Engraulisin: A novel marine derived cell penetrating peptide with activity against drug resistant bacteria.

Cell penetrating peptides (CPP) with their intrinsic ability to penetrate plasma membranes facilitate intracellular uptake of various macromolecules. Although a substantial number of CPPs have been reported over the last three decades, the number is still inadequate when compared to the theoretically feasible peptides with similar physicochemical composition. Marine organisms, due to their hostile environment, are an immense source of several high-valued therapeutically relevant peptides. Various marine derived antibacterial, antimycotic and anticancer peptides have demonstrated improved activity in comparison to peptides of terrestrial origin. While a significant number of marine bioactive peptides exist, cell penetrating peptides from marine organisms remain unravelled. In this study, we report Engraulisin from Engraulis japonicus, a computationally derived novel cell penetrating peptide of marine origin. Engraulisin manifest successful uptake in mammalian cells at 5 µM concentration with negligible cytotoxicity observed through MTT assay. Analysis of its cellular uptake mechanism revealed significant inhibition at 4 °C suggesting endocytosis as the major route of cellular entry. Interestingly, the novel peptide also demonstrated selective antimicrobial activity against Methicillin-resistant Staphylococcus aureus (MRSA). Additionally, molecular dynamics simulation with POPC and POPG bilayer system unveiled significance of positively charged residues in forming a stable membrane interaction. Engraulisin represents a novel marine-derived cell penetrating peptide which can be explored for cellular delivery of pharmaceutically relevant molecules.

An efficient computational protocol for template-based design of peptides that inhibit interactions involving SARS-CoV-2 proteins.

The RNA-dependent RNA polymerase (RdRp) complex of SARS-CoV-2 lies at the core of its replication and transcription processes. The interfaces between holo-RdRp subunits are highly conserved, facilitating the design of inhibitors with high affinity for the interaction interface hotspots. We, therefore, take this as a model protein complex for the application of a structural bioinformatics protocol to design peptides that inhibit RdRp complexation by preferential binding at the interface of its core subunit nonstructural protein, nsp12, with accessory factor nsp7. Here, the interaction hotspots of the nsp7-nsp12 subunit of RdRp, determined from a long molecular dynamics trajectory, are used as a template. A large library of peptide sequences constructed from multiple hotspot motifs of nsp12 is screened in-silico to determine sequences with high geometric complementarity and interaction specificity for the binding interface of nsp7 (target) in the complex. Two lead designed peptides are extensively characterized using orthogonal bioanalytical methods to determine their suitability for inhibition of RdRp complexation. Binding affinity of these peptides to accessory factor nsp7, determined using a surface plasmon resonance (SPR) assay, is slightly better than that of nsp12: dissociation constant of 133nM and 167nM, respectively, compared to 473nM for nsp12. A competitive ELISA is used to quantify inhibition of nsp7-nsp12 complexation, with one of the lead peptides giving an IC50 of 25µM . Cell penetrability and cytotoxicity are characterized using a cargo delivery assay and MTT cytotoxicity assay, respectively. Overall, this work presents a proof-of-concept of an approach for rational discovery of peptide inhibitors of SARS-CoV-2 protein-protein interactions.

Peptide cargo administration: current state and applications.

Effective delivery of drug molecules to the target site is a challenging task. In the last decade, several innovations in the drug delivery system (DDS) have tremendously improved the therapeutic efficacy of drug molecules. Among various DDS, cell-penetrating peptides (CPPs) based DDS have gathered notable attention owing to their safety, efficacy, selectivity, specificity, and ease of synthesis. CPPs are emerging as an efficient and effective pharmaceutical nanocarriers-based platforms for successful management of various important human health disorders. Failure of several current chemotherapeutic strategies is attributed to low solubility, reduced bioavailability, and off-target delivery of several anti-cancer drugs. Similarly, development of therapeutics for vision-threatening disorders is challenged by the anatomical as well as physiological complexity of the eye. Such therapeutic challenges in cancer and ocular disease management can be overcome by developing cell-penetrating peptide (CPP) based peptide drug conjugates (PDCs). CPPs can be used to deliver various types of cargo molecules including nucleic acids, small molecules, and peptides/proteinaceous agents. In this review, we have briefly introduced CPPs and the linker strategies employed for the development of PDCs. Furthermore, recent studies employing CPP-based PDCs for cancer and ocular disease management have been discussed in detail highlighting their significance over conventional DDS. Later sections of the review are focused on the current status of clinical trials and future implications of CPP-based PDCs in vaccine development. KEY POINTS: • Cell-penetrating peptides (CPPs) can deliver a variety of cargo macromolecules via covalent and non-covalent conjugation. • CPP-based peptide drug conjugates (PDCs) can overcome drawbacks of conventional drug delivery methods such as biocompatibility, solubility, stability, and specificity. • Various PDCs are in clinical trial phase for cancer and ocular therapeutics.

