Pharmacological and biological antiviral therapeutics for cardiac coxsackievirus infections

Subtype B coxsackieviruses (CVB) represent the most commonly identified infectious agents associated with acute and chronic myocarditis, with CVB3 being the most common variant. Damage to the heart is induced both directly by virally mediated cell destruction and indirectly due to the immune and autoimmune processes reacting to virus infection. This review addresses antiviral therapeutics for cardiac coxsackievirus infections discovered over the last 25 years. One group represents pharmacologically active low molecular weight substances that inhibit virus uptake by binding to the virus capsid (e.g., pleconaril) or inactivate viral proteins (e.g., NO-metoprolol and ribavirin) or inhibit cellular proteins which are essential for viral replication (e.g., ubiquitination inhibitors). A second important group of substances are interferons. They have antiviral but also immunomodulating activities. The third and most recently discovered group includes biological and cellular therapeutics. Soluble receptor analogues (e.g., sCAR-Fc) bind to the virus capsid and block virus uptake. Small interfering RNAs, short hairpin RNAs and antisense oligonucleotides bind to and led to degradation of the viral RNA genome or cellular RNAs, thereby preventing their translation and viral replication. Most recently mesenchymal stem cell transplantation has been shown to possess antiviral activity in CVB3 infections. Taken together, a number of antiviral therapeutics has been developed for the treatment of myocardial CVB infection in recent years. In addition to low molecular weight inhibitors, biological therapeutics have become promising anti-viral agents.     

TAT peptide internalization: seeking the mechanism of entry

During the last decade several peptides have been extensively studied for their ability to translocate across the plasma membrane. These peptides have been called "cell penetrating peptides" (CPP) or "protein transduction domains" (PTD). These peptides also promote the cellular uptake of various cargo molecules. Their mechanism of cellular entry appeared very intriguing since most publications in the field highlighted an energy-independent process. Indeed, cellular uptake of these peptides was still observed by fluorescence microscopy at low temperature or in the presence of several drugs known to inhibit active transport. In addition, internalization was reported to be much faster than known endocytic processes. However the involvement of a specific cellular component responsible for this uptake process appeared unlikely following intensive structure activity relationship studies using a wide panel of Tat analogues. Several reports about a possible artefactual redistribution of CPPs, and their associated cargos, during the cell fixation step commonly used for fluorescence microscopy have recently emerged in the literature. Moreover strong ionic interactions of CPPs with the cell surface also led to an overestimation of the recorded cell-associated fluorescent signal. It now seems well established that arginine-rich peptides are internalized by an energy dependent process involving endocytosis. Whatever the case, however, an increasing number of data indicate that the conjugation of non-permeant molecules to these CPPs allows their cellular uptake and leads to the expected biological responses, thus pointing to the interest of this delivery strategy. However, initial structure activity relationship studies of these CPPs will have to be reconsidered and the relative potency of each peptide (and their analogues) to vectorize the cargos to their most appropriate subcellular compartment will require careful re-evaluation.     

A Modular Synthesis of Modified Phosphoanhydrides

Phosphoanhydrides (P‐anhydrides) are ubiquitously occurring modifications in nature. Nucleotides and their conjugates, for example, are among the most important building blocks and signaling molecules in cell biology. To study and manipulate their biological functions, a diverse range of analogues have been developed. Phosphate‐modified analogues have been successfully applied to study proteins that depend on these abundant cellular building blocks, but very often both the preparation and purification of these molecules are challenging. This study discloses a general access to P‐anhydrides, including different nucleotide probes, that greatly facilitates their preparation and isolation. The convenient and scalable synthesis of, for example, ¹⁸O labeled nucleoside triphosphates holds promise for future applications in phosphoproteomics.     

