Angler Peptides: Macrocyclic Conjugates Inhibit p53:MDM2/X Interactions and Activate Apoptosis in Cancer Cells.

Peptides are being developed as targeted anticancer drugs to modulate cytosolic protein-protein interactions involved in cancer progression. However, their use as therapeutics is often limited by their low cell membrane permeation and/or inability to reach cytosolic targets. Conjugation to cell penetrating peptides has been successfully used to improve the cytosolic delivery of high affinity binder peptides, but cellular uptake does not always result in modulation of the targeted pathway. To overcome this limitation, we developed "angler peptides" by conjugating KD3, a noncell permeable but potent and specific peptide inhibitor of p53:MDM2 and p53:MDMX interactions, with a set of cyclic cell-penetrating peptides. We examined their binding affinity for MDM2 and MDMX, the cell entry mechanism, and role in reactivation of the p53 pathway. We identified two angler peptides, cTAT-KD3 and cR10-KD3, able to activate the p53 pathway in cancer cells. cTAT-KD3 entered cells via endocytic pathways, escaped endosomes, and activated the p53 pathway in breast (MCF7), lung (A549), and colon (HCT116) cancer cell lines at concentrations in the range of 1-12 µM. cR10-KD3 reached the cytosol via direct membrane translocation and activated the p53 pathway at 1 µM in all the tested cell lines. Our work demonstrates that nonpermeable anticancer peptides can be delivered into the cytosol and inhibit intracellular cancer pathways when they are conjugated with stable cell penetrating peptides. The mechanistic studies suggest that direct translocation leads to less toxicity, higher cytosol delivery at lower concentrations, and lower dependencies on the membrane of the tested cell line than occurs for an endocytic pathway with endosomal escape. The angler strategy can rescue high affinity peptide binders identified from high throughput screening and convert them into targeted anticancer therapeutics, but investigation of their cellular uptake and cell death mechanisms is essential to confirming modulation of the targeted cancer pathways.

Palmitoylation of Membrane-Penetrating Magainin Derivatives Reinforces Necroptosis in A549 Cells Dependent on Peptide Conformational Propensities.

Anticancer lipopeptides (ACLPs) are considered promising alternatives to combat resistant cancer cells, but the influence of peptide conformational propensity alone on their selectivity and mechanism remains obscure. In this study, we developed N-palmitoylated MK5E (P1MK5E) and MEK5 (P1MEK5) that have the same composition of 23 residues undergoing the pH-dependent structural alterations but differ in the conformational tendency of their amino acid composites. Nonlipidated peptides were readily accumulated in the A549 cell nucleus by the direct membrane translocation and the heparan sulfate-mediated endocytosis than the lipid-raft-dependent pathway. The increased hydrophobicity favored the amino acid-position-dependent folding of P1MK5E and P1MEK5, respectively, toward the α-helical coiled-coil nanofibrils and amyloidlike ß-protofibrils. At the close concentrations (∼7.5 µM) to the toxic effects of doxorubicin (DOX), P1MK5E exhibited (i) an increased anticancer toxicity through a time-dependent S-phase arrest, (ii) enhanced plasma membrane permeability, and (iii) dose-dependent changes in the cell death characteristic features in the A549 cells relative to P1MEK5 that was almost inactive at ∼75 µM. These observations were in accordance with the TNF-α-mediated necroptotic signaling in the c-MYC/PARP1-overexpressed A549 cells exposed to P1MK5E and accompanied by the ultrastructure of plasma membrane protrusions, extensive endoplasmic reticulum (ER) membrane expansion, mitochondrial swelling, and the formation of distinct cytoplasmic vacuolation. The structural results and the bioactivity behaviors, herein, declared the significance of α-helical propensity in the peptide sequence and the nanostructure morphologies of self-assembling ACLPs upon the selectivity and enhanced anticancer effectiveness, which notably holds promise in the design and development of efficient therapeutics for cancer.

Fluorescent conjugated polymer nanovector for in vivo tracking and regulating the fate of stem cells for restoring infarcted myocardium.

