Showing papers in "ChemBioChem in 2009"
TL;DR: The covalent conjugation of drug and aptamer creates alternative opportunities for targeted therapy, as multiple yet specific aptamers can be “generated” relatively easily by cell‐SELEX for any target cells; this demonstrates the full potential of cell‐ SELEX as a molecular discovery tool for biomedical studies and drug development.
Abstract: The conjugation of antitumor drugs to targeting reagents such as antibodies is a promising method that can increase the efficacy of chemotherapy and reduce the overall toxicity of the drugs. In this study, we covalently link the antitumor agent doxorubicin (Dox) to the DNA aptamer sgc8c, which was selected by the cell-SELEX method. In doing so, we expected that this sgc8c-Dox conjugate would specifically kill the target CCRF-CEM (T-cell acute lymphoblastic leukemia, T-cell ALL) cells, but with minimal toxicity towards nontarget cells. The results demonstrated that the sgc8c-Dox conjugate possesses many of the properties of the sgc8c aptamer, including high binding affinity (K(d)=2.0+/-0.2 nM) and the capability to be efficiently internalized by target cells. Moreover, due to the specific conjugation method, the acid-labile linkage connecting the sgc8c-Dox conjugate can be cleaved inside the acidic endosomal environment. Cell viability tests demonstrate that the sgc8c-Dox conjugates not only possess potency similar to unconjugated Dox, but also have the required molecular specificity that is lacking in most current targeted drug delivery strategies. Furthermore, we found that nonspecific uptake of membrane-permeable Dox to nontarget cell lines could also be inhibited by linking the drug with the aptamer; thus, the conjugates are selective for cells that express higher amounts of target proteins. Compared to the less effective Dox-immunoconjugates, these sgc8c-Dox conjugates make targeted chemotherapy more feasible with drugs having various potencies. When combined with the large number of recently created DNA aptamers that specifically target a wide variety of cancer cells, this drug-aptoconjugation method will have broad implications for targeted drug delivery.
359 citations
TL;DR: This review will focus primarily on recent developments in the study of WTAs in Bacillus subtilis and Staphylococcus aureus, and will include a discussion of strategies for the discovery of WTA inhibitors and prospects for these inhibitors as antibiotics.
Abstract: One of the major differences between Gram-negative and Gram-positive organisms is the presence or absence of an outer membrane (Figure 1). In Gram-negative organisms, the outer membrane protects the organism from the environment. It filters out toxic molecules and establishes a compartment, the periplasm, which retains extracytoplasmic enzymes required for cell-wall growth and degradation. It also serves as a scaffold to which proteins and polysaccharides that mediate interactions between the organism and its environment are anchored.[1] In addition, in ways that are not completely understood, the outer membrane functions along with a thin layer of peptidoglycan to help stabilize the inner membrane so that it can withstand the high osmotic pressures within the cell.[2]
Figure 1
Simplified depiction of Gram-positive and Gram-negative bacterial cell envelopes. Gram-negative organisms have a distinct periplasm; Gram-positive organisms do not, but recent studies have suggested that they have a periplasmic-like compartment between ...
Gram-positive organisms, in contrast, lack an outer membrane and a distinct periplasm (Figure 1). The peptidoglycan layers are consequently very thick compared to those in Gram-negative organisms.[4] These thick layers of peptidoglycan stabilize the cell membrane and also provide many sites to which other molecules can be attached. Gram-positive peptidoglycan is heavily modified with carbohydrate-based anionic polymers that play an important role in membrane integrity.[5] These anionic polymers appear to perform some of the same functions as the outer membrane: they influence membrane permeability, mediate extracellular interactions, provide additional stability to the plasma membrane, and, along with peptidoglycan, act as scaffolds for extracytoplasmic enzymes required for cell-wall growth and degradation.
A major class of these cell surface glycopolymers are the teichoic acids (TAs), which are phosphate-rich molecules found in a wide range of Gram-positive bacteria, pathogens and nonpathogens alike. There are two types of TAs: the lipo-TAs (LTAs), which are anchored to the plasma membrane and extend from the cell surface into the peptidoglycan layer;[6] and the wall TAs (WTAs), which are covalently attached to peptidoglycan and extend through and beyond the cell wall (Figure 1).[7] Together, LTAs and WTAs create what has been aptly described as a “continuum of negative charge” that extends from the bacterial cell surface beyond the outermost layers of peptidoglycan.[5] Neuhaus and Baddiley comprehensively reviewed both LTAs and WTAs in 2003.[5] Since then, however, new functions for WTAs in pathogenesis have been uncovered and it has been suggested that the biosynthetic enzymes that make these polymers are targets for novel antibacterial agents.[8,9] Indeed, the first WTA-active antibiotic has just been reported.[10] This review will focus primarily on recent developments in the study of WTAs in Bacillus subtilis and Staphylococcus aureus, and will include a discussion of strategies for the discovery of WTA inhibitors and prospects for these inhibitors as antibiotics.
319 citations
TL;DR: New drugs from silent gene clusters: Analysis of genome sequence data has identified numerous “cryptic” gene clusters encoding novel natural product biosynthetic assembly lines; this suggests that many new bioactive metabolites remain to be discovered, even in extensively investigated organisms.
