Artificial peptides to induce membrane denaturation and disruption and modulate membrane composition and fusion

Membranes consisting of phospholipid bilayers are an essential constituent of eukaryotic cells and their compartments. The alteration of their composition, structure, and morphology plays an important role in modulating physiological processes, such as transport of molecules, cell migration, or signaling, but it can also lead to lethal effects. The three main classes of membrane-active peptides that are responsible for inducing such alterations are cell-penetrating peptides (CPPs), antimicrobial peptides (AMPs), and fusion peptides (FPs). These peptides are able to interact with lipid bilayers in highly specific and tightly regulated manners. They can either penetrate the membrane, inducing nondestructive, transient alterations, or disrupt, permeabilize, or translocate through it, or induce membrane fusion by generating attractive forces between two bilayers. Because of these properties, membrane-active peptides have attracted the attention of the pharmaceutical industry, and naturally occurring bioactive structures have been used as a platform for synthetic modification and the development of artificial analogs with optimized therapeutic properties to transport biologically active cargos or serve as novel antimicrobial agents. In this review, we focus on synthetic membrane interacting peptides with bioactivity comparable with their natural counterparts and describe their mechanism of action.     

γ-Secretase inhibitors and modulators

γ-Secretase is a fascinating, multi-subunit, intramembrane cleaving protease that is now being considered as a therapeutic target for a number of diseases. Potent, orally bioavailable γ-secretase inhibitors (GSIs) have been developed and tested in humans with Alzheimer's disease (AD) and cancer. Preclinical studies also suggest the therapeutic potential for GSIs in other disease conditions. However, due to inherent mechanism based-toxicity of non-selective inhibition of γ-secretase, clinical development of GSIs will require empirical testing with careful evaluation of benefit versus risk. In addition to GSIs, compounds referred to as γ-secretase modulators (GSMs) remain in development as AD therapeutics. GSMs do not inhibit γ-secretase, but modulate γ-secretase processivity and thereby shift the profile of the secreted amyloid β peptides (Aβ) peptides produced. Although GSMs are thought to have an inherently safe mechanism of action, their effects on substrates other than the amyloid β protein precursor (APP) have not been extensively investigated. Herein, we will review the current state of development of GSIs and GSMs and explore pertinent biological and pharmacological questions pertaining to the use of these agents for select indications. This article is part of a Special Issue entitled: Intramembrane Proteases.     

Membrane Fusion

The fusion of biological membranes results in two bilayer-based membranes merging into a single membrane. In this process the lipids have to undergo considerable rearrangement. The nature of the intermediates that are formed during this rearrangement has been investigated. Certain fusion proteins facilitate this process. In many cases short segments of these fusion proteins have a particularly important role in accelerating the fusion process. Studies of the interaction of model peptides with membranes have allowed for increased understanding at the molecular level of the mechanism of the promotion of membrane fusion by fusion proteins. There is an increased appreciation of the roles of several independent segments of fusion proteins in promoting the fusion process.Many of the studies of the fusion of biological membranes have been done with the fusion of enveloped viruses with other membranes. One reason for this is that the number of proteins involved in viral fusion is relatively simple, often requiring only a single protein. For many enveloped viruses, the structure of their fusion proteins has certain common elements, suggesting that they all promote fusion by an analogous mechanism. Some aspects of this mechanism also appears to be common to intracellular fusion, although several proteins are involved in that process which is more complex and regulated than is fusion.     

Secondary structure of cell-penetrating peptides controls membrane interaction and insertion

The clinical use of efficient therapeutic agents is often limited by the poor permeability of the biological membranes. In order to enhance their cell delivery, short amphipathic peptides called cell-penetrating peptides (CPPs) have been intensively developed for the last two decades. CPPs are based either on protein transduction domains, model peptide or chimeric constructs and have been used to deliver cargoes into cells through either covalent or non-covalent strategies. Although several parameters are simultaneously involved in their internalization mechanism, recent focuses on CPPs suggested that structural properties and interactions with membrane phospholipids could play a major role in the cellular uptake mechanism. In the present work, we report a comparative analysis of the structural plasticity of 10 well-known CPPs as well as their ability to interact with phospholipid membranes. We propose a new classification of CPPs based on their structural properties, affinity for phospholipids and internalization pathways already reported in the literature.     