Stem cell therapy holds great promise for cardiac regeneration. However, the lack of ability to control stem cell fate after in vivo transplantation greatly restricts its therapeutic outcomes. MicroRNA delivery has emerged as a powerful tool to control stem cell fate for enhanced cardiac regeneration. However, the clinical translation of therapy based on gene-transfected stem cells remains challenging, due to the unknown in vivo behaviors of stem cells. Here, we developed a nano-platform (i.e., PFBT@miR-1-Tat NPs) that can achieve triggered release of microRNA-1 to promote cardiac differentiation of mesenchymal stem cells (MSCs), and long-term tracking of transplanted MSCs through bright and ultra-stable fluorescence of conjugated polymer poly(9,9-dioctylfluorene-alt-benzothiadiazole) (PFBT). We found that PFBT@miR-1-Tat NP-treated MSCs significantly restored the infarcted myocardium by promoting stem cell cardiac differentiation and integration with the in situ cardiac tissues. Meanwhile, MSCs without gene delivery improved the infarcted heart functions mainly through a paracrine effect and blood vessel formation. The developed conjugated polymer nanovector should be a powerful tool for manipulating as well as revealing the fate of therapeutic cells in vivo, which is critical for optimizing the therapeutic route of gene and cell combined therapy and therefore for accelerating clinical translation. STATEMENT OF SIGNIFICANCE: The lack of controllability in stem cell fate and the unclear in vivo cellular behaviors restrict the therapeutic outcomes of stem cell therapy. Herein, we engineered fluorescent conjugated polymer nanoparticles as gene delivery nanovectors with controlled release and high intracellular delivery capability to harness the fate of mesenchymal stem cells (MSCs) in vivo, meanwhile to reveal the cellular mechanism of gene-treated stem cell therapy. As compared with only MSC treatment that improves infarcted myocardium functions through paracrine effect, treatment with conjugated polymer nanovector-treated MSCs significantly restored infarcted myocardium through enhancing MSC cardiac differentiation and integration with the in-situ cardiac tissues. These findings demonstrate that the conjugated polymer nanovector would be a powerful tool in optimizing gene and cell combined therapy.

Interference with ERK-dimerization at the nucleocytosolic interface targets pathological ERK1/2 signaling without cardiotoxic side-effects.

Dysregulation of extracellular signal-regulated kinases (ERK1/2) is linked to several diseases including heart failure, genetic syndromes and cancer. Inhibition of ERK1/2, however, can cause severe cardiac side-effects, precluding its wide therapeutic application. ERKT188-autophosphorylation was identified to cause pathological cardiac hypertrophy. Here we report that interference with ERK-dimerization, a prerequisite for ERKT188-phosphorylation, minimizes cardiac hypertrophy without inducing cardiac adverse effects: an ERK-dimerization inhibitory peptide (EDI) prevents ERKT188-phosphorylation, nuclear ERK1/2-signaling and cardiomyocyte hypertrophy, protecting from pressure-overload-induced heart failure in mice whilst preserving ERK1/2-activity and cytosolic survival signaling. We also examine this alternative ERK1/2-targeting strategy in cancer: indeed, ERKT188-phosphorylation is strongly upregulated in cancer and EDI efficiently suppresses cancer cell proliferation without causing cardiotoxicity. This powerful cardio-safe strategy of interfering with ERK-dimerization thus combats pathological ERK1/2-signaling in heart and cancer, and may potentially expand therapeutic options for ERK1/2-related diseases, such as heart failure and genetic syndromes.

Cell penetrating peptides: the potent multi-cargo intracellular carriers.

