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Volume 9
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Pharmaceuticals, Volume 9, Issue 3 (September 2016) – 26 articles

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2182 KiB  
Review
Antimicrobial Peptides Targeting Gram-Positive Bacteria
by Nermina Malanovic and Karl Lohner
Pharmaceuticals 2016, 9(3), 59; https://doi.org/10.3390/ph9030059 - 20 Sep 2016
Cited by 293 | Viewed by 18442
Abstract
Antimicrobial peptides (AMPs) have remarkably different structures as well as biological activity profiles, whereupon most of these peptides are supposed to kill bacteria via membrane damage. In order to understand their molecular mechanism and target cell specificity for Gram-positive bacteria, it is essential [...] Read more.
Antimicrobial peptides (AMPs) have remarkably different structures as well as biological activity profiles, whereupon most of these peptides are supposed to kill bacteria via membrane damage. In order to understand their molecular mechanism and target cell specificity for Gram-positive bacteria, it is essential to consider the architecture of their cell envelopes. Before AMPs can interact with the cytoplasmic membrane of Gram-positive bacteria, they have to traverse the cell wall composed of wall- and lipoteichoic acids and peptidoglycan. While interaction of AMPs with peptidoglycan might rather facilitate penetration, interaction with anionic teichoic acids may act as either a trap for AMPs or a ladder for a route to the cytoplasmic membrane. Interaction with the cytoplasmic membrane frequently leads to lipid segregation affecting membrane domain organization, which affects membrane permeability, inhibits cell division processes or leads to delocalization of essential peripheral membrane proteins. Further, precursors of cell wall components, especially the highly conserved lipid II, are directly targeted by AMPs. Thereby, the peptides do not inhibit peptidoglycan synthesis via binding to proteins like common antibiotics, but form a complex with the precursor molecule, which in addition can promote pore formation and membrane disruption. Thus, the multifaceted mode of actions will make AMPs superior to antibiotics that act only on one specific target. Full article
(This article belongs to the Special Issue Antimicrobial Peptides: Expanded Activity Spectrum and Applications)
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Figure 1

Figure 1
<p>Percentage of hydrophobic residues of frog antimicrobial peptides (AMPs) in relation to the number of total peptides acting only against Gram-positive (<b>A</b>) and Gram-negative; (<b>B</b>) bacteria, respectively. The Antimicrobial Peptide Database [<a href="#B16-pharmaceuticals-09-00059" class="html-bibr">16</a>] displays 401 peptides from diverse genera of frog active against both, Gram-positive and Gram-negative bacteria. Sixty one AMPs are found to be specific for Gram-positive and 42 for Gram-negative bacteria, whereby only peptides are considered with MIC &lt; 100 μM or 100 μg/mL.</p> Full article ">Figure 2
<p>Schematic representation of membrane organization and morphology of selected Gram-positive bacteria. Gram-positive bacteria are characterized by a phospholipid bilayer of different composition (white ellipsoid, neutral phospholipid; grey ellipsoid, anionic phospholipid; black ellipsoid, cholesterol; black squares, neutral lipid). With the exception of L-form bacteria and mycoplasma, the plasma membrane (PM) of most Gram-positive bacteria is covered by peptidoglycan (PGN), which is a major component of the bacterial cell wall. The cell wall of cocci and bacilli contain also lipoteichoic acid (LTA), whereas mycobacteria and corynebacteria contain arabinogalactan (AG) and outer membrane (OM) composed of mycolic acid, sugar and lipids.</p> Full article ">Figure 3
<p>Schematic illustration of the cell envelope and main components of cell wall and plasma membrane (PM) of <span class="html-italic">S. aureus,</span> as a representative of Gram-positive bacteria.</p> Full article ">Figure 4
<p>Proposed interaction sites of AMPs interfering with PGN biosynthesis. Nisin is typical of a number of lantibiotics and defensins that specifically bind to lipid II, though different binding motifs may apply. In addition, nisin can form a distinct peptide-lipid II pore indicated on the right hand side of the scheme. Short linear and cyclic RW-rich peptides are representative for peptides that delocalize peripheral membrane proteins like MurG, thereby interfering with physiological processes. Lipid II structure is simplified showing in addition to peptide and carbon chains the phosphate (P) with negative charges, <span class="html-italic">N</span>-acetylglucosoamine (G) and <span class="html-italic">N</span>-acetylmuramic acid (M).</p> Full article ">Figure 5
<p>Scheme of proposed pore formation of daptomycin modified from [<a href="#B231-pharmaceuticals-09-00059" class="html-bibr">231</a>]. Insertion of the peptide in the membrane interface and oligomerization creates a void in the hydrophobic core (<b>A</b>) inducing local positive curvature accompanied by deeper penetration of the peptide complex (<b>B</b>). This results in a dimple formation with a locally strongly decreased membrane thickness [<a href="#B236-pharmaceuticals-09-00059" class="html-bibr">236</a>]. The oligomeric daptomycin may flip to the inner membrane leaflet and eventually combine with a complex of the outer leaflet to form a toroidal pore (<b>C</b>). Note, in the gel phase the void can be compensated by interdigitation of the lipid’s acyl chains (<b>D</b>) as shown for several AMPs [<a href="#B234-pharmaceuticals-09-00059" class="html-bibr">234</a>].</p> Full article ">
4037 KiB  
Review
TRPV3 in Drug Development
by Lisa M. Broad, Adrian J. Mogg, Elizabeth Eberle, Marcia Tolley, Dominic L. Li and Kelly L. Knopp
Pharmaceuticals 2016, 9(3), 55; https://doi.org/10.3390/ph9030055 - 9 Sep 2016
Cited by 51 | Viewed by 10701
Abstract
Transient receptor potential vanilloid 3 (TRPV3) is a member of the TRP (Transient Receptor Potential) super-family. It is a relatively underexplored member of the thermo-TRP sub-family (Figure 1), however, genetic mutations and use of gene knock-outs and selective pharmacological tools are [...] Read more.
Transient receptor potential vanilloid 3 (TRPV3) is a member of the TRP (Transient Receptor Potential) super-family. It is a relatively underexplored member of the thermo-TRP sub-family (Figure 1), however, genetic mutations and use of gene knock-outs and selective pharmacological tools are helping to provide insights into its role and therapeutic potential. TRPV3 is highly expressed in skin, where it is implicated in skin physiology and pathophysiology, thermo-sensing and nociception. Gain of function TRPV3 mutations in rodent and man have enabled the role of TRPV3 in skin health and disease to be particularly well defined. Pre-clinical studies provide some rationale to support development of TRPV3 antagonists for therapeutic application for the treatment of inflammatory skin conditions, itch and pain. However, to date, only one compound directed towards block of the TRPV3 receptor (GRC15300) has progressed into clinical trials. Currently, there are no known clinical trials in progress employing a TRPV3 antagonist. Full article
(This article belongs to the Special Issue Transient Receptor Potential (TRP) Channels in Drug Discovery: Old Concepts & New Thoughts)
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Figure 1

Figure 1
<p>Total publications per year around TRPV3 (orange bars) relative to other thermo-TRPs (TRPV1, TRPV2, TRPV4, TRPM8 and TRPA1). Data as of 25/05/2016. Searches were conducted using PubMed and, where applicable, included alternative nomenclature (e.g., TRPV1 and VR1).</p> Full article ">Figure 2
<p>Membrane topology of TRPV3. Residues involved in heat activation (N643, I644, N647, L658 and Y661), activation by 2-APB (H426 and R696), Camphor (C612 and C619) and modulation by phosphatidylinositol 4,5-bisphosphate (PI(4,5)P<sub>2</sub>; R696 and K705), ATP (K169 and K174), Mg<sup>2+</sup> (D641) and Ca<sup>2+</sup> (R696) are highlighted, in addition to the location of ankyrin repeats [<a href="#B7-pharmaceuticals-09-00055" class="html-bibr">7</a>,<a href="#B8-pharmaceuticals-09-00055" class="html-bibr">8</a>,<a href="#B9-pharmaceuticals-09-00055" class="html-bibr">9</a>,<a href="#B10-pharmaceuticals-09-00055" class="html-bibr">10</a>,<a href="#B11-pharmaceuticals-09-00055" class="html-bibr">11</a>,<a href="#B12-pharmaceuticals-09-00055" class="html-bibr">12</a>,<a href="#B13-pharmaceuticals-09-00055" class="html-bibr">13</a>].</p> Full article ">Figure 3
<p>Activators, inhibitors and modulators of TRPV3. Quaternary structure of TRPV3 with compounds and signalling pathways known to activate, inhibit or modulate the receptor. See Introduction for references–additional references [<a href="#B23-pharmaceuticals-09-00055" class="html-bibr">23</a>,<a href="#B24-pharmaceuticals-09-00055" class="html-bibr">24</a>,<a href="#B25-pharmaceuticals-09-00055" class="html-bibr">25</a>,<a href="#B26-pharmaceuticals-09-00055" class="html-bibr">26</a>].</p> Full article ">Figure 4
<p>Summary of TRPV3 expression and function in epidermal keratinocytes. TRPV3 protein has been found throughout the epidermis and around hair follicles, with protein elevated under certain inflammatory skin conditions.</p> Full article ">Figure 5
<p>Structure of TRPV3 selective antagonist FTP-THQ [<a href="#B63-pharmaceuticals-09-00055" class="html-bibr">63</a>].</p> Full article ">Figure 6
<p>FTP-THQ [1-({[3-fluoro-5-(trifluoromethyl)pyridine-2-yl]sulfanyl}acetyl)-8-methyl-1,2,3,4-tetrahydroquinoline], a potent and selective TRPV3 receptor antagonist blocked TRPV3 mediated release of ATP (<b>A</b> &amp; <b>B</b>) and GM-CSF (<b>C</b>) from mouse keratinocytes in vitro. Results are mean ± SEM of 3 independent experiments. Statistical significance was assessed using the paired Student’s <span class="html-italic">t</span>-test, * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.001.</p> Full article ">Figure 7
<p>Effects of FTP-THQ on histamine-induced scratching behavior. Harlan CD-1 mice (<span class="html-italic">n</span> = 7–8/treatment group), 4–5 weeks old were acclimated to testing room for 1 h. FTP-THQ was administered at 30, 100, or 200 mg/kg i.p., 1 h prior to histamine, while diphenhyramine was administered at 20 mg/kg, 30 min prior to histamine. Animals were then placed inside a clear plexiglass chamber and the number of scratching bouts was scored for 20 min. Data were collected via Abacus software; one-way ANOVA with post-hoc Dunnett’s was used for analysis. * <span class="html-italic">p</span> &lt; 0.05 vs. vehicle control.</p> Full article ">Figure 8
<p>Effects of FTP-THQ on formalin-induced nocifensive behavior. Harlan Sprague Dawley Rats (<span class="html-italic">n</span> = 7–8/treatment group), were acclimated to the testing room for 1 h. FTP-THQ was administered at 50, 100, or 200 mg/kg i.p. 15 min prior to formalin, while the positive control tramadol was administered at 40 mg/kg i.p. 30 min prior to formalin. The animals were placed in Startle Behavior Chambers and behavior events (licking, guarding, flinching) binned in 5-min intervals and plotted as Early and Late Phase. Data were analyzed using 1-way ANOVA, and comparisons of drug treatment groups were compared with control groups using a post-hoc Dunnett’s comparison. * <span class="html-italic">p</span> &lt; 0.05 vs. vehicle control.</p> Full article ">Figure 9
<p>Timeline of major TRPV3 development activities.</p> Full article ">Figure 10
<p>Number of patents applied for and granted per year for each of the thermo-TRPs compared to TRPV3 (orange bars).</p> Full article ">Figure 11
<p>Examples of patented TRPV3 antagonists from Glenmark Pharmaceuticals.</p> Full article ">Figure 12
<p>Representatives from Hydra Biosciences patented TRPV3 antagonist series.</p> Full article ">Figure 13
<p>A leading representative from Abbvie’s TRPV3 antagonist series.</p> Full article ">
985 KiB  
Article
Preparation of Temozolomide-Loaded Nanoparticles for Glioblastoma Multiforme Targeting—Ideal Versus Reality
by Chooi Yeng Lee and Ing Hong Ooi
Pharmaceuticals 2016, 9(3), 54; https://doi.org/10.3390/ph9030054 - 8 Sep 2016
Cited by 33 | Viewed by 7308
Abstract
Temozolomide (TMZ) is one of the most effective chemotherapeutic agents for glioblastoma multiforme, but the required high administration dose is accompanied by side effects. To overcome this problem and to further improve TMZ’s efficacy, targeted delivery of TMZ by using polymeric nanoparticles has [...] Read more.
