Salt-Induced Linker Dehydration Modulates Micellar Structure in Ether-Linked Sulfate Surfactants.

Ether-linked surfactants are widely used in formulations such as liquid soaps, but despite their ubiquity, it is unclear how n-ethylene glycol linkers in surfactants, such as sodium lauryl n-(ethylene glycol) sulfate (SLEnS), influence micellar packing in the presence of NaCl. In the present work, we probe the structure and hydration of ether linkers in micelles comprising monodisperse SLEnS surfactants using contrast-variation small-angle neutron scattering (CV-SANS) and small-angle X-ray scattering (SAXS). Using SAXS, changes in micellar structure were observed for SLEnS (n = 1, 2, or 3) arising from the extent of ethoxylation. Scattering profiles indicated a clear transition from elongated cylindrical micelles to shorter ellipsoidal micelles with increasing ethoxylation. With CV-SANS, micellar structure and linker geometries of SLE3S were able to be resolved, indicating that a change in micellar architecture is modulated by dehydration of the tri(ethylene glycol) linker, offering new insights into the role of water and ions in the self-assembly of this key class of surfactants.

Influence of Surfactant Structure on Polydisperse Formulations of Alkyl Ether Sulfates and Alkyl Amidopropyl Betaines.

Surfactants provide detergency, foaming, and texture in personal care formulations, yet the micellization of typical industrial primary and cosurfactants is not well understood, particularly in light of the polydisperse nature of commercial surfactants. Synergistic interactions are hypothesized to drive the formation of elongated wormlike self-assemblies in these mixed surfactant systems. Small-angle neutron scattering, rheology, and pendant drop tensiometry are used to examine surface adsorption, viscoelasticity, and self-assembly structure for wormlike micellar formulations comprising cocoamidopropyl betaine, and its two major components laurylamidopropyl betaine and oleylamidopropyl betaine, with sodium alkyl ethoxy sulfates. The tail length of sodium alkyl ethoxy sulfates was related to their ability to form wormlike micelles in electrolyte solutions, indicating that a tail length greater than 10 carbons is required to form wormlike micelles in NaCl solutions, with the decyl homologue unable to form elongated micelles and maintaining a low viscosity even at 20 wt % surfactant loading with 4 wt % NaCl present. For these systems, the incorporation of a disperse ethoxylate linker does not enable shorter chain surfactants to elongate into wormlike micelles for single-component systems; however, it could increase the interactions between surfactants in mixed surfactant systems. For synergy in surfactant mixing, the nonideal regular solution theory is used to study the sulfate/betaine mixtures. Tail mismatch appears to drive lower critical micelle concentrations, although tail matching improves synergy with larger relative reductions in critical micelle concentrations and greater micelle elongation, as seen by both tensiometric and scattering measurements.

Dual-Labelling Strategies for Nuclear and Fluorescence Molecular Imaging: Current Status and Future Perspectives.

Molecular imaging offers the possibility to investigate biological and biochemical processes non-invasively and to obtain information on both anatomy and dysfunctions. Based on the data obtained, a fundamental understanding of various disease processes can be derived and treatment strategies can be planned. In this context, methods that combine several modalities in one probe are increasingly being used. Due to the comparably high sensitivity and provided complementary information, the combination of nuclear and optical probes has taken on a special significance. In this review article, dual-labelled systems for bimodal nuclear and optical imaging based on both modular ligands and nanomaterials are discussed. Particular attention is paid to radiometal-labelled molecules for single-photon emission computed tomography (SPECT) and positron emission tomography (PET) and metal complexes combined with fluorescent dyes for optical imaging. The clinical potential of such probes, especially for fluorescence-guided surgery, is assessed.

Inhibition of N-type calcium ion channels by tricyclic antidepressants - experimental and theoretical justification for their use for neuropathic pain.

A number of tricyclic antidepressants (TCAs) are commonly prescribed off-label for the treatment of neuropathic pain. The blockade of neuronal calcium ion channels is often invoked to partially explain the analgesic activity of TCAs, but there has been very limited experimental or theoretical evidence reported to support this assertion. The N-type calcium ion channel (CaV2.2) is a well-established target for the treatment of neuropathic pain and in this study a series of eleven TCAs and two closely related drugs were shown to be moderately effective inhibitors of this channel when endogenously expressed in the SH-SY5Y neuroblastoma cell line. A homology model of the channel, which matches closely a recently reported Cryo-EM structure, was used to investigate via docking and molecular dynamics experiments the possible mode of inhibition of CaV2.2 channels by TCAs. Two closely related binding modes, that occur in the channel cavity that exists between the selectivity filter and the internal gate, were identified. The TCAs are predicted to position themselves such that their ammonium side chains interfere with the selectivity filter, with some, such as amitriptyline, also appearing to hinder the channel's ability to open. This study provides the most comprehensive evidence to date that supports the notion that the blockade of neuronal calcium ion channels by TCAs is at least partially responsible for their analgesic effect.