Introduction: Cell penetrating peptides (CPPs) known as protein translocation domains (PTD), membrane translocating sequences (MTS), or Trojan peptides (TP) are able to cross biological membranes without clear toxicity using different mechanisms, and facilitate the intracellular delivery of a variety of bioactive cargos. CPPs could overcome some limitations of drug delivery and combat resistant strains against a broad range of diseases. Despite delivery of different therapeutic molecules by CPPs, they lack cell specificity and have a short duration of action. These limitations led to design of combined cargo delivery systems and subsequently improvement of their clinical applications. Areas covered: This review covers all our studies and other researchers in different aspects of CPPs such as classification, uptake mechanisms, and biomedical applications. Expert opinion: Due to low cytotoxicity of CPPs as compared to other carriers and final degradation to amino acids, they are suitable for preclinical and clinical studies. Generally, the efficiency of CPPs was suitable to penetrate the cell membrane and deliver different cargos to specific intracellular sites. However, no CPP-based therapeutic approach has approved by FDA, yet; because there are some disadvantages for CPPs including short half-life in blood, and nonspecific CPP-mediated delivery to normal tissue. Thus, some methods were used to develop the functions of CPPs in vitro and in vivo including the augmentation of cell specificity by activatable CPPs, specific transport into cell organelles by insertion of corresponding localization sequences, incorporation of CPPs into multifunctional dendrimeric or liposomal nanocarriers to improve selectivity and efficiency especially in tumor cells.

A fluorescent nanoprobe based on azoreductase-responsive metal-organic frameworks for imaging VEGF mRNA under hypoxic conditions.

As VEGF mRNA is an endothelial cell-specific mitogen and a key regulator of angiogenesis in a variety of physiological and pathological processes, high expression levels of VEGF messenger RNA (mRNA) contribute to VEGF-driven angiogenesis in the hypoxic areas of solid tumors and then disrupt the vascular barrier, which may potentiate tumor cell extravasation. Thus, monitoring the changes in VEGF mRNA is necessary to understand the genetic programme under hypoxic conditions and thus facilitate risk assessment and risk reduction in hypoxic environments. Herein, a new fluorescent nanoprobe based on azoreductase-responsive functional metal-organic frameworks (AMOFs) was developed to realize the imaging of VEGF mRNA under hypoxic conditions. Since the azobenzene units in the AMOFs can be reduced to amines by the highly expressed azoreductase in an oxygen-deficient environment, the VEGF mRNA-targeted molecular beacon (MB), which is adsorbed on the surface of AMOFs via electrostatic interactions, can be released due to the structural damage of AMOFs. Moreover, TAMRA (carboxytetramethylrhodamine, donor) and Cy5 (acceptor) were close to each other due to the stem-loop conformation of MB, thus inducing high fluorescence energy resonance transfer (FRET) efficiency. Upon the addition of VEGF mRNA, the hybridization of VEGF mRNA destroyed the stem-loop conformation of MB, and then, the two fluorophores labeled on MB were separated with low FRET efficiency. This constructed fluorescent nanoprobe enables the quantitative analysis and in situ imaging of the VEGF mRNA level in living cells under hypoxic conditions. We expect that it will offer a potentially rich opportunity to understand the physiological processes of genetic programme.

Cell Membrane Composition Drives Selectivity and Toxicity of Designed Cyclic Helix-Loop-Helix Peptides with Cell Penetrating and Tumor Suppressor Properties.

The tumor suppressor protein p53 is inactive in a large number of cancers, including some forms of sarcoma, breast cancer, and leukemia, due to overexpression of its intrinsic inhibitors MDM2 and MDMX. Reactivation of p53 tumor suppressor activity, via disruption of interactions between MDM2/X and p53 in the cytosol, is a promising strategy to treat cancer. Peptides able to bind MDM2 and/or MDMX were shown to prevent MDM2/X:p53 interactions, but most possess low cell penetrability, low stability, and/or high toxicity to healthy cells. Recently, the designed peptide cHLH-p53-R was reported to possess high affinity for MDM2, resistance toward proteases, cell-penetrating properties, and toxicity toward cancer cells. This peptide uses a stable cyclic helix-loop-helix (cHLH) scaffold, which includes two helices connected with a Gly loop and cyclized to improve stability. In the current study, we were interested in examining the cell selectivity of cHLH-p53-R, its cellular internalization, and ability to reactivate the p53 pathway. We designed analogues of cHLH-p53-R and employed biochemical and biophysical methodologies using in vitro model membranes and cell-based assays to compare their structure, activity, and mode-of-action. Our studies show that cHLH is an excellent scaffold to stabilize and constrain p53-mimetic peptides with helical conformation, and reveal that anticancer properties of cHLH-p53-R are mediated by its ability to selectively target, cross, and disrupt cancer cell membranes, and not by activation of the p53 pathway. These findings highlight the importance of examining the mode-of-action of designed peptides to fully exploit their potential to develop targeted therapies.