Membrane-acting biomimetic peptoids against visceral leishmaniasis.

Visceral leishmaniasis (VL) is among the most neglected tropical diseases in the world. Drug cell permeability is essential for killing the intracellular residing parasites responsible for VL, making cell-permeating peptides a logical choice to address VL. Unfortunately, the limited biological stability of peptides restricts their usage. Sequence-specific oligo-N-substituted glycines ('peptoids') are a class of peptide mimics that offers an excellent alternative to peptides in terms of ease of synthesis and good biostability. We tested peptoids against the parasite Leishmania donovani in both forms, that is, intracellular amastigotes and promastigotes. N-alkyl hydrophobic chain addition (lipidation) and bromination of oligopeptoids yielded compounds with good antileishmanial activity against both forms, showing the promise of these antiparasitic peptoids as potential drug candidates to treat VL.

Peptide-mediated leishmaniasis management strategy: Tachyplesin emerges as an effective anti-leishmanial peptide against Leishmania donovani.

Visceral leishmaniasis is one of the neglected tropical diseases caused by an intracellular parasite, Leishmania donovani. Drug resistance, adverse side effects and long treatment regimes are important limitations in achieving the effective elimination of visceral leishmaniasis. In the absence of any vaccine, chemotherapy remains a viable treatment for leishmaniasis. For effective killing of leishmania parasite, the drug molecule needs to cross the cell membrane. In the present study, marine membrane-active peptide Tachyplesin has been used against Leishmania donovani. Further, the mechanism of action and importance of cysteine amino acids of Tachyplesin in anti-leishmanial activity has been assessed. The cargo-carrying ability of Tachyplesin in L. donovani has been established. Thus, dual-use of Tachyplesin as an anti-leishmanial peptide as well as a cargo delivery vehicle makes the marine peptide an attractive therapeutic target against visceral leishmaniasis.

Efficient delivery of anti-miR-210 using Tachyplesin, a cell penetrating peptide, for glioblastoma treatment.

The levels of microRNAs (miRNAs) are altered in various diseases including glioblastoma (GBM) and this alteration may have widespread effects on various hallmarks of cancer cells. MiR210 is overexpressed in GBM and functions as an oncogenic miRNA. Anti-miR210 therapy holds great promise but its efficient delivery remains a major challenge. Our work here explores a novel role of Tachyplesin (Tpl), a cell-penetrating antimicrobial peptide, as a nanocarrier for anti-miR210. Tpl electrostatically interacts with anti-miR210 at 1:25 and 1:50 (anti-miR:Tpl) weight ratios to form a complex and efficiently delivers anti-miR210 inside GBM cells cultured as 2D and 3D spheroid model. Treatment of GBM cells with the complex significantly inhibited miR210 levels (~90%), proliferation, migration and spheroid formation ability and induced apoptosis as evident by increased levels of caspase 3/7 and ROS. GBM cells pre-treated with anti-miR210:Tpl complex were also found to be sensitive to TMZ mediated action. Uptake of the complex in GBM cells induced the levels of miR210 targeted tumor suppressor genes, NeuroD2 and HIF3A. Overall, our work reveals a novel and efficient miRNA delivery ability of Tpl in glioma cells, holding a great promise for treatment of GBM and potentially for other cancers.

Antibacterial properties of Latarcin 1 derived cell-penetrating peptides.

Cell-penetrating peptides (CPPs) and antimicrobial peptides (AMPs) share certain physicochemical parameters such as amphipathicity, hydrophobicity, cationicity and pI, due to which these two groups of peptides also exhibit overlapping functional characteristics. In our current work, we have evaluated antimicrobial properties of cell-penetrating peptides derived from Latarcin1. Latarcin derived peptide (LDP) exhibited antimicrobial activity against representative microorganisms tested and bactericidal effect against methicillin resistant Staphylococcus aureus (MRSA), which was used as model organism of study in the present work. However, LDP exhibited cytotoxicity against HeLa cells. Further, nuclear localization sequence (NLS) was fused to LDP and interestingly, LDP-NLS showed antimicrobial effect against bacteria, showed bactericidal effect against MRSA and also did not exhibit cytotoxicity in HeLa cells till the highest concentrations tested. Thus, our results inferred that fusion of NLS to LDP significantly reduced cytotoxicity of LDP against HeLa cells (Ponnappan and Chugh, 2017) and exhibited significantly higher cell-penetrating activity in MRSA in comparison to LDP alone. Consolidated results of uptake assays, time-kill assays and PI membrane damage assays show that LDP killed MRSA mainly by membrane damage, where as LDP-NLS might have intracellular targets. Owing to its cell-penetrating activity in HeLa cells and antimicrobial activity against MRSA, LDP-NLS efficiently inhibited intracellular infection of MRSA in HeLa cells as observed in invasion assays. Hence, our results suggest that LDP-NLS is a dual action peptide with AMP and CPP activity and could be potential candidate as peptide antibiotic and drug delivery vector in both mammalian and bacterial cells.