Abstract: Natural products have a very broad spectrum of applications. Many natural products are used clinically as antibacterial, antifungal, antiparasitic, anticancer and immunosuppressive agents and are therefore of utmost importance for our society. When in the 1940s the golden age of antibiotics was ushered in, a "gold rush fever" of natural product discovery in the pharmaceutical industry ensued for many decades. However, the traditional process of discovering new bioactive natural products is generally long and laborious, and known natural products are frequently rediscovered. A mass-withdrawal of pharmaceutical companies from new natural product discovery and natural products research has thus occurred in recent years. In this article, the concept of genome mining for novel natural product discovery, which promises to provide a myriad of new bioactive natural compounds, is summarized and discussed. Genome mining for new natural product discovery exploits the huge and constantly increasing quantity of DNA sequence data from a wide variety of organisms that is accumulating in publicly accessible databases. Genes encoding enzymes likely to be involved in natural product biosynthesis can be readily located in sequenced genomes by use of computational sequence comparison tools. This information can be exploited in a variety of ways in the search for new bioactive natural products.
298 citations
TL;DR: This work has shown that light emission by V. fischeri depends upon the bacterial cell density, and only occurs at high cell densities, which explains why V.fischeri only produces light in the fish organ where it lives at very high densities.
Abstract: Vibrio fischeri is a remarkable Gram-negative marine bacterium. First, it emits light when it colonizes the fish or squid organs dedicated to this purpose. Second, it turns this light on when entering these organs and switches it off when leaving them. In V. fischeri, the production of light, also termed bioluminescence, results from the activity of enzymes that are encoded by the lux operon. [1] Bioluminescence results from the hydrolysis of decanal, or other fatty aldehydes, by luciferase/flavin reductase enzymes. This process is costly in terms of energy because lux genes also encode fatty aldehyde synthesis. [2] Nonetheless, this bioluminescence has been selected and maintained during the course of evolution. One possible reason for this is that the fish–V. fischeri association resembles symbiosis. [3] The animal provides the bacteria with an exclusive, carbon-rich ecological niche. The bacterium emits photons, a function that both attracts prey and deters predators in the light-deprived environment of the deep ocean. [4] Bioluminescence can be produced in vitro in the appropriate culture medium. Remarkably, light emission by V. fischeri depends upon the bacterial cell density, and only occurs at high cell densities. [5] This phenomenon, termed “quorum sensing” (QS), [6] explains why V. fischeri only produces light in the fish organ where it lives at very high densities. It does not produce light in the open sea where the bacteria are extremely diluted
278 citations
TL;DR: In this paper, a review summarizes the most exciting mitochondrial transporters and discusses their applications, including their application in the mito-specific delivery of bioactive cargos.
Abstract: Mitochondria are the energy factories of the cell and also serve as a checkpoint regulating programmed cell death. Not surprisingly, dysfunctional mitochondria are implicated in a variety of diseases ranging from metabolic disorders to cancer. Treatment of these diseases through the delivery of targeted drugs, however, is impeded by the difficulty of penetrating the membranes that define this organelle. The properties of this barrier serve as a major obstacle to drug delivery and a lack of effective transporters has hindered the advancement of mitochondrial medicine. Recently, however, synthetic transporters that show promise for the mito-specific delivery of bioactive cargos have begun to emerge. This review summarizes the most exciting of these developments and discusses their applications.
276 citations
TL;DR: The mechanism of ROS production by Cu–Aβ in relation to its aggregations state, as well as the metal‐transfer reaction from and to Aβ are crucial in order to understand why A β oligomers are highly toxic and why Aβ seems to bind Cu and Zn only in AD.
Abstract: The metal ions copper, zinc and iron have been shown to be involved in Alzheimer's disease (AD). Cu, Zn and Fe ions are proposed to be implicated in two key steps of AD pathology: 1) aggregation of the peptide amyloid-β (Aβ), and 2) production of reactive oxygen species (ROS) induced by Aβ. There is compelling evidence that Cu and Zn bind directly to Aβ in AD. This formation of Cu/Zn–Aβ complexes is thought to be aberrant as they have been detected only in AD, but not under healthy conditions. In this context, the understanding of how these metal ions interact with Aβ, their influence on structure and oligomerization become an important issue for AD. Moreover, the mechanism of ROS production by Cu–Aβ in relation to its aggregations state, as well as the metal-transfer reaction from and to Aβ are crucial in order to understand why Aβ oligomers are highly toxic and why Aβ seems to bind Cu and Zn only in AD.
254 citations
TL;DR: This article outlines the technique, sortase‐mediated ligation, and its applications in protein engineering, which include the introduction of unnatural molecules into proteins, protein immobilization, protein–protein conjugation, protein cyclization, as a self‐cleavable tag for protein expression, protein-PNA hybrids, neoglycoconjugates, and cell‐surface protein labeling, etc.
Abstract: A new enzymatic protein ligation tool, sortase, has recently emerged from Gram-positive bacteria. This article outlines the technique, sortase-mediated ligation, and its applications in protein engineering, which include the introduction of unnatural molecules into proteins, protein immobilization, protein–protein conjugation, protein cyclization, as a self-cleavable tag for protein expression, protein–PNA hybrids, neoglycoconjugates, and cell-surface protein labeling, etc.
226 citations
TL;DR: The interaction of MT with physiologically relevant molecules and its effect on zincthiolate bonds are discussed, linked to recent progress in the functional role of MT in cellular zinc transport, energy production, and protection of the organism against oxidative stress and neurodegenerative diseases.