Colorimetric Detection of Extracellular Hydrogen Peroxide Using an Integrated Microfluidic Device.

It is well known that hydrogen peroxide (H2O2) is a signaling molecule essential for vital physiological reactions in mammalian cells, such as cell survival, intercellular communication, and cancer metabolism. However, to fully understand the function of H2O2, it is critical to monitor its intracellular and/or extracellular concentrations. Current techniques implemented to address this need require large sample volumes, expensive instrumentation, and long sample preparation and analysis times, inapplicable to inline or online monitoring. In this paper, a new integrated microfluidic device capable of overcoming these limitations is demonstrated for the colorimetric detection of extracellular hydrogen peroxide H2O2. The device contains an optical waveguide to determine absorbance changes and micromixers to enable complete mixing of reagents using a passive approach. This novel H2O2-sensing device has allowed the detection of H2O2 in the range of 0.5-60 µM with a detection limit of 167 ± 5.8 nM and a sensitivity of 13.5 ± 0.1 AU/mM. Proof of concept of the device was demonstrated by quantifying H2O2 release from benign prostatic epithelial (BPH-1) cells upon stimulation with phorbol 12-myristate 13-acetate (PMA). Results show that this integrated device can be potentially utilized to continuously monitor cell-released metabolites autonomously without constant human supervision during the process. Furthermore, this can be achieved without interfering with the cell culture conditions, as only a very small volume of conditioned media (less than 0.4 µL), and not the cells, is required.

The neuronal calcium ion channel activity of constrained analogues of MONIRO-1.

Structural modifications of the neuronal calcium channel blocker MONIRO-1, including constraining the phenoxyaniline portion of the molecule and replacing the guanidinium functionality with tertiary amines, led to compounds with significantly improved affinities for the endogenously expressed CaV2.2 channel in the SH-SY5Y neuroblastoma cell line. These analogues also showed promising activity towards the CaV3.2 channel, recombinantly expressed in HEK293T cells. Both of these ion channels have received attention as likely targets for the treatment of neuropathic pain. The dibenzoazepine and dihydrobenzodiazepine derivatives prepared in this study show an encouraging combination of neuronal calcium ion channel inhibitory potency, plasma stability and potential to cross the blood-brain-barrier.

Cellular Uptake and Photo-Cytotoxicity of a Gadolinium(III)-DOTA-Naphthalimide Complex "Clicked" to a Lipidated Tat Peptide.

A new bifunctional macrocyclic chelator featuring a conjugatable alkynyl-naphthalimide fluorophore pendant group has been prepared and its Gd(III) complex coupled to a cell-penetrating lipidated azido-Tat peptide derivative using Cu(I)-catalysed "click" chemistry. The resulting fluorescent conjugate is able to enter CAL-33 tongue squamous carcinoma cells, as revealed by confocal microscopy, producing a very modest anti-proliferative effect (IC50 = 93 µM). Due to the photo-reactivity of the naphthalimide moiety, however, the conjugate's cytotoxicity is significantly enhanced (IC50 = 16 µM) upon brief low-power UV-A irradiation.

Bioactive Mimetics of Conotoxins and other Venom Peptides.

Ziconotide (Prialt®), a synthetic version of the peptide ω-conotoxin MVIIA found in the venom of a fish-hunting marine cone snail Conus magnus, is one of very few drugs effective in the treatment of intractable chronic pain. However, its intrathecal mode of delivery and narrow therapeutic window cause complications for patients. This review will summarize progress in the development of small molecule, non-peptidic mimics of Conotoxins and a small number of other venom peptides. This will include a description of how some of the initially designed mimics have been modified to improve their drug-like properties.

M2 macrophage accumulation in the aortic wall during angiotensin II infusion in mice is associated with fibrosis, elastin loss, and elevated blood pressure.