Temozolomide (TMZ) is one of the most effective chemotherapeutic agents for glioblastoma multiforme, but the required high administration dose is accompanied by side effects. To overcome this problem and to further improve TMZ’s efficacy, targeted delivery of TMZ by using polymeric nanoparticles has been explored. We synthesised the PLGA-PEG-FOL copolymer and attempted encapsulation of TMZ into PLGA-PEG-FOL nanoparticles using the emulsion solvent evaporation method and the nanoprecipitation method. Conjugation of PEG and FOL to PLGA has been reported to be able to increase the delivery of TMZ to the brain as well as targeting the glioma cells. However, despite making numerous modifications to these methods, the loading of TMZ in the nanoparticles only ranged between 0.2% and 2%, and the nanoparticles were between 400 nm and 600 nm in size after freeze-drying. We proceed with determining the release profile of TMZ in phosphate buffered saline (PBS). Our initial data indicated that TMZ was slowly released from the nanoparticles. The metabolite of TMZ rather than the parent compound was detected in PBS. Our study suggests that while PLGA-PEG-FOL can be used as a polymeric or encapsulation material for central delivery of TMZ, a practical and cost effective formulation method is still far from reach. Full article
(This article belongs to the Special Issue Nanobiotechnology in Medicinal Chemistry)
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Figure 1

Figure 1
<p>The release of the metabolic product of TMZ, 5-aminoimidazole-4-carboxamide (AIC) in vitro from the TMZ-loaded PLGA-PEG-FOL nanoparticles.</p> Full article ">Figure 2
<p>(<b>Top</b>) The NMR spectrum of PLGA-PEG-FOL. (<b>Bottom</b>) The numbering in the chemical structure of PLGA-PEG-FOL refers to the corresponding proton resonance peaks in the NMR spectrum.</p> Full article ">Scheme 1
<p>Reaction scheme for the synthesis of PLGA-PEG-COOH from PLGA-COOH.</p> Full article ">Scheme 2
<p>Reaction scheme for the synthesis of PLGA-PEG-FOL.</p> Full article ">
1831 KiB  
Article
1H and 15N NMR Analyses on Heparin, Heparan Sulfates and Related Monosaccharides Concerning the Chemical Exchange Regime of the N-Sulfo-Glucosamine Sulfamate Proton
by Vitor H. Pomin
Pharmaceuticals 2016, 9(3), 58; https://doi.org/10.3390/ph9030058 - 7 Sep 2016
Cited by 11 | Viewed by 7001
Abstract
Heparin and heparan sulfate are structurally related glycosaminoglycans (GAGs). Both GAGs present, although in different concentrations, N-sulfo-glucosamine (GlcNS) as one of their various composing units. The conditional fast exchange property of the GlcNS sulfamate proton in these GAGs has been pointed as [...] Read more.
Heparin and heparan sulfate are structurally related glycosaminoglycans (GAGs). Both GAGs present, although in different concentrations, N-sulfo-glucosamine (GlcNS) as one of their various composing units. The conditional fast exchange property of the GlcNS sulfamate proton in these GAGs has been pointed as the main barrier to its signal detection via NMR experiments, especially 1H-15N HSQC. Here, a series of NMR spectra is collected on heparin, heparan sulfate and related monosaccharides. The N-acetyl glucosamine-linked uronic acid types of these GAGs were properly assigned in the 1H-15N HSQC spectra. Dynamic nuclear polarization (DNP) was employed in order to facilitate 1D spectral acquisition of the sulfamate 15N signal of free GlcNS. Analyses on the multiplet pattern of scalar couplings of GlcNS 15N has helped to understand the chemical properties of the sulfamate proton in solution. The singlet peak observed for GlcNS happens due to fast chemical exchange of the GlcNS sulfamate proton in solution. Analyses on kinetics of alpha-beta anomeric mutarotation via 1H NMR spectra have been performed in GlcNS as well as other glucose-based monosaccharides. 1D 1H and 2D 1H-15N HSQC spectra recorded at low temperature for free GlcNS dissolved in a proton-rich solution showed signals from all exchangeable protons, including those belonging to the sulfamate group. This work suits well to the current grand celebration of one-century-anniversary of the discovery of heparin. Full article
(This article belongs to the Special Issue Grand Celebration: 100th Anniversary of the Discovery of Heparin)
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Figure 1

Figure 1
<p>Structural representation of the main disaccharide building blocks of heparan sulfate [→4)-β-<span class="html-small-caps">d</span>-GlcA-(1→4)-α-<span class="html-small-caps">d</span>-GlcNAc-(1→] (<b>A</b>) and heparin [→4)-α-<span class="html-small-caps">l</span>-IdoA2S-(1→4)-α-<span class="html-small-caps">d</span>-GlcNS6S-(1→] (<b>B</b>). Although each unit can be seen in both GAG types, the first disaccharide is dominant in heparan sulfate while the second is more abundant in heparin. Structures highlight the different chemical exchange properties of the <sup>15</sup>N-linked protons of the <span class="html-italic">N</span>-acetyl and <span class="html-italic">N</span>-sulfated groups with the protons from the bulk solvent. Analysis on the scalar coupling multiplet pattern related to direct observation of <sup>15</sup>N in GlcNS is diagnostic of the chemical exchange regime of the amide proton.</p> Full article ">Figure 2
<p>2D NMR <sup>1</sup>H-<sup>15</sup>N HSQC spectra of heparin (<b>A</b>,<b>B</b>) and heparan sulfates isolated from Chinese Hamster Ovarian cells (<b>C</b>) and from the bivalve <span class="html-italic">Nodipecten nodosus</span> (<b>D</b>). Expansions are δ<sub>H</sub>/δ<sub>N</sub> 3.0–9.5/77.0–147.0 ppm (<b>A</b>) and δ<sub>H</sub>/δ<sub>N</sub> 8.1–8.5/118.0–124.6 ppm (<b>B</b>–<b>D</b>) for samples (10 mg/mL) dissolved in 50 mM sodium acetate buffer, 12.5% D<sub>2</sub>O (pH 4.5), 0.1% sodium azide. The solid and dashed circles highlight the observed and theoretical regions for the <sup>1</sup>H-<sup>15</sup>N cross-peaks of <span class="html-italic">N</span>-acetyl glucosamine (GlcNAc) and <span class="html-italic">N</span>-sulfo-glucosamine (GlcNS) units, respectively. Spectra were recorded at 18.8 T and 25 °C.</p> Full article ">Figure 3
<p>Comparison of the 1D NMR spectra <sup>15</sup>N direct-observe of the <sup>15</sup>N-isotopically labeled side chain glutamine (<sup>15</sup>N-Gln) at 23 mM with 98% <sup>15</sup>N abundance (<b>A</b>,<b>B</b>) versus <span class="html-italic">N</span>-sulfo-glucosamine (GlcNS) at 20 mM with <sup>15</sup>N natural abundance (0.37%) (<b>C</b>) at non-polarized (<b>A</b>) and hyperpolarized conditions (<b>B</b>,<b>C</b>). All spectra were recorded at 11.7 T and 37 °C.</p> Full article ">Figure 4
<p>Kinetics of anomeric mutarotation observed for glucose (Glc) and Glc-based standards such as glucosamine (GlcN), <span class="html-italic">N</span>-acetyl glucosamine (GlcNAc) and <span class="html-italic">N</span>-sulfo-glucosamine (GlcNS) measured through 1D <sup>1</sup>H NMR spectra recorded within different time courses after dissolution in 100% D<sub>2</sub>O (10 mg/mL). Anomeric proton signals (αH1 and βH1), their respective relative percentages and ring proton signals are indicated in the panels. HOD denotes residual water signals. Spectra were recorded at 18.8 T and 25 °C.</p> Full article ">Figure 5
<p>1D <sup>1</sup>H NMR spectra of <span class="html-italic">N</span>-sulfo-glucosamine (GlcNS). Expansions are 2.9–7.3 ppm (<b>A</b>) and 3.10–3.55 ppm (<b>B</b>,<b>C</b>) for GlcNS (10 mg/mL) dissolved in 10%:20%:70% D<sub>2</sub>O/acetone/H<sub>2</sub>O for spectra recorded in 18.8 T NMR instrument at 3 °C (<b>A</b>,<b>B</b>) or 37 °C (<b>C</b>). Spectrum in panel B is just a close-up window of the correspondent region in spectrum of panel A.</p> Full article ">
1067 KiB  
Review
Targeting TRPM2 in ROS-Coupled Diseases
by Shinichiro Yamamoto and Shunichi Shimizu
Pharmaceuticals 2016, 9(3), 57; https://doi.org/10.3390/ph9030057 - 7 Sep 2016
Cited by 34 | Viewed by 8435
Abstract
Under pathological conditions such as inflammation and ischemia-reperfusion injury large amounts of reactive oxygen species (ROS) are generated which, in return, contribute to the development and exacerbation of disease. The second member of the transient receptor potential (TRP) melastatin subfamily, TRPM2, is a [...] Read more.
Under pathological conditions such as inflammation and ischemia-reperfusion injury large amounts of reactive oxygen species (ROS) are generated which, in return, contribute to the development and exacerbation of disease. The second member of the transient receptor potential (TRP) melastatin subfamily, TRPM2, is a Ca2+-permeable non-selective cation channel, activated by ROS in an ADP-ribose mediated fashion. In other words, TRPM2 functions as a transducer that converts oxidative stress into Ca2+ signaling. There is good evidence that TRPM2 plays an important role in ROS-coupled diseases. For example, in monocytes the influx of Ca2+ through TRPM2 activated by ROS contributes to the aggravation of inflammation via chemokine production. In this review, the focus is on TRPM2 as a molecular linker between ROS and Ca2+ signaling in ROS-coupled diseases. Full article
(This article belongs to the Special Issue Transient Receptor Potential (TRP) Channels in Drug Discovery: Old Concepts & New Thoughts)
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Figure 1

Figure 1
<p>ROS production during inflammation and ischemia-reperfusion. (<b>A</b>) In resting state, cytosolic activators such as p40<sup>phox</sup>, p47<sup>phox</sup>, p67<sup>phox</sup> and small G protein RAC do not interact with NOX2-p22<sup>phox</sup> complex. These activators translocate to the plasma membrane during phagocytosis and interact with the NOX2-p22<sup>phox</sup> complex. Electrons derived from NADPH are transferred through the complex to molecular oxygen, leading to <sup>·</sup>O<sub>2</sub><sup>−</sup> production; (<b>B</b>) Oxidative phosphorylation is initiated by electron transport from NADH and/or FADH<sub>2</sub> to the electron transport chain in the mitochondrial inner membrane. The electron transport chain is composed of complexes I–IV. Electrons derived from NADH and FADH<sub>2</sub> are fed to complex I and complex II, respectively. They are then transferred to complexes in ascending order of the redox potential, which release free energy. Molecular oxygen accepts electrons for the formation of H<sub>2</sub>O. On the other hand, the electron transport chain uses free energy derived from electron transport to pump H<sup>+</sup> out of the matrix, thereby creating proton gradient across the mitochondrial inner membrane. By utilizing energy released by the influx of H<sup>+</sup> into the matrix, ADP is phosphorylated, resulting in the generation of ATP. <sup>·</sup>O<sub>2</sub><sup>−</sup> is generated by the leakage of electrons from complexes I and III in the electron transport chain. The activity of the electron transport chain generates a relatively small amount of <sup>·</sup>O<sub>2</sub><sup>−</sup> under normal conditions, but its production may be greatly magnified by events occurring during ischemia-reperfusion. The expression of NOX isoforms is up-regulated by HIF1α during ischemia, and then NADPH oxidase then generates large amounts of ROS by reoxygenation during reperfusion. During ischemia, ATP is catabolized into hypoxanthine.</p> Full article ">Figure 2
<p>Does administration with TRPM2 inhibitors during ROS-coupled disease development improve the grade of these diseases? Pathological mouse model studies have been performed under <span class="html-italic">Trpm2</span>-disrupted conditions, and suggested that <span class="html-italic">Trpm2</span> KO mice are protected from ROS-coupled diseases. However, in terms of cure, it is important that the grade of these diseases is improved by the inhibition of TRPM2 during disease development. Therefore, the studies whether the inhibition of TRPM2 during ROS-coupled disease development has curative effects on the diseases should be done in the future.</p> Full article ">
1953 KiB  
Article
Therapeutic Potential of Gramicidin S in the Treatment of Root Canal Infections
by Marina Berditsch, Hannah Lux, Oleg Babii, Sergii Afonin and Anne S. Ulrich
Pharmaceuticals 2016, 9(3), 56; https://doi.org/10.3390/ph9030056 - 7 Sep 2016
Cited by 26 | Viewed by 6693
Abstract
An intrinsic clindamycin-resistant Enterococcus faecalis, the most common single species present in teeth after failed root canal therapy, often possesses acquired tetracycline resistance. In these cases, root canal infections are commonly treated with Ledermix® paste, which contains demeclocycline, or the new [...] Read more.