Development of 2-aminoisobutyric acid (Aib)-rich cell-penetrating foldamers for efficient siRNA delivery.

We have designed and synthesized a set of cell-penetrating foldamers (CPFs), Blocks 1-8, composed of the common amino acids Leu, Arg, and Gly, as well as the helicogenic amino acid 2-aminoisobutyric acid. The findings showed that Block 3 could deliver siRNA into cells without significant cytotoxicity. We also demonstrated that Block 3 could be applied to selectively kill the oncogene-driven cancer cells.

Pharmacological inhibition of Bax-induced cell death: Bax-inhibiting peptides and small compounds inhibiting Bax.

IMPACT STATEMENT: Bax induces mitochondria-dependent programed cell death. While cytotoxic drugs activating Bax have been developed for cancer treatment, clinically effective therapeutics suppressing Bax-induced cell death rescuing essential cells have not been developed. This mini-review will summarize previously reported Bax inhibitors including peptides, small compounds, and antibodies. We will discuss potential applications and the future direction of these Bax inhibitors.

Designing and enhancing the antifungal activity of corneal specific cell penetrating peptide using gelatin hydrogel delivery system.

BACKGROUND: Fungal keratitis is a major cause of corneal blindness accounting for more than one-third of microbiologically proven cases. The management of fungal keratitis is through topical or systemic antifungal medications alone or in combination with surgical treatment. Topical medications such as natamycin and voriconazole pose major challenges due to poor penetration across the corneal epithelium. To address the issue various carrier molecules like nanoparticles, lipid vesicles, and cell penetrating peptides were explored. But the major drawback such as non-specificity and lack of bioavailability remains. PURPOSE: In this study, we have attempted to design corneal specific cell penetrating peptide using subtractive proteomic approach from the published literature and tried to improve its bioavailability through gelatin hydrogel delivery system. MATERIAL AND METHODS: Using subtractive proteomic approach two peptides VRF005 and VRF007 were identified on the basis of solubility, cell permeability and amphipathicity. The peptides were modeled for three-dimensional structure and simulated for membrane penetration. The peptides were characterized using circular dichroism spectroscopy, dynamic light scattering and native polyacrylamide gel electrophoresis. Further uptake studies were performed on primary corneal epithelial cells and the stability was analyzed in corneal epithelial tissue lysates. Insilico prediction of peptides showed it to have antifungal activity which was further validated using colony forming assay and time killing kinetics. The duration of antifungal activity of peptide was improved using gelatin hydrogel through sustained delivery. RESULTS: VRF005 and VRF007 showed α-helical structure and was within the allowed region of Ramachandran plot. The simulation study showed their membrane penetration. The peptide uptake was found to be specific to corneal epithelial cells and also showed intracellular localization in Candida albicans and Fusarium solani. Peptides were found to be stable up to 2 hours when incubated with corneal epithelial tissue lysate. Dynamic light scattering, and native polyacrylamide gel electrophoresis revealed aggregation of peptides. VRF007 showed antifungal activity up to 24 hour whereas VRF005 showed activity up to 4 hours. Hence gelatin hydrogel-based delivery system was used to improve the activity. Actin staining of corneal epithelial cells showed that the cells were attached on gelatin hydrogel. CONCLUSION: We have designed corneal specific cell penetrating peptides using subtractive proteomic approach. Bioavailability and delivery of peptide was enhanced using gelatin hydrogel system.

Histidine-Rich Cell-Penetrating Peptide for Cancer Drug Delivery and Its Uptake Mechanism.

