Positional Scanning Synthesis of a Peptoid Library Yields New Inducers of Apoptosis that Target Karyopherins and Tubulin

We describe the synthesis of a library of 11 638 N‐alkylglycine peptoid trimers in a positional scanning format with adjustment of reaction conditions to account for different reactivities of the monomer building blocks. Evaluation of the library by high‐content phenotypic screening for modulators of the cytoskeleton and mitosis resulted in the identification of two apoptosis‐inducing peptoids, which, despite their structural similarity, target different proteins and cellular mechanisms. Whereas one peptoid binds to karyopherins, which mediate nuclear transport, the other N‐alkylglycine trimer binds tubulin at the vinca alkaloid binding site.     

1,2,3‐Triazolium‐Based Cationic Amphipathic Peptoid Oligomers Mimicking Antimicrobial Helical Peptides

Amphipathic cationic peptoids (N‐substituted glycine oligomers) represent a promising class of antimicrobial peptide mimics. The aim of this study is to explore the potential of the triazolium group as a cationic moiety and helix inducer to develop potent antimicrobial helical peptoids. Herein we report the first solid‐phase synthesis of peptoid oligomers incorporating 1,2,3‐triazolium‐type side chains and their evaluation against Escherichia coli, Enterococcus faecalis, and Staphylococcus aureus. Several triazolium‐based oligomers, even of short length, selectively kill bacteria over mammalian cells. SEM visualization of S. aureus cells treated with a dodecamer and a hexamer reveals severe cell membrane damage and suggests that the longer oligomer acts by pore formation.     

Using the peptide BP100 as a cell-penetrating tool for the chemical engineering of actin filaments within living plant cells

The delivery of externally applied macromolecules or nanoparticles into living cells still represents a critically limiting step before the full capabilities of chemical engineering can be explored. Molecular transporters such as cell-penetrating peptides, peptoids, and other mimetics can be used to carry cargo across the cellular membrane, but it is still difficult to find suitable sequences that operate efficiently for any particular type of cell. Here we report that BP100 (KKLFKKILKYL-amide), originally designed as an antimicrobial peptide against plant pathogens, can be employed as a fast and efficient cell-penetrating agent to transport fluorescent test cargoes into the cytosol of walled plant cells. The uptake of BP100 proceeds slightly more slowly than the endocytosis of fluorescent dextranes, but BP100 accumulates more efficiently and to much higher levels (by an order of magnitude). The entry of BP100 can be efficiently blocked by latrunculin B; this suggests that actin filaments are essential to the uptake mechanism. To test whether this novel transporter can also be used to deliver functional cargoes, we designed a fusion construct of BP100 with the actin-binding Lifeact peptide (MGVADLIKKFESISKEE). We demonstrated that the short BP100 could transport the attached 17-residue sequence quickly and efficiently into tobacco cells. The Lifeact construct retained its functionality as it successfully labeled the actin bundles that tether the nucleus in the cell center.     

Chivosazoles A and F,Cytostatic Macrolides from Myxobacteria,Interfere with Actin

Chivosazoles A and F, isolated from Sorangium cellulosum, showed high antiproliferative activity with different mammalian cell lines including human cancer cells. The chivosazoles caused a delay in G2/M phase of the cell cycle, and treated cells often contained two nuclei. By labeling F‐actin it was shown that the actin cytoskeleton of the cells starts to break down after a few minutes of treatment. In vitro polymerization assays with purified G‐actin revealed that the chivosazoles inhibit actin polymerization and also cause a depolymerization of pyrene‐labeled F‐actin microfilaments prepared in vitro. Chivosazoles are new tools for the investigation of issues concerning the actin cytoskeleton and they have a different mode of action from the known microfilament‐disrupting compounds like rhizopodin and cytochalasin D.     

A comparison of linear and cyclic peptoid oligomers as potent antimicrobial agents

We investigated the antimicrobial activities of N-substituted glycine "peptoid" oligomers incorporating cationic and hydrophobic side chains. Head-to-tail macrocyclization was employed to enhance antimicrobial activity. Both linear and cyclic peptoids, ranging from six to ten residues, demonstrate potent antimicrobial activity against Gram-positive and Gram-negative bacteria. These peptoids do not cause significant lysis of human erythrocytes, indicating selective antimicrobial activity. Conformational ordering established upon macrocyclization is generally associated with an enhanced capacity to inhibit bacterial cell growth. Moreover, increased hydrophobic surface area also plays a role in improving antimicrobial activity. We demonstrate the potency of a cyclic peptoid in exerting antimicrobial activity against clinical strains of S. aureus while deterring the emergence of antimicrobial resistance.     

