Its Effect against Clavibacter michiganensis subsp. michiganensis.

Solanum lycopersicum L. is affected among other pests and diseases, by the actinomycete Clavibacter michiganensis subsp. michiganensis (Cmm), causing important economic losses worldwide. Antimicrobial peptides (AMPs) are amphipathic cationic oligopeptides with which the development of pathogenic microorganisms has been inhibited. Therefore, in this study, we evaluate antimicrobial activity of mesoporous silica nanoparticles (MSN5.4) loaded with human β-defensin-2 (hβD2) and two mutants (TRX-hβD2-M and hβD2-M) against Cmm. hβD2, TRX-hβD2-M and hβD2-M presented a half-maximum inhibitory concentration (IC50) of 3.64, 1.56 and 6.17 μg/mL, respectively. MSNs had average particle sizes of 140 nm (SEM) and a tunable pore diameter of 4.8 up to 5.4 nm (BJH). AMPs were adsorbed more than 99% into MSN and a first release after 24 h was observed. The MSN loaded with the AMPs inhibited the growth of Cmm in solid and liquid media. It was also determined that MSNs protect AMPs from enzymatic degradation when the MSN/AMPs complexes were exposed to a pepsin treatment. An improved AMP performance was registered when it was adsorbed in the mesoporous matrix. The present study could expand the applications of MSNs loaded with AMPs as a biological control and provide new tools for the management of phytopathogenic microorganisms.

Enhanced Binding Affinity of siRNA Overhang-Binding Fluorescent Probes by Conjugation with Cationic Oligopeptides for Improved Analysis of the siRNA Delivery Process.

Carrier-mediated delivery of small interfering RNAs (siRNAs) into the living cells is important for the realization of siRNA therapeutics that can silence target genes through RNA interference. We recently proposed a new strategy for analyzing the siRNA delivery process based on affinity labeling with a peptide nucleic acid (PNA)-based fluorescent probe (PyAATO; Py: pyrene, A: adenine; TO: thiazole orange) capable of selectively binding to the overhanging structures of siRNAs. We have prepared new probes with improved binding affinity by conjugation with a cationic oligopeptide. The probe, carrying six lysine residues (PyAATO-Lys6 (Lys6)), displayed a 39-fold increase in affinity, compared with that of the parent probe containing no oligopeptides. Thermodynamic characterization revealed that enhanced affinity resulted from the favorable polyelectrolyte effect, due to the electrostatic interaction between lysine residues and phosphate anions of the RNA duplexes near the overhanging structure. Lys6 showed the improved imaging ability of the carrier-mediated siRNA delivery process in living cells, in which 20 nm siRNA could be analyzed and was considered to show the minimal off-target effects.

Enhancement in RNase H activity of a DNA/RNA hybrid duplex using artificial cationic oligopeptides.

This study assessed the effects of artificial cationic oligopeptides on a DNA/RNA hybrid duplex. An oligopeptide containing the octamer of l-2,4-diaminobutyric acid (Dab8) was found to enhance both the RNase A resistance and RNase H activity of the DNA/RNA hybrid, which are important for developing nucleic acid drugs.

Correction: Enhancement in RNase H activity of a DNA/RNA hybrid duplex using artificial cationic oligopeptides.

Correction for 'Enhancement in RNase H activity of a DNA/RNA hybrid duplex using artificial cationic oligopeptides' by Rintaro Iwata Hara et al., Chem. Commun., 2018, 54, 8526-8529.

カチオン性オリゴペプチドによる卵白アルブミンのナノ粒子化

カチオン性オリゴペプチドによる卵白アルブミンのナノ粒子化

Fluorescent Peptide Beacons for the Selective Ratiometric Detection of Heparin.

Heparin is extensively used as an anticoagulant drug during surgery. Two fluorophore-functionalized cationic oligopeptides HS 1 and HS 2 were developed to monitor heparin ratiometrically in aqueous media. Upon binding to heparin, HS 1 and HS 2 undergo a conformational change from an open form to a folded form, which leads to a distinct change in the fluorescence properties. HS 1 switches from pyrene monomer emission to an excimer emission. For HS 2, a fluorescence resonance energy transfer (FRET) process is enabled between a naphthalene donor and a dansyl acceptor. This method is highly selective for heparin relative to other similar biological analytes such as hyaluronic acid or chondroitin sulfate. HS 1 and HS 2 could also detect heparin ratiometrically in diluted bovine serum. The strong ratiometric emission color change can also be observed by the naked eye. Addition of the polycationic protein protamine releases both HS 1 and HS 2 from their heparin complex, which simultaneously restores pyrene monomer emission for the first case and decreases the FRET process for the latter case, respectively. Dynamic light scattering (DLS) and AFM studies confirm aggregate formation of heparin with HS 1 and HS 2.