Abstract: Metallothioneins (MTs) were discovered more than 50 years ago and identified as low-molecular weight, sulfhydryl-rich proteins that were subsequently found to bind zinc predominantly. The binding of seemingly redox inactive zinc ions allows MT to play a central role in oxidoreductive cellular metabolism, cellular zinc distribution and homeostasis. In this interpretive study, we discuss the interaction of MT with physiologically relevant molecules and its effect on zinc-thiolate bonds. These interactions are linked to recent progress in the functional role of MT in cellular zinc transport, energy production, and protection of the organism against oxidative stress and neurodegenerative diseases.
195 citations
TL;DR: Recent advances both in the understanding of the mechanism of UGM and in the synthesis of galactofuranose and its derivatives are highlighted in this review.
Abstract: The thermodynamically less stable form of galactose-galactofuranose (Galf)-is essential for the viability of several pathogenic species of bacteria and protozoa but absent in this form in mammals, so the biochemical pathways by which Galf-containing glycans are assembled and catabolysed are attractive sites for drug action This potential has led to increasing interest in the synthesis of molecules containing Galf residues, their subsequent use in studies directed towards understanding the enzymes that process these residues and the identification of potential inhibitors of these pathways Major achievements of the past several years have included an in-depth understanding of the mechanism of UDP-galactopyranose mutase (UGM), the enzyme that produces UDP-Galf, which is the donor species for galactofuranosyltransferases A number of methods for the synthesis of galactofuranosides have also been developed, and practitioners in the field now have many options for the initiation of a synthesis of glycoconjugates containing either alpha- or beta-Galf residues UDP-Galf has also been prepared by a number of approaches, and it appears that a chemoenzymatic approach is currently the most viable method for producing multi-milligram amounts of this important intermediate Recent advances both in the understanding of the mechanism of UGM and in the synthesis of galactofuranose and its derivatives are highlighted in this review
181 citations
TL;DR: FACS‐based screening for enzymatic activity and its potential application for the directed evolution of enzymes, ribozymes, and catalytic antibodies are discussed.
Abstract: Directed enzyme evolution has proven to be a powerful tool for improving a range of properties of enzymes through consecutive rounds of diversification and selection. However, its success depends heavily on the efficiency of the screening strategy employed. Fluorescence-activated cell sorting (FACS) has recently emerged as a powerful tool for screening enzyme libraries due to its high sensitivity and its ability to analyze as many as 10(8) mutants per day. Applications of FACS screening have allowed the isolation of enzyme variants with significantly improved activities, altered substrate specificities, or even novel functions. This review discusses FACS-based screening for enzymatic activity and its potential application for the directed evolution of enzymes, ribozymes, and catalytic antibodies.
149 citations
TL;DR: The basic concepts and requirements of controlling biological (especially cellular) function with light, including photochemical regulation of biologically active molecules, are focused on.
Abstract: Biological processes, particularly gene function, are naturally regulated with high spatiotemporal resolution in single cells and multicellular organisms. The activity of genes, proteins, and other biological molecules is precisely controlled in timing and location. This is especially evident during the complex biological processes observed in the development of an organism. In order to understand and to study these processes and their misregulation in human disease, it is imperative to control them with the same level of spatiotemporal resolution found in nature. Here, light irradiation represents a unique tool, because it can be easily and precisely controlled in timing, location, and amplitude; thus, light enables the precise activation and deactivation of biological function.
Rather than providing a comprehensive literature review, this article focuses on the basic concepts and requirements of controlling biological (especially cellular) function with light. Recent examples are used to illustrate these concepts. The interested reader can find additional excellent and comprehensive reviews regarding the photochemical regulation of biologically active molecules in the literature.[1]
TL;DR: A class of mitochondria‐penetrating peptides was studied in an effort to optimize their applications in the delivery of bioactive cargo to this therapeutically important organelle, and it was discovered that while an alternating cationic/hydrophobic residue motif is not required, the inclusion of a stretch of adjacent cATIONic amino acids can impede access to the organelle.
Abstract: A class of mitochondria-penetrating peptides (MPPs) was studied in an effort to optimize their applications in the delivery of bioactive cargo to this therapeutically important organelle. The sequence requirements for mitochondrial entry were monitored, and it was discovered that while an alternating cationic/hydrophobic residue motif is not required, the inclusion of a stretch of adjacent cationic amino acids can impede access to the organelle. In addition, a variety of N- and C-terminal cargo were tested to determine if there are limitations to the lipophilicity, charge, or polarity of compounds that can be transported to mitochondria by MPPs. The results reported demonstrate that these peptide sequences are versatile transporters that will have a range of biological applications.
TL;DR: The ubiquitous nature of bacteria in the environment, and the role they play in infectious disease has been one of the most extensively researched areas in biomedical science, and upwards of 75 % of microbial infections that occur in the human body are underpinned by the formation and persistence of biofilms.
