Landscape phages and their fusion proteins targeted to breast cancer cells

Breast cancer is a leading cause of death among women in the USA. The efficacy of existing anticancer therapeutics can be improved by targeting them through conjugation with ligands binding to cellular receptors. Recently, we developed a novel drug targeting strategy based on the use of pre-selected cancer-specific 'fusion pVIII proteins' (fpVIII), as targeting ligands. To study the efficiency of this approach in animal models, we developed a panel of breast cancer cell-binding phages as a source of targeted fpVIIIs. Two landscape phage peptide libraries (8-mer f8/8 and 9-mer f8/9) were screened to isolate 132 phage variants that recognize breast carcinoma cells MCF-7 and ZR-75-1 and internalize into the cells. When tested for their interaction with the breast cancer cells in comparison with liver cancer cells HepG2, human mammary cells MCF-10A cells and serum, 16 of the phage probes selectively interacted with the breast cancer cells whereas 32 bound both breast and liver cancer cells. The most prominent cancer-specific phage DMPGTVLP, demonstrating sub-nanomolar Kd in interaction with target cells, was used for affinity chromatography of cellular membrane molecules to reveal its potential binding receptor. The isolated protein was identified by direct sequencing as cellular surface nucleolin. This conclusion was confirmed by inhibition of the phage-cell interaction with nucleolin antibodies. Other prominent phage binders VPTDTDYS, VEEGGYIAA, and DWRGDSMDS demonstrate consensus motifs common to previously identified cancer-specific peptides. Isolated phage proteins exhibit inherent binding specificity towards cancer cells, demonstrating the functional activity of the selected fused peptides. The selected phages, their peptide inserts and intact fusion proteins can serve as promising ligands for the development of targeted nanomedicines and their study in model mice with xenograft of human cells MCF-7 and ZR-75-1.     

Cover Feature: Where in the Cell Is our Cargo? Methods Currently Used To Study Intracellular Cytosolic Localisation (ChemBioChem 4/2019)

The internalisation and delivery of active substances into cells is a field of growing interest for chemical biology and therapeutics. As we move from small-molecule-based drugs towards bigger cargos, such as antibodies, enzymes, nucleases or nucleic acids, the development of efficient delivery systems becomes critical for their practical application. Different strategies and synthetic carriers have been developed; these include cationic lipids, gold nanoparticles, polymers, cell-penetrating peptides (CPPs), protein surface modification etc. However, all of these methodologies still present limitations relating to the precise targeting of the different intracellular compartments and, in particular, difficulties in access to the cellular cytosol. Additionally, the precise quantification of the cellular uptake of a compound is not enough to demonstrate delivery and/or functional activity. Therefore, methods to determine cellular distributions of cargos and carriers are of critical importance for identifying the barriers that are blocking the activity. Herein we survey the different techniques that can currently be used to track and to monitor the subcellular localisation of the synthetic compounds that we deliver inside cells.     

Cytotoxicity of human RNase-based immunotoxins requires cytosolic access and resistance to ribonuclease inhibition

Immunotoxins are targeted therapeutics designed to kill cancer cells. The targeting moiety of an immunotoxin selectively binds to a tumor cell and targets it for death via an attached toxin. Because the toxins are typically of plant or bacterial origin, their clinical use is limited by immunogenicity and nonspecific toxicity. To circumvent these problems, we have begun to engineer immunotoxins containing human pancreatic ribonuclease. Here we describe the generation of ribonuclease mutants designed to evade a ubiquitous cytosolic inhibitor that would otherwise block cytotoxicity. Two mutants retained catalytic activity and were relatively resistant to the inhibitor. To deliver them to human T leukemic cells, these ribonuclease variants were fused to a single chain Fv fragment specific for CD 7.The ribonuclease-sFv fusion proteins bound CD 7(+) T cells and were internalized yet were not cytotoxic. Transfection of the proteins directly into the cytosol reduced cell viability, suggesting that the failure of the immunotoxins to kill cells when added externally resulted from the inability of the ribonuclease moiety to access the cytosol efficiently. Our results indicate appropriate intracellular routing, as well as resistance to inhibition, is critical to the cytotoxicity of human ribonuclease-based immunotoxins.     