Branched amphiphilic cationic oligopeptides form peptiplexes with DNA: a study of their biophysical properties and transfection efficiency.

Over the past decade, peptides have emerged as a new family of potential carriers in gene therapy. Peptides are easy to synthesize and quite stable. Additionally, sequences shared by the host proteome are not expected to be immunogenic or trigger inflammatory responses, which are commonly observed with viral approaches. We recently reported on a new class of branched amphiphilic peptide capsules (BAPCs) that self-assemble into extremely stable nanospheres. These capsules are capable of retaining and delivering alpha-emitting radionuclides to cells. Here we report that, in the presence of double stranded plasmid DNA, BAPCs are unable to form. Instead, depending of the peptide/DNA ratios, the peptides either coat the plasmid surface forming nanofibers (high peptide to DNA ratio) or condense the plasmid into nanometer-sized compacted structures (at low peptide to DNA ratios). Different gene delivery efficiencies are observed for the two types of assemblies. The compacted nanometer-sized structures display much higher transfection efficiencies in HeLa cells. This level of transfection is greater than that observed for a lipid-based reagent when the total number of viable transfected cells is taken into account.

Molecular Simulations of Polycation–DNA Binding Exploring the Effect of Peptide Chemistry and Sequence in Nuclear Localization Sequence Based Polycations

Gene therapy relies on the delivery of DNA into cells, and polycations are one class of vectors enabling efficient DNA delivery. Nuclear localization sequences (NLS), cationic oligopeptides that target molecules for nuclear entry, can be incorporated into polycations to improve their gene delivery efficiency. We use simulations to study the effect of peptide chemistry and sequence on the DNA-binding behavior of NLS-grafted polycations by systematically mutating the residues in the grafts, which are based on the SV40 NLS (peptide sequence PKKKRKV). Replacing arginine (R) with lysine (K) reduces binding strength by eliminating arginine-DNA interactions, but placing R in a less hindered location (e.g., farther from the grafting point to the polycation backbone) has surprisingly little effect on polycation-DNA binding strength. Changing the positions of the hydrophobic proline (P) and valine (V) residues relative to the polycation backbone changes hydrophobic aggregation within the polycation and, consequently, changes the conformational entropy loss that occurs upon polycation-DNA binding. Since conformational entropy loss affects the free energy of binding, the positions of P and V in the grafts affect DNA binding affinity. The insight from this work guides synthesis of polycations with tailored DNA binding affinity and, in turn, efficient DNA delivery.

Synthesis and properties of cationic oligopeptides with different side chain lengths that bind to RNA duplexes

A series of artificial peptides bearing cationic functional groups with different side chain lengths were designed, and their ability to increase the thermal stability of nucleic acid duplexes was investigated. The peptides with amino groups selectively increased the stability of RNA/RNA duplexes, and a relationship between the side chain length and the melting temperature (Tm) of the peptide–RNA complexes was observed. On the other hand, while peptides with guanidino groups exhibited a similar tendency with respect to the peptide structure and thermal stability of RNA/RNA duplexes, those with longer side chain lengths, such as l-2-amino-4-guanidinobutyric acid (Agb) or l-arginine (Arg) oligomers, stabilized both RNA/RNA and DNA/DNA duplexes, and those with shorter side chain lengths exhibited a higher ability to selectively stabilize RNA/RNA duplexes. In addition, peptides were designed with different levels of flexibility by introducing glycine (Gly) residues into the l-2-amino-3-guanidinopropionic acid (Agp) oligomers. It was found that insertion of Gly did not affect the thermal stability of the peptide–RNA complexes, but an alternate arrangement of Gly and Agp apparently decreased the thermal stability. Therefore, in the Agp oligomer, consecutive Agp sequences are essential for increasing the stability of RNA/RNA duplexes.