Abstract: The ubiquitous nature of bacteria in the environment, and the role they play in infectious disease has been one of the most extensively researched areas in biomedical science. It has led to tremendous scientific breakthroughs aimed at eradicating a myriad of diseases and improving the overall quality of life. However, within the past 20–30 years, there has been an ACHTUNGTRENNUNGincreased understanding that bacterial biofilms are a major factor in the morbidity and mortality of most infectious diseases. This is significant because bacterial biofilms are resistant to common therapeutic approaches that would eliminate their free-floating (planktonic) counterparts. Biofilms are described as surface-associated communities of microorganisms encased in a protective extracellular matrix. Approximately 80 % of the world’s microbial biomass resides in the biofilm state, and the National Institutes of Health (NIH) estimates that upwards of 75 % of microbial infections that occur in the human body are underpinned by the formation and persistence of biofilms. Common diseases associated with the formation of biofilms include lung infections of individuals who suffer from cystic fibrosis (CF), burn wound infections, otitis media, bacterial endocarditis, and tooth decay (Table 1). 6] Additionally, the
TL;DR: The potential of synthetic chemistry to contribute to the further development of DNA nanomaterials is described and the structure of the predesigned structure can be built up by self‐assembly of the designed DNA strands.
Abstract: Due to its self-assembling nature, DNA is undoubtedly an excellent molecule for the creation of various multidimensional nanostructures and the placement of functional molecules and materials. DNA molecules behave according to the programs of their sequences. Mixtures of numbers of DNA molecules can be placed precisely and organized into single structures to form nanoarchitectures. Once the appropriate sequences for the target nanostructure are established, the predesigned structure can be built up by self-assembly of the designed DNA strands. DNA nanotechnology has already reached the stage at which the organization of desired functional molecules and nanomaterials can be programmed on a defined DNA scaffold. In this review, we will focus on DNA nanotechnology and describe the potential of synthetic chemistry to contribute to the further development of DNA nanomaterials.
TL;DR: Olefin metathesis has emerged as a viable strategy for site‐selective protein modification and its development from early peptide models and met athesis in water to its ultimate application to protein substrates is traced.
Abstract: For a reaction to be generally useful for protein modification, it must be site-selective and efficient under conditions compatible with proteins: aqueous media, low to ambient temperature, and at or near neutral pH. To engineer a reaction that satisfies these conditions is not a simple task. Olefin metathesis is one of most useful reactions for carbon-carbon bond formation, but does it fit these requirements? This minireview is an account of the development of olefin metathesis for protein modification. Highlighted below are examples of olefin metathesis in peptidic systems and in aqueous media that laid the groundwork for successful metathesis on protein substrates. Also discussed are the opportunities in protein engineering for the genetic introduction of amino acids suitable for metathesis and the related challenges in chemistry and biology.
TL;DR: Being the smallest synthetic bioreactor, such a model suggests that very small compartments can sustain (minimal) cellular life, and a possible super‐concentration effect is proposed.
Abstract: The question of the minimal size of a cell that is still capable of endorsing life has been discussed extensively in the literature, but it has not been tackled experimentally by a synthetic-biology approach. This is the aim of the present work; in particular, we examined the question of the minimal physical size of cells using liposomes that entrapped the complete ribosomal machinery for expression of enhanced green fluorescence protein, and we made the assumption that this size would also correspond to a full fledged cell. We found that liposomes with a radius of about 100 nm, which is the smallest size ever considered in the literature for protein expression, are still capable of protein expression, and surprisingly, the average yield of fluorescent protein in the liposomes was 6.1-times higher than in bulk water. This factor would become even larger if one would refer only to the fraction of liposomes that are fully viable, which are those that contain all the molecular components (about 80). The observation of viable liposomes, which must contain all macromolecular components, indeed represents a conundrum. In fact, classic statistical analysis would give zero or negligible probability for the simultaneous entrapment of so many different molecular components in one single 100 nm radius spherical compartment at the given bulk concentration. The agreement between theoretical statistical predictions and experimental data is possible with the assumption that the concentration of solutes in the liposomes becomes larger by at least a factor twenty. Further investigation is required to understand the over-concentration mechanism, and to identify the several biophysical factors that could play a role in the observed activity enhancement. We conclude by suggesting that these entrapment effects in small-sized compartments, once validated, might be very relevant in the origin-of-life scenario.
TL;DR: Eleven variants demonstrated either a strong shift or improved regio‐ or stereoselectivity during oxidation of at least one substrate as compared to CYP102A1 wild type.
Abstract: A minimal CYP102A1 mutant library of only 24 variants plus wild type was constructed by combining five hydrophobic amino acids (alanine, valine, phenylalanine, leucine and isoleucine) in two positions. Both positions are located close to the centre of the haem group. The first, position 87, has been shown to mediate substrate specificity and regioselectivity in CYP102A1. The second hotspot, position 328, was predicted to interact with all substrates during oxidation and has previously been identified by systematic analysis of 31 crystal structures and 6300 sequences of cytochrome P450 monooxygenases. By systematically altering the size of the side chains, a broad range of binding site shapes was generated. All variants were functionally expressed in E. coli. The library was screened with four terpene substrates geranylacetone, nerylacetone, (4R)-limonene and (+)-valencene. Only three variants showed no activity towards all four terpenes, while eleven variants demonstrated either a strong shift or improved regio- or stereoselectivity during oxidation of at least one substrate as compared to CYP102A1 wild type.
TL;DR: The markedly enhanced cell survival in the presence of resveratrol indicates that the small oligomeric structures that are observed during β‐sheet formation are not toxic and could be off‐pathway assembly products.
Abstract: GRAPES FOR AMYLOIDS: The red wine compound resveratrol can effectively inhibit the formation of IAPP amyloid that is found in type II diabetes. Our in vitro inhibition results do not depend on the antioxidant activity of resveratrol. Further, the markedly enhanced cell survival in the presence of resveratrol also indicates that the small oligomeric structures that are observed during beta-sheet formation are not toxic and could be off-pathway assembly products.
