Development of a penetratin-conjugated stapled peptide that inhibits Wnt/ß-catenin signaling.

Wnt/ß-catenin pathway triggers the formation of a complex between ß-catenin and T cell-specific transcription factor (TCF), which induces transcriptional activation. Excessive transcriptional activation of this pathway is associated with the development, cause, and deterioration of various cancers. Therefore, the Wnt/ß-catenin pathway is an attractive drug target for cancer therapeutics and small molecule- and peptide-based protein-protein interaction (PPI) inhibitors have been developed. However, peptide-based PPI inhibitors generally have low cell-membrane permeability because of their large molecular size. To improve cell-membrane permeability, conjugating cell-penetrating peptides (CPPs) to PPI-inhibiting peptides is a useful method for developing intracellularly targeted PPI inhibitors. In this study, we focused on the interaction between ß-catenin and liver receptor homologue-1 (LRH-1) and designed and synthesized a series of LRH-1-derived peptides to develop inhibitors against Wnt/ß-catenin signaling. The results showed that a penetratin-conjugated LRH-1-derived peptide (Penetratin-st7) predominantly inhibited DLD-1 cell growth at 20 µM treatment via inhibition of the Wnt signaling pathway. This result suggests that Penetratin-st7 is one of promising PPI inhibitors between TCF and ß-catenin.

Study of the 99m Tc-labeling conditions of 6-hydrazinonicotinamide-conjugated peptides from a new perspective: Introduction to the term radio-stoichiometry.

Specific tumor uptake of peptide radiopharmaceuticals depends on tumor binding affinity and their radiochemical purity. Several important parameters that influence the 99m Tc-labeling and consequently the radiochemical purity of 6-hydrazinonicotinamide (HYNIC)-conjugated peptide are radionuclide activity, the amount of peptide, the amount of coligands, and the amount of reducing agents (stannous ion). In this review article, we have attempted studying these parameters in the HYNIC-conjugated peptides (somatostatin, cholecystokinin/gastrin, bombesin, and RGD analogs) from a new perspective to obtain most used and optimized radio-stoichiometric relationships. One of the most important results in this review is that for 99m Tc-labeling of HYNIC-conjugated peptides, it is better to consider the most calculated mole ratio between technetium-99m and the peptide (mole ratio of technetium-99m to the peptide 1:200-400). The statistical results also show that among these 99m Tc-labeled peptides, the most used and favorable coligand is tricine/EDDA with two to one (2:1) mole ratio. These optimized radio-stoichiometric relationships, favorable coligand mole ratio, and applicable radiolabeling points can greatly improve the labeling process of the HYNIC-conjugated peptides, by reducing trial and error, increasing specific activity, and saving materials.

Rhodamine B-conjugated encrypted vipericidin nonapeptide is a potent toxin to zebrafish and associated with in vitro cytotoxicity.

BACKGROUND: Animal venoms contain a diverse array of proteins and enzymes that are toxic toward various physiological systems. However, there are also some practical medicinal uses for these toxins including use as anti-bacterial and anti-tumor agents. METHODS: In this study, we identified a nine-residue cryptic oligopeptide, KRFKKFFKK (EVP50) that is repeatedly encoded in tandem within vipericidin sequences. RESULTS: EVP50 displayed in vivo potent lethal toxicity to zebrafish larvae (LD50=6 µM) when the peptide's N-terminus was chemically conjugated to rhodamine B (RhoB). In vitro, RhoB-conjugated EVP50 (RhoB-EVP50) exhibited a concentration-dependent cytotoxic effect toward MCF-7 and MDA-MB-231 breast cancer cells. In MCF-7 cells, the RhoB-EVP50 nonapeptide accumulated inside the cells within minutes. In the cytoplasm, the RhoB-EVP50 induced extracellular calcium influx and intracellular calcium release. Membrane budding was also observed after incubation with micromolar concentrations of the fluorescent EVP50 conjugate. CONCLUSIONS: The conjugate's interference with calcium homeostasis, its intracellular accumulation and its induced membrane dysfunction (budding and vacuolization) seem to act in concert to disrupt the cell circuitry. Contrastively, unconjugated EVP50 peptide did not display neither toxic nor cytotoxic activities in our in vivo and in vitro models. GENERAL SIGNIFICANCE: The synergic mechanism of toxicity was restricted to the structurally modified encrypted vipericidin nonapeptide.