The Epidemiology of Thyroid Cancer in Los Angeles County

More than 300 new cases of thyroid cancer are diagnosed in Los Angeles County every year. The age-adjusted annual incidence rates of this disease for all races combined are 2.4 for males and 6.1 for females. Rates for women are more than twice rates for men in each major ethnic group. Blacks of both sexes have the lowest rates; Japanese, Chinese, other Asians and Spanish-surnamed whites all have rates that are as high as or higher than rates among non-Spanish-surnamed whites. Other demographic patterns include the excess of thyroid cancer among Jewish residents of Los Angeles.There have been an increase in thyroid cancer incidence and a decline in mortality for this disease in the United States over the past several decades. Several possible explanations can be made for these trends. Also, the risk factors for thyroid cancer deserve review.     

Correlation between simulated physicochemical properties and hemolycity of protegrin-like antimicrobial peptides: predicting experimental toxicity

The therapeutic, antibiotic potential of antimicrobial peptides can be prohibitively diminished because of the cytotoxicity and hemolytic profiles they exhibit. Quantifying and predicting antimicrobial peptide toxicity against host cells is thus an important goal of AMP related research. In this work, we present quantitative structure activity relationships for toxicity of protegrin-like antimicrobial peptides against human cells (epithelial and red blood cells) based on physicochemical properties, such as interaction energies and radius of gyration, calculated from molecular dynamics simulations of the peptides in aqueous solvent. The hypothesis is that physicochemical properties of peptides, as manifest by their structure and interactions in a solvent and as captured by atomistic simulations, are responsible for their toxicity against human cells. Protegrins are beta-hairpin peptides with high activity against a wide variety of microbial species, but in their native state are toxic to human cells. Sixty peptides with experimentally determined toxicities were used to develop the models. We test the resulting relationships to determine their ability to predict the toxicity of several protegrin-like peptides. The developed QSARs provide insight into the mechanism of cytotoxic action of antimicrobial peptides. In a subsequent blind test, the QSAR correctly ranked four of five protegrin analogues newly synthesized and tested for toxicity.     

Synthetic Oligopeptides Mimicking γ-Core Regions of Cysteine-Rich Peptides of Solanum lycopersicum Possess Antimicrobial Activity against Human and Plant Pathogens

Plant cysteine-rich peptides (CRPs) represent a diverse group of molecules involved in different aspects of plant physiology. Antimicrobial peptides, which directly suppress the growth of pathogens, are regarded as promising templates for the development of next-generation pharmaceuticals and ecologically friendly plant disease control agents. Their oligopeptide fragments are even more promising because of their low production costs. The goal of this work was to explore the antimicrobial activity of nine short peptides derived from the γ-core-containing regions of tomato CRPs against important plant and human pathogens. We discovered antimicrobial activity in peptides derived from the defensin-like peptides, snakins, and MEG, which demonstrates the direct involvement of these CRPs in defense reactions in tomato. The CRP-derived short peptides appeared particularly active against the gram-positive bacterium Clavibacter michiganensis, which causes bacterial wilt—opening up new possibilities for their use in agriculture to control this dangerous disease. Furthermore, high inhibitory potency of short oligopeptides was demonstrated against the yeast Cryptococcus neoformans, which causes serious diseases in humans, making these peptide molecules promising candidates for the development of next-generation pharmaceuticals. Studies of the mode of action of the two most active peptides indicate fungal membrane permeabilization as a mechanism of antimicrobial action.     

Isolation and biochemical characterization of peptides presenting antimicrobial activity from the skin of Phyllomedusa hypochondrialis

Amphibian antimicrobial peptides have been known for many decades and several of them have been isolated from anuran species. Dermaseptins are among the most studied antimicrobial peptides and are found in the skin secretion of tree frogs from the Phyllomedusinae subfamily. These peptides exert a lytic action on bacteria, protozoa, yeast, and filamentous fungi at micromolar concentrations, but unlike polylysines, present little hemolytic activity. In this work, two antimicrobial peptides were isolated from the crude skin secretion of Phyllomedusa hypochondrialis and tested against Gram-positive and Gram-negative bacteria, presenting no hemolytic activity at the tested concentrations. One of them was identified with the recently reported peptide PS-7 belonging to the phylloseptin family, and another was a novel peptide, named DPh-1, which was fully purified, sequenced by ‘de novo’ mass spectrometry and grouped into Dermaseptins (DPh-1).     