Hypoxia Modulates Cellular Endocytic Pathways and Organelles with Enhanced Cell Migration and 3D Cell Invasion**

Hypoxia, a decrease in cellular or tissue level oxygen content, is characteristic of most tumors and has been shown to drive cancer progression by altering multiple subcellular processes. We hypothesized that the cancer cells in a hypoxic environment might have slower proliferation rates and increased invasion and migration rates with altered endocytosis compared to the cancer cells in the periphery of the tumor mass that experience normoxic conditions. We induced cellular hypoxia by exposing cells to cobalt chloride, a chemical hypoxic mimicking agent. This study measured the effect of hypoxia on cell proliferation, migration, and invasion. Uptake of fluorescently labeled transferrin, galectin3, and dextran that undergo endocytosis through major endocytic pathways (Clathrin-mediated pathway (CME), Clathrin-independent pathway (CIE), Fluid phase endocytosis (FPE)) were analyzed during hypoxia. Also, the organelle changes associated with hypoxia were studied with organelle trackers. We found that the proliferation rate decreased, and the migration and invasion rate increased in cancer cells in hypoxic conditions compared to normoxic cancer cells. A short hypoxic exposure increased galectin3 uptake in hypoxic cancer cells, but a prolonged hypoxic exposure decreased clathrin-independent endocytic uptake of galectin 3. Subcellular organelles, such as mitochondria, increased to withstand the hypoxic stress, while other organelles, such as Endoplasmic reticulum (ER), were significantly decreased. These data suggest that hypoxia modulates cellular endocytic pathways with reduced proliferation and enhanced cell migration and invasion.     

冷冻保存中影响胞内冰生长的因素

细胞胞内冰的形成会导致严重的细胞损伤从而导致低温贮存中的诸多问题。以蚕豆为研究对象,用细胞松弛素B溶解细胞骨架,使用低温显微系统在不同的冷却速率下进行冷冻实验。实验结果表明,使用细胞松弛素B处理过的细胞在冷冻过程中结晶温度更高,结晶时间更短,但细胞骨架对胞内冰的生长过程影响较小。外界条件起着关键作用,接种冰晶影响细胞内冰晶的形成温度及冰晶的生长速率。最后,通过光强度图对细胞的损伤程度进行了分析。     

Cationic DOPC–Detergent Conjugates for Safe and Efficient in Vitro and in Vivo Nucleic Acid Delivery

The ability of a nonviral nucleic acid carrier to deliver its cargo to cells with low associated toxicity is a critical issue for clinical applications of gene therapy. We describe biodegradable cationic DOPC–C₁₂E₄ conjugates in which transfection efficiency is based on a Trojan horse strategy. In situ production of the detergent compound C₁₂E₄ through conjugate hydrolysis within the acidic endosome compartment was expected to promote endosome membrane destabilization and subsequent release of the lipoplexes into cytosol. The transfection efficiency of the conjugates has been assessed in vitro, and associated cytotoxicity was determined. Cellular uptake and intracellular distribution of the lipoplexes have been investigated. The results show that direct conjugation of DOPC with C₁₂E₄ produces a versatile carrier that can deliver both DNA and siRNA to cells in vitro with high efficiency and low cytotoxicity. SAR studies suggest that this compound might represent a reasonable compromise between the membrane activity of the released detergent and susceptibility of the conjugate to degradation enzymes in vitro. Although biodegradability of the conjugates had low impact on carrier efficiency in vitro, it proved critical in vivo. Significant improvement of transgene expression was obtained in the mouse lung tuning biodegradability of the carrier. Importantly, this also allowed reduction of the inflammatory response that invariably characterizes cationic‐lipid‐mediated gene transfer in animals.     

Cooperative, heparan sulfate-dependent cellular uptake of dimeric guanidinoglycosides