The Ubiquitin System: An Emerging Therapeutic Target for Lung Cancer

The ubiquitin system, present in all eukaryotes, contributes to regulating multiple types of cellular protein processes such as cell signaling, cell cycle, and receptor trafficking, and it affects the immune response. In most types of cancer, unusual events in ubiquitin-mediated signaling pathway modulation can lead to a variety of clinical outcomes, including tumor formation and metastasis. Similarly, ubiquitination acts as a core component, which contributes to the alteration of cell signaling activity, dictating biosignal turnover and protein fates. As lung cancer acquires the most commonly mutated proteins, changes in the ubiquitination of the proteins contribute to the development of lung cancer. Various inhibitors targeting the ubiquitin system have been developed for clinical applications in lung cancer treatment. In this review, we summarize the current research advances in therapeutics for lung cancer by targeting the ubiquitin system.     

Proteome editing using engineered proteins that hijack cellular quality control machinery

Protein silencing is an important aspect of both scientific investigation of native protein function and therapeutic targeting of aberrant protein activity. Many techniques for silencing proteins at the DNA or RNA level exist such as CRISPR, RNAi, or TALEN. Cellular proteins can also be selectively removed at the posttranslational level using proteome editing techniques, many of which employ engineered proteins to engage natural cellular quality control machinery for accelerating the removal of otherwise stable proteins. Here, we summarize recent progress in the development of such engineered proteins, comparing them to analogous microbial-, viral-, and small molecule-based strategies for selectively degrading proteins of interest. Finally, we highlight the advantages and limitations of proteome editing technologies and discuss how the application of directed evolution could alleviate current challenges and usher in a new wave of designer proteins for diagnostic and therapeutic application.     

Expression of intein‐tagged fusion protein and its applications in downstream processing

The conventional methods of downstream purification of a recombinant protein are not only complicated and delicate but time consuming, and need to be improved. Since the intein, the protein splicing element, was discovered, this self‐cleaving element has been exploited and applied to the purification of recombinant proteins which could significantly simplify the purification procedure. Intein has the unique property that when it is combined with an affinity tag, it enables a target protein to be purified in a single chromatographic step. This review elucidates the properties of intein (the mechanism that unravels the intein‐based protein splicing), the advantages of an intein affinity expression system, the progress of intein‐based protein purification procedures, and recent advances in the applications of intein. Further development of the intein‐based purification system may lead to the applications of this system to industrial‐scale production of recombinant proteins. Copyright © 2009 Society of Chemical Industry     

Extracellular Vesicles as Potential Therapeutics for Inflammatory Diseases

Extracellular vesicles (EV) deliver cargoes such as nucleic acids, proteins, and lipids between cells and serve as an intercellular communicator. As it is revealed that most of the functions associated to EVs are closely related to the immune response, the important role of EVs in inflammatory diseases is emerging. EVs can be functionalized through EV surface engineering and endow targeting moiety that allows for the target specificity for therapeutic applications in inflammatory diseases. Moreover, engineered EVs are considered as promising nanoparticles to develop personalized therapeutic carriers. In this review, we highlight the role of EVs in various inflammatory diseases, the application of EV as anti-inflammatory therapeutics, and the current state of the art in EV engineering techniques.     

Engineered pH-inducible intein Mtu ΔI-CM variants with markedly reduced premature cleavage activity

Inteins have been widely exploited for the purification of tagless proteins. Among them, pH-inducible C-terminal-cleavage inteins enable the preparation of proteins and peptides with an authentic N-terminus. However, a severe premature cleavage around neutral pH has limited the application of these inteins, especially when used in recombinant hosts such as Escherichia coli. By targeting the microenvironment of the two key histidine residues H429 and H439, we engineered Mtu ΔI-CM intein to markedly reduce its premature cleavage. Kinetic analyses suggested that although the variants retained the pH dependence, they indeed cleaved slower, especially at pH 7.6. These variants resulted in higher yields for two model polypeptides than the original Mtu ΔI-CM intein, when used in conjunction with a cleavable self-assembling tag. This work suggests that more controllable pH-inducible inteins can be obtained by manipulating the residues in the self-cleavage sites and provide better performance for tag-based protein preparation strategies.     

Site-specific chemical modification of proteins with a prelabelled cysteine tag using the artificially split Mxe GyrA intein

The selective modification of proteins with a synthetic probe is of central interest for many aspects of protein chemistry. We have recently reported a new approach in which a short cysteine-containing tag (CysTag) fused to one part of a split intein is first modified with a sulfhydryl-reactive probe. In a second step, protein trans-splicing is used to link the labelled CysTag to a target protein that has been expressed in fusion with the complementary split intein fragment. Here, we present the generation and biochemical characterisation of the artificially split Mycobacterium xenopi GyrA intein. We show that this split intein is active without a renaturation step and that it provides a significant improvement for the CysTag protein-labelling approach in terms of product yields and target protein tolerance. Two proteins with multiple cysteine residues, human growth hormone and a multidomain nonribosomal peptide synthetase, were site-specifically modified with high yields. Our approach combines the benefits of the plethora of commercially available cysteine-reactive probes with a straightforward route for their site-specific incorporation even into complex and cysteine-rich proteins.     