In this work, we report a drug delivery system based on the pH-responsive self-assembly and -disassembly behaviors of peptides. Here, a systematically designed histidine-rich lipidated peptide (NP1) is presented to encapsulate and deliver an anticancer drug ellipticine (EPT) into two model cells: non-small-cell lung carcinoma and Chinese hamster ovary cells. The mechanism of pH-responsive peptide self-assembly and -disassembly involved in the drug encapsulation and release process are extensively investigated. We found that NP1 could self-assemble as a spherical nanocomplex (diameter = 34.43 nm) in a neutral pH environment with EPT encapsulated and positively charged arginine amino acids aligned outward and EPT is released in an acidic environment due to the pH-triggered disassembly. Furthermore, the EPT-encapsulating peptide could achieve a mass loading ability of 18% (mass of loaded-EPT/mass of NP1) with optimization. More importantly, it is revealed that the positively charged arginine on the periphery of the NP1 peptides could greatly facilitate their direct translocation through the negatively charged plasma membrane via electrostatic interaction, instead of via endocytosis, which provides a more efficient uptake pathway.

Potent Protein Delivery into Mammalian Cells via a Supercharged Polypeptide.

The efficient delivery of proteins into cells is needed to fully realize the potential of protein-based therapeutics. Current protein delivery strategies generally suffer from poor endosomal escape and low tolerance for serum. Here, the genetic fusion of a supercharged polypeptide, called SCP, to a protein provides a generic method for intracellular protein delivery. It allows efficient protein endocytosis and endosomal escape and is capable of potently delivering various proteins with a range of charges, sizes, and bioactivities into the nucleus of living cells. SCP is discovered to bind directly to the nuclear import protein importin ß1 and gains access to the nucleus. Furthermore, SCP shows minimal hemolytic activity and stability in serum and lacks toxicity and immunogenicity in vivo. Effective gene editing can be achieved by SCP-mediated delivery of Cas9 protein and guide RNA. This study may provide an efficient and useful tool for the design and development of cell-nuclear-targeted drug delivery.

An anthraquinone-enzyme-peptide hybrid as a photo-switchable enzyme.

A purpose-designed anthraquinone-horseradish peroxidase (HRP)-cell penetrating peptide hybrid 1 showed high and effective cell permeability. Furthermore, 1 exhibited enzymatic activity without photo-irradiation, whereas significant self-degradation and loss of enzymatic activity occurred upon photo-irradiation in living cells.

Oligoalanine helical callipers for cell penetration.

Even for short peptides that are enriched in basic amino acids, the large chemical space that can be spanned by combinations of natural amino acids hinders the rational design of cell penetrating peptides. We here report on short oligoalanine scaffolds for the fine-tuning of peptide helicity in different media and the study of cell penetrating properties. This strategy allowed the extraction of the structure/activity features required for maximal membrane interaction and cellular penetration at minimal toxicity. These results confirmed oligoalanine helical callipers as optimal scaffolds for the rational design and the identification of cell penetrating peptides.

Indoloazepinone-Constrained Oligomers as Cell-Penetrating and Blood-Brain-Barrier-Permeating Compounds.

Non-cationic and amphipathic indoloazepinone-constrained (Aia) oligomers have been synthesized as new vectors for intracellular delivery. The conformational preferences of the [l-Aia-Xxx]n oligomers were investigated by circular dichroism (CD) and NMR spectroscopy. Whereas Boc-[l-Aia-Gly]2,4 -OBn oligomers 12 and 13 and Boc-[l-Aia-ß3 -h-l-Ala]2,4 -OBn oligomers 16 and 17 were totally or partially disordered, Boc-[l-Aia-l-Ala]2 -OBn (14) induced a typical turn stabilized by C5 - and C7 -membered H-bond pseudo-cycles and aromatic interactions. Boc-[l-Aia-l-Ala]4 -OBn (15) exhibited a unique structure with remarkable T-shaped π-stacking interactions involving the indole rings of the four l-Aia residues forming a dense hydrophobic cluster. All of the proposed FITC-6-Ahx-[l-Aia-Xxx]4 -NH2 oligomers 19-23, with the exception of FITC-6-Ahx-[l-Aia-Gly]4 -NH2 (18), were internalized by MDA-MB-231 cells with higher efficiency than the positive references penetratin and Arg8 . In parallel, the compounds of this series were successfully explored in an in vitro blood-brain barrier (BBB) permeation assay. Although no passive diffusion permeability was observed for any of the tested Ac-[l-Aia-Xxx]4 -NH2 oligomers in the PAMPA model, Ac-[l-Aia-l-Arg]4 -NH2 (26) showed significant permeation in the in vitro cell-based human model of the BBB, suggesting an active mechanism of cell penetration.