Oligoarginine and guanidinium-rich molecular transporters have been shown to facilitate the intracellular delivery of a diverse range of biologically relevant cargos. Several such transporters have been suggested to interact with cell-surface heparan sulfate proteoglycans as part of their cell-entry pathway. Unlike for other guanidinium-rich transporters, the cellular uptake of guanidinoglycosides at nanomolar concentrations is exclusively heparan sulfate dependent. As distinct cells differ in their expression levels and/or the composition of cell-surface heparan sulfate proteoglycans, one might be able to exploit such differences to selectively target certain cell types. To systematically investigate the nature of their cell-surface interactions, monomeric and dimeric guanidinoglycosides were synthesized by using neomycin, paromomycin, and tobramycin as scaffolds. These transporters differ in the number and 3D arrangement of their guanidinium groups. Their cellular uptake was measured by flow cytometry in wild-type and mutant Chinese hamster ovary cells after the corresponding fluorescent streptavidin-phycoerythrin-Cy5 conjugates had been generated. All derivatives showed negligible uptake in mutant cells lacking heparan sulfate. Decreasing the number of guanidinium groups diminished uptake, but the three dimensional arrangement of these groups was less important for cellular delivery. Whereas conjugates prepared with the monomeric carriers showed significantly reduced uptake in mutant cells expressing heparan sulfate chains with altered patterns of sulfation, conjugates prepared with the dimeric guanidinoglycosides could overcome this deficiency and maintain high levels of uptake in such deficient cells. This finding suggests that cellular uptake depends on the valency of the transporter and both the content and arrangement of the sulfate groups on the cell-surface receptors. Competition studies with chemically desulfated or carboxy-reduced heparin derivatives corroborated these observations. Taken together, these findings show that increasing the valency of the transporters retains heparan sulfate specificity and provides reagents that could distinguish different cell types based on the specific composition of their cell-surface heparan sulfate proteoglycans.     

Synthesis and Biological Evaluation of Fluorescent Bryostatin Analogues

To investigate the cellular distribution of tumor‐promoting vs. non‐tumor‐promoting bryostatin analogues, we synthesized fluorescently labeled variants of two bryostatin derivatives that have previously shown either phorbol ester‐like or bryostatin‐like biological activity in U937 leukemia cells. These new fluorescent analogues both displayed high affinity for protein kinase C (PKC) binding and retained the basic properties of the parent unlabeled compounds in U937 assays. The fluorescent compounds showed similar patterns of intracellular distribution in cells, however; this argues against an existing hypothesis that various patterns of intracellular distribution are responsible for differences in biological activity. Upon further characterization, the fluorescent compounds revealed a slow rate of cellular uptake; correspondingly, they showed reduced activity for cellular responses that were only transient upon treatment with phorbol ester or bryostatin 1.     

Accessible Synthetic Probes for Staining Actin inside Platelets and Megakaryocytes by Employing Lifeact Peptide

Lifeact is a 17‐residue peptide that can be employed in cell microscopy as a probe for F‐actin when fused to fluorescent proteins, but therefore is not suitable for all cell types. We have conjugated fluorescently labelled Lifeact to three different cell‐penetrating systems (a myristoylated carrier (myr), the pH low insertion peptide (pHLIP) and the cationic peptide TAT) as a strategy to deliver Lifeact into cells and developed new tools for actin staining with improved synthetic accessibility and low toxicity, focusing on their suitability in platelets and megakaryocytes. Using confocal microscopy, we characterised the cell distribution of the new hybrids in fixed cells, and found that both myr– and pHLIP–Lifeact conjugates provide efficient actin staining upon cleavage of Lifeact from the carriers, without affecting cell spreading. This new approach could facilitate the design of new tools for actin visualisation.     

A Moisture Transmembrane Transfer Model for Pore Network Simulation of Plant Materials Drying

The drying of plant materials with cellular tissue is often viewed as drying of porous media that is assumed to consist of cell cytoskeleton and intercellular space. Various approaches have been reported in the literature to describe heat and mass transfer during drying of such porous materials. However, the fact remains that the water in a cellular tissue is mostly intracellular and it should be driven out of the cells across cell membranes before transporting in cell gaps, as in a general porous media. In the present study, the transport process of moisture in a cellular tissue was analyzed. A mathematical model for moisture transport across the cell membrane was established, which was correlated to a self-developed, dual-scale pore network model (cell and pore network) for drying of plant materials. The relationship between mass volumetric flux and average intracellular moisture content was developed based on the microscopic images and the drying experiments.     

Peptoid-Peptide hybrid ligands targeting the polo box domain of polo-like kinase 1

We replaced the amino terminal Pro residue of the Plk1 polo-box-domain-binding pentapeptide (PLHSpT) with a library of N-alkyl-Gly "peptoids", and identified long-chain tethered phenyl moieties giving greater than two-orders-of-magnitude affinity enhancement. Further simplification by replacing the peptoid residue with appropriate amides gave low-nanomolar affinity N-acylated tetrapeptides. Binding of the N-terminal long-chain phenyl extension was demonstrated by X-ray co-crystal data.     