Computing Metal-Binding Proteins for Therapeutic Benefit

Over one third of biomolecules rely on metal ions to exert their cellular functions. Metal ions can play a structural role by stabilizing the structure of biomolecules, a functional role by promoting a wide variety of biochemical reactions, and a regulatory role by acting as messengers upon binding to proteins regulating cellular metal-homeostasis. These diverse roles in biology ascribe critical implications to metal-binding proteins in the onset of many diseases. Hence, it is of utmost importance to exhaustively unlock the different mechanistic facets of metal-binding proteins and to harness this knowledge to rationally devise novel therapeutic strategies to prevent or cure pathological states associated with metal-dependent cellular dysfunctions. In this compendium, we illustrate how the use of a computational arsenal based on docking, classical, and quantum-classical molecular dynamics simulations can contribute to extricate the minutiae of the catalytic, transport, and inhibition mechanisms of metal-binding proteins at the atomic level. This knowledge represents a fertile ground and an essential prerequisite for selectively targeting metal-binding proteins with small-molecule inhibitors aiming to (i) abrogate deregulated metal-dependent (mis)functions or (ii) leverage metal-dyshomeostasis to selectively trigger harmful cells death.     

Direct Intracellular Delivery of Cell‐Impermeable Probes of Protein Glycosylation by Using Nanostraws

Bioorthogonal chemistry is an effective tool for elucidating metabolic pathways and measuring cellular activity, yet its use is currently limited by the difficulty of getting probes past the cell membrane and into the cytoplasm, especially if more complex probes are desired. Here we present a simple and minimally perturbative technique to deliver functional probes of glycosylation into cells by using a nanostructured “nanostraw” delivery system. Nanostraws provide direct intracellular access to cells through fluid conduits that remain small enough to minimize cell perturbation. First, we demonstrate that our platform can deliver an unmodified azidosugar, N‐azidoacetylmannosamine, into cells with similar effectiveness to a chemical modification strategy (peracetylation). We then show that the nanostraw platform enables direct delivery of an azidosugar modified with a charged uridine diphosphate group (UDP) that prevents intracellular penetration, thereby bypassing multiple enzymatic processing steps. By effectively removing the requirement for cell permeability from the probe, the nanostraws expand the toolbox of bioorthogonal probes that can be used to study biological processes on a single, easy‐to‐use platform.     

A suspension cell-based interaction platform for interrogation of membrane proteins

The majority of clinically approved therapeutics target membrane proteins (MPs), highlighting the need for tools to study this important category of proteins. To overcome limitations with recombinant MP expression, whole cell screening techniques have been developed that present MPs in their native conformations. Whereas many such platforms utilize adherent cells, here we introduce a novel suspension cell-based platform termed “biofloating” that enables quantitative analysis of interactions between proteins displayed on yeast and MPs expressed on mammalian cells, without need for genetic fusions. We characterize and optimize biofloating and illustrate its sensitivity advantage compared to an adherent cell-based platform (biopanning). We further demonstrate the utility of suspension cell-based approaches by iterating rounds of magnetic-activated cell sorting selections against MP-expressing mammalian cells to enrich for a specific binder within a yeast-displayed antibody library. Overall, biofloating represents a promising new technology that can be readily integrated into protein discovery and development workflows.     

Synthesis of cationic magnetite nanoparticles for intracellular protein delivery

Intracellular protein delivery shows great promise in the treatment of various diseases. However, therapeutic applications of this method are limited by its low delivery efficiency and poor targeting ability. As one of most important drug delivery cargoes, Fe₃O₄ nanoparticles (nFe₃O₄) have attracted much attention for both therapeutic and diagnostic applications, especially for targeting drug delivery. To use nFe₃O₄ for protein delivery, a simple but effective modification of nFe₃O₄ is critical to attach proteins on its surface. In this work, by designing and synthesizing cationic poly(2‐(dimethylamino)ethyl methacrylate) (PDMA)‐grafted nFe₃O₄ via in situ atom transfer radical polymerization (ATRP), we demonstrate a simple solution to improve interactions between nFe₃O₄ and proteins. With the grafted PDMA on the surface, nFe₃O₄ exhibits not only significant enhancement in dispersibility and stability in aqueous phase, but also an excellent capability to attach negative‐charged proteins. Moreover, with the assistance of external magnetic field, PDMA‐grafted nFe₃O₄ can be used as a targetable vector to deliver proteins into specific cells. This work provides a novel platform based on cationic magnetite nanoparticles that can deliver therapeutic proteins into specific sites for the treatment of various diseases. © 2013 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014 , 131, 40260.     