Cation exchange as a single polishing step for conjugated peptides.

Purification of peptides typically includes expensive reverse phase (RP) processes, which utilize high pressure and large volumes of solvent. For two conjugated peptides, chromatography process development targeted a low-pressure aqueous process that could achieve target product purities of ≥95%, comparable to purities seen with traditional RP. A high throughput screening approach of different modalities was used to identify binding and elution conditions on a cation exchange resin and small-scale columns were used in order to assess impurity removal and process yield. The parameters for load and gradient elution were optimized to increase product purity and process productivity with a wide operating window identified where high purity and productivity are achieved. Computational modeling was then used to validate experimental chromatography results and to gain insight on the effect of the chemical modifications on the surface properties of the two peptides. Both modeling and experimental data showed that with optimization, cation exchange could be utilized as a single polishing step for conjugated peptides. Similar purities were achieved as those seen with RP with up to double the productivity.

The Rhodamine B-encrypted Vipericidin Peptide, RhoB-Ctn[1-9], Displays In vitro Antimicrobial Activity Against Opportunistic Bacteria and Yeasts.

BACKGROUND: Crotalicidin (Ctn), a snake venom cathelicidin-related antimicrobial peptide, is a 34-residue-long linear lysine-rich vipericidin obtained from the South American rattlesnake, Crotalus durissus terrificus. Ctn contains tandem repeats of nine amino acid residues (1KRFKKFFKK9 and 16KRLKKIFKK24; consensus: 1KRhKKhFKK9, h = hydrophobic amino acid) as an integral part of its structure. OBJECTIVES: The aim of this study was to evaluate the antimicrobial activity of the encrypted vipericidin nonapeptide KRFKKFFKK, designated as Ctn[1-9], and its structural analogue, rhodamine- B‒conjugated Ctn[1-9], designated as RhoB-Ctn[1-9]. METHODS: The susceptibility of representative pathogenic bacteria and yeasts to antimicrobial agents was determined using the broth microdilution minimum inhibitory concentration (MIC) method. Cytotoxicity was estimated using a hemolytic assay. The accumulation of RhoB-Ctn[1-9] in microbial cells was observed by fluorescence microscopy. The antimicrobial synergism of RhoB-Ctn[1-9] with antimicrobials was evaluated using a checkerboard analysis. RESULTS: RhoB-conjugated Ctn[1-9] displayed selective antimicrobial activity against infectious gram-negative bacteria such as Escherichia coli, Pseudomonas aeruginosa, and pathogenic species of Candida with low hemolytic effects on human erythrocytes which were not observed with unconjugated Ctn[1-9]. RhoB-Ctn[1-9] could permeate cell membranes and accumulate intracellularly in microbial cells. RhoB-Ctn[1-9] exhibits synergistic effects when used with antibiotics or antifungal agents and reduced the MICs of the peptide and antimicrobials. CONCLUSION: These findings indicate the potential of crotalicidin-related short peptides as structural motifs for the diversification of biological functionalities. Further, they set the stage to investigate the molecular mechanisms by which chemically modified vipericidin repeats modulate cell fate.

Smart Titanium Coating Composed of Antibiotic Conjugated Peptides as an Infection-Responsive Antibacterial Agent.

Antibacterial coating is rapidly emerging as a pivotal strategy for mitigating spread of bacterial pathogens. However, many challenges still need to be overcome in order to develop a smart coating that can achieve on-demand antibacterial effects. In this study, a Staphylococcus aureus (S. aureus) sensitive peptide sequence is designed, and an antibiotic is then conjugated with this tailor-made peptide. The antibiotic-peptide conjugate is then linked to the surface of a titanium implant, where the peptide can be recognized and cleaved by an enzyme secreted by S. aureus. This allows for the release of antibiotics in the presence of S. aureus, thus achieving delivery of an antibacterial specifically when an infection occurs.

Antioxidant-Conjugated Peptide Attenuated Metabolic Reprogramming in Pulmonary Hypertension.