Kinetic analysis of cellular internalization and expulsion of unstructured D-chirality cell penetrating peptides

Most cell penetrating peptides (CPPs) are unstructured and susceptible to proteolytic degradation. One alternative is to incorporate D-chirality amino acids into unstructured CPPs to allow for enhanced uptake and intracellular stability. This work investigates CPP internalization using a series of time, concentration, temperature, and energy dependent studies, resulting in a three-fold increase in uptake and 50-fold increase in stability of D-chirality peptides over L-chirality counterparts. CPP internalization occurred via a combination of direct penetration and endocytosis, with a percentage of internalized CPP expelling from cells in a time-dependent manner. Mechanistic studies identified that cells exported the intact internalized D-chirality CPPs via an exocytosis independent pathway, analogous to a direct penetration method out of the cells. These findings highlight the potential of a D-chirality CPP as bio-vector in therapeutic and biosensing applications, but also identify a new expulsion method suggesting a relationship between uptake kinetics, intracellular stability, and export kinetics.     

Efficient intracellular delivery of nucleic acid pharmaceuticals using cell-penetrating peptides

Over the last 20 years, researchers have designed or discovered peptides that can permeate membranes and deliver exogenous molecules inside a cell. These peptides, known as cell-penetrating peptides (CPPs), typically consist of 6-30 residues, including HIV TAT peptide, penetratin, oligoarginine, transportan, and TP10. Through chemical conjugation or noncovalent complex formation, these structures successfully deliver bioactive and membrane-impermeable molecules into cells. CPPs have also gained attention as an attractive vehicle for the delivery of nucleic acid pharmaceuticals (NAPs), including genes/plasmids, short oligonucleotides, and small interference RNAs and their analogues, due to their high internalization efficacy, low cytotoxicity, and flexible structural design. In this Account, we survey the potential of CPPs for the design and optimization of NAP delivery systems. First, we describe the impact of the N-terminal stearylation of CPPs. Endocytic pathways make a major contribution to the cellular uptake of NAPs. Stearylation at the N-terminus of CPPs with stearyl-octaarginine (R8), stearyl-(RxR)(4), and stearyl-TP10 prompts the formation of a self-assembled core-shell nanoparticle with NAPs, a compact structure that promotes cellular uptake. Researchers have designed modifications such as the addition of trifluoromethylquinoline moieties to lysine residues to destabilize endosomes, as exemplified by PepFect 6, and these changes further improve biological responsiveness. Alternatively, stearylation also allows implantation of CPPs onto the surface of liposomes. This feature facilitates "programmed packaging" to establish multifunctional envelope-type nanodevices (MEND). The R8-MEND showed high transfection efficiency comparable to that of adenovirus in non-dividing cells. Understanding the cellular uptake mechanisms of CPPs will further improve CPP-mediated NAP delivery. The cellular uptake of CPPs and their NAP complex involves various types of endocytosis. Macropinocytosis, a mechanism which is also activated in response to stimuli such as growth factors or viruses, is a primary pathway for arginine-rich CPPs because high cationic charge density promotes this endocytic pathway. The use of larger endosomes (known as macropinosomes) rather than clathrin- or caveolae-mediated endocytosis has been reported in macropinocytosis which would also facilitate the endocytosis of NAP nanoparticles into cells.     

Extracellular Matrix Composition Modulates the Responsiveness of Differentiated and Stem Pancreatic Cancer Cells to Lipophilic Derivate of Gemcitabine

Background: Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal disease. Gemcitabine (GEM) is used as the gold standard drug in PDAC treatment. However, due to its poor efficacy, it remains urgent to identify novel strategies to overcome resistance issues. In this context, an intense stroma reaction and the presence of cancer stem cells (CSCs) have been shown to influence PDAC aggressiveness, metastatic potential, and chemoresistance. Methods: We used three-dimensional (3D) organotypic cultures grown on an extracellular matrix composed of Matrigel or collagen I to test the effect of the new potential therapeutic prodrug 4-(N)-stearoyl-GEM, called C18GEM. We analyzed C18GEM cytotoxic activity, intracellular uptake, apoptosis, necrosis, and autophagy induction in both Panc1 cell line (P) and their derived CSCs. Results: PDAC CSCs show higher sensitivity to C18GEM treatment when cultured in both two-dimensional (2D) and 3D conditions, especially on collagen I, in comparison to GEM. The intracellular uptake mechanisms of C18GEM are mainly due to membrane nucleoside transporters’ expression and fatty acid translocase CD36 in Panc1 P cells and to clathrin-mediated endocytosis and CD36 in Panc1 CSCs. Furthermore, C18GEM induces an increase in cell death compared to GEM in both cell lines grown on 2D and 3D cultures. Finally, C18GEM stimulated protective autophagy in Panc1 P and CSCs cultured on 3D conditions. Conclusion: We propose C18GEM together with autophagy inhibitors as a valid alternative therapeutic approach in PDAC treatment.     