An intrinsic clindamycin-resistant Enterococcus faecalis, the most common single species present in teeth after failed root canal therapy, often possesses acquired tetracycline resistance. In these cases, root canal infections are commonly treated with Ledermix® paste, which contains demeclocycline, or the new alternative endodontic paste Odontopaste, which contains clindamycin; however, these treatments are often ineffective. We studied the killing activity of the cyclic antimicrobial peptide gramicidin S (GS) against planktonic and biofilm cells of tetracycline-resistant clinical isolates of E. faecalis. The high therapeutic potential of GS for the topical treatment of problematic teeth is based on the rapid bactericidal effect toward the biofilm-forming, tetracycline-resistant E. faecalis. GS reduces the cell number of planktonic cells within 20–40 min at a concentration of 40–80 μg/mL. It kills the cells of pre-grown biofilms at concentrations of 100–200 μg/mL, such that no re-growth is possible. The translocation of the peptide into the cell interior and its complexation with intracellular nucleotides, including the alarmon ppGpp, can explain its anti-biofilm effect. The successful treatment of persistently infected root canals of two volunteers confirms the high effectiveness of GS. The broad GS activity towards resistant, biofilm-forming E. faecalis suggests its applications for approval in root canal medication. Full article
(This article belongs to the Special Issue Antimicrobial Peptides: Expanded Activity Spectrum and Applications)
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Figure 1

Figure 1
<p>Anti-biofilm activity of GS. (<b>A</b>) When eight <span class="html-italic">E. faecalis</span> strains were compared using a crystal violet staining assay, the tetracycline-resistant clinical isolate TRE2 exhibited the strongest biofilm formation in all three nutrient media; (<b>B1</b>) SEM image of a TRE2 biofilm formed on a hydroxyapatite disk, which served as a control and as a starting point for antibiotic treatment; (<b>B2</b>) when B1 was incubated in TH broth, the biofilm is found to re-grow in a suspended form; (<b>C1</b>) SEM image of B1 after treatment with 400 μg/mL demeclocycline, and (<b>C2</b>) re-growth of a biofilm in suspension; (<b>D1</b>) B1 after treatment with 400 μg/mL GS; and (<b>D2</b>) no biofilm re-growth was observed after treatment, even when only 200 μg/mL of GS were used in D1. Reproducible results were obtained in two independent experiments. The scale on the SEM images is 1 μm.</p> Full article ">Figure 2
<p>Reduction of the number of <span class="html-italic">E. faecalis</span> bacteria during exposure to GS: (<b>A</b>) control strain DSM 2570; (<b>B</b>) cheese isolate WW4; (<b>C</b>) root canal isolate WW6; and (<b>D</b>–<b>F</b>) clinical tetracycline-resistant isolates TRE1, TRE2, and TRE4, respectively. The bacterial number remained unchanged in the controls (dotted lines), but decreased drastically during exposure to 5 × MIC of GS (solid lines). The dashed lines show the faster bactericidal effect of 10 × MIC for DSM 2570 and WW6 strains; 10 CFU/mL was taken as the detection limit although no colonies were grown from undiluted sample. The standard deviations were calculated from at least two independent experiments.</p> Full article ">Figure 3
<p>Translocation of GS into the cells and its binding affinity to the bacterial alarmon ppGpp. (<b>A</b>) Green fluorescence of the <span class="html-italic">E. faecalis</span> cell envelope upon staining with the dye CFSE, which cannot penetrate the cellular membrane. (<b>B</b>) Fluorescence throughout the interior of the cells is seen upon staining with a fluorescent GS-sw(FP) analog, confirming that the peptide can translocate across the cell membrane into the cytoplasm. (<b>C</b>) Co-precipitation of GS and ppGpp is seen to occur at roughly equimolar ratios, and a stable opalescent suspension is formed when GS is in large (i.e., electrostatic) excess. (<b>D</b>) SEM showed that the aggregation ppGpp by GS led to the formation of short nano-rods of about 50 nm width and 200 nm length. (<b>E</b>) When GS was added to ppGpp at different molar ratios, the <sup>31</sup>P-NMR signals of the latter disappeared successively. The pure ppGpp sample (1:0) is shown in red. The scale on the fluorescent images is 5 μm.</p> Full article ">Figure 4
<p>Molar 36 of a 25-year-old male patient before and after treatment. (<b>A</b>) The radiolucent area in the X-ray image indicates decay. (<b>B</b>) The sealed root canals after treatment with GS. The red arrowheads mark the decayed area and the sealed root canals, respectively.</p> Full article ">
250 KiB  
Review
The Role of Antimicrobial Peptides in Influenza Virus Infection and Their Potential as Antiviral and Immunomodulatory Therapy
by I-Ni Hsieh and Kevan L. Hartshorn
Pharmaceuticals 2016, 9(3), 53; https://doi.org/10.3390/ph9030053 - 6 Sep 2016
Cited by 67 | Viewed by 8093
Abstract
Influenza A virus (IAV) remains a major threat that can cause severe morbidity and mortality due to rapid genomic variation. Resistance of IAVs to current anti-IAV drugs has been emerging, and antimicrobial peptides (AMPs) have been considered to be potential candidates for novel [...] Read more.
Influenza A virus (IAV) remains a major threat that can cause severe morbidity and mortality due to rapid genomic variation. Resistance of IAVs to current anti-IAV drugs has been emerging, and antimicrobial peptides (AMPs) have been considered to be potential candidates for novel treatment against IAV infection. AMPs are endogenous proteins playing important roles in host defense through direct antimicrobial and antiviral activities and through immunomodulatory effects. In this review, we will discuss the anti-IAV and immunomodulatory effects of classical AMPs (defensins and cathelicidins), and proteins more recently discovered to have AMP-like activity (histones and Alzheimer’s associated β-amyloid). We will discuss the interactions between AMPs and other host defense proteins. Major emphasis will be placed on novel synthetic AMPs derived from modification of natural proteins, and on potential methods of increasing expression of endogenous AMPs, since these approaches may lead to novel antiviral therapeutics. Full article
(This article belongs to the Special Issue Antimicrobial Peptides: Expanded Activity Spectrum and Applications)
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Review
TRPV1: A Target for Rational Drug Design
by Vincenzo Carnevale and Tibor Rohacs
Pharmaceuticals 2016, 9(3), 52; https://doi.org/10.3390/ph9030052 - 23 Aug 2016
Cited by 84 | Viewed by 12780
Abstract
Transient Receptor Potential Vanilloid 1 (TRPV1) is a non-selective, Ca2+ permeable cation channel activated by noxious heat, and chemical ligands, such as capsaicin and resiniferatoxin (RTX). Many compounds have been developed that either activate or inhibit TRPV1, but none of them are [...] Read more.
Transient Receptor Potential Vanilloid 1 (TRPV1) is a non-selective, Ca2+ permeable cation channel activated by noxious heat, and chemical ligands, such as capsaicin and resiniferatoxin (RTX). Many compounds have been developed that either activate or inhibit TRPV1, but none of them are in routine clinical practice. This review will discuss the rationale for antagonists and agonists of TRPV1 for pain relief and other conditions, and strategies to develop new, better drugs to target this ion channel, using the newly available high-resolution structures. Full article
(This article belongs to the Special Issue Transient Receptor Potential (TRP) Channels in Drug Discovery: Old Concepts & New Thoughts)
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Figure 1

Figure 1
<p>Molecular architecture of the Transient Receptor Potential Vanilloid 1 channel (TRPV1). Cartoon representation of the structure of the transmembrane region of TRPV1 as determined via cryoEM in lipid nanodiscs: (<b>A</b>) side view; and (<b>B</b>) top view. For clarity, only two subunits are highlighted in solid color while the other two are rendered as grey shading. Different colors highlight the major structural elements described in the text: S1–S4 domain (blue), linker domain (red), S5 (orange), pore helix and selectivity filter (yellow), S6 (brown) and TRP domain (grey).</p> Full article ">Figure 2
<p>Binding mode of TRPV1 modulators. Structure of TRPV1 in complex with: RTX (<b>A</b>); and capsazepine <b>(B</b>). For clarity only the structural element surrounding the vanilloid binding site are shown in cartoon representation using the same color code used in <a href="#pharmaceuticals-09-00052-f001" class="html-fig">Figure 1</a>. Amino acid side chains contacting the ligands are shown as sticks; to highlight the location of ligand in the two structures, the carbon atoms of RTX and capsezepine are highlighted by the cyan color. Note how the conformations of RTX and capsazepine are very similar, except for a phenyl group, which, in RTX, contacts the side chains of S6.</p> Full article ">
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Article
Acceptability, Safety, and Efficacy of Oral Administration of Extracts of Black or Red Maca (Lepidium meyenii) in Adult Human Subjects: A Randomized, Double-Blind, Placebo-Controlled Study
by Carla Gonzales-Arimborgo, Irma Yupanqui, Elsa Montero, Dulce E. Alarcón-Yaquetto, Alisson Zevallos-Concha, Lidia Caballero, Manuel Gasco, Jianping Zhao, Ikhlas A. Khan and Gustavo F. Gonzales
Pharmaceuticals 2016, 9(3), 49; https://doi.org/10.3390/ph9030049 - 18 Aug 2016
Cited by 45 | Viewed by 19483
Abstract
The plant maca, grown at 4000 m altitude in the Peruvian Central Andes, contains hypocotyls that have been used as food and in traditional medicine for centuries. The aim of this research was to provide results on some health effects of oral administration [...] Read more.
The plant maca, grown at 4000 m altitude in the Peruvian Central Andes, contains hypocotyls that have been used as food and in traditional medicine for centuries. The aim of this research was to provide results on some health effects of oral administration of spray-dried extracts of black or red maca (Lepidium meyenii) in adult human subjects living at low (LA) and high altitude (HA). A total of 175 participants were given 3 g of either placebo, black, or red maca extract daily for 12 weeks. Primary outcomes were changes in sexual desire, mood, energy, health-related quality of life score (HRQL), and chronic mountain sickness (CMS) score, or in glycaemia, blood pressure, and hemoglobin levels. Secondary outcomes were acceptability and safety, assessed using the Likert test and side effect self-recording, respectively, and the effect of altitude. At low altitude, 32, 30, and 32 participants started the study receiving placebo, red maca, or black maca, respectively. At high altitudes, 33, 35, and 31 participants started the study receiving placebo, red maca, and black maca, respectively. Consumption of spray-dried extracts of red and black maca resulted in improvement in mood, energy, and health status, and reduced CMS score. Fatty acids and macamides were higher in spray-dried extracts of black maca than in red maca. GABA predominated in spray-dried extracts of red maca. Effects on mood, energy, and CMS score were better with red maca. Black maca and, in smaller proportions, red maca reduced hemoglobin levels only in highlanders with abnormally high hemoglobin levels; neither variety of maca reduced hemoglobin levels in lowlanders. Black maca reduced blood glucose levels. Both varieties produced similar responses in mood, and HRQL score. Maca extracts consumed at LA or HA had good acceptability and did not show serious adverse effects. In conclusion, maca extract consumption relative to the placebo improved quality of life parameters. Differences in the level of improvement between red and black maca are probably due to differences in the composition of these two plant varieties. Both maca extracts were well tolerated and safe. Full article
(This article belongs to the Special Issue Nutraceuticals and Botanicals: Bioactive Molecules and Therapeutic Properties for Human Health)
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<p>Percentage of subjects perceiving an increase in sexual desire following a 12-week treatment with the placebo or a spray-dried extract of red or black maca at low (<b>Upper</b>) and high altitudes (<b>Bottom</b>). Bars are standard error. At LA, consumption of spray-dried extract of red maca increases the percentage of subjects with increased sexual desire over time (<span class="html-italic">p</span> = 0.0055). <span class="html-italic">p</span> &gt; 0.05 between placebo and maca-treated groups. HA: <span class="html-italic">p</span> = 0.0027, <span class="html-italic">p</span> = 0.0000, and <span class="html-italic">p</span> = 0.0019 over time for treatments with placebo, red maca, and black maca, respectively. <span class="html-italic">p</span> = 0.03 (chi square = 6.96) at week 12 between treatment with spray-dried extract of red maca and placebo or spray-dried extract of black maca.</p> Full article ">Figure 2
<p>Percentage of subjects perceiving an increase in mood after treatment for 12 weeks with a placebo, spray-dried extract of red maca, and spray-dried extract of black maca. Percentage of subjects with a score for mood of 4–5 (in agreement or completely in agreement that consumption of the product increased their mood) at (<b>Upper</b>) low and (<b>Bottom</b>) high altitude. LA: <span class="html-italic">p</span> = 0.01; 0.01; 0.029; 0.0026 using chi square test at weeks 3, 4, 8, and 12 comparing treatment with placebo, spray-dried extract of red maca, and spray-dried extract of black maca. Placebo: <span class="html-italic">p</span> &gt; 0.05 over time (weeks 1 to 12); red maca <span class="html-italic">p</span> = 0.0001 over time; black maca <span class="html-italic">p</span> &gt; 0.05 over time (weeks 1 to 12). HA: <span class="html-italic">p</span> = 0.019; 0.04; and 0.05 using chi square test at weeks 1,2, and 12 comparing treatment with placebo, spray-dried extract of red maca and spray-dried extract of black maca. Placebo: <span class="html-italic">p</span> = 0.0001; red maca: <span class="html-italic">p</span> = 0.003; black maca: <span class="html-italic">p</span> = 0.0002 over time.</p> Full article ">Figure 3
<p>Percentage of subjects perceiving an increase in energy after treatment for 12 weeks with a placebo, red maca, or black maca at low (<b>Upper</b>) and high (<b>Bottom</b>) altitude. LA: <span class="html-italic">p</span> = 0.07; 0.02; 0.01; 0.003; 0.001 and &lt;0.00001 using chi square test at weeks 1, 2, 3, 4, 8, and 12 comparing treatment with placebo, red maca, or black maca. Placebo group: <span class="html-italic">p</span> &gt; 0.05 over time (weeks 1 to 12); red maca group: <span class="html-italic">p</span> = 0.0001 over time (weeks 1 to 12); black maca group: <span class="html-italic">p</span> &gt; 0.05 over time (weeks 1 to 12). HA: <span class="html-italic">p</span> &gt; 0.05 between placebo group and groups with red or black maca (chi square test at weeks 1, 2, 3, 4, 8 and 12). Placebo: <span class="html-italic">p</span> = 0.0001; red maca: <span class="html-italic">p</span> = 0.0001; black maca: <span class="html-italic">p</span> = 0.0001 over time (weeks 1 to 12).</p> Full article ">Figure 4
<p>Heath-Related Quality of Life Score (HRQL) in subjects receiving a 12-week treatment of a placebo, spray-dried extract of red maca, or spray-dried extract of black maca at low (<b>Upper</b>) or high altitude (<b>Bottom</b>). Data are mean ± standard error of the mean. <b>Upper</b>: LA: Placebo group: <span class="html-italic">p</span> &gt; 0.05 with respect to time 0. Red maca group: <span class="html-italic">p</span> &lt; 0.05 at week 8 and <span class="html-italic">p</span> &lt; 0.01 at week 12 with respect to time 0 (one tail). Black maca group: <span class="html-italic">p</span> &lt; 0.05 at week 4 with respect to time 0 and <span class="html-italic">p</span> &lt; 0.01 at weeks 8 and 12 (one tail). <span class="html-italic">p</span> &lt; 0.05 comparing red maca group with placebo group at eight and 12 weeks of treatment and <span class="html-italic">p</span> &lt; 0.05 at week 4, and <span class="html-italic">p</span> &lt; 0.01 at weeks 8 and 12, comparing the group treated with black maca with the group treated with a placebo; <b>Bottom</b>: HA: placebo group: <span class="html-italic">p</span> &gt; 0.05 with respect to time 0. Red Maca group: <span class="html-italic">p</span> &lt; 0.05 at week 8 and <span class="html-italic">p</span> &lt; 0.01 at week 12 with respect to time 0. Black maca group: <span class="html-italic">p</span> &lt; 0.01 at weeks 8 and 12 with respect to time 0. <span class="html-italic">p</span> &lt; 0.05 black and red maca groups at weeks 8 and 12 with respect to the placebo group.</p> Full article ">Figure 5
<p>Delta of Qinghai score for Chronic Mountain Sickness (CMS) diagnosis in adult human subjects after a 12-week treatment with spray-dried extract of maca (red or black) or with a placebo at high altitude. Data are mean ± standard error of the mean. Placebo: <span class="html-italic">p</span> &gt; 0.05 over time. Red maca: <span class="html-italic">p</span> &lt; 0.05 at week 4 and <span class="html-italic">p</span> &lt; 0.01 at weeks 8 and 12 with respect to values at time 0. Black maca: <span class="html-italic">p</span> &lt; 0.05 at weeks 8 and 12 with respect to values at time 0. Red maca group: <span class="html-italic">p</span> &lt; 0.05 at weeks 4 and 8 with respect to the placebo and <span class="html-italic">p</span> &lt; 0.01 with respect to the placebo at week 12.</p> Full article ">Figure 6
<p>Flow diagram following the recruitment, enrollment, and number of subjects that finished treatment of the clinical trial at low altitude (<b>A</b>) and high altitude (<b>B</b>). Low altitude: Chi square: 0.11; <span class="html-italic">p</span> = 0.99 with respect to subjects who failed to complete the follow-up among different groups of treatment. High altitude: Chi square: 0.6; <span class="html-italic">p</span> = 0.99 with respect to subjects who failed to complete the follow-up among different treatment groups.</p> Full article ">Figure 6 Cont.