Pulmonary arterial hypertension (PAH) is a chronic cardiopulmonary disorder instigated by pulmonary vascular cell proliferation. Activation of Akt was previously reported to promote vascular remodeling. Also, the irreversible nitration of Y350 residue in Akt results in its activation. NitroAkt was increased in PAH patients and the SU5416/Hypoxia (SU/Hx) PAH model. This study investigated whether the prevention of Akt nitration in PAH by Akt targeted nitroxide-conjugated peptide (NP) could reverse vascular remodeling and metabolic reprogramming. Treatment of the SU/Hx model with NP significantly decreased nitration of Akt in lungs, attenuated right ventricle (RV) hypertrophy, and reduced RV systolic pressure. In the PAH model, Akt-nitration induces glycolysis by activation of the glucose transporter Glut4 and lactate dehydrogenase-A (LDHA). Decreased G6PD and increased GSK3ß in SU/Hx additionally shunted intracellular glucose via glycolysis. The increased glycolytic rate upregulated anaplerosis due to activation of pyruvate carboxylase in a nitroAkt-dependent manner. NP treatment resolved glycolytic switch and activated collateral pentose phosphate and glycogenesis pathways. Prevention of Akt-nitration significantly controlled pyruvate in oxidative phosphorylation by decreasing lactate and increasing pyruvate dehydrogenases activities. Histopathological studies showed significantly reduced pulmonary vascular proliferation. Based on our current observation, preventing Akt-nitration by using an Akt-targeted nitroxide-conjugated peptide could be a useful treatment option for controlling vascular proliferation in PAH.

Development of a receptor-based inhibitory penta-unit-conjugated peptide to enhance anthrax toxin neutralization.

Anthrax toxin is a key virulence factor for Bacillus anthracis. The cell-binding component of anthrax toxin, protective antigen (PA), mediates the entry of the toxin into cells by first binding to the extracellular von Willebrand factor A (VWA) domain of the cellular anthrax toxin receptor (ATR). Herein, we targeted the VWA domain of the cellular receptor to develop a more effective antitoxin agent for neutralization of anthrax toxin. We selected ATR-binding peptides by using a phage display: among these, we identified two novel peptides binding to the ATR with high affinity and specificity, and that neutralized anthrax toxicity in cells. Furthermore, to enhance the functional efficiency of the probes, the peptides were modified and conjugated to three polyvalent probe backbones: a 17 amino-acid-based cyclic form penta-unit, poly-d-lysine (PDL), or the M13 bacteriophage. One of the functionally modified polyvalent peptide probes, the penta-unit-conjugated probe (PUCP) produced the most potent neutralization of anthrax toxin, with half-maximal inhibitory concentration (IC50) of 20 nM. The PUCP disrupted anthrax toxin binding to its receptor and reduced endocytosis of anthrax toxin. This peptide-based approach may, therefore, represent a promising strategy to combat anthrax toxicosis and other bacterial diseases and may be efficient for disease treatment.

Venom Peptides and Toxins - A Prospective Spearhead in Cancer Treatment.

BACKGROUND & AIM: Cancer is a condition of genetically or environmentally mutated, uncontrollable cell growth that directly affects human morbidity and mortality. Many treatments have been adopted to reduce cancer cell proliferation; however, new mutated developments of some cancer cells have started to show resistance towards current therapies and treatments that cause some drugs to lose their efficacy. Additionally, deleterious side-effects of some hard application methods like radiation therapy, chemotherapy, and surgery are less favorable. Accumulative research effort has revealed that peptides and toxins identified from underutilized natural sources including venomous reptiles, amphibians, insects, arachnids, marine organisms and plants are increasingly being employed in cancer treatment. This demands more peptides / toxins to be identified from underutilized natural sources as an alternative therapeutic approach. METHOD & RESULTS: Accumulative research effort has revealed that peptides and toxins identified from underutilized natural sources including venomous reptiles, amphibians, insects, arachnids, marine organisms and plants are increasingly being employed in cancer treatment. Secondary structures / pharmacophore modifications have proven to be an important criterion for raising the efficacy level and anticancer effects. Structure specificity and structural-related cytotoxicity have successfully allowed these peptides to target and cause sufficient damage to malignant cells with minimal cytotoxicity effects towards healthy cells. On top of that, some these pure peptides had adopted multiple anticancer mechanisms and demonstrated collective anticancer effects within a single application. CONCLUSION: Our review exclusively selected peptides and toxins found identified from various natural sources in combating malignant cells, their selectivity towards specific anticancer mechanisms, and the prospective of conjugated peptide as a single entity for a new therapeutic strategy.