Evaluation of Stable LifeAct-mRuby2- and LAMP1-NeonGreen Expressing A549 Cell Lines for Investigation of Aspergillus fumigatus Interaction with Pulmonary Cells

Inhaled Aspergillus fumigatus spores can be internalized by alveolar type II cells. Cell lines stably expressing fluorescently labeled components of endocytic pathway enable investigations of intracellular organization during conidia internalization and measurement of the process kinetics. The goal of this report was to evaluate the methodological appliance of cell lines for studying fungal conidia internalization. We have generated A549 cell lines stably expressing fluorescently labeled actin (LifeAct-mRuby2) and late endosomal protein (LAMP1-NeonGreen) following an evaluation of cell-pathogen interactions in live and fixed cells. Our data show that the LAMP1-NeonGreen cell line can be used to visualize conidia co-localization with LAMP1 in live and fixed cells. However, caution is necessary when using LifeAct-mRuby2-cell lines as it may affect the conidia internalization dynamics.     

Control of growth and inflammatory response of macrophages and foam cells with nanotopography

ABSTRACT: Macrophages play an important role in modulating the immune function of the human body, while foam cells differentiated from macrophages with subsequent fatty streak formation play a key role in atherosclerosis. We hypothesized that nanotopography modulates the behavior and function of macrophages and foam cells without bioactive agent. In the present study, nanodot arrays ranging from 10 to 200 nm were used to evaluate the growth and function of macrophages and foam cells. In the quantitative analysis, the cell adhesion area in macrophages increased with 10- to 50-nm nanodot arrays compared to the flat surface, while it decreased with 100- and 200-nm nanodot arrays. A similar trend of adhesion was observed in foam cells. Immunostaining, specific to vinculin and actin filaments, indicated that a 50-nm surface promoted cell adhesion and cytoskeleton organization. On the contrary, 200-nm surfaces hindered cell adhesion and cytoskeleton organization. Further, based on quantitative real-time polymerase chain reaction data, expression of inflammatory genes was upregulated for the 100- and 200-nm surfaces in macrophages and foam cells. This suggests that nanodots of 100 and 200 nm triggered immune inflammatory stress response. In summary, nanotopography controls cell morphology, adhesions, and proliferation. By adjusting the nanodot diameter, we could modulate the growth and expression of function-related genes in the macrophages and foam cell system. The nanotopography-mediated control of cell growth and morphology provides potential insight for designing cardiovascular implants.     

Increase of the Intracellular Zinc Concentration Leads to an Activation and Internalisation of the Epidermal Growth Factor Receptor in A549 Cells

(1) Background: Zinc is suggested to play a major role in epidermal growth factor (EGF)-induced cell regeneration and proliferation. To deepen the knowledge on the underlying mechanisms zinc’s effects on the epidermal growth factor receptor (EGFR) activation and its endocytosis was investigated in the alveolar carcinoma cell line A549. (2) Methods: An increase of intracellular zinc was generated by adding zinc extracellularly compared to the intracellular release of zinc from zinc-binding proteins by stimulation with a nitric oxide donor. Zinc-initiated EGFR phosphorylation was checked by Western blotting and receptor endocytosis assays were performed by using flow cytometry. (3) Results: Besides a dose-dependent EGFR phosphorylation, a dose- and time dependent significant receptor internalisation was initiated by both types of zinc increases. In addition, both increased intracellular zinc levels further promoted EGF-induced EGFR phosphorylation and internalisation. (4) Conclusion: This report confirms a transactivating effect of zinc on the EGFR for A549 cells and is the first describing an influence of zinc on the EGFR endocytosis. The transferability of the fine-tuning of EGFR-induced signalling by zinc needs to be verified in vivo, but the presented data underline that zinc might be helpful during treatment of disturbed regeneration and tissue repair.     

Experimental Methods for Evaluating the Bacterial Uptake of Trojan Horse Antibacterials

The field of antibacterial siderophore conjugates, referred to as Trojan Horse antibacterials, has received increasing attention in recent years, driven by the rise of antimicrobial resistance. Trojan Horse antibacterials offer an opportunity to exploit the specific pathways present in bacteria for active iron uptake, potentially allowing the drugs to bypass membrane-associated resistance mechanisms. Hence, the Trojan Horse approach might enable the redesigning of old antibiotics and the development of antibacterials that target specific pathogens. Critical parts of evaluating such Trojan Horse antibacterials and improving their design are the quantification of their bacterial uptake and the identification of the pathways by which this occurs. In this minireview, we highlight a selection of the biological and chemical methods used to study the uptake of Trojan Horse antibacterials, exemplified with case studies, some of which have led to drug candidates in clinical development or approved antibiotics.