TL;DR: The results obtained with manno-GNPs are presented as inhibitors of DC-SIGN-mediated HIV trans-infection of human activated peripheral blood mononuclear cells (PBMCs) and GNPs that display multiple copies of the disacchaACHTUNGTRENNUNG(GlcNAc)2 of gp120 are observed.
Abstract: Viral entry is a critical step in human immunodeficiency virus (HIV) infection that takes place through a series of sequential interactions between the envelope glycoprotein gp120, cellular receptor CD4, and coreceptors CCR5 or CXCR4 on T cells. Through a mechanism named trans-infection, dendritic cells (DCs) efficiently transfer the virus to T lymphocytes where viral replication occurs. Interference in HIV transmission in the DC–lymphocyte synapse is a preferential but complex target in the development of new compounds displaying anti-HIV activity. HIV–DC interaction is mediated by the glycans of gp120 and the C-type lectin DC-SIGN (dendritic cell-specific ICAM 3grabbing nonintegrin) expressed on DCs. DC-SIGN is tetrameric and specifically recognizes N-linked high-mannose oligosaccharides (Man9GlcNAc2) through multivalent and Ca -dependent protein–carbohydrate interactions. Mimicking the cluster presentation of the oligomannosides on the surface of the virus is a strategy for designing carbohydrate-based antiviral agents. Mannose multivalent systems based on proteins, peptides, liposomes, and dendrimers as scaffolds have recently been prepared for targeting DCs and tested as inhibitors of DC-SIGN binding to gp120 in ELISA and SPR experiments. Nevertheless, none of these systems has so far been tested in cell-based trans-infection models. Glyconanoparticles (GNPs) are polyvalent, biocompatible sugar-functionalized gold nanoclusters. 10] Glyconanoparticle platforms offer the unique potential for simultaneous incorporation of different ligands in variable densities on a single gold cluster. We have prepared a small library of GNPs (mannoGNPs) coated with sets of different structural motifs of the Nlinked high-mannose undecasaccharide Man9ACHTUNGTRENNUNG(GlcNAc)2 of gp120 (or with an unnatural heptasaccharide) and have observed that GNPs that display multiple copies of the disacchaACHTUNGTRENNUNGride Mana1-2Mana block DC-SIGN/gp120 binding at 120 nm in surface plasmon resonance (SPR) experiments. The mannoGNPs were designed to target DC-SIGN receptors present on DCs by mimicking the clustered carbohydrate display of gp120. In this work we present the results obtained with manno-GNPs (Scheme 1) as inhibitors of DC-SIGN-mediated HIV trans-infection of human activated peripheral blood mononuclear cells (PBMCs). We show that manno-GNPs coated with the linear tetrasaccharide Mana1-2Mana1-2Mana1-3Mana inhibit HIV trans-infection of human T cells similarly to GNPs coated with more complex branched pentaand heptaoligomannosides. Hybrid gold manno-GNPs displaying different densities of linear and branched mannose oligosaccharides were prepared (Scheme 1). The manno-conjugates 6–10 were synthesized by conjugation of ethylamino mannosides 1–5, obtained by the protocol of Wong et al. , with isothiocyanate linker 11 and subsequent removal of the acetyl group (Scheme 1 A). A shorter five-carbon atom aliphatic linker was used for the preparation of 5’-mercaptopentyl b-d-glucopyranoside (GlcC5S), and was hidden internally in order to allow correct presentation of the oligomannosides. Manno-GNPs with 100, 50, or 10 % densities of dimannoside (D-100, D-50, and D-10, respectively), trimannoside (T-50 and T-10), tetramannoside (Te-50 and Te-10), pentamannoside (P-50 and P-10), and heptamannoside (H-50) were prepared from mixtures of manno-conjugates 6–10 and gluco-conjugate GlcC5S by using a previously described methodology (Scheme 1 B). GNPs were characterized by transmission electron microscopy (TEM), H NMR, IR, UV/Visible, and elemental analysis as previously reported. The average number of (oligo)mannosides per GNP and their average molecular weights (Table 1) were calculated from elemental analysis and gold cluster size (1–2 nm as determined by TEM; see the Supporting Information). For trans-infection studies, we used Raji DC-SIGN transfected lymphoblastoid B cells, which can capture and transmit HIV with an efficiency similar to that of monocyte-derived DCs. Manno-GNPs are nontoxic to Raji DC-SIGN and to human activated PBMCs at concentrations of 100 mg mL , as determined by the CellTiter cell viability assay (Figure S9 in the Supporting Information). The activities of manno-GNPs against R5 or X4 HIV-1 were evaluated through an original DC-SIGN transfer assay in which inhibition of HIV-1 infection by GNPs was assessed by use of recombinant viruses carrying the Renilla reporter genes in their genomes. In this assay, inhibition of viral replication is proportional to Renilla-luciferase activity in cell lysates. Briefly, Raji DC-SIGN cells were preincubated with GNPs for 1 h and were then pulsed with JR-Renilla (R5) or NL4.3-Renilla (X4) recombinant viruses for 2 h. Afterwards, the cell cultures were extensively washed and co-cultured with activated PBMCs that would be infected through transfer of the virus bound to DC-SIGN in Raji cells. Viral replication was as[a] Dr. O. Mart nezvila, Dr. M. Marradi, Dr. C. Clavel, Prof. Dr. S. Penad s Laboratory of GlycoNanotechnology, CIC biomaGUNE/CIBER-BBN P8 Miram n 182, 20009 San Sebasti n (Spain) Fax: (+ 34) 943-00-53-01 E-mail : spenades@cicbiomagune.es [b] Dr. L. M. Bedoya, Dr. J. Alcam AIDS Immunopathology Unit, National Center of Microbiology Instituto de Salud Carlos III, Ctra.Majadahonda-Pozuelo Km. 2,200 Madrid (Spain) E-mail : ppalcami@isciii.es [c] Dr. C. Clavel Current address : IBMM, UMR 5247CNRS-Universit s Montpellier 2 et 1 ENSCM, Rue de l’Ecole Normale 8, 34296 Montpellier Cedex 5 (France) Supporting information for this article is available on the WWW under http ://dx.doi.org/10.1002/cbic.200900294.