<p>Flow diagram following the recruitment, enrollment, and number of subjects that finished treatment of the clinical trial at low altitude (<b>A</b>) and high altitude (<b>B</b>). Low altitude: Chi square: 0.11; <span class="html-italic">p</span> = 0.99 with respect to subjects who failed to complete the follow-up among different groups of treatment. High altitude: Chi square: 0.6; <span class="html-italic">p</span> = 0.99 with respect to subjects who failed to complete the follow-up among different treatment groups.</p> Full article ">
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Communication
Design and Prototype of an Automated Column-Switching HPLC System for Radiometabolite Analysis
by Neil Vasdev and Thomas Lee Collier
Pharmaceuticals 2016, 9(3), 51; https://doi.org/10.3390/ph9030051 - 17 Aug 2016
Cited by 4 | Viewed by 6946
Abstract
Column-switching high performance liquid chromatography (HPLC) is extensively used for the critical analysis of radiolabeled ligands and their metabolites in plasma. However, the lack of streamlined apparatus and consequently varying protocols remain as a challenge among positron emission tomography laboratories. We report here [...] Read more.
Column-switching high performance liquid chromatography (HPLC) is extensively used for the critical analysis of radiolabeled ligands and their metabolites in plasma. However, the lack of streamlined apparatus and consequently varying protocols remain as a challenge among positron emission tomography laboratories. We report here the prototype apparatus and implementation of a fully automated and simplified column-switching procedure to allow for the easy and automated determination of radioligands and their metabolites in up to 5 mL of plasma. The system has been used with conventional UV and coincidence radiation detectors, as well as with a single quadrupole mass spectrometer. Full article
(This article belongs to the Special Issue New Challenges in Radiochemistry)
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<p>Flow chart of the processing of a metabolite sample. The plasma sample loaded on to the Sample loop S1 is transferred to the trapping cartridge C1. The materials retained on the trapping cartridge are then separated on the Analytical column C2. All materials from the trapping cartridge and the analytical column are passed through the coincidence detector D1 and UV and/or Mass Spectrometer D2 and collected in the fraction collector F1 for analysis in a well counter.</p> Full article ">Figure 2
<p>Comparison of high performance liquid chromatography (HPLC) chromatogram and plasma fractions counted in a gamma counter for a [<sup>18</sup>F] flurpiridaz sample. (<b>a</b>) Raw chromatogram from inline coincidence detector (60% CH<sub>3</sub>CN in phosphate buffered saline eluted through a C-18 column (Waters Xbridge BEH, 130Å; 100 × 4.6 mm, 3.5 μm) at 1 mL/min; (<b>b</b>) 2 mL fractions from the HPLC analysis were counted on the gamma counter, then plotted as smoothed line for comparison.</p> Full article ">Figure 3
<p>Equilibration of the trapping cartridge and analytical column.</p> Full article ">Figure 4
<p>The loading of the plasma from the sample loop to the trapping cartridge while the analytical column continues to be equilibrated with the eluting solvent.</p> Full article ">Figure 5
<p>The elution of the plasma sample from the trapping cartridge to the analytical column which is analyzed by the coincidence detector, UV detector and/or mass spectrometer.</p> Full article ">Figure 6
<p>If the pressure of the analytical column exceeds the set pressure limit, the 10-port column direction valve V3 is switched, the analytical column flow is reversed to remove materials obstructing the flow of solvent. Once the pressure drops the valve is switched and the flow returned to normal flow, as per Step 3.</p> Full article ">
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Review
TRP Channels as Therapeutic Targets in Diabetes and Obesity
by Andrea Zsombok and Andrei V. Derbenev
Pharmaceuticals 2016, 9(3), 50; https://doi.org/10.3390/ph9030050 - 17 Aug 2016
Cited by 36 | Viewed by 6819
Abstract
During the last three to four decades the prevalence of obesity and diabetes mellitus has greatly increased worldwide, including in the United States. Both the short- and long-term forecasts predict serious consequences for the near future, and encourage the development of solutions for [...] Read more.
During the last three to four decades the prevalence of obesity and diabetes mellitus has greatly increased worldwide, including in the United States. Both the short- and long-term forecasts predict serious consequences for the near future, and encourage the development of solutions for the prevention and management of obesity and diabetes mellitus. Transient receptor potential (TRP) channels were identified in tissues and organs important for the control of whole body metabolism. A variety of TRP channels has been shown to play a role in the regulation of hormone release, energy expenditure, pancreatic function, and neurotransmitter release in control, obese and/or diabetic conditions. Moreover, dietary supplementation of natural ligands of TRP channels has been shown to have potential beneficial effects in obese and diabetic conditions. These findings raised the interest and likelihood for potential drug development. In this mini-review, we discuss possibilities for better management of obesity and diabetes mellitus based on TRP-dependent mechanisms. Full article
(This article belongs to the Special Issue Transient Receptor Potential (TRP) Channels in Drug Discovery: Old Concepts & New Thoughts)
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Article
Nutraceutical Improvement Increases the Protective Activity of Broccoli Sprout Juice in a Human Intestinal Cell Model of Gut Inflammation
by Simonetta Ferruzza, Fausta Natella, Giulia Ranaldi, Chiara Murgia, Carlotta Rossi, Kajetan Trošt, Fulvio Mattivi, Mirella Nardini, Mariateresa Maldini, Anna Maria Giusti, Elisabetta Moneta, Cristina Scaccini, Yula Sambuy, Giorgio Morelli and Simona Baima
Pharmaceuticals 2016, 9(3), 48; https://doi.org/10.3390/ph9030048 - 12 Aug 2016
Cited by 23 | Viewed by 9066
Abstract
Benefits to health from a high consumption of fruits and vegetables are well established and have been attributed to bioactive secondary metabolites present in edible plants. However, the effects of specific health-related phytochemicals within a complex food matrix are difficult to assess. In [...] Read more.
Benefits to health from a high consumption of fruits and vegetables are well established and have been attributed to bioactive secondary metabolites present in edible plants. However, the effects of specific health-related phytochemicals within a complex food matrix are difficult to assess. In an attempt to address this problem, we have used elicitation to improve the nutraceutical content of seedlings of Brassica oleracea grown under controlled conditions. Analysis, by LC-MS, of the glucosinolate, isothiocyanate and phenolic compound content of juices obtained from sprouts indicated that elicitation induces an enrichment of several phenolics, particularly of the anthocyanin fraction. To test the biological activity of basal and enriched juices we took advantage of a recently developed in vitro model of inflamed human intestinal epithelium. Both sprouts’ juices protected intestinal barrier integrity in Caco-2 cells exposed to tumor necrosis factor α under marginal zinc deprivation, with the enriched juice showing higher protection. Multivariate regression analysis indicated that the extent of rescue from stress-induced epithelial dysfunction correlated with the composition in bioactive molecules of the juices and, in particular, with a group of phenolic compounds, including several anthocyanins, quercetin-3-Glc, cryptochlorogenic, neochlorogenic and cinnamic acids. Full article
(This article belongs to the Special Issue Nutraceuticals and Botanicals: Bioactive Molecules and Therapeutic Properties for Human Health)
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Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>Assessment of growth conditions on composition and biological activity of broccoli sprouts. Schematic representation of the experimental workflow used to evaluate and compare the composition and the biological activity of juices prepared from broccoli sprouts grown under two different conditions.</p> Full article ">Figure 2
<p>Box plot of LC-MS untargeted metabolomic fingerprinting. Distribution of LC-MS normalized (Z-scores) data from three biological replicates of juice from sprouts grown under two experimental conditions (basal, BJ; enriching, EJ). (<b>A</b>) Negative ion mode (mean values of <span class="html-italic">n</span> = 598 peaks); (<b>B</b>) Positive ion mode (mean values of <span class="html-italic">n</span> = 855 peaks). Center line shows the median; box limits indicate the 25th and 75th percentiles; whiskers extend to the most extreme data point within 1.5 times the interquartile range. *** <span class="html-italic">p</span> &lt; 0.0001 EJ vs. BJ.</p> Full article ">Figure 3
<p>Broccoli sprout juices protect epithelial barrier integrity of zinc-depleted Caco-2 cells exposed to TNFα. Caco-2 cells differentiated on filters for 21 days were pre-incubated for 14 h with experimental medium containing the indicated amount of broccoli juice in the AP compartment (red bars: EJ; blue bars: BJ; green bar: experimental medium without juice addition, noJ). Cells were then zinc deprived by TPEN incubation and exposed to TNFα for 5 h. As control, a set of filters was pre-incubated with the addition of 250 μL/mL of BJ or EJ for 14 h and then maintained in experimental medium for the whole experiment. TEER values, measured at the end of the experiment, are expressed as means ± SD from three experiments performed in triplicate. Statistical analysis was performed by one-way ANOVA followed by Fischer post hoc test. Different letters above bars indicate significant differences vs. TPEN/TNFα-treated cells without juice pre-treatement (green bar). a: <span class="html-italic">p</span> &lt; 0.0001; b: <span class="html-italic">p</span> &lt; 0.01; c: <span class="html-italic">p</span> &lt; 0.05.</p> Full article ">Figure 4
<p>Time course of the protective effects of broccoli sprout juices in TPEN/TNFα treated Caco-2 cells. Caco-2 cells differentiated on filters for 21 days were pre-incubated for 14 h in experimental medium with BJ (green triangles), EJ (purple circles) or without juice (red squares) addition in the AP compartment. Cells were zinc deprived by TPEN incubation and exposed to TNFα except untreated control cells (blue diamonds) that were maintained in experimental medium throughout the experiment. TEER was monitored in the last 5 h of the experiment during TNFα treatment. Data are the mean ± SD from three experiments performed in triplicate. One-way ANOVA was performed on mean-centered data followed by Fischer post hoc test. Different letters above bars indicate significant differences (<span class="html-italic">p</span> &lt; 0.05) among treatments.</p> Full article ">Figure 5
<p>Juices from independendent sprouts growths show similar effects in TPEN/TNFα treated Caco-2 cells. Caco-2 cells differentiated on filters for 21 days were pre-incubated for 14 h in experimental medium with BJ (full bars) or EJ (striped bars) obtained from three indipendent sprouts growths (green, blue and orange bars) or without juice (red bar) addition in the AP compartment. Cells were then zinc deprived by TPEN incubation and exposed to TNFα. TEER values were measured after 5 h of TNFα treatment. Statistical analysis was performed by one-way ANOVA followed by Fischer post hoc test. Different letters above bars indicate significant differences (<span class="html-italic">p</span> &lt; 0.01) among treatments.</p> Full article ">Figure 6
<p>TPEN/TNFα-treated Caco-2 cell protection is correlated to phytochemical enrichment of broccoli sprout juice. Partial least square (PLS) regression analysis was used to predict the biological effect exerted on Caco-2 intestinal cells exposed to TPEN/TNFα from composition data (see <a href="#pharmaceuticals-09-00048-t001" class="html-table">Table 1</a>) of six juice samples corresponding to three independent replicates of sprouts grown under two different conditions. (<b>A</b>) Scores plot showing the distribution of juice samples: BJ-1 to 3, juices from sprouts grown in basal conditions; EJ-1 to 3, juices from sprouts grown in enriching conditions; (<b>B</b>) Loadings plot showing the correlation between phytochemical descriptors (X-variables) and biological effect (Y-variable). The secondary metabolites that significantly affect response prediction are circled.</p> Full article ">
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Review
Resiniferatoxin: The Evolution of the “Molecular Scalpel” for Chronic Pain Relief
by Dorothy Cimino Brown
Pharmaceuticals 2016, 9(3), 47; https://doi.org/10.3390/ph9030047 - 11 Aug 2016
Cited by 53 | Viewed by 8808
Abstract
Control of chronic pain is frequently inadequate or can be associated with debilitating side effects. Ablation of certain nociceptive neurons, while retaining all other sensory modalities and motor function, represents a new therapeutic approach to controlling severe pain while avoiding off-target side effects. [...] Read more.