Design,Synthesis and Bioevaluation of an EphA2 Receptor‐Based Targeted Delivery System

Because of its overexpression in a range of solid tumors, the EphA2 receptor is a validated target for cancer therapeutics. We recently described a new targeted delivery system based on specific EphA2‐targeting peptides conjugated with the chemotherapeutic agent paclitaxel. Here, we investigate the chemical determinants responsible for the stability and degradation of these agents in plasma. Introducing modifications in both the peptide and the linker between the peptide and paclitaxel resulted in drug conjugates that are both long‐lived in rat plasma and that markedly decrease tumor size in a prostate cancer xenograft model compared with paclitaxel alone treatment. These studies identify critical rate‐limiting degradation sites on the peptide–drug conjugates, enabling the design of agents with increased stability and efficacy. These results provide support for our central hypothesis that peptide–drug conjugates targeting EphA2 represent an innovative and potentially effective strategy to selectively deliver cytotoxic drugs to cancer cells.     

Live-Cell Copper-Induced Fluorescence Quenching of the Flavin-Binding Fluorescent Protein CreiLOV

CreiLOV is a flavin-binding fluorescent protein derived from the blue-light photoreceptor protein family that contains light-oxygen-voltage (LOV) sensing domains. Flavin-binding fluorescent proteins represent a promising foundation for new fluorescent reporters and biosensors that can address limitations of the well-established green fluorescent protein (GFP) family. Flavin-binding fluorescent proteins are smaller than GFPs, are stable over a wider pH range, offer rapid chromophore incorporation, and are oxygen-independent so can be applied to live anaerobic organisms. Among the flavin-binding fluorescent proteins, CreiLOV has a high quantum yield and excellent photophysical properties, making it promising for cellular applications. Here, we investigated the suitability of CreiLOV as an intensity- and fluorescence-lifetime-based metal sensor. CreiLOV selectively binds copper(II) over other biologically relevant metals with low-micromolar affinity, resulting in fluorescence quenching and a decrease in the fluorescence lifetime that can be observed in cuvettes and live bacterial cells.     

Chimeras of the homing endonuclease PI-SceI and the homologous Candida tropicalis intein: a study to explore the possibility of exchanging DNA-binding modules to obtain highly specific endonucleases with altered specificity

Homing endonucleases are extremely specific endodeoxyribonucleases. In vivo, these enzymes confer mobility on their genes by inducing a very specific double-strand cut in cognate alleles that lack the cooling sequence for the homing endonuclease; the cellular repair of the double-strand break with the endonuclease-containing allele as a template leads to integration of the endonuclease gene, completing the homing process. As a result of their extreme sequence specificity, homing endonucleases are promising tools for genome engineering. For this purpose, it is desirable to design enzymes with defined new specificities. To analyse which DNA-binding elements are potential candidates for use in the design of enzymes with modified or even new specificity, we produced several chimeric proteins derived from the Saccharomyces cerevisiae VMA1 intein (PI-SceI) and the related Candida tropicalis VMA1 intein. Although the mature Candida intein is devoid of endonucleolytic activity, the exchange of two DNA-binding modules of PI-SceI with the homologous elements from the Candida intein results in an active endonuclease. The low sequence homology in these modules indicates that different protein-DNA contacts are responsible for the recognition of related DNA sequences. This flexibility in DNA recognition should, in principle, allow endonucleases to be produced with new specificities useful for genome engineering.     