TL;DR: The stability of the SA captured by this fixation strategy (DNA wells and two biotin linkers) was directly compared on the same molecule with the stability of SA captured with other possible strategies that do not employ wells or two linkers.
Abstract: A new punched DNA origami assembly with periodic nanometer-scale wells has been successfully designed and constructed. Through the attachment of two biotins at the two edges of each well, just one streptavidin (SA) tetramer (d = 5 nm) was size-selectively captured in each 6.8 x 12 x 2.0 nm well; this allowed formation of a 28 nm-period SA nanoarray of individual molecules. The position of SA capture can be fully controlled by placement of biotins in the nanoarray well. Moreover, construction of a 2D nanoarray of individual SA tetramers through selective positioning of SA tetramers in any desired wells in a complex of such punched origami motifs is also possible. The stability of the SA captured by this fixation strategy (DNA wells and two biotin linkers) was directly compared on the same molecule with the stability of SA captured with other possible strategies that do not employ wells or two linkers. In this way, the robustness of this means of fixation was clearly established.
TL;DR: A historical perspective is provided of the more than 100 years of research that led to the current picture of the interesting biosynthesis of phenazine natural products, including the discovery of P. aeruginosa and its role in human opportunistic infections.
Abstract: Physicians of the 19th century were familiar with the conspicuous occurrence of “blue pus”, which they sometimes observed in patients with severe purulent wounds. Even older are reports of and folk remedies against “blue milk”, a coloration that sometimes developed in fresh milk after some days. Key insight into these phenomena was provided in 1859—exactly 150 years ago—when Mathurin-Joseph Fordos, at a session of the Societ d’ mulation pour les Sciences Pharmaceutiques, reported the isolation of the blue pigment “pyocyanine” (from the Greek words for “pus” and “blue”; pyocyanine is nowadays more commonly spelled as pyocyanin) from purulent wound dressings by chloroform extraction. Pyocyanin (5-N-methyl-1hydroxophenazinium betaine) was the first example of a phenazine natural product, a compound class that has grown to well over 100 members since this first report. Due to the improved understanding of their importance to the phenazinegenerating and also to commensal species, the phenazines have been reviewed several times in recent years. Here, we provide a historical perspective of the more than 100 years of research that led us to our current picture of the interesting biosynthesis of phenazine natural products. The details of Fordos’ pyocyanin isolation method, chloroform extraction followed by acidification and partition into an aqueous phase, were published one year later and are still in use today, but it took until 1882 for the French pharmacist Carle Gessard to show that the blue coloration in pus was due to the presence of a microorganism that he then termed Bacillus pyocyaneus. B. pyocyaneus is nowadays known as Pseudomonas aeruginosa, and the Latin term still reflects this strain’s capacity to secrete colored compounds in the modern name: “aerugo” is the Latin word for verdigris, the blue–green coating that develops on copper after long exposure to air. P. aeruginosa is an important human opportunistic pathogen responsible for a large number of nosocomial infections, and it is also the main course of low life expectancy in patients with cystic fibrosis due to chronic infections of the lungs. The production of pyocyanin is used both for identification in the clinic and as a reporter signal in P. aeruginosa research until today. The occurrence of blue milk, on the other hand, is probably due to an environmental strain of P. fluorescens, and it is not yet clear if this coloration also is a consequence of phenazine production. Gessard’s discovery of P. aeruginosa was resonated in many publications from the medical field, but it required more than 50 years before the correct chemical structure of pyocyanin was established. The chemical composition was first studied by Ledderhose, who derived a formula that was later corrected by McCombie and Scarborough and by Wrede and Strack. Wrede and Strack were also the first to discover a phenazine moiety in a breakdown product of pyocyanin, but their studies were hampered by the fact that they could only obtain a defined molecular weight when working in glacial acetic acid, under which circumstances they obtained a pyocyanin dimer. This dimer was questioned by the results of electrochemical studies by Elema and by Friedheim and Michaelis, before Hillemann finally derived the correct structure in 1938. In retrospect, it seems possible that the conditions employed by Wrede and Strack induced a 1:1 charge-transfer complex of reduced and oxidized pyocyanin, similar to the phenazine derivative chlororaphin, which is also produced by P. aeruginosa (Figure 1). Jensen and Holten later measured the dipole moment of pyocyanin and found that its zwitterionic mesomer is present in considerable amounts. In the course of these studies, it became clear that pyocyanin is a redox-active compound that changes its color depending on pH and oxidation state. This also explained the “chameleon phenomenon”, which describes a temporary color change of P. aeruginosa cultures on solid media after exposure to air by disturbance with a platinum needle. Since the first isolation by Fordos, more than 100 phenazine derivatives modified at all positions of the ring system and colored in all shades of
TL;DR: The conventional notion that peptides are poor candidates for orally available drugs because of protease‐sensitive peptide bonds, intrinsic hydrophilicity, and ionic charges contrasts with the diversity of antibiotic natural products with peptide‐based frameworks that are synthesized and utilized by Nature.