Control of chronic pain is frequently inadequate or can be associated with debilitating side effects. Ablation of certain nociceptive neurons, while retaining all other sensory modalities and motor function, represents a new therapeutic approach to controlling severe pain while avoiding off-target side effects. transient receptor potential cation channel subfamily V member 1 (TRPV1) is a calcium permeable nonselective cation channel expressed on the peripheral and central terminals of small-diameter sensory neurons. Highly selective chemoablation of TRPV1-containing peripheral nerve endings, or the entire TRPV1-expressing neuron itself, can be used to control chronic pain. Administration of the potent TRPV1 agonist resiniferatoxin (RTX) to neuronal perikarya or nerve terminals induces calcium cytotoxicity and selective lesioning of the TRPV1-expressing nociceptive primary afferent population. This selective neuroablation has been coined “molecular neurosurgery” and has the advantage of sparing motor, proprioceptive, and other somatosensory functions that are so important for coordinated movement, performing activities of daily living, and maintaining quality of life. This review examines the mechanisms and preclinical data underlying the therapeutic use of RTX and examples of such use for the management of chronic pain in clinical veterinary and human pain states. Full article
(This article belongs to the Special Issue Transient Receptor Potential (TRP) Channels in Drug Discovery: Old Concepts & New Thoughts)
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<p>An example of canine bone cancer: (<b>a</b>) A radiograph of the left radius and ulna reveals a severe moth eaten osteolytic lesion of the ulna (white arrows); (<b>b</b>) Compared to the right forelimb there is marked swelling of the left forelimb (white arrows) due to edema associated with the underlying bone tumor.</p> Full article ">
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Review
Differential Activation of TRP Channels in the Adult Rat Spinal Substantia Gelatinosa by Stereoisomers of Plant-Derived Chemicals
by Eiichi Kumamoto and Tsugumi Fujita
Pharmaceuticals 2016, 9(3), 46; https://doi.org/10.3390/ph9030046 - 28 Jul 2016
Cited by 16 | Viewed by 5800
Abstract
Activation of TRPV1, TRPA1 or TRPM8 channel expressed in the central terminal of dorsal root ganglion (DRG) neuron increases the spontaneous release of l-glutamate onto spinal dorsal horn lamina II (substantia gelatinosa; SG) neurons which play a pivotal role in regulating nociceptive [...] Read more.
Activation of TRPV1, TRPA1 or TRPM8 channel expressed in the central terminal of dorsal root ganglion (DRG) neuron increases the spontaneous release of l-glutamate onto spinal dorsal horn lamina II (substantia gelatinosa; SG) neurons which play a pivotal role in regulating nociceptive transmission. The TRP channels are activated by various plant-derived chemicals. Although stereoisomers activate or modulate ion channels in a distinct manner, this phenomenon is not fully addressed for TRP channels. By applying the whole-cell patch-clamp technique to SG neurons of adult rat spinal cord slices, we found out that all of plant-derived chemicals, carvacrol, thymol, carvone and cineole, increase the frequency of spontaneous excitatory postsynaptic current, a measure of the spontaneous release of l-glutamate from nerve terminals, by activating TRP channels. The presynaptic activities were different between stereoisomers (carvacrol and thymol; (−)-carvone and (+)-carvone; 1,8-cineole and 1,4-cineole) in the extent or the types of TRP channels activated, indicating that TRP channels in the SG are activated by stereoisomers in a distinct manner. This result could serve to know the properties of the central terminal TRP channels that are targets of drugs for alleviating pain. Full article
(This article belongs to the Special Issue Transient Receptor Potential (TRP) Channels in Drug Discovery: Old Concepts & New Thoughts)
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Figure 1

Figure 1
<p>Effects of thymol and carvacrol on glutamatergic spontaneous excitatory transmission in rat substantia gelatinosa (SG) neurons. (<b>A</b>,<b>B</b>) The chemical structures of thymol (<b>A</b>) and carvacrol (<b>B</b>). (<b>C</b>,<b>D</b>) The frequency and amplitude of sEPSC under the action of thymol (<b>C</b>) or carvacrol (<b>D</b>), relative to those before drug superfusion, which were plotted against the logarithm of drug concentration. This thymol (carvacrol) effect was measured for 0.5 min around 5 min (3.5 min) after the beginning of its superfusion. The results in (<b>C</b>) were obtained from all neurons tested, while those in (<b>D</b>) were obtained from neurons where carvacrol (1 mM) increased sEPSC frequency &gt; 5%. The continuous curves in (<b>C</b>) and (<b>D</b>) were drawn according to the Hill equation [half-maximal effective concentration (EC<sub>50</sub>) and Hill coefficient (n<sub>H</sub>) in (<b>C</b>) and (<b>D</b>): 0.18 mM, 4.9 and 0.69 mM, 2.1, respectively]. (<b>Ea</b>–<b>c</b>) Chart recordings showing sEPSCs and holding currents in the absence and presence of thymol in Krebs solution without (left) or with a TRPV1 antagonist capsazepine (<b>Ea</b>), a TRPA1 antagonist HC-030031 (<b>Eb</b>) or a TRPM8 antagonist BCTC (<b>Ec</b>; right). In each of (<b>Ea</b>)–(<b>Ec</b>), the right recording was obtained about 30 min after the left one from the same neuron. (<b>Ea</b>–<b>c</b>) Chart recordings showing sEPSCs in the absence and presence of carvacrol in Krebs solution without (<b>Fa</b>) and with capsazepine (<b>Fb</b>) or HC-030031 (<b>Fc</b>); these recordings were obtained from the same neuron at an interval of 30 min. In this and subsequent figures, value in parentheses indicates the number of neurons tested; each point with vertical bars represents the mean values and standard error of the mean (SEM); if the SEM of the values is less than the size of symbol, the vertical bar is not shown; control level (1) is indicated by horizontal dotted line; the duration of drug superfusion is shown by a horizontal bar above the chart recording. Holding potential (V<sub>H</sub>) = −70 mV. This research was originally published in [<a href="#B88-pharmaceuticals-09-00046" class="html-bibr">88</a>,<a href="#B89-pharmaceuticals-09-00046" class="html-bibr">89</a>].</p> Full article ">Figure 2
<p>Effects of (−)-carvone and (+)-carvone on glutamatergic spontaneous excitatory transmission in rat SG neurons. (<b>A</b>,<b>B</b>) The chemical structures of (−)-carvone (<b>A</b>) and (+)-carvone (<b>B</b>). (<b>C</b>,<b>D</b>) The frequency and amplitude of sEPSC under the action of (−)-carvone (<b>C</b>) or (+)-carvone (<b>D</b>), relative to those before drug superfusion, which were plotted against the logarithm of drug concentration. This carvone effect was measured for 0.5 min around 3 min after the beginning of its superfusion. The continuous curves in (<b>C</b>) and (<b>D</b>) were drawn according to the Hill equation (EC<sub>50</sub> and n<sub>H</sub> in (<b>C</b>) and (<b>D</b>): 0.70 mM, 2.2 and 0.72 mM, 2.4, respectively). (<b>Ea,b,Fa,b</b>) Chart recordings showing sEPSCs in the absence and presence of (−)-carvone (<b>E</b>) or (+)-carvone (<b>F</b>) in Krebs solution without (left) and with capsazepine (<b>Ea,Fa</b>) or HC-030031 (<b>Eb,Fb</b>; right). In each of (<b>Ea,b,Fa,b</b>), the right recording was obtained about 20 min after the left one from the same neuron. V<sub>H</sub> = −70 mV. This research was originally published in [<a href="#B101-pharmaceuticals-09-00046" class="html-bibr">101</a>].</p> Full article ">Figure 3
<p>Effects of 1,8-cineole and 1,4-cineole on glutamatergic spontaneous excitatory transmission in rat SG neurons. (<b>A</b>,<b>B</b>) The chemical structures of 1,8-cineole (<b>A</b>) and 1,4-cineole (<b>B</b>). (<b>C</b>,<b>D</b>) The frequency and amplitude of sEPSC under the action of 1,8-cineole (<b>C</b>) or 1,4-cineole (<b>D</b>), relative to those before drug superfusion, which were plotted against the logarithm of drug concentration. This cineole effect was measured for 0.5 min around 3.5 min after the addition of its drug. The continuous curves in (<b>C</b>) and (<b>D</b>) were drawn according to the Hill equation (EC<sub>50</sub> and n<sub>H</sub> in (<b>C</b>) and (<b>D</b>): 3.2 mM, 1.3 and 0.42 mM, 1.7, respectively). (<b>Ea</b>–<b>e,Fa</b>–<b>e</b>) Chart recordings showing sEPSCs in the absence and presence of 1,8-cineole (<b>E</b>) or 1,4-cineole (<b>F</b>) in Krebs solution without (left) and with capsazepine (<b>a</b>), SB-366791 (<b>b</b>), HC-030031 (<b>c</b>), mecamylamine (<b>d</b>) or BCTC (<b>e</b>; right). In each of (<b>Ea</b>–<b>e,Fa</b>–<b>e</b>), the right recording was obtained about 20 min after the left one from the same neuron. V<sub>H</sub> = −70 mV. This research was originally published in [<a href="#B27-pharmaceuticals-09-00046" class="html-bibr">27</a>].</p> Full article ">
219 KiB  
Review
TRPV1 and TRPM8 in Treatment of Chronic Cough
by Eva Millqvist
Pharmaceuticals 2016, 9(3), 45; https://doi.org/10.3390/ph9030045 - 28 Jul 2016
Cited by 25 | Viewed by 8237
Abstract
Chronic cough is common in the population, and among some there is no evident medical explanation for the symptoms. Such a refractory or idiopathic cough is now often regarded as a neuropathic disease due to dysfunctional airway ion channels, though the knowledge in [...] Read more.
Chronic cough is common in the population, and among some there is no evident medical explanation for the symptoms. Such a refractory or idiopathic cough is now often regarded as a neuropathic disease due to dysfunctional airway ion channels, though the knowledge in this field is still limited. Persistent coughing and a cough reflex easily triggered by irritating stimuli, often in combination with perceived dyspnea, are characteristics of this disease. The patients have impaired quality of life and often reduced work capacity, followed by social and economic consequences. Despite the large number of individuals suffering from such a persisting cough, there is an unmet clinical need for effective cough medicines. The cough treatment available today often has little or no effect. Adverse effects mostly follow centrally acting cough drugs comprised of morphine and codeine, which demands the physician’s awareness. The possibilities of modulating airway transient receptor potential (TRP) ion channels may indicate new ways to treat the persistent cough “without a reason”. The TRP ion channel vanilloid 1 (TRPV1) and the TRP melastin 8 (TRPM8) appear as two candidates in the search for cough therapy, both as single targets and in reciprocal interaction. Full article
(This article belongs to the Special Issue Transient Receptor Potential (TRP) Channels in Drug Discovery: Old Concepts & New Thoughts)
197 KiB  
Review
TRPM8 Puts the Chill on Prostate Cancer
by Guillaume P. Grolez and Dimitra Gkika
Pharmaceuticals 2016, 9(3), 44; https://doi.org/10.3390/ph9030044 - 9 Jul 2016
Cited by 31 | Viewed by 6429
Abstract
Prostate cancer (PCa) is one of the most frequently diagnosed cancers in developed countries. Several studies suggest that variations in calcium homeostasis are involved in carcinogenesis. Interestingly, (Transient Receptor Potential Melastatin member 8) TRPM8 calcium permeable channel expression is differentially regulated during prostate [...] Read more.