Supramolecular Bioconjugates for Protein and Small Drug Delivery

Supramolecular conjugation techniques have been developed to produce novel nanosized systems by assembling materials with diverse physicochemical and biological features. These techniques have been adapted to obtain innovative bioconjugates to deliver drugs with poor biopharmaceutical properties and nano-devices with potential “theranostic” activity. Supramolecular drug delivery systems include polymer therapeutics such as drug–polymer bioconjugates, and colloidal carriers such as micelles, liposomes, polyplexes, and organic and inorganic nanoparticles. By virtue of their wide array of chemical composition and properties, polymers represent key elements for the construction of novel supramoelcular formulations. Polymer bioconjugation is a fledged technique for fabrication of protein–polymer conjugates. PEGylation, in particular, produces derivatives with enhanced pharmacokinetic, immunological, and stability properties as compared to the parent protein. Over the years, new methods have been set up to obtain site-directed polymer conjugation. In this review we report few grafting to and growing from PEGylation examples for the preparation of therapeutically effective protein bioconjugates. Supramolecular formulations with unique properties can be also obtained by assembling functional polymers, targeting agents, physicochemical modifiers, and biomodulators. These systems may be designed for disease tissue disposition and cell recognition/penetration. Cyclodextrins, for example, have been functionalized with polyethylene glycol and folic acid to produce tumor-targeted drug carriers. Interesting results have been obtained with this novel class of drug delivery systems. In addition, responsive polymers have been conjugated to gold nanoparticles to endow a new colloidal platform with triggerable cell disposition properties, which can be exploited either in biomedicine or diagnosis.     

Lipoprotein-inspired nanoparticles for cancer theranostics

Over hundreds of millions of years, animals have evolved endogenous lipoprotein nanoparticles for shuttling hydrophobic molecules to different parts of the body. In the last 70 years, scientists have developed an understanding of lipoprotein function, often in relationship to lipid transport and heart disease. Such biocompatible, lipid-protein complexes are also ideal for loading and delivering cancer therapeutic and diagnostic agents, which means that lipoprotein and lipoprotein-inspired nanoparticles also offer opportunities for cancer theranostics. By mimicking the endogenous shape and structure of lipoproteins, the nanocarrier can remain in circulation for an extended period of time, while largely evading the reticuloendothelial cells in the body's defenses. The small size (less than 30 nm) of the low-density (LDL) and high-density (HDL) classes of lipoproteins allows them to maneuver deeply into tumors. Furthermore, lipoproteins can be targeted to their endogenous receptors, when those are implicated in cancer, or to other cancer receptors. In this Account, we review the field of lipoprotein-inspired nanoparticles related to the delivery of cancer imaging and therapy agents. LDL has innate cancer targeting potential and has been used to incorporate diverse hydrophobic molecules and deliver them to tumors. Nature's method of rerouting LDL in atherosclerosis provides a strategy to extend the cancer targeting potential of lipoproteins beyond its narrow purview. Although LDL has shown promise as a drug nanocarrier for cancer imaging and therapy, increasing evidence indicates that HDL, the smallest lipoprotein, may also be of use for drug targeting and uptake into cancer cells. We also discuss how synthetic HDL-like nanoparticles, which do not include human or recombinant proteins, can deliver molecules directly to the cytoplasm of certain cancer cells, effectively bypassing the endosomal compartment. This strategy could allow HDL-like nanoparticles to be used to deliver drugs that have increased activity in the cytoplasm. Lipoprotein nanoparticles have evolved to be ideal delivery vehicles, and because of that specialized function, they have the potential to improve cancer theranostics.     

A TAT-streptavidin fusion protein directs uptake of biotinylated cargo into mammalian cells

The HIV-1 TAT peptide has been used extensively for directing the intracellular delivery of an assortment of cargo, including DNA, liposomes and macromolecules. For protein delivery, a variety of TAT-fusion proteins have been described which link the TAT coding sequence to the protein coding sequence of interest. Streptavidin represents a potentially useful TAT-fusion protein because it could be used to deliver a wide array of biotinylated cargo. Here we have characterized a TAT-streptavidin (TAT-SA) fusion protein, which retains the ability to bind biotinylated cargo while directing their efficient cellular uptake. Fluorescence activated cell sorting (FACS) analysis and confocal microscopy characterization showed that TAT-SA is internalized by Jurkat T-cells and NIH 3T3 cells alone and when complexed to phycoerythrin, whereas the native streptavidin is not. Additionally, biotinylated alkaline phosphatase is successfully internalized and retains its activity when complexed to TAT-SA and incubated with Jurkat T-cells. Confocal microscopy suggested, however, that internalized TAT-SA and TAT-SA complexes were largely compartmentalized in vesicular compartments, rather than freely diffusing in the cytoplasmic compartment. To effect cytoplasmic delivery, the endosomal releasing polymer, poly(propylacrylic acid) (PPAA), was biotinylated and complexed to TAT-SA. Endosomal release and cytoplasmic delivery of fluorescently labeled TAT-SA complexes with PPAA was shown by the diffuse distribution of fluorescent protein in the cytoplasm. Taken together, these results demonstrate that TAT-SA can be used to direct intracellular delivery of large biotinylated cargo to intracellular compartments and that biotinylated PPAA can direct cytoplasmic delivery where desired.