Effects of N-terminal and C-terminal modification on cytotoxicity and cellular uptake of amphiphilic cell penetrating peptides.

PURPOSE: To assess the effect of "N-Acetylation and C-Amidation" on the cellular uptake, cytotoxicity and performance of amphiphilic cell penetrating peptides (CPP) loaded with methotrexate (MTX). METHODS: Several CPPs were synthesized by solid phase peptide synthesis method. Some of these sequences were modified with pyroglutamic acid at N-terminus and benzylamine or memantine at C-terminus. The resultant nanomaterials were prepared due to the physical linkage between CPPs and MTX. The internalization and cytotoxicity of both CPP-MTX bioconjugates and unmodified CPPs against MCF-7 human breast adenocarcinoma cells was evaluated. RESULTS: N-l and C-terminal modification did not alter the toxicity of CPPs. Physical linkage of CPPs with MTX resulted in a lower drug loading efficiency in comparison with chemically conjugated CPP-MTX bio-conjugates. Both nano-complexes increase the toxic effect of MTX on MCF-7 cells. Furthermore, N- and C-terminal modification may cause a tangible reduction in cellular uptake of CPPs. CONCLUSION: In conclusion, it was shown that cytotoxicity of modified peptides which were physically linked with MTX, considerably higher than both physically loaded unmodified peptides and chemically conjugated peptides with MTX. Also, cell internalization was reduced after peptide end-protection. These findings confirmed the effectiveness of N- and C-terminal modifications on cell viability and CPPs internalization.

The use of electronic-neutral penetrating peptides cyclosporin A to deliver pro-apoptotic peptide: A possibly better choice than positively charged TAT.

Cell-penetrating peptides (CPPs) are increasingly important in transporting macromolecules across cell membranes, but their use remains confined to narrow clinical applications due to the systemic toxicity induced by their positive charges. Several newly discovered electronic neutral penetrating peptides are not attracting much attention because their penetrating capacity is normally far less powerful than cationic or amphiphilic CPPs. In this study, we found the electronic neutral cyclic peptide cyclosporin A (CsA) exhibited 5.6-fold and 19.1-fold stronger penetrating capacity, respectively, than two reported electronic neutral peptides PFVYLI (PFV) and pentapeptide VPTLQ (VPT) in MCF-7 human breast cancer cells. To systematically evaluate the efficiency and toxicity of CsA, we utilized CsA to deliver a membrane-impenetrable pro-apoptotic peptide (PAD) and compared this to the well-established cationic penetrating peptide TAT (RKKRRQRRR). By conjugating CsA to PAD, the internalization of PAD increased 2.2- to 4.7-fold in four different tumor cell lines, and that of CsA-PAD conjugate was significantly higher than TAT-PAD conjugate in MCF-7 and HeLa human cervical cancer cells. Cytotoxicity studies demonstrated that CsA-PAD exhibited a large increase in cell cytotoxicity compared to PAD in four different tumor cell lines, with the effect being similar or greater than the effect of TAT-PAD, depending upon the cell type. The mechanistic studies demonstrated that modifying CsA or TAT did not change the cytotoxicity mechanism of PAD, which occurred via mitochondrial membrane damage related to apoptosis. In vivo studies showed that CsA-PAD could achieve similar anti-tumor efficacy to TAT-PAD but with much lower systemic toxicity, especially to the heart and liver. In conclusion, our study demonstrates for the first time that the electronic-neutral penetrating peptide CsA can be used as a powerful tool to deliver peptide drugs with similar efficiency and less toxicity than the positively charged TAT peptide.