Natural and grafted cyclotides in cancer therapy: An insight

Cyclotides is a rapidly growing class of plant‐derived cyclic peptides exhibiting several bioactivities with potential applications in the agricultural and pharmaceutical sectors. Both natural and grafted cyclotides have shown promise in cancer therapy. Approximately 70 natural cyclotides belonging to three plant families (Fabaceae, Rubiaceae, and Violaceae) have shown cytotoxicity against several cancer cell lines. Cyclotides exhibit considerable stability against thermal and enzymatic proteolysis, owing to their unique structure with knotted topology and head to tail cyclization. Further, their small size, high stability, oral bioavailability, and tolerance to amino acid substitution in structural loops make them an ideal platform for designing peptide‐based drugs for cancer. Thus, cyclotides provide ideal scaffolds for bioactive epitope grafting and facilitating drug delivery in cancer treatment. Many anticancer linear peptides have been grafted in cysteine knotted cyclic framework of cyclotide for enhancing their cell permeability across cellular membranes, thereby improving their delivery and pharmacokinetics. The present review comprehensively discusses the distribution, toxicity, and anticancer bioactivity of natural cyclotides. Further, it systematically elaborates on the role and action of epitopes' into grafted cyclotides in targeting cancer. The review also encompasses related patents landscape study and future challenges in peptide‐based cancer therapy.     

Serum peptidome patterns that distinguish metastatic thyroid carcinoma from cancer-free controls are unbiased by gender and age

Serum peptidomics is a special form of functional proteomics. The small number of blood proteins that are the source of most prominent peptides in human serum serve as a substrate pool for commonly occurring and/or cancer-derived proteases. Exoprotease activities in particular, when superimposed on the ex vivo coagulation and complement degradation pathways, contribute to generation of not only cancer-specific but also "cancer type"-specific serum peptides. Following development of a unique, semiautomated serum peptide profiling platform and after completing investigations to eliminate common experimental bias, we have now studied possible effects of gender and age on serum peptidomes of 200 healthy men and women, ages 20-80, and of 60 patients (30 men and 30 women) with metastatic thyroid carcinomas. Extensive MALDI-TOF MS and data analysis suggested negligible contributions of both age and gender to the serum peptidome patterns except that healthy men and women under 35 years, but not older individuals, could be distinguished with approximately 70% accuracy. Considering the more advanced age of most patients, this finding is unlikely to interfere with peptidomics analysis of most cancers. By examining patient samples and age/gender-matched controls followed by variability analysis of either demographic or disease (versus control) groups, we could conclusively rule out demographic bias. An optimized, 12-peptide ion thyroid cancer signature was then developed, enabling classification of an independent validation set with 95% sensitivity and 95% specificity (binomial confidence intervals, 75.1-99.9%). Ten of these peptides had previously been assigned to signature patterns of other solid tumor cancers. One of the two newly discovered peptides was dehydro-Ala(3)-fibrinopeptide A. As we expand this study to include hundreds of thyroid cancer patients, the peptide signature will be adjusted, further validated, and then evaluated in a clinical setting used either independently or in combination with existing markers.     

细胞穿透肽:一种新型非病毒载体

细胞膜的选择通透性对维持细胞内环境的稳定起着非常重要的作用,但细胞膜的这种特性限制了一些生物大分子和药物进入细胞内,不利于对一些细胞内疾病的诊断和药物靶向治疗的应用。如何将一些具有诊断和治疗潜力的生物大分子、药物通过细胞膜进入细胞内一直是医学界研究的热点和难点。细胞穿透肽是一类能够携带多肽、蛋白质、核酸、纳米颗粒、病毒颗粒及药物等穿过细胞膜进入细胞,导致完整载物内化的短肽,为生物大分子和药物进入细胞内部提供了有力的运载工具,其作为载体具有的高转导效率和低毒性特点,已经得到了广泛关注和大量研究。目前,细胞穿透肽作为生物分子和药物细胞内化的运载体已经在荧光成像,肿瘤治疗,抗炎治疗及药物靶向治疗中发挥了潜在的诊断和治疗作用,显示出其诱人的应用前景。     

Activity and stability of human kallikrein-2-specific linear and cyclic peptide inhibitors.