Solution structure of a HNA-RNA hybrid

BACKGROUND: Synthetic nucleic acid analogues with a conformationally restricted sugar-phosphate backbone are widely used in antisense strategies for biomedical and biochemical applications. The modified backbone protects the oligonucleotides against degradation within the living cell, which allows them to form stable duplexes with sequences in target mRNAs with the aim of arresting their translation. The biologically most active antisense oligonucleotides also trigger cleavage of the target RNA through activation of endogenous RNase H. Systematic studies of synthetic oligonucleotides have also been conducted to delineate the origin of the chirality of DNA and RNA that are both composed of D-nucleosides. RESULTS: Hexitol nucleic acids (HNA) are the first example of oligonucleotides with a six-membered carbohydrate moiety that can bind strongly and selectively to complementary RNA oligomers. We present the first high resolution nuclear magnetic resonance structure of a HNA oligomer bound to a complementary RNA strand. The HNA-RNA complex forms an anti-parallel heteroduplex and adopts a helical conformation that belongs to the A-type family. Possibly, due to the rigidity of the rigid chair conformation of the six-membered ring both the HNA and RNA strand in the duplex are well defined. The observed absence of end-fraying effects also indicate a reduced conformational flexibility of the HNA-RNA duplex compared to canonical dsRNA or an RNA-DNA duplex. CONCLUSIONS: The P-P distance across the minor groove, which is close to A-form, and the rigid conformation of the HNA-RNA complex, explain its resistance towards degradation by Rnase H. The A-form character of the HNA-RNA duplex and the reduced flexibility of the HNA strand is possibly responsible for the stereoselectivity of HNA templates in non-enzymatic replication of oligonucleotides, supporting the theory that nucleosides with six-membered rings could have existed at some stage in molecular evolution.     

Discovery of bioactive small-molecule inhibitor of poly adp-ribose polymerase: implications for energy-deficient cells

Poly (ADP-ribose) polymerase (PARP1) is a nuclear protein that, when overactivated by oxidative stress-induced DNA damage, ADP ribosylates target proteins leading to dramatic cellular ATP depletion. We have discovered a biologically active small-molecule inhibitor of PARP1. The discovered compound inhibited PARP1 enzymatic activity in vitro and prevented ATP loss and cell death in a surrogate model of oxidative stress in vivo. We also investigated a new use for PARP1 inhibitors in energy-deficient cells by using Huntington's disease as a model. Our results showed that insult with the oxidant hydrogen peroxide depleted cellular ATP in mutant cells below the threshold of viability. The protective role of PARP1 inhibitors against oxidative stress has been shown in this model system.     

Cellular targets of natural products

Natural products have evolved, at least in part, to bind to biological macromolecules, particularly proteins. As a result, natural products are able to interact with many specific targets within the cell. Indeed for many years this has been central in the drug development process. Today, however, natural products are finding increasing use as probes to interrogate biological systems as part of chemical genomics and related research. In order to demonstrate the utility of natural products in these efforts, the biological activities of many of the major classes of natural products is discussed, according to the cellular organelle and localisation of their specific molecular targets. Emphasis is given to newly discovered compounds and activities that either provide interesting insights into a specific biological function, or that form the basis for potentially new therapeutic approaches.     

Planar Chiral Ligands:Design and Applications in Asymmetric Synthesis

Chiral ligands play an important role in asymmetric synthesis. Among them the ligands having planar chirality attract more interesting of organic chemists because of their unique structure. Recently, some new types of planar chiral ligands, including 1,1'-disubstituted ferrocene 1, bis(ferrocene carboxylic)diaminocyclohexane 2, and benzylic substituted cyclophane 3, are synthesized (Scheme 1)^[1]. These chiral ligands have been successfully used in asymmetric allylic alkylation, Heck reaction, etc. The role of planar chirality in asymmetric induction by using NMR and X-ray are also studied.     

Phosphoric Acid-Catalyzed Enantioselective Synthesis of Axially Chiral Anthrone-based Compounds

Anthrones and analogues are structural cores shared by diverse pharmacologically active natural and synthetic compounds. The sp²-rich nature imposes inherent obstruction to introduce stereogenic element onto the tricyclic aromatic backbone. In our pursuit to expand the chemical space of axial chirality, a novel type of axially chiral anthrone-derived skeleton was discovered. This work establishes oxime ether as suitable functionality to furnish axial chirality on symmetric anthrone skeletons through stereoselective condensation of the carbonyl entity with long-range chirality control. The enantioenriched anthrones could be elaborated into dibenzo-fused seven-membered N-heterocycles containing well-defined stereogenic center via Beckmann rearrangement with axial-to-point chirality conversion.     

Cell-penetrating peptides as delivery vehicles for biology and medicine

Cell-penetrating peptides (CPPs) have found numerous applications in biology and medicine since the first synthetic cell-permeable sequence was identified two decades ago. Numerous types of drugs have been transported into cells using CPPs, including small-molecule pharmaceuticals, therapeutic proteins, and antisense oligonucleotides. Improved agents for medical imaging have been generated by conjugation with CPPs, with the appended peptides promoting cellular uptake and in some cases, cell-type specificity. Organelle-specific CPPs have also been generated, providing a means to target specific subcellular sites. This review highlights achievements in this area and illustrates the numerous examples where peptide chemistry was exploited as a means to provide new tools for biology and medicine.     