Prostate cancer (PCa) is one of the most frequently diagnosed cancers in developed countries. Several studies suggest that variations in calcium homeostasis are involved in carcinogenesis. Interestingly, (Transient Receptor Potential Melastatin member 8) TRPM8 calcium permeable channel expression is differentially regulated during prostate carcinogenesis, thereby suggesting a potential functional role for this channel in those cell processes, which are important for PCa evolution. Indeed, several studies have shown that TRPM8 plays a key role in processes such as the proliferation, viability and cell migration of PCa cells. Where cell migration is concerned, TRPM8 seems to have a protective anti-invasive effect and could be a particularly promising therapeutic target. The goal of this review is to inventory advances in understanding of the role of TRPM8 in the installation and progression of PCa. Full article
(This article belongs to the Special Issue Transient Receptor Potential (TRP) Channels in Drug Discovery: Old Concepts & New Thoughts)
909 KiB  
Communication
Convergent Synthesis of Two Fluorescent Ebselen-Coumarin Heterodimers
by Jim Küppers, Anna Christina Schulz-Fincke, Jerzy Palus, Mirosław Giurg, Jacek Skarżewski and Michael Gütschow
Pharmaceuticals 2016, 9(3), 43; https://doi.org/10.3390/ph9030043 - 8 Jul 2016
Cited by 14 | Viewed by 8438
Abstract
The organo-seleniumdrug ebselen exhibits a wide range of pharmacological effects that are predominantly due to its interference with redox systems catalyzed by seleno enzymes, e.g., glutathione peroxidase and thioredoxin reductase. Moreover, ebselen can covalently interact with thiol groups of several enzymes. According to [...] Read more.
The organo-seleniumdrug ebselen exhibits a wide range of pharmacological effects that are predominantly due to its interference with redox systems catalyzed by seleno enzymes, e.g., glutathione peroxidase and thioredoxin reductase. Moreover, ebselen can covalently interact with thiol groups of several enzymes. According to its pleiotropic mode of action, ebselen has been investigated in clinical trials for the prevention and treatment of different ailments. Fluorescence-labeled probes containing ebselen are expected to be suitable for further biological and medicinal studies. We therefore designed and synthesized two coumarin-tagged activity-based probes bearing the ebselen warhead. The heterodimers differ by the nature of the spacer structure, for which—in the second compound—a PEG/two-amide spacer was introduced. The interaction of this probe and of ebselen with two cysteine proteases was investigated. Full article
(This article belongs to the Special Issue Selected Papers from the 1st International Electronic Conference on Medicinal Chemistry)
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<p>Structure of the fluorescently labeled ebselen derivatives.</p> Full article ">Figure 2
<p>Absorption (10 μM, 1% DMSO, dashed lines) and emission (1 μM, 1% DMSO, solid lines) spectra of the ebselen-coumarin heterodimer <b>15</b>. The extinction and the fluorescence units, respectively, were plotted versus the wavelength. Spectra recorded in buffer (50 mM sodium phosphate, pH 7.8, with 500 mM NaCl) (<b>blue</b>), MeOH (<b>violet</b>) and CH<sub>2</sub>Cl<sub>2</sub> (<b>green</b>) are shown. Wavelengths of absorption were used for excitation. The heterodimer <b>9</b> was measured similarly (see <a href="#pharmaceuticals-09-00043-t001" class="html-table">Table 1</a>). All fluorescent measurements were carried out with a PMT value of 200 V.</p> Full article ">Scheme 1
<p>Synthetic route to the coumarin-labeled ebselen derivative <b>9</b>.</p> Full article ">Scheme 2
<p>Synthetic route to the coumarin-labeled ebselen derivative <b>15</b>.</p> Full article ">
5198 KiB  
Article
Polylactic Acid—Lemongrass Essential Oil Nanocapsules with Antimicrobial Properties
by Ioannis L. Liakos, Alexandru Mihai Grumezescu, Alina Maria Holban, Iordache Florin, Francesca D’Autilia, Riccardo Carzino, Paolo Bianchini and Athanassia Athanassiou
Pharmaceuticals 2016, 9(3), 42; https://doi.org/10.3390/ph9030042 - 7 Jul 2016
Cited by 48 | Viewed by 11298
Abstract
Polylactic acid was combined with lemongrass essential oil (EO) to produce functional nanocapsules (NCs). The obtained polylactic acid nanoparticles showed antimicrobial activity both with and without the presence of lemongrass oil; however, the presence of EO improved the activity of the NCs. The [...] Read more.
Polylactic acid was combined with lemongrass essential oil (EO) to produce functional nanocapsules (NCs). The obtained polylactic acid nanoparticles showed antimicrobial activity both with and without the presence of lemongrass oil; however, the presence of EO improved the activity of the NCs. The presence of lemongrass assisted the formation of well-separated NCs and also provided enhanced antimicrobial properties, since lemongrass is known for its antimicrobial character. Fluorescence microscopy was used to optically observe the nanoparticles and NCs and revealed the attachment of lemongrass oil with the polylactic acid NCs. Dynamic light scattering was used to determine their size. UV absorption was used to determine the exact amount of lemongrass oil found in the polylactic acid—lemongrass oil NCs, which was important for understanding the minimum inhibitory concentration for the antimicrobial experiments. A series of clinically important microbial species were used in the study and the obtained NCs proved to have very good antimicrobial properties against all tested strains. Such NCs can be used for the design of ecological strategies, based on natural alternatives, which may be efficient against severe infections, including those that involve resistant pathogens and biofilms or those with difficult to reach localization. Full article
(This article belongs to the Special Issue Nanobiotechnology in Medicinal Chemistry)
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<p>Transmission andfluorescence microscopy images of (<b>a</b>,<b>b</b>) polylactic acid nanoparticles (PLA NPs) and (<b>c</b>,<b>d</b>) polylactic acid/5-lemongrass oil nanocapsules (PLA/5-LG NCs) repsectively (scale bars, 1 µm).</p> Full article ">Figure 2
<p>Dynamic light scattering (DLS) analysis revealing the size and Z-potential of PLA NPs and PLA/5-LG NCs.</p> Full article ">Figure 3
<p>Raman spectra of PLA NPs (<b>blue</b> line), PLA/5-LG NCs (<b>green</b> dashed line) and LG oil (<b>black</b> dots) (<b>a</b>) whole spectrum and (<b>b</b>) zoomed spectrum.</p> Full article ">Figure 4
<p>(<b>a</b>) UV absorption spectra of increasing amount of PLA/5-LG NCs solution (5, 10, 20, 30, 40 and 50 μL): the peak at 275 nm corresponds to PLA while the shoulder at 240 nm corresponds to LG absorption; (<b>b</b>) intensities of the peak at 240 nm for increasing amounts of PLA/5-LG NCs; (<b>c</b>) calibration curve of LG essential oil; and (<b>d</b>) calculation of LG oil content in 5 μL of PLA/5-LG NCs.</p> Full article ">Figure 5
<p>Graphic representation revealing the growth inhibition diameters of tested microbial strains grown in the presence of PLA NPs, PLA/5-LG NCs and control LG.</p> Full article ">Figure 6
<p>Graphic representation revealing the minimum inhibitory concentration values (MICs) of PLA NPs, PLA/5-LG NCs and control LG oil on the tested microbial strains after 24 h or incubation.</p> Full article ">Figure 7
<p>Graphic representation revealing the biofilm inhibition MICs of PLA NPs, PLA/5-LG NCs and LG oil control on the tested microbial strains.</p> Full article ">Figure 8
<p>The viability of human amniotic fluid stem cells (AFSC) grown in the presence of PLA/5-LG NCs and in control conditions. The MTT assay shows that, after 72 h, the cells present a normal metabolism and growth capacity compared to control (<span class="html-italic">n</span> = 3, <span class="html-italic">p</span> &lt; 0.05%).</p> Full article ">Figure 9
<p>Fluorescence microscopy images of the human AFSC monolayer in the presence of (<b>A</b>) PLA NPs; (<b>B</b>) PLA/5-LG NCs and (<b>C</b>) control. The cells present a normal morphology, are adherent, and have a relative uniform distribution after five days of PLA/5-LG NC stimulation.</p> Full article ">Figure 10
<p>UV absorption spectra of 0.94 μL LG essential oil in 3 mL of acetonitrile.</p> Full article ">Figure 11
<p>UV absorption spectra of 250 μL PLA NPs water solution in 3 mL of acetonitrile.</p> Full article ">
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Review
Multiple Functions of the New Cytokine-Based Antimicrobial Peptide Thymic Stromal Lymphopoietin (TSLP)
by Louise Bjerkan, Andreas Sonesson and Karl Schenck
Pharmaceuticals 2016, 9(3), 41; https://doi.org/10.3390/ph9030041 - 5 Jul 2016
Cited by 29 | Viewed by 9377
Abstract
Thymic stromal lymphopoietin (TSLP) is a pleiotropic cytokine, hitherto mostly known to be involved in inflammatory responses and immunoregulation. The human tslp gene gives rise to two transcription and translation variants: a long form (lfTSLP) that is induced by inflammation, and a short, [...] Read more.
Thymic stromal lymphopoietin (TSLP) is a pleiotropic cytokine, hitherto mostly known to be involved in inflammatory responses and immunoregulation. The human tslp gene gives rise to two transcription and translation variants: a long form (lfTSLP) that is induced by inflammation, and a short, constitutively-expressed form (sfTSLP), that appears to be downregulated by inflammation. The TSLP forms can be produced by a number of cell types, including epithelial and dendritic cells (DCs). lfTSLP can activate mast cells, DCs, and T cells through binding to the lfTSLP receptor (TSLPR) and has a pro-inflammatory function. In contrast, sfTSLP inhibits cytokine secretion of DCs, but the receptor mediating this effect is unknown. Our recent studies have demonstrated that both forms of TSLP display potent antimicrobial activity, exceeding that of many other known antimicrobial peptides (AMPs), with sfTSLP having the strongest effect. The AMP activity is primarily mediated by the C-terminal region of the protein and is localized within a 34-mer peptide (MKK34) that spans the C-terminal α-helical region in TSLP. Fluorescent studies of peptide-treated bacteria, electron microscopy, and liposome leakage models showed that MKK34 exerted membrane-disrupting effects comparable to those of LL-37. Expression of TSLP in skin, oral mucosa, salivary glands, and intestine is part of the defense barrier that aids in the control of both commensal and pathogenic microbes. Full article
(This article belongs to the Special Issue Antimicrobial Peptides: Expanded Activity Spectrum and Applications)
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<p>TSLP transcript variants and protein isoforms. (<b>A</b>) Graphics showing the TSLP gene (green), the long form, and short form transcripts (blue), and the protein products (red). Long (NM_033035.4, NP_149024.1) and short (NM_138551.3, NP_612561.2) transcript and protein variants, respectively, are indicated (NCBI). (<b>B</b>) Amino acid sequence of human TSLP isoforms. The putative signal sequences of the TSLP isoforms are marked in blue, and the mature protein in black. N-linked glycosylation sites are marked green, and methionine start codons are marked red. Bold black characters indicate the position of MKK34. (<b>C</b>) JNet secondary structure prediction of lfTSLP based on the amino acid sequence. Helices are marked as red tubes, and sheets are marked as green arrows. JNETCONF: The confidence estimate for the prediction, high values indicate high confidence. Modified from the web-based application Jpred (The Barton Group, School of Life Sciences, University of Dundee, UK). (<b>D</b>) Three-dimensional structure (Swiss-model, [<a href="#B9-pharmaceuticals-09-00041" class="html-bibr">9</a>]) of lfTSLP (<b>left</b>) and sfTSLP (<b>right</b>).</p> Full article ">Figure 1 Cont.