Human glandular kallikrein (KLK2) is a highly prostate-specific serine protease, which is mainly excreted into the seminal fluid, but part of which is also secreted into circulation from prostatic tumors. Since the expression level of KLK2 is elevated in aggressive tumors and it has been suggested to mediate the metastasis of prostate cancer, inhibition of the proteolytic activity of KLK2 is of potential therapeutic value. We have previously identified several KLK2-specific linear peptides by phage display technology. Two of its synthetic analogs, A R R P A P A P G (KLK2a) and G A A R F K V W W A A G (KLK2b), show specific inhibition of KLK2 but their sensitivity to proteolysis in vivo may restrict their potential use as therapeutic agents. In order to improve the stability of the linear peptides for in vivo use, we have prepared cyclic analogs and compared their biological activity and their structural stability. A series of cyclic variants with cysteine bridges were synthesized. Cyclization inactivated one peptide (KLK2a) and its derivatives, while the other peptide (KLK2b) and its derivatives remained active. Furthermore, backbone cyclization of KLK2b improved significantly the resistance against proteolysis by trypsin and human plasma. Nuclear magnetic resonance studies showed that cyclization of the KLK2b peptides does not make the structures more rigid. In conclusion, we have shown that backbone cyclization of KLK2 inhibitory peptides can be used to increase stability without losing biological activity. This should render the peptides more useful for in vivo applications, such as tumor imaging and prostate cancer targeting.     

抗菌肽在宿主防御中作用

抗菌肽广泛地存在于自然界中,其中许多抗菌肽具有直接抗微生物活性,能作用于G-、 G+细菌、真菌、寄生虫甚至是包膜病毒,并且在宿主先天免疫和适应性反应中有重要的调节作用。近来,越来越多的证据表明抗菌肽是有效的免疫辅助因子,能够与其他的众多免疫效应子协同作用,从而起始适应性免疫,促进伤口愈合,抑制前炎症反应以及诱导和调节细胞因子和趋化因子的产生。另外,随着抗菌肽作用机理逐渐被揭开,将这些内源性肽及其衍生物制成抗感染治疗药剂将会有广阔的应用前景。     

Identification of PSA peptide mimotopes using phage display peptide library

Prostate cancer (PCa) is one of the most common types of cancer in men in the United States and is the second leading cause of cancer related death in men. Clinically, secreted prostate specific antigen (PSA) has gained recognition because of its proteolytic activity being directly linked to PCa cell proliferation leading to disease initiation and progression. Using phage display technology, we identified four distinct cyclical peptides. These peptides apart from differences in their amino acid sequence, elicited minimal cross reactive antibody responses against each other. One of the four peptides analyzed produced an antibody response that recognizes the PSA protein. We demonstrate that the synthetic PSA peptide mimics identified in our study are immunologically active and produce neutralizing activity and this has relevance and utility for prostate cancer disease progression.     

A plasmid display platform for the selection of peptides exhibiting a functional cell-penetrating phenotype

Cell-penetrating peptides (CPPs) represent a promising nonviral platform for the delivery of therapeutic cargos to cells and tissues. However, these peptides are often nonspecific, and their mechanism of action is still a subject of debate, which hinders the design of new CPPs. The alternative to rational protein design is the combinatorial approach to protein engineering, whereby large libraries of peptides are created and a screening or selection procedure is used to identify members with the desired phenotype(s). Here we describe a novel procedure for selecting peptides with a CPP phenotype using a plasmid display (PD) platform to link the peptides to their encoding DNA sequences. The PD system is based on genetic fusions to a DNA binding domain. The plasmid was designed to concomitantly express a fluorescent reporter protein to serve as a mock therapeutic cargo indicating its functional delivery into a cell. We have demonstrated this selection strategy using a control CPP (the TAT peptide) in the PC12 neuronal-like cell line. In the absence of transfection reagents, TAT was unable to deliver the protein/DNA complexes. The inclusion of the HA2 peptide from the hemagglutinin protein and the addition of polyethylenimine (PEI) were similarly ineffective. The addition of Lipofectamine, however, enabled the TAT-mediated delivery of the protein/DNA complexes, which was significantly better than control experiments without a CPP. This new PD selection platform will be a valuable new approach for use in identifying unique CPPs from randomized libraries with novel abilities and specificities.     

VDAC1-based peptides: novel pro-apoptotic agents and potential therapeutics for B-cell chronic lymphocytic leukemia