The thin line between cell‐penetrating and antimicrobial peptides: the case of Pep‐1 and Pep‐1‐K

Cell-penetrating peptides (CPPs) are cationic oligopeptides able to translocate across biological membranes without perturbing them, while antimicrobial peptides (AMPs) kill bacteria mainly by disrupting their membranes. The two peptide classes share several characteristics (charge, amphipathicity, helicity, and length), and therefore the molecular properties discriminating between the two different bioactivities are not clear. Pep-1-K (KKTWWKTWWTKWSQPKKKRKV) is a new AMP derived from the widely studied CPP Pep-1 (KETWWETWWTEWSQPKKKRKV), or 'Chariot', known for its ability to carry large cargoes across biological membranes. Pep-1-K was obtained from Pep-1 by substituting the three Glu residues with Lys, to increase its cationic character. Previous studies showed that these modifications endow Pep-1-K with a potent antimicrobial activity, with MICs in the low micromolar range. Here, we characterized the interaction of Pep-1 and Pep-1-K with model membranes to understand the reason for the antimicrobial activity of Pep-1-K. The data show that this peptide causes vesicle aggregation, perturbs membrane order, and induces the leakage of ions, but not of larger solutes, while these effects were not observed for Pep-1. These differences are likely due, at least in part, to the higher affinity of Pep-1-K toward anionic bilayers, which mimick the composition of bacterial membranes.

Contributions of the Histidine Side Chain and the N-Terminal α-Amino Group to the Binding Thermodynamics of Oligopeptides to Nucleic Acids as a Function of pH

Interactions of histidine with nucleic acid phosphates and histidine pK(a) shifts make important contributions to many protein-nucleic acid binding processes. To characterize these phenomena in simplified systems, we quantified binding of a histidine-containing model peptide HWKK ((+)NH(3)-His-Trp-Lys-Lys-NH(2)) and its lysine analogue KWKK ((+)NH(3)-Lys-Trp-Lys-Lys-NH(2)) to a single-stranded RNA model, polyuridylate (polyU), by changes in tryptophan fluorescence as a function of salt concentration and pH. For both HWKK and KWKK, equilibrium binding constants, K(obs), and magnitudes of log-log salt derivatives, SK(obs) identical with (partial differential logK(obs)/partial differential log[Na(+)]), decreased with increasing pH in the manner expected for a titration curve model in which deprotonation of the histidine and alpha-amino groups weakens binding and reduces its salt-dependence. Fully protonated HWKK and KWKK exhibit the same K(obs) and SK(obs) within uncertainty, and these SK(obs) values are consistent with limiting-law polyelectrolyte theory for +4 cationic oligopeptides binding to single-stranded nucleic acids. The pH-dependence of HWKK binding to polyU provides no evidence for pK(a) shifts nor any requirement for histidine protonation, in stark contrast to the thermodynamics of coupled protonation often seen for these cationic residues in the context of native protein structure where histidine protonation satisfies specific interactions (e.g., salt-bridge formation) within highly complementary binding interfaces. The absence of pK(a) shifts in our studies indicates that additional Coulombic interactions across the nonspecific-binding interface between RNA and protonated histidine or the alpha-amino group are not sufficient to promote proton uptake for these oligopeptides. We present our findings in the context of hydration models for specific vs nonspecific nucleic acid binding.

Cationic oligopeptides with the repeating sequence L‐lysyl‐L‐alanyl‐L‐alanine: conformational and thermal stability study using optical spectroscopic methods

The infrared (IR), vibrational circular dichroism (VCD), and electronic circular dichroism (ECD) spectra of short cationic sequential peptides (L-Lys-L-Ala-L-Ala)(n) (n = 1, 2, and 3) were measured over a range of temperatures (20-90 degrees C) in aqueous solution at near-neutral pH values in order to investigate their solution conformations and thermally induced conformational changes. VCD spectra of all three oligopeptides measured in the amide I' region indicate the presence of extended helical polyproline II (PPII)-like conformation at room temperature. UV-ECD spectra confirmed this conclusion. Thus, the oligopeptides adopt a PPII-like conformation, independent of the length of the peptide chain. However, the optimized dihedral angles phi and psi are within the range -82 to -107 degrees and 143-154 degrees , respectively, and differ from the canonical PPII values. At elevated temperatures, the observed intensity and bandshape variations in the VCD and ECD spectra show that the PPII-like conformation of the Lys-Ala-Ala sequence is still preferred, being in equilibrium with an unordered conformer at near-neutral pH values within the range of temperatures from 20 to 90 degrees C. This finding was obtained from analysis of the temperature-dependent spectra using the singular value decomposition method. The study presents KAA-containing oligopeptides as conformationally stable models of biologically important cationic peptides and proteins.