Cell-penetrating and cargo-delivery ability of a spider toxin-derived peptide in mammalian cells.

Cell-penetrating peptides are short cationic peptides with inherent ability to cross the plasma membrane barrier as well as intracellularly deliver cargo molecules conjugated to them. Venoms from snakes, scorpions and spiders are rich in membrane-active peptides. Crotamine from snake venom as well as maurocalcine and imperatoxin isolated from scorpion venoms have been reported to possess cell-penetrating property in mammalian cells. Latarcins, a group of spider venom toxins, has also been reported to possess antimicrobial property. However, cell-penetrating ability of Latarcins is still not elucidated. This is the first report where cell-penetrating ability of a peptide derived from spider toxin, Latarcin 1 has been demonstrated. Interestingly, the structurally minimized sequence of Latarcin 1 (LDP - Latarcin-derived peptide) when conjugated with nuclear localization sequence from Simian Virus T40 antigen (LDP-NLS) translocates across cell membrane in HeLa cells. The chimeric LDP-NLS peptide also did not exhibit cytotoxicity towards mammalian cells in contrast to the LDP that showed lesser uptake and higher cytotoxicity. LDP-NLS also successfully delivered macromolecular protein cargo inside the cells.

A pH-dependent charge reversal peptide for cancer targeting.

Naturally occurring cationic antimicrobial peptides exhibit not only antimicrobial activity, but also anticancer activity and are expected to be new weapons in cancer treatment. The selectivity for cancer cells over normal cells is at least partly due to the more negative surface of cancer cells. A lower pH in tumor tissue (pH 6.2-6.9) than that in normal tissues (pH 7.3-7.4) has also been utilized to develop anticancer agents. However, cytotoxicity against normal cells at physiological pH is often an issue. Furthermore, acidic regions can be found in some normal tissues such as the kidneys. Therefore, existing approaches to cancer targeting are not fully satisfactory. In this study, we designed a peptide, HE (GIHHWLHSAHEFGEHFVHHIMNS-amide), with a charge that reverses from -1.5 at pH 7.4 to +6 at pH 5.5 for cancer targeting at low pH based on the antimicrobial peptide magainin 2 by introducing 6 His, an additional Glu, and an amidated terminal. HE interacted with cancer-mimicking negatively charged liposomes in a pH-dependent fashion with a midpoint with a pH of 6.5 just above the membrane surface. The peptide killed human renal adenocarcinoma ACHN cells at pH 6.0, but not at pH 7.4, and was nontoxic against human normal glomerular mesangial cells even at this low pH. Thus, the novel peptide may be a promising lead peptide for cancer therapy, although this derivatization resulted in weakened cytotoxicity.

How charge distribution influences the function of membrane-active peptides: Lytic or cell-penetrating?

Lytic and cell-penetrating peptides (CPPs) are both membrane-active peptides sharing similar physicochemical properties. Although their respective functions have been intensively investigated, the difference of intrinsic properties between these two types of peptides is rarely discussed. In this study, we designed a series of analogs of a recently discovered CPP ZXR-1 (FKIGGFIKKLWRSKLA) by varying the charge distributions both on the helical wheel projection and along the sequence. These peptides showed different functions on cell membranes, including membrane lytic (peptide Z1), cell-penetrating (peptide ZXR-1, Z2 and Z3), and inactive (peptide Z4) peptides. The three groups of peptides displayed different interactions with model lipid monolayer, and found that peptide insertion might be an important dynamic step to distinguish lytic and cell penetrating functions. Based on the analysis of charge distribution patterns, it was proposed that the charge distributions on the helical wheel and along the sequence are both able to influence the functions of the membrane-active peptides. This finding provides a further understanding about the effect of charge distribution on the functions of membrane-active peptides, and will be helpful for the design of functional peptides.