The voltage-dependent anion channel 1 (VDAC1), localized in the outer mitochondrial membrane, mediates metabolic cross-talk between the mitochondrion and the cytoplasm and thus serves a fundamental role in cell energy metabolism. VDAC1 also plays a key role in mitochondria-mediated apoptosis, interacting with anti-apoptotic proteins. Resistance of cancer cells to apoptosis involves quenching the mitochondrial apoptotic pathway by over-expression of anti-apoptotic/pro-survival hexokinase (HK) and Bcl-2 family proteins, proteins that mediate their anti-apoptotic activities via interaction with VDAC1. Using specifically designed VDAC1-based cell-penetrating peptides, we targeted these anti-apoptotic proteins to prevent their pro-survival/anti-apoptotic activities. Anti-apoptotic proteins are expressed at high levels in B-cell chronic lymphocytic leukemia (CLL), an incurable disease requiring innovative new approaches to improve therapeutic outcome. CLL is characterized by a clonal accumulation of mature neoplastic B cells that are resistant to apoptosis. Specifically, we demonstrate that the VDAC1-based peptides (Antp-LP4 and N-Terminal-Antp) selectively kill peripheral blood mononuclear cells (PBMCs) obtained from CLL patients, yet spare those obtained from healthy donors. The cell death induction competence of the peptides was well correlated with the amount of double positive CD19/CD5 cancerous CLL PBMCs, further illustrating their selectivity toward cancer cells. Moreover, these VDAC1-based peptides induced apoptosis by activating the mitochondria-mediated pathway, reflected in membrane blebbing, condensation of nuclei, DNA fragmentation, release of mitochondrial cytochrome c, loss of mitochondrial membrane potential, decreased cellular ATP levels and detachment of HK, all leading to apoptotic cell death. Thus, the mode of action of the peptides involves decreasing energy production and inducing apoptosis. Over 27 versions of cell-penetrating VDAC1-based peptides were designed and screened to identify the most stable, short and apoptosis-inducing peptides toward CLL-derived lymphocytes. In this manner, three optimized peptides suitable for in vivo studies were identified. This study thus reveals the potential of VDAC1-based peptides as an innovative and effective anti-CLL therapy.     

The Ig VH complementarity-determining region 3-containing Rb9 peptide,inhibits melanoma cells migration and invasion by interactions with Hsp90 and an adhesion G-protein coupled receptor

The present work aims at investigating the mechanism of action of the Rb9 peptide, which contains the V_HCDR 3 sequence of anti-sodium-dependent phosphate transport protein 2B (NaPi2B) monoclonal antibody RebMab200 and displayed antitumor properties. Short peptides corresponding to the hypervariable complementarity-determining regions (CDRs) of immunoglobulins have been associated with antimicrobial, antiviral, immunomodulatory and antitumor activities regardless of the specificity of the antibody. We have shown that the CDR derived peptide Rb9 induced substrate hyperadherence, inhibition of cell migration and matrix invasion in melanoma and other tumor cell lines. Rb9 also inhibited metastasis of murine melanoma in a syngeneic mouse model. We found that Rb9 binds to and interferes with Hsp90 chaperone activity causing attenuation of FAK-Src signaling and downregulation of active Rac1 in B16F10-Nex2 melanoma cells. The peptide also bound to an adhesion G-protein coupled receptor, triggering a concentration-dependent synthesis of cAMP and activation of PKA and VASP signaling as well as IP-3 dependent Ca²⁺ release. Hsp90 is highly expressed on the cell surface of melanoma cells, and synthetic agents that target Hsp90 are promising cancer therapeutic drugs. Based on their remarkable antitumor effects, the CDR-H3-derived peptides from RebMab200, and particularly the highly soluble and stable Rb9, are novel candidates to be further studied as potential antitumor drugs, selectively acting on cancer cell motility and invasion.     

Antiangiogenic peptides and proteins: from experimental tools to clinical drugs

The formation of a 'tumor-associated vasculature', a process referred to as tumor angiogenesis, is a stromal reaction essential for tumor progression. Inhibition of tumor angiogenesis suppresses tumor growth in many experimental models, thereby indicating that tumor-associated vasculature may be a relevant target to inhibit tumor progression. Among the antiangiogenic molecules reported to date many are peptides and proteins. They include cytokines, chemokines, antibodies to vascular growth factors and growth factor receptors, soluble receptors, fragments derived from extracellular matrix proteins and small synthetic peptides. The polypeptide tumor necrosis factor (TNF, Beromun) was the first drug registered for the regional treatment of human cancer, whose mechanisms of action involved selective disruption of the tumor vasculature. More recently, bevacizumab (Avastin), an antibody against vascular endothelial growth factor (VEGF)-A, was approved as the first systemic antiangiogenic drug that had a significant impact on the survival of patients with advanced colorectal cancer, in combination with chemotherapy. Several additional peptides and antibodies with antiangiogenic activity are currently tested in clinical trials for their therapeutic efficacy. Thus, peptides, polypeptides and antibodies are emerging as leading molecules among the plethora of compounds with antiangiogenic activity. In this article, we will review some of these molecules and discuss their mechanism of action and their potential therapeutic use as anticancer agents in humans.