Macrophages accumulate in blood vessels during hypertension. However, their contribution to vessel remodeling is unknown. In the present study, we examined the polarization state of macrophages (M1/M2) in aortas of mice during hypertension and investigated whether antagonism of chemokine receptors involved in macrophage accumulation reduces vessel remodeling and blood pressure (BP). Mice treated with ANG II (0.7 mg·kg(-1)·day(-1), 14 days) had elevated systolic BP (158 ± 3 mmHg) compared with saline-treated animals (122 ± 3 mmHg). Flow cytometry revealed that ANG II infusion increased numbers of CD45(+)CD11b(+)Ly6C(hi) monocytes and CD45(+)CD11b(+)F4/80(+) macrophages by 10- and 2-fold, respectively. The majority of macrophages were positive for the M2 marker CD206 but negative for the M1 marker inducible nitric oxide synthase. Expression of other M2 genes (arginase-1, Fc receptor-like S scavenger receptor, and receptor-1) was elevated in aortas from ANG II-treated mice, whereas M1 genes [TNF and chemokine (C-X-C motif) ligand 2] were unaltered. A PCR array to identify chemokine receptor targets for intervention revealed chemokine (C-C motif) receptor 2 (CCR2) to be upregulated in aortas from ANG II-treated mice, while flow cytometry identified Ly6C(hi) monocytes as the main CCR2-expressing cell type. Intervention with a CCR2 antagonist (INCB3344; 30 mg·kg(-1)·day(-1)), 7 days after the commencement of ANG II infusion, reduced aortic macrophage numbers. INCB334 also reduced aortic collagen deposition, elastin loss, and BP in ANG II-treated mice. Thus, ANG II-dependent hypertension in mice is associated with Ly6C(hi) monocyte and M2 macrophage accumulation in the aorta. Inhibition of macrophage accumulation with a CCR2 antagonist prevents ANG II-induced vessel fibrosis and elevated BP, highlighting this as a promising approach for the future treatment of vessel remodeling/stiffening in hypertension.

Inhibition of N-type calcium channels by fluorophenoxyanilide derivatives.

A set of fluorophenoxyanilides, designed to be simplified analogues of previously reported ω-conotoxin GVIA mimetics, were prepared and tested for N-type calcium channel inhibition in a SH-SY5Y neuroblastoma FLIPR assay. N-type or Cav2.2 channel is a validated target for the treatment of refractory chronic pain. Despite being significantly less complex than the originally designed mimetics, up to a seven-fold improvement in activity was observed.

Enantioselective total syntheses of the proposed structures of prevezol B and evaluation of anti-cancer activity.

The first enantioselective total syntheses of the proposed structures of the natural product prevezol B are reported. The reported syntheses complement the previously-reported syntheses of the proposed structures of prevezol C, a stereoisomer of prevezol B. It was previously shown that the structure of the naturally occurring prevezol C had been incorrectly assigned. This work has led us to conclude that the proposed structures of prevezol B are also incorrect and major revision of both of the structures of the prevezols B and C is required. Cytotoxicity studies on the human cervical cancer cell line HeLa revealed that the synthesized prevezol B and C compounds were not active even at the highest concentration used (100 µM). However, one of the synthetic precursors was shown to have modest potency against HeLa cells (IC50 = 23.5 ± 1.8 µM).

An iterative in silico and modular synthetic approach to aqueous soluble tercyclic α-helix mimetics.

Tercyclic scaffolds, designed to have improved synthetic accessibility and aqueous solubility, were evaluated as structural α-helix mimetics by using an iterative in silico approach. The synthesis of these tercyclic scaffolds was accomplished using a modular synthetic approach by employing functionalised methoxyphenyl units which were readily manipulated to allow the introduction of various nitrogen-based heterocycles. The ability of these scaffolds to mimic the key i, i + 3 and i + 7 residues of a polyalanine α-helix was ratified by in silico studies, X-ray crystallographic and NOESY analysis, and their aqueous solubility was measured by a kinetic turbidimetric method.

Quantitative preparative gas chromatography of caffeine with nuclear magnetic resonance spectroscopy.

Caffeine test solute was employed in combination with an internal standard (IS), 1,4-dimethoxybenzene, in preparative-gas chromatography (prep-GC), with nuclear magnetic resonance (NMR) experiments. The IS served to: (i) quantify the trapping efficiency of an external trapping assembly, consisting of a capillary column cryotrap at the end of the analytical column; (ii) quantify the solute response in different NMR samples; and (iii) permit correlation of expected level of response of a compound in the NMR experiment, based on relative responses of the IS and solute in the GC result. The recovery rate of caffeine from multiple injections of sample (1×, 2×, 5× and 10×) was 69.6 ± 1.3%, which correlated well (R(2) = 0.999) with the number of injections of compound. The (1)H-NMR spectrum was sufficient to enable structural characterisation of the reference caffeine compound, and was achieved with recovery of amounts of ≤10 µg from a single aliquot. Less than 400 µg of collected caffeine (40 replicate injections) was sufficient for structural characterisation by (13)C-NMR spectral analysis. The method allows development of approaches to separate unknown compounds in complex samples, and to separately use MS and NMR for their characterisation.