Abstract: The conventional notion that peptides are poor candidates for orally available drugs because of protease-sensitive peptide bonds, intrinsic hydrophilicity, and ionic charges contrasts with the diversity of antibiotic natural products with peptide-based frameworks that are synthesized and utilized by Nature. Several of these antibiotics, including penicillin and vancomycin, are employed to treat bacterial infections in humans and have been best-selling therapeutics for decades. Others might provide new platforms for the design of novel therapeutics to combat emerging antibiotic-resistant bacterial pathogens.
TL;DR: This review covers the main developments of the chemistry and biology of modified oligonucleotides, including recent examples of chemical modifications, conjugation and derivatisation, antisense, RNAi and other applications, such as gene functional analysis and bioanalysis.
Abstract: Chemical modification of oligonucleotides came to prominence in the early 1990s with the advent of the antisense approach to control gene expression. Whilst the antisense field suffered to some extent from unrealistically high expectations for clinical applications, in some quarters, efforts to develop modified oligonucleotides and nucleic acid mimic has continued at a pace. Further impetus for the field came with the discovery of RNA interference, which has reinvigorated research into chemical modification of oligonucleotides to stabilise and improve the efficacy of siRNAs. In addition to this, modified oligonucleotides and mimics have found a wide range of other applications. Whilst clinical development of antisense oligonucleotides (ASOs) has been slow, the use of ASOs to probe gene function, for fundamental studies, which do not necessarily have therapeutic applications, has accelerated considerably. For example, one of the earliest examples of an oligonucleotide mimic, the PMO, is now widely used in gene functional analysis, in a range of organisms from human cells through to zebrafish. Many conjugation strategies have been developed to aid delivery, localisation and imaging of modified oligonucleotides. Notably nanoparticles have proven to be very useful not only as transfection agents, but also as a means to aid the detection of specific sequences, such as in their use with surface plasmon resonance and nanoflares. In addition to this, modified oligonucleotides and mimics are finding wide-spread applications as bioanalytical and diagnostic tools, allowing more stringent detection of SNPs from increasingly small concentrations of sample DNA. Finally, modified oligonucleotides and mimics are proving useful as building blocks in the assembly of higher-order nanostructures. In this review we cover the main developments of the chemistry and biology of modified oligonucleotides, including recent examples of chemical modifications, conjugation and derivatisation, antisense, RNAi and other applications, such as gene functional analysis and bioanalysis. This review is not intended to present an exhaustive account of all the chemical modifications, targets and applications for modified oligonucleotides, but rather highlights the recent and ever increasing diversity and possibilities that such oligonucleotides and derivatives possess. This review also highlights how well-known limitations, such as stability, transfection, targeting etc. , are being overcome. To a large extent the review covers the most recent literature and the reader is referred to our previous review of this field for details of earlier work in this area. 2. Chemical Modifications
TL;DR: Replacement of the helix with corresponding lid regions from CALB homologues from Neurospora crassa and Gibberella zeae resulted in new CALB chimeras with novel biocatalytic properties.
Abstract: The best of both worlds. Long molecular dynamics (MD) simulations of Candida antarctica lipase B (CALB) confirmed the function of helix alpha5 as a lid structure. Replacement of the helix with corresponding lid regions from CALB homologues from Neurospora crassa and Gibberella zeae resulted in new CALB chimeras with novel biocatalytic properties. The figure shows a snapshot from the MD simulation. The Candida antarctica lipase B (CALB) has found very extensive use in biocatalysis reactions. Long molecular dynamics simulations of CALB in explicit aqueous solvent confirmed the high mobility of the regions lining the channel that leads into the active site, in particular, of helices alpha5 and alpha10. The simulation also confirmed the function of helix alpha5 as a lid of the lipase. Replacing it with corresponding lid regions from the CALB homologues from Neurospora crassa and Gibberella zeae resulted in two new CALB mutants. Characterization of these revealed several interesting properties, including increased hydrolytic activity on simple esters, specifically substrates with C(alpha) branching on the carboxylic side, and much increased enantioselectivity in hydrolysis of racemic ethyl 2-phenylpropanoate (E>50), which is a common structure of the profen drug family.
TL;DR: Despite significant advances in Stat3 inhibition, truly potent (in vivo), isoform-selective, small molecule Stat3 agents have not been readily forthcoming; this is likely due in part to the challenge of disrupting protein–protein interactions.
Abstract: Signal transducer and activator of transcription 3 (Stat3) protein is a cytosolic transcription factor that relays signals from receptors in the plasma membrane directly to the nucleus, and is routinely hyperactivated in many human cancers and diseases.[1] Regarded as an oncogene, Stat3 is well-recognized as a master regulator of cellular events that lead to the cancer phenotype, making this protein viable target for molecular therapeutic design.[2] Stat3 inhibitors have included peptides,[3–4] peptidomimetics,[5–9] small molecules[10–14] and metal complexes.[15] Despite significant advances in Stat3 inhibition,[1] truly potent (in vivo), isoform-selective, small molecule Stat3 agents have not been readily forthcoming; this is likely due in part to the challenge of disrupting protein–protein interactions.[16]
TL;DR: The structural diversity of acidic lipopeptides, including daptomycin, which is already in clinical use, is depicted in this review.