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.

CyLoP-1: Membrane-active peptide with cell-penetrating and antimicrobial properties.

CyLoP-1 is a cysteine-rich cell-penetrating peptide derived from nuclear localization sequence of snake toxin, crotamine. The peptide has shown cytoplasmic uptake in mammalian cells at lower concentrations. In the present study, the cell-penetrating and antimicrobial activity of the peptide has been studied by employing mammalian cells, plant cells as well as bacterial and fungal pathogens. The study shows that the peptide acts as an effective CPP and a cargo-delivery vector for not only mammalian cells but also for plant cells. Besides this, the peptide also possesses antimicrobial activity against representative pathogens tested. It is shown to be effective in killing methicillin-resistant Staphylococcus aureus. We have observed that the presence of cysteine residues in the peptide play a major role in conferring cell-penetrating as well as antimicrobial activity to the peptide since there is a significant decline in these activities when cysteine residues are replaced with serine residues. Our findings are significant for the proposition that CyLoP-1 is an efficient membrane-active peptide with both cell-penetrating and antimicrobial activity. Hence, it can be further evaluated for its application in the field of drug-delivery, plant biotechnology and as a peptide-antibiotic.

Membrane-active peptides from marine organisms--antimicrobials, cell-penetrating peptides and peptide toxins: applications and prospects.

Marine organisms are known to be a rich and unique source of bioactive compounds as they are exposed to extreme conditions in the oceans. The present study is an attempt to briefly describe some of the important membrane-active peptides (MAPs) such as antimicrobial peptides (AMPs), cell-penetrating peptides (CPPs) and peptide toxins from marine organisms. Since both AMPs and CPPs play a role in membrane perturbation and exhibit interchangeable role, they can speculatively fall under the broad umbrella of MAPs. The study focuses on the structural and functional characteristics of different classes of marine MAPs. Further, AMPs are considered as a potential remedy to antibiotic resistance acquired by several pathogens. Peptides from marine organisms show novel post-translational modifications such as cysteine knots, halogenation and histidino-alanine bridge that enable these peptides to withstand harsh marine environmental conditions. These unusual modifications of AMPs from marine organisms are expected to increase their half-life in living systems, contributing to their increased bioavailability and stability when administered as drug in in vivo systems. Apart from AMPs, marine toxins with membrane-perturbing properties could be essentially investigated for their cytotoxic effect on various pathogens and their cell-penetrating activity across various mammalian cells. The current review will help in identifying the MAPs from marine organisms with crucial post-translational modifications that can be used as template for designing novel therapeutic agents and drug-delivery vehicles for treatment of human diseases.

Cell-penetrating peptides: Nanocarrier for macromolecule delivery in living cells.

Novel classes and applications of cell-penetrating peptides (CPPs) are being constantly discovered since they were first identified 2 decades ago. These short cationic peptides (nanomolecules) either by covalent binding or by noncovalent binding can traverse cell membranes and deliver a variety of molecules that are unable to overcome the permeability barrier in their own capacity. The ability of the CPPs to deliver variety of macromolecules, such as oligonucleotides, therapeutic drugs, proteins, and medical imaging agents, by forming nanoparticulate carriers in a range of cells has led them to emerge as a potential tool for both macromolecule delivery application and to gain insight into the fundamentals of mechanism of cellular uptake across the plasma membrane. This review explores the recent advances, challenges, and future prospects in the field of CPP-mediated cargo delivery in mammalian and plant cells. Studies have been conducted into the peptide chemistry and stability of CPP-macromolecular complexes. Most of the CPPs have been shown to be nontoxic and do not interfere with the functionality of the macromolecules delivered across the cell membrane. The mechanism of uptake of CPP-cargo complexes and the uptake of CPPs alone across the plasma membrane remains unresolved. As the world of CPPs is rapidly advancing in both mammalian and plant system, there is a promising future for the various applications of transduction and transfection into intact cells.

Translocation of cell-penetrating peptides and delivery of their cargoes in triticale microspores.