<p>TSLP transcript variants and protein isoforms. (<b>A</b>) Graphics showing the TSLP gene (green), the long form, and short form transcripts (blue), and the protein products (red). Long (NM_033035.4, NP_149024.1) and short (NM_138551.3, NP_612561.2) transcript and protein variants, respectively, are indicated (NCBI). (<b>B</b>) Amino acid sequence of human TSLP isoforms. The putative signal sequences of the TSLP isoforms are marked in blue, and the mature protein in black. N-linked glycosylation sites are marked green, and methionine start codons are marked red. Bold black characters indicate the position of MKK34. (<b>C</b>) JNet secondary structure prediction of lfTSLP based on the amino acid sequence. Helices are marked as red tubes, and sheets are marked as green arrows. JNETCONF: The confidence estimate for the prediction, high values indicate high confidence. Modified from the web-based application Jpred (The Barton Group, School of Life Sciences, University of Dundee, UK). (<b>D</b>) Three-dimensional structure (Swiss-model, [<a href="#B9-pharmaceuticals-09-00041" class="html-bibr">9</a>]) of lfTSLP (<b>left</b>) and sfTSLP (<b>right</b>).</p> Full article ">Figure 2
<p>Immunohistochemical (IHC) staining and in situ hybridization (ISH) of sections of oral mucosa (<b>A</b>–<b>C</b>), skin (<b>D</b>–<b>F</b>), salivary gland (<b>G</b>–<b>I</b>), and smokeless tobacco (“snus”; <b>J</b>,<b>K</b>) for TSLP variants. Left column: IHC staining with anti-TSLP antibody recognizing both lfTSLP and sfTSLP (brown color). Middle column: IHC staining with anti-TSLP antibody recognizing lfTSLP only. As no specific staining is detected in (<b>B</b>,<b>E</b>,<b>H</b>), this means that the staining in (<b>A</b>,<b>D</b>,<b>G</b>) represents sfTSLP. In oral mucosa exposed to smokeless tobacco, lfTSLP is seen (<b>K</b>). Right column: ISH staining by use of sfTSLP-specific probe (blue color) which confirms strong expression of sfTSLP in oral mucosa and salivary gland, and weak expression in skin. Modified from [<a href="#B5-pharmaceuticals-09-00041" class="html-bibr">5</a>].</p> Full article ">Figure 3
<p>STAT5 phosphorylation in response to lfTSLP, 60 aa sfTSLP, 63 aa sfTSLP, or lfTSLP combined with sfTSLP in blood-derived CD1c myeloid DCs incubated with poly(I:C) for 24 h, and then treated with sfTSLP or/and lfTSLP for 15 min. Phosphorylation of STAT5 was assessed by flow cytometry. From [<a href="#B5-pharmaceuticals-09-00041" class="html-bibr">5</a>].</p> Full article ">Figure 4
<p>Plot of hydrophobic moment (μH) for the mature lfTSLP (131 amino acids).</p> Full article ">Figure 5
<p>Antimicrobial activity of short and long forms of thymic stromal lymphopoietin (sfTSLP and lfTSLP). (<b>A</b>) lfTSLP exhibited a larger zone of inhibition of growth of <span class="html-italic">Escherichia coli</span> ATCC 25922 in comparison with LL-37: (a) control; (b) 10 µM LL-37; and (c) 10µM TSLP. Mean values and standard deviations (n = 4). (<b>B</b>) In a viable count assay, indicated bacterial (<span class="html-italic">Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus</span>, and <span class="html-italic">Staphylococcus epidermidis</span>) and fungal isolates (<span class="html-italic">Candida albicans</span> and <span class="html-italic">Candida parapsilosis</span>) were subjected to 2 µM of TSLP. The number of cfu was registered. (<b>C</b>) Suspensions of the indicated bacterial and fungal species were treated for 2 h with 60 amino acid (aa) sfTSLP, and 63 aa sfTSLP or lfTSLP peptide at a concentration of 1.35 mM before being plated on agar. Colony-forming units per ml were determined after incubation overnight. The values were normalized to the levels obtained without the addition of test peptides (broken line). (<b>D</b>) Suspensions of <span class="html-italic">Streptococcus mitis</span> were treated with equimolar concentrations of 60 aa sfTSLP, LL-37, or lfTSLP and analyzed as in C. From: [<a href="#B7-pharmaceuticals-09-00041" class="html-bibr">7</a>] (<b>A</b>,<b>B</b>) and [<a href="#B5-pharmaceuticals-09-00041" class="html-bibr">5</a>] (<b>C</b>,<b>D</b>).</p> Full article ">Figure 6
<p>Helical structure of MKK34. A helical wheel projection was constructed using the amino acid sequence of MKK34.</p> Full article ">Figure 7
<p>Enzymatic digestion of TSLP. (<b>A</b>) TSLP was digested with S. aureus V8 proteinase, and cleavage products were visualized by Western blot analysis using polyclonal antibodies against human TSLP. Products produced by V8 cleavage of TSLP revealed a major immunoreactive protein fragment at about 16 kDa. (<b>B</b>) TSLP and LL-37 were incubated with and without human neutrophil (leukocyte) elastase (HLE), <span class="html-italic">S. aureus</span> V8 proteinase or <span class="html-italic">Pseudomonas aeruginosa</span> elastase (PAE) and analyzed under non-reducing conditions by SDS-PAGE. From ref. [<a href="#B7-pharmaceuticals-09-00041" class="html-bibr">7</a>].</p> Full article ">Figure 8
<p>Electron microscopy analysis. <span class="html-italic">Staphylococcus aureus</span> and <span class="html-italic">Pseudomonas aeruginosa</span> were incubated with 30 μM of MKK34 and LL-37 for 2 h at 37 °C and visualized by negative staining. Scale bar 1 μm. Control: buffer control. From [<a href="#B7-pharmaceuticals-09-00041" class="html-bibr">7</a>], image courtesy of Matthias Mörgelin, Lund University, Lund, Sweden.</p> Full article ">
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Review
Functions of Cationic Host Defense Peptides in Immunity
by Mahadevappa Hemshekhar, Vidyanand Anaparti and Neeloffer Mookherjee
Pharmaceuticals 2016, 9(3), 40; https://doi.org/10.3390/ph9030040 - 4 Jul 2016
Cited by 70 | Viewed by 6485
Abstract
Cationic host defense peptides are a widely distributed family of immunomodulatory molecules with antimicrobial properties. The biological functions of these peptides include the ability to influence innate and adaptive immunity for efficient resolution of infections and simultaneous modulation of inflammatory responses. This unique [...] Read more.
Cationic host defense peptides are a widely distributed family of immunomodulatory molecules with antimicrobial properties. The biological functions of these peptides include the ability to influence innate and adaptive immunity for efficient resolution of infections and simultaneous modulation of inflammatory responses. This unique dual bioactivity of controlling infections and inflammation has gained substantial attention in the last three decades and consequent interest in the development of these peptide mimics as immunomodulatory therapeutic candidates. In this review, we summarize the current literature on the wide range of functions of cationic host defense peptides in the context of the mammalian immune system. Full article
(This article belongs to the Special Issue Antimicrobial Peptides: Expanded Activity Spectrum and Applications)
663 KiB  
Communication
Odanacatib, a Cathepsin K Cysteine Protease Inhibitor, Kills Hookworm In Vivo
by Jon J. Vermeire, Brian M. Suzuki and Conor R. Caffrey
Pharmaceuticals 2016, 9(3), 39; https://doi.org/10.3390/ph9030039 - 4 Jul 2016
Cited by 5 | Viewed by 7567
Abstract
Hookworm infection is chief among soil-transmitted helminthiases (STHs) for the chronic morbidly inflicted. Deworming via mass drug administration (MDA) programs most often employs single doses of benzimidazole drugs to which resistance is a constant threat. To discover new drugs, we employ a hamster [...] Read more.
Hookworm infection is chief among soil-transmitted helminthiases (STHs) for the chronic morbidly inflicted. Deworming via mass drug administration (MDA) programs most often employs single doses of benzimidazole drugs to which resistance is a constant threat. To discover new drugs, we employ a hamster model of hookworm infection with Ancylostoma ceylanicum and use albendazole (ABZ; 10 mg/kg orally) as the gold standard therapy. We previously showed that a single oral 100 mg/kg dose of the cathepsin cysteine protease (CP) inhibitor, K11777, offers near cure of infection that is associated with a 95% reduction in the parasite’s resident CP activity. We confirm these findings here and demonstrate that odanacatib (ODN), Merck’s cathepsin K inhibitor and post-clinical Phase III drug candidate for treatment of osteoporosis, decreases worm burden by 73% at the same dose with a 51% reduction in the parasite’s CP activity. Unlike K11777, ODN is a modest inhibitor of both mammalian cathepsin B and the predominant cathepsin B-like activity measureable in hookworm extracts. ODN’s somewhat unexpected efficacy, therefore, may be due to its excellent pharmacokinetic (PK) profile which allows for sustained plasma exposure and, possibly, sufficient perturbation of hookworm cathepsin B activity to be detrimental to survival. Accordingly, identifying a CP inhibitor(s) that combines the inhibition potency of K11777 and the PK attributes of ODN could lead to a drug that is effective at a lower dose. Achieving this would potentially provide an alternative or back-up to the current anti-hookworm drug, albendazole. Full article
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<p>Structures of K11777 and ODN.</p> Full article ">Figure 2
<p>K11777 and ODN reduce <span class="html-italic">Ancylostoma</span> <span class="html-italic">ceylanicum</span> burdens in Golden Syrian hamsters and decrease the parasite’s resident CP activity. (<b>A</b>) Groups of hamsters (<span class="html-italic">n</span> = 3) were infected with 75 third stage <span class="html-italic">A. ceylanicum</span> larvae. At 18 days post-infection (DPI) hamsters were treated once orally with K11777 (100 mg/kg) dissolved in water, or with ODN (100 mg/kg) or ABZ (10 mg/kg) dissolved in PEG400. At 24 DPI, all hamsters were sacrificed and intestinal worms counted. Reductions in worm burdens by ODN and K11777 were statistically significant (one-way ANOVA: <span class="html-italic">p</span> &lt; 0.05 and <span class="html-italic">p</span> &lt; 0.01, respectively); (<b>B</b>) Hamsters (<span class="html-italic">n</span> = 1) were treated with single oral doses of K11777, ODN or the PEG400 vehicle as described in (<b>A</b>). Worms were harvested 8 h later and soluble extracts prepared. Specific cysteine protease activity (relative fluorescence units/min/mg soluble extract) was measured using the fluorogenic substrate Z-Phe-Arg-AMC. Data points are expressed as means ± S.D. values from a single experiment performed in triplicate.</p> Full article ">Figure 3
<p>Inhibition of hookworm cysteine protease activity by K11777 and ODN. Soluble extracts of female (black bars) and male (grey bars) <span class="html-italic">A. ceylanicum</span> were incubated for 10 min with 1 μM inhibitor, as described in the text. Residual cysteine protease activity was measured with the fluorogenic substrate Z-Phe-Arg-AMC. Data were generated from two experiments each in duplicate; one experiment is shown.</p> Full article ">
702 KiB  
Review
Heparin: Past, Present, and Future
by Eziafa I. Oduah, Robert J. Linhardt and Susan T. Sharfstein
Pharmaceuticals 2016, 9(3), 38; https://doi.org/10.3390/ph9030038 - 4 Jul 2016
Cited by 192 | Viewed by 21124
Abstract
Heparin, the most widely used anticoagulant drug in the world today, remains an animal-derived product with the attendant risks of adulteration and contamination. A contamination crisis in 2007–2008 increased the impetus to provide non-animal-derived sources of heparin, produced under cGMP conditions. In addition, [...] Read more.
Heparin, the most widely used anticoagulant drug in the world today, remains an animal-derived product with the attendant risks of adulteration and contamination. A contamination crisis in 2007–2008 increased the impetus to provide non-animal-derived sources of heparin, produced under cGMP conditions. In addition, recent studies suggest that heparin may have significant antineoplastic activity, separate and distinct from its anticoagulant activity, while other studies indicate a role for heparin in treating inflammation, infertility, and infectious disease. A variety of strategies have been proposed to produce a bioengineered heparin. In this review, we discuss several of these strategies including microbial production, mammalian cell production, and chemoenzymatic modification. We also propose strategies for creating “designer” heparins and heparan-sulfates with various biochemical and physiological properties. Full article
(This article belongs to the Special Issue Grand Celebration: 100th Anniversary of the Discovery of Heparin)
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Figure 1

Figure 1
<p>Heparin mechanisms within the coagulation cascade. Box A: AT (red) bound with heparin fragments (green) of any length within the unique pentasaccharide sequence can inhibit factor Xa. Box B: AT (red) bound with heparin (green) with chain length &gt;17 disaccharide units can inhibit thrombin (Factor IIa).</p> Full article ">Figure 2
<p>Heparin biosynthesis. The glycosaminoglycan-protein linkage region is first formed under the action of glycosyltransferases. The repeating disaccharide units are then elongated by GlcA and GlcNAc transferases. Chain modifications including <span class="html-italic">N</span>-deacetylation and <span class="html-italic">N</span>-sulfonation, <span class="html-italic"><span class="html-small-caps">O</span></span>-sulfonations, and epimerization then occur under the actions of the specified enzymes. Monosaccharide symbols in this figure follow the SNFG (Symbol Nomenclature for Glycans) system [<a href="#B15-pharmaceuticals-09-00038" class="html-bibr">15</a>]</p> Full article ">
1783 KiB  
Review
Strategies to Overcome Heparins’ Low Oral Bioavailability
by Ana Rita Neves, Marta Correia-da-Silva, Emília Sousa and Madalena Pinto
Pharmaceuticals 2016, 9(3), 37; https://doi.org/10.3390/ph9030037 - 29 Jun 2016
Cited by 25 | Viewed by 8721
Abstract
Even after a century, heparin is still the most effective anticoagulant available with few side effects. The poor oral absorption of heparins triggered the search for strategies to achieve oral bioavailability since this route has evident advantages over parenteral administration. Several approaches emerged, [...] Read more.