Abstract: Acidic lipopeptide antibiotics are a new class of potent antibiotics, which includes daptomycin, A54145, calcium-dependent antibiotics (CDAs), friulimicins/amphomycins, laspartomycin/glycinocins and others. The importance of this novel class is exemplified by the success story of the clinically approved daptomycin, which is used for the treatment of skin infections and bacteremia caused by multidrug-resistant bacteria, including methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci. The potency of acidic lipopeptides is inherent in their chemical structure. The nonribosomally synthesized peptide cores consist of eleven to 13 amino acids, which are rigidified by the formation of a ten-membered ring. An N-terminal fatty acid, which facilitates insertion into the lipid bilayer of bacterial membranes, completes the structure. All these antibiotics contain multiple nonproteinogenic amino acids as well as different lipid tails; this yields remarkable structural diversity. This review summarizes the observed structural variety through a detailed description of the composition of the acidic lipopeptides. Furthermore, engineering approaches towards novel lipopeptides are presented. Recent discoveries in the field of tailoring enzymes, which enable structural plurality mainly by amino and fatty acid precursor biosynthesis, are highlighted.
TL;DR: The results show that these compounds display varying responses to the respective sensors; this underlines the importance of determining the whole bouquet of AHLs and its function to fully understand their importance for regulatory functions in bacteria.
Abstract: N-Acylhomoserine lactones (AHLs) are used by a wide variety of bacteria for cell-cell communication in "quorum-sensing". These compounds are derived from L-homoserine lactone and a fatty acid, which varies in chain-length, degree of saturation, and the presence or absence of an oxygen atom at C-3. In this study we describe for the first time the occurrence of acyl chains carrying a methyl branch, and present a GC-MS-based method that can be used to distinguish these compounds from unbranched isomers. The bacterium Aeromonas culicicola produces several methyl branched AHLs. In Jannaschia helgolandensis--a marine bacterium of the Roseobacter clade--a doubly unsaturated AHL, (2E,9Z)-N-(2,9-hexadecadienoyl)-L-homoserine lactone, occurs. The location and configuration of the double bonds was proven by spectrometric investigation and synthesis. Finally, a method was developed to establish the absolute configuration of 3-hydroxyalkanoyl-HSLs by mild cleavage and chiral gas chromatography. The AHLs synthesized during this study were tested in sensor systems specific for certain AHL types. The results show that these compounds display varying responses to the respective sensors; this underlines the importance of determining the whole bouquet of AHLs and its function to fully understand their importance for regulatory functions in bacteria.
TL;DR: The proline scanning and solid‐state NMR studies suggested that aggregates both of wild‐type Aβ42 and of E22K‐Aβ42 contain two conformers: a major one with a turn at positions 25 and 26, and a minor one at positions 22 and 23, which might be a key event in the pathogenesis of Alzheimer's disease.
Abstract: Aggregation of the 42-residue amyloid beta-protein (Abeta42) plays a crucial role in the pathogenesis of Alzheimer's disease (AD). Despite numerous structural studies on Abeta aggregates, the relationship between tertiary structure and toxicity remains unclear. Our proline scanning and solid-state NMR studies suggested that aggregates both of wild-type Abeta42 and of E22K-Abeta42 (one of the mutants related to cerebral amyloid angiopathy) contain two conformers: a major one with a turn at positions 25 and 26, and a minor one with a turn at positions 22 and 23. To identify the toxic conformer, the derivative Abeta42-lactam(22K-23E), in which the side chains at positions 22 and 23 were covalently linked, was synthesized as a minor conformer surrogate, along with Abeta42-lactam(25K-26E) as a major conformer surrogate. The Abeta42-lactam(22K-23E) showed stronger aggregation, neurotoxicity, radical generation, and oligomerization than wild-type Abeta42, whereas in Abeta42-lactam(25K-26E) were weak. The transition from the physiological conformation with a turn at positions 25 and 26 to the toxic conformation with a turn at positions 22 and 23 might be a key event in the pathogenesis of AD.
TL;DR: In this review, the recent progress of protein‐based fluorescent biosensors is described on the basis of their platform and construction strategy, which are primarily divided into the genetically encoded fluorescent bios Sensors and chemically constructed biosensor.
Abstract: The creation of novel bioanalytical tools for the detection and monitoring of a range of important target substances and biological events in vivo and in vitro is a great challenge in chemical biology and biotechnology. Protein-based fluorescent biosensors--integrated devices that convert a molecular-recognition event to a fluorescent signal--have recently emerged as a powerful tool. As the recognition units various proteins that can specifically recognize and bind a variety of molecules of biological significance with high affinity are employed. For the transducer, fluorescent proteins, such as green fluorescent protein (GFP) or synthetic fluorophores, are mostly adopted. Recent progress in protein engineering and organic synthesis allows us to manipulate proteins genetically and/or chemically, and a library of such protein scaffolds has been significantly expanded by genome projects. In this review, we briefly describe the recent progress of protein-based fluorescent biosensors on the basis of their platform and construction strategy, which are primarily divided into the genetically encoded fluorescent biosensors and chemically constructed biosensors.