Lanthanide tags for site-specific ligation to an unnatural amino acid and generation of pseudocontact shifts in proteins.

Pseudocontact shifts (PCS) from paramagnetic lanthanide ions present powerful long-range structural restraints for structural biology by NMR spectroscopy, but site-specific tagging of proteins with lanthanides remains a challenge, as most of the available lanthanide tags require proteins with single cysteine residues. We show that cyclen-based paramagnetic lanthanide tags can be attached to proteins in a site-specific manner by Cu(I)-catalyzed azide-alkyne cycloaddition to a genetically encoded p-azido-l-phenylalanine residue with a tether that proved sufficiently short and rigid for the observation of PCSs in several proteins. Despite the sterically demanding conditions associated with bulky tags and reactions close to the protein surface, ligation yields consistently above 50% and approaching 100% were obtained with the help of the Cu(I)-stabilizing ligand BTTAA. The yields were high independent of the presence of cysteine residues, thereby avoiding the need for cysteine mutations associated with conventional lanthanide-tagging strategies.

The RpoB H481Y rifampicin resistance mutation and an active stringent response reduce virulence and increase resistance to innate immune responses in Staphylococcus aureus.

The occurrence of mutations in methicillin-resistant Staphylococcus aureus (MRSA) during persistent infection leads to antimicrobial resistance but may also impact host-pathogen interactions. Here, we investigate the host-pathogen consequences of 2 mutations arising in clinical MRSA during persistent infection: RpoB H481Y, which is linked to rifampicin resistance, and RelA F128Y, which is associated with an active stringent response. Allelic exchange experiments showed that both mutations cause global transcriptional changes, leading to upregulation of capsule production, with attenuated virulence in a murine bacteremia model and reduced susceptibility to both antimicrobial peptides and whole-blood killing. Disruption of capsule biosynthesis reversed these impacts on innate immune function. These data clearly link MRSA persistence and reduced virulence to the same mechanisms that alter antimicrobial susceptibility. Our study highlights the wider consequences of suboptimal antimicrobial use, where drug resistance and immune escape mechanisms coevolve, thus increasing the likelihood of treatment failure.

ω-Conotoxin GVIA mimetics that bind and inhibit neuronal Ca(v)2.2 ion channels.

The neuronal voltage-gated N-type calcium channel (Ca(v)2.2) is a validated target for the treatment of neuropathic pain. A small library of anthranilamide-derived ω-Conotoxin GVIA mimetics bearing the diphenylmethylpiperazine moiety were prepared and tested using three experimental measures of calcium channel blockade. These consisted of a ¹²5I-ω-conotoxin GVIA displacement assay, a fluorescence-based calcium response assay with SH-SY5Y neuroblastoma cells, and a whole-cell patch clamp electrophysiology assay with HEK293 cells stably expressing human Ca(v)2.2 channels. A subset of compounds were active in all three assays. This is the first time that compounds designed to be mimics of ω-conotoxin GVIA and found to be active in the ¹²5I-ω-conotoxin GVIA displacement assay have also been shown to block functional ion channels in a dose-dependent manner.

pH-tuneable binding of 2'-phospho-ADP-ribose to ketopantoate reductase: a structural and calorimetric study.

The crystal structure of Escherichia coli ketopantoate reductase in complex with 2'-monophosphoadenosine 5'-diphosphoribose, a fragment of NADP+ that lacks the nicotinamide ring, is reported. The ligand is bound at the enzyme active site in the opposite orientation to that observed for NADP+, with the adenine ring occupying the lipophilic nicotinamide pocket. Isothermal titration calorimetry with R31A and N98A mutants of the enzyme is used to show that the unusual ;reversed binding mode' observed in the crystal is triggered by changes in the protonation of binding groups at low pH. This research has important implications for fragment-based approaches to drug design, namely that the crystallization conditions and the chemical modification of ligands can have unexpected effects on the binding modes.

The design and synthesis of inhibitors of pantothenate synthetase.

Pantothenate synthetase catalyses the ATP-dependent condensation of D-pantoate and beta-alanine to form pantothenate. Ten analogues of the reaction intermediate pantoyl adenylate, in which the phosphodiester is replaced by either an ester or sulfamoyl group, were designed as potential inhibitors of the enzyme. The esters were all modest competitive inhibitors, the sulfamoyls were more potent, consistent with their closer structural similarity to the pantoyl adenylate intermediate.