Calcium ions effectively enhance the effect of antisense peptide nucleic acids conjugated to cationic tat and oligoarginine peptides

Cell-penetrating peptides have been widely used to improve cellular delivery of a variety of proteins and antisense agents. However, recent studies indicate that such cationic peptides are predominantly entering cells via an endosomal pathway. We now show that the nuclear antisense effect in HeLa cells of a variety of peptide nucleic acid (PNA) peptide conjugates is significantly enhanced by addition of 6 mM Ca(2+) (as well as by the lysosomotrophic agent chloroquine). In particular, the antisense activities of Tat(48-60) and heptaarginine-conjugated PNAs were increased 44-fold and 8.5-fold, respectively. Evidence is presented that the mechanism involves endosomal release. The present results show that Ca(2+) can be used as an effective enhancer for in vitro cellular delivery of cationic peptide-conjugated PNA oligomers, and also emphasize the significance of the endosomal escape route for such peptides.     

Cellular Cations Control Conformational Switching of Inositol Pyrophosphate Analogues

The inositol pyrophosphate messengers (PP‐InsPs) are emerging as an important class of cellular regulators. These molecules have been linked to numerous biological processes, including insulin secretion and cancer cell migration, but how they trigger such a wide range of cellular responses has remained unanswered in many cases. Here, we show that the PP‐InsPs exhibit complex speciation behaviour and propose that a unique conformational switching mechanism could contribute to their multifunctional effects. We synthesised non‐hydrolysable bisphosphonate analogues and crystallised the analogues in complex with mammalian PPIP5K2 kinase. Subsequently, the bisphosphonate analogues were used to investigate the protonation sequence, metal‐coordination properties, and conformation in solution. Remarkably, the presence of potassium and magnesium ions enabled the analogues to adopt two different conformations near physiological pH. Understanding how the intrinsic chemical properties of the PP‐InsPs can contribute to their complex signalling outputs will be essential to elucidate their regulatory functions.     

Calixarenes and calix[4]resorcinarenes as chiral NMR solvating agents

Calixarenes (CAs) and calix[4]resorcinarenes are cavity compounds, chiral analogues of which have the potential to be used as reagents for the differentiation of enantiomers in NMR spectroscopy. The nature of the substituent groups attached to the cavity permits the preparation of organic- or water-soluble analogues. In NMR applications, chirality of the CAs or calix[4]resorcinarene is usually achieved through the attachment of enantiomerically pure substituent groups. The use of inherently chiral analogues for chiral differentiation is less common. The range of CAs and calix[4]resorcinarenes that have been used for chiral analysis in NMR spectroscopy is reviewed.     

Synthesis of New Chiral Building Blocks for Novel Peptide Nucleic Acids

IntroductionAbouttenyearsago ,PNA ,astructuralmimicofDNAinwhichthesugar phosphatebackboneisreplacedbyN (2 aminoethyl)glycine (aeg)linkageemergedasapotentialanti sensetherapeuticagent.1PNAhassomeadvantages:(1)itisstabletocellularnucleasesandproteases,(2 )ithybridizeswithcomplementaryDNAorRNA (cDNA/RNA)sequenceswithhighaffinity ,(3)ithaslownon specificinteractionwithcellularcontentsand (4 )itiseasilysynthesizedbyadoptionofsolidphasepeptidesynthesischemistry .However,thema jorlimitationo…     

Marked small molecule libraries: a truncated approach to molecular probe design

A truncated approach to the design of molecular probes from small molecule libraries is outlined, based upon the incorporation of a bioorthogonal marker. The applicability of this strategy to small molecule chemical genetics screens has been demonstrated using analogues of the known stress activated protein kinase (SAPK) pathway activator, anisomycin. Compounds marked with a propargyl group have shown activation of the SAPK pathways comparable to that induced by their parent structures, as demonstrated by immunoblot assays against the downstream target JNK1/2. The considerable advantages of this new approach to molecular probe design have been illustrated through the rapid development of a functionally active fluorescent molecular probe, through coupling of the marked analogues to fluorescent azides using the copper(i)-catalyzed Huisgen 1,3-dipolar cycloaddition reaction. Active molecular probes generated in this study were used to investigate cellular uptake through FACS analysis and confocal microscopy.     