Microspore culture is contributing significantly in the field of plant breeding for crop improvement in general and cereals, in particular. In the present study, we investigated the uptake of fluorescently labeled cell-penetrating peptides (CPP; Tat, Tat(2), M-Tat, peptide vascular endothelial-cadherin, transportan) in the freshly isolated triticale microspores (mid-late uninucleate stage). We demonstrated that Tat (RKKRRQRRR) and Tat(2) (RKKRRQRRRRKKRRQRRR) are able to efficiently transduce GUS enzyme (272 kDa) in its functional form in 5 and 14% of the microspores, respectively, in a noncovalent manner. Pep-1, a synthetic CPP, was able to transduce GUS enzyme in its active form in 31% of the microspores. The effect of various endocytic and macropinocytic inhibitors on Tat(2)-mediated GUS enzyme delivery was studied and revealed a preferred micropinocytosis entry. DNase I protection assay and confocal laser microscopy was carried out to recommend a ratio of 4:1 Tat(2)-linear plasmid DNA (pActGUS) in complex preparation for microspore transfection. We further show that Tat(2) can successfully deliver GUS gene in near to 2% triticale microspores. The negative control mutated Tat (M-Tat: AKKRRQRRR) failed to transducer the GUS protein and transfect the GUS gene in microspore nucleus. The ability of CPPs to deliver macromolecules (protein as well as linear plasmid DNA) noncovalently has been demonstrated in triticale isolated microspores. It further confirms potential applications of CPPs in developing simple, time saving, cost effective plant genetic engineering technologies.

Study of uptake of cell penetrating peptides and their cargoes in permeabilized wheat immature embryos.

The uptake of five fluorescein labeled cell-penetrating peptides (Tat, Tat(2), mutated-Tat, peptide vascular endothelial-cadherin and transportan) was studied in wheat immature embryos. Interestingly, permeabilization treatment of the embryos with toluene/ethanol (1 : 20, v/v with permeabilization buffer) resulted in a remarkably higher uptake of cell-penetrating peptides, whereas nonpermeabilized embryos failed to show significant cell-penetrating peptide uptake, as observed under fluorescence microscope and by fluorimetric analysis. Among the cell-penetrating peptides investigated, Tat monomer (Tat) showed highest fluorescence uptake (4.2-fold greater) in permeabilized embryos than the nonpermeabilized embryos. On the other hand, mutated-Tat serving as negative control did not show comparable fluorescence levels even in permeabilized embryos. A glucuronidase histochemical assay revealed that Tat peptides can efficiently deliver functionally active beta-glucuronidase (GUS) enzyme in permeabilized immature embryos. Tat(2)-mediated GUS enzyme delivery showed the highest number of embryos with GUS uptake (92.2%) upon permeabilization treatment with toluene/ethanol (1 : 40, v/v with permeabilization buffer) whereas only 51.8% of nonpermeabilized embryos showed Tat(2)-mediated GUS uptake. Low temperature, endocytosis and macropinocytosis inhibitors reduced delivery of the Tat(2)-GUS enzyme cargo complex. The results suggest that more than one mechanism of cell entry is involved simultaneously in cell-penetrating peptide-cargo uptake in wheat immature embryos. We also studied Tat(2)-plasmid DNA (carrying Act-1GUS) complex formation by gel retardation assay, DNaseI protection assay and confocal laser microscopy. Permeabilized embryos transfected with Tat(2)-plasmid DNA complex showed 3.3-fold higher transient GUS gene expression than the nonpermeabilized embryos. Furthermore, addition of cationic transfecting agent Lipofectamine 2000 to the Tat(2)-plasmid DNA complex resulted in 1.5-fold higher transient GUS gene expression in the embryos. This is the first report demonstrating translocation of various cell-penetrating peptides and their potential to deliver macromolecules in wheat immature embryos in the presence of a cell membrane permeabilizing agent.

Cellular uptake of cell-penetrating peptides pVEC and transportan in plants.

Internalization of fluorescently labeled CPPs, pVEC, transportan and scrambled pVEC, in a range of plant cells was investigated. Cellular uptake of the peptides was found to be tissue dependent. pVEC and transportan were distinctly internalized in triticale mesophyll protoplasts, onion epidermal cells, leaf bases and root tips of seven-day old triticale seedlings but showed negligible florescence in coleoptile and leaf tips as observed under a fluorescence microscope. Further, pVEC and transportan uptake studies were focused on mesophyll protoplasts as a system of investigation. In fluorimetric studies transportan showed 2.3 times higher cellular internalization than pVEC in protoplasts, whereas scrambled pVEC failed to show any significant fluorescence. Effect of various factors on cellular internalization of pVEC and transportan in protoplasts was also investigated. The cellular uptake of both the peptides was concentration dependent and nonsaturable. The cellular uptake of pVEC and transportan was enhanced at low temperature (4 degrees C). The presence of endocytic/macropinocytosis inhibitors did not reduce the cellular uptake of the peptides, suggesting direct cell penetration, receptor-independent internalization of pVEC and transportan into the plant cells.