Impact of the Core Chemistry of Self-Assembled Spherical Nucleic Acids on their In Vitro Fate

Nucleic acid therapeutics (NATs), such as mRNA, small interfering RNA or antisense oligonucleotides are extremely efficient tools to modulate gene expression and tackle otherwise undruggable diseases. Spherical nucleic acids (SNAs) can efficiently deliver small NATs to cells while protecting their payload from nucleases, and have improved biodistribution and muted immune activation. Self-assembled SNAs have emerged as nanostructures made from a single DNA-polymer conjugate with similar favorable properties as well as small molecule encapsulation. However, because they maintain their structure by non-covalent interactions, they might suffer from disassembly in biologically relevant conditions, especially with regard to their interaction with serum proteins. Here, we report a systematic study of the factors that govern the fate of self-assembled SNAs. Varying the core chemistry and using stimuli-responsive disulfide crosslinking, we show that extracellular stability upon binding with serum proteins is important for recognition by membrane receptors, triggering cellular uptake. At the same time, intracellular dissociation is required for efficient therapeutic release. Disulfide-crosslinked SNAs combine these two properties and result in efficient and non-toxic unaided gene silencing therapeutics. We anticipate these investigations will help the translation of promising self-assembled structures towards in vivo gene silencing applications.     

In Vitro Antioxidant and Anticancer Properties of Various E. senegalensis Extracts

Although Erythrina senegalensis is a plant widely used in traditional medicine in sub-Saharan Africa, its biological properties have been poorly investigated to date. We first characterized by conventional reactions the composition of several stem bark extracts and evaluated in acellular and cellular assays their pro- or antioxidant properties supported by their high phenolic and flavonoid content, particularly with the methanolic extract. The pro- or antioxidant effects observed did not correlate with their IC₅₀ concentrations against five cancer cell lines determined by MTT assay. Indeed, the CH₂Cl₂ extract and its ethyl acetate (EtOAc) subfraction appeared more potent although they harbored lower pro- or antioxidant effects. Nevertheless, at equipotent concentration, both extracts induced ER- and mitochondria-derived vacuoles observed by fluorescent microscopy that further led to non-apoptotic cell death. LC coupled to high resolution MS investigations have been performed to identify chemical compounds of the extracts. These investigations highlighted the presence of compounds formerly isolated from E. senegalensis including senegalensein that could be retrieved only in the EtOAc subfraction but also thirteen other compounds, such as 16:3-Glc-stigmasterol and hexadecanoic acid, whose anticancer properties have been previously reported. Nineteen other compounds remain to be identified. In conclusion, E. senegalensis appeared rich in compounds with antioxidant and anticancer properties, supporting its use in traditional practice and its status as a species of interest for further investigations in anticancer drug research.     

Synthesis of New Chrial Building Blocks for Novel Peptide Nucleic Acids

N-Boc protected amino acids of analogues of peptide nucleic acid (PNA),which are a class of conformationally constrained building blocks based on 4-aminoproline backbone with chirality at 2-c and 4-c,have been synthesized.Those monomers can be used for the construction of novel peptide nucleic acid analogues.     

A long-pitch orthoconic antiferroelectric mixture modified by isomeric and racemic homostructural dopants

Orthoconic Antiferroelectric Liquid Crystals (OAFLC) are recognised as a promising medium for display and photonic applications due to their unique electrooptical properties. The application of OAFLC is still hampered by a number of parasitic effects deteriorating the electrooptical performance of contemporary available materials. An attempt elaborating of a working OAFLC mixture with the helical pitch longer than the typical cell gap is reported. Using this mixture, near-perfect optical uniformity and excellent dark state at the zero electric field applied were obtained. Basic OAFLC mixture was doped with two homostructural analogues to study the influence of the molecular polarity and chirality on the electrooptical performance and chosen physical parameters. The physical and structural properties of admixtures and their electrooptical performance are presented and discussed.     

Compression and shear surface rheology in spread layers of beta-casein and beta-lactoglobulin

We investigate the surface viscoelasticity of beta-lactoglobulin and beta-casein spread surface monolayers using a recently discovered method. Step compressions are performed, and the surface pressure is measured as a function of time. This is a common experiment for surface monolayers. However in our experiments the pressure is recorded by two perpendicular sensors, parallel and perpendicular to the compression direction. This enables us to clearly measure the time relaxation of both the compression and shear moduli, at the same time, in a single experiment, and with a standard apparatus. beta-Lactoglobulin and beta-casein monolayers are interesting because of their importance in food science and because they exhibit universally slow dynamical behavior that is still not fully understood. Our results confirm that the compressional modulus dominates the total viscoelastic response in both proteins. Indeed for beta-casein we confirm that the shear modulus is always negligible, i.e., the layer is in a fluid state. In beta-lactoglobulin a finite shear modulus emerges above a critical concentration. We emphasize that in Langmuir trough dynamic experiments the surface pressure should be measured in both the compression and the perpendicular directions.