Even after a century, heparin is still the most effective anticoagulant available with few side effects. The poor oral absorption of heparins triggered the search for strategies to achieve oral bioavailability since this route has evident advantages over parenteral administration. Several approaches emerged, such as conjugation of heparins with bile acids and lipids, formulation with penetration enhancers, and encapsulation of heparins in micro and nanoparticles. Some of these strategies appear to have potential as good delivery systems to overcome heparin’s low oral bioavailability. Nevertheless, none have reached the market yet. Overall, this review aims to provide insights regarding the oral bioavailability of heparin. Full article
(This article belongs to the Special Issue Grand Celebration: 100th Anniversary of the Discovery of Heparin)
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Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>Structural features of heparins that limit their oral bioavailability. UFH—Unfractioned heparin.</p> Full article ">Figure 2
<p>Advantages of heparins over vitamin K antagonists (VKA) and new oral anticoagulants (NOACs) drugs.</p> Full article ">Figure 3
<p>Chemical structure of deoxycholic acid (DOCA), fatty acids, and cholesterol.</p> Full article ">Figure 4
<p>Structure of different heparins-DOCA conjugates. (<b>A</b>) Heparin-DOCA conjugate in which both carboxylic groups of heparin and DOCA were conjugated through a linker [<a href="#B22-pharmaceuticals-09-00037" class="html-bibr">22</a>,<a href="#B23-pharmaceuticals-09-00037" class="html-bibr">23</a>,<a href="#B24-pharmaceuticals-09-00037" class="html-bibr">24</a>,<a href="#B30-pharmaceuticals-09-00037" class="html-bibr">30</a>,<a href="#B31-pharmaceuticals-09-00037" class="html-bibr">31</a>,<a href="#B33-pharmaceuticals-09-00037" class="html-bibr">33</a>]; (<b>B</b>) Heparin-DOCA conjugate in which a carboxylic group of heparin and a hydroxyl group of DOCA were conjugated through a linker [<a href="#B29-pharmaceuticals-09-00037" class="html-bibr">29</a>]; (<b>C</b>) Heparin-DOCA conjugate in which an amine group of heparin and a carboxylic group of DOCA were conjugated without a linker [<a href="#B25-pharmaceuticals-09-00037" class="html-bibr">25</a>,<a href="#B26-pharmaceuticals-09-00037" class="html-bibr">26</a>,<a href="#B27-pharmaceuticals-09-00037" class="html-bibr">27</a>,<a href="#B28-pharmaceuticals-09-00037" class="html-bibr">28</a>,<a href="#B32-pharmaceuticals-09-00037" class="html-bibr">32</a>]; (<b>D</b>) Heparin-tetrameric DOCA conjugate [<a href="#B37-pharmaceuticals-09-00037" class="html-bibr">37</a>].</p> Full article ">Figure 5
<p>Chemical structure of penetration enhancers that increase oral bioavailability of heparins. MCC—Mono-<span class="html-italic">N</span>-carboxymethyl chitosan; SNOCC: <span class="html-italic">N</span>,<span class="html-italic">O</span>-Carboxymethyl chitosan; PCP-Cys: Polycarbophil-cystein; DMC—<span class="html-italic">N</span>,<span class="html-italic">N</span>-Dimethyl chitosan; SNAD—Sodium <span class="html-italic">N</span>-[10-(2-hydroxybenzoyl) amino decanoate; SNAC: Sodium <span class="html-italic">N</span>-(8-[2-hydroxybenzoyl] amino) caprylate.</p> Full article ">
4407 KiB  
Article
Site-Specific Labeling of Protein Kinase CK2: Combining Surface Display and Click Chemistry for Drug Discovery Applications
by Christian Nienberg, Anika Retterath, Kira-Sophie Becher, Thorsten Saenger, Henning D. Mootz and Joachim Jose
Pharmaceuticals 2016, 9(3), 36; https://doi.org/10.3390/ph9030036 - 27 Jun 2016
Cited by 13 | Viewed by 8880
Abstract
Human CK2 is a heterotetrameric constitutively active serine/threonine protein kinase and is an emerging target in current anti-cancer drug discovery. The kinase is composed of two catalytic CK2α subunits and two regulatory CK2β subunits. In order to establish an assay to identify protein-protein-interaction [...] Read more.
Human CK2 is a heterotetrameric constitutively active serine/threonine protein kinase and is an emerging target in current anti-cancer drug discovery. The kinase is composed of two catalytic CK2α subunits and two regulatory CK2β subunits. In order to establish an assay to identify protein-protein-interaction inhibitors (PPI) of the CK2α/CK2β interface, a bioorthogonal click reaction was used to modify the protein kinase α-subunit with a fluorophore. By expanding the genetic code, the unnatural amino acid para azidophenylalanine (pAzF) could be incorporated into CK2α. Performing the SPAAC click reaction (Strain-Promoted Azide-Alkyne Cycloaddition) by the use of a dibenzylcyclooctyne-fluorophore (DBCO-fluorophore) led to a specifically labeled human protein kinase CK2α. This site-specific labeling does not impair the phosphorylation activity of CK2, which was evaluated by capillary electrophoresis. Furthermore a dissociation constant (KD) of 631 ± 86.2 nM was determined for the substrate αS1-casein towards CK2α. This labeling strategy was also applied to CK2β subunit on Escherichia coli, indicating the site-specific modifications of proteins on the bacterial cell surface when displayed by Autodisplay. Full article
(This article belongs to the Special Issue Selected Papers from the 1st International Electronic Conference on Medicinal Chemistry)
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Graphical abstract
Full article ">Figure 1
<p>Comparison of the phosphorylation activity of the heterotetrameric CK2 before and after reaction with FITC. The CE-based assay as described before by Gratz et al. [<a href="#B23-pharmaceuticals-09-00036" class="html-bibr">23</a>] was used to determine the CK2 activity. Electropherogram of the phosphorylation of the substrate peptide RRRDDDSDDD (114 µM) by unlabeled (I, 2.6 µg) and fluorescein-conjugated CK2 (II, 2.6 µg) after an incubation time of 30 min is shown. Substrate (S) and product (P) peaks were detected after 3.7 min and 4.3 min, respectively.</p> Full article ">Figure 2
<p>Ribbon diagram illustrating the structure of heterotetrameric human protein kinase CK2. For this purpose CK2 structure (PDB identification number 1JWH) was processed with the UCSF Chimera 1.10.2 software package [<a href="#B26-pharmaceuticals-09-00036" class="html-bibr">26</a>]. The catalytic CK2α subunit binds to the regulatory CK2β subunit. Dimerization of two β-subunits is mediated by a zinc finger. The non-hydrolysable ATP analogue adenosine 5´-[β,γ-imido]triphosphate (AMPPNP) is bound in the ATP binding pocket of one catalytic α-subunit. A tyrosine in position 239 (Y239) was chosen to be replaced by the unnatural amino acid pAzF into CK2α.</p> Full article ">Figure 3
<p>SDS-PAGE analysis of gene expression and incorporation of pAzF into CK2α. The addition or the omission of the unnatural amino acid pAzF, the inducers IPTG and arabinose to <span class="html-italic">E. coli</span> BL21(DE3) cells with the plasmids CK2α<sup>Y239Stop</sup> and pEVOL-pAzF, expressing the mutated CK2α (IPTG) and the amber suppressor tRNA (constitutive)/aminoacyl-tRNA synthetase (constitutive/arabinose), were proven in a volume of 1 mL minimal medium for each case. Cells were boiled for 20 min at 95 °C and protein lysates were separated on 10% acrylamide. The apparent molecular mass of the marker proteins is shown in lane M. Full-length CK2α (40 kDa) could be synthesized in lane 6 and 8, i.e., when all components were present. Because of the stop codon UAG and the lack of pAzF, the truncated CK2α (28 kDa) appeared in lane 2 and 4.</p> Full article ">Figure 4
<p>SPAAC click reaction of CK2α-pAzF with the two dibenzylcyclooctyne-fluorophores DBCO545 and DBCO-Sulfo -Cy5, respectively (R<sub>1</sub> = N-terminal sequence of CK2α-pAzF, R<sub>2</sub> = C-terminal sequence of CK2α-pAzF). For both cases, beside the regioisomer 1,4 as shown in the reaction scheme, the regioisomer 1,5 can be obtained as well.</p> Full article ">Figure 5
<p>SDS-PAGE analysis of the SPAAC click reaction between CK2α-pAzF and the fluorophore DBCO545. Protein solutions were separated on 10% acrylamide. In lane M, the apparent molecular mass of the marker proteins is given. Purified and concentrated CK2α-pAzF (11 µg) in presence and absence of DBCO545 in a final concentration of 50 µM are shown in lane 2 and lane 1, respectively. As control purified full length CK2α (2.8 µg) without incorporated pAzF was also incubated with DBCO545 (lane 3). (<b>A</b>) Proteins were stained with Coomassie brilliant blue G250. (<b>B</b>) Visualization of the fluorescent protein band of CK2α-DBCO545 by LED-illuminator (470 nM).</p> Full article ">Figure 6
<p>Proof of interaction between CK2β and the CK2α-DBCO-subunit. The activity of CK2α-DBCO545 [<b>□</b>] alone and by addition of purified CK2β [<b>■</b>] was analyzed by CE assay. There were significant differences in activity (<span class="html-italic">n</span> = 3, error bars ± SEM, *** <span class="html-italic">p</span> &lt; 0.0001, unpaired <span class="html-italic">t</span> test).</p> Full article ">Figure 7
<p>Phosphorylation activity of the heterotetrameric CK2 with or without coupling to DBCO545. (<b>A</b>) Comparison of the phosphorylated product between the holoenzyme including CK2α-DBCO545 (I, 2.6 µg) and CK2α-pAzF (II, 2.6 µg) is shown in an electropherogram after 30 min incubation with the substrate RRRDDDSDDD. Substrate (S) and product (P) peaks were detected after 3.7 min and 4.3 min, respectively. (<b>B</b>) The activities of the holoenzymes consisting of CK2α-pAzF [<b>□</b>] as well as CK2α-DBCO545 [<b>■</b>] were analyzed after 15, 30 and 45 min for each sample by CE. Mean values ± standard errors of the means (SEM) from three independent experiments are given (not significant, <span class="html-italic">p</span> &gt; 0.05).</p> Full article ">Figure 8
<p>Proof of interaction between surface-displayed CK2β and CK2α-DBCO545-subunit. CK2β, which was translocated on the surface of <span class="html-italic">E. coli</span> by Autodisplay, was incubated for 1 h at 37 °C with purified specifically labeled CK2α-DBCO545. The binding affinity of CK2β and CK2α-DBCO545 (red, mF = 3800) was analyzed by flow cytometry. As a non-binding control, surface-displayed sorbitol dehydrogenase [<a href="#B33-pharmaceuticals-09-00036" class="html-bibr">33</a>] was used (grey, mF = 108).</p> Full article ">Figure 9
<p>SPAAC reaction of CK2β-AT-pAzF and DBCO545 on the surface of <span class="html-italic">E. coli</span>. Cells (OD<sub>578</sub> = 1) displaying CK2β-AT-pAzF were incubated with the fluorophore DBCO545 (50 µM) for 1h at RT. After three washing steps, <span class="html-italic">E. coli</span> displaying CK2β-AT-DBCO545 (red, mF = 1495) were analyzed by flow cytometry. As a control, surface translocated CK2β-AT without incorporated unnatural amino acid pAzF was applied and treated identically (grey, mF = 120).</p> Full article ">Figure 10
<p>Interaction of CK2α-DBCO-Sulfo-Cy5 and human α<sub>S1</sub>-casein. To a constant amount of CK2α (65 nM) α<sub>S1</sub>-casein was titrated in different concentrations, ranging from 0.76 nM to 12.50 µM. (<b>A</b>) The normalized fluorescence signals of the thermophoresis of 15 different dilutions of α<sub>S1</sub>-casein in presence of the CK2α subunit were recorded. (<b>B</b>) The <span class="html-italic">K<sub>D</sub></span> value of 631 ± 86.2 nM was determined from three independent experiments using NT Analysis 1.5.41 software (NanoTemper Technologies GmbH, München, Germany).</p> Full article ">
337 KiB  
Editorial
Announcing the 2016 Pharmaceuticals Travel Award for Young Investigators
by Jean Jacques Vanden Eynde
Pharmaceuticals 2016, 9(3), 35; https://doi.org/10.3390/ph9030035 - 27 Jun 2016
Cited by 2 | Viewed by 3570
Abstract
For the first time in its short history, our journal is able, this year, to support a young researcher in the field of medicinal chemistry by offering a travel grant of 800 CHF.[...] Full article
3085 KiB  
Article
Relationship between Surface Properties and In Vitro Drug Release from a Compressed Matrix Containing an Amphiphilic Polymer Material
by Cristhian J. Yarce, Diego Pineda, Clara E. Correa and Constain H. Salamanca
Pharmaceuticals 2016, 9(3), 34; https://doi.org/10.3390/ph9030034 - 24 Jun 2016
Cited by 35 | Viewed by 5744
Abstract
The performance of compressed tablet drug delivery systems made using polymeric materials depend on multiple factors, such as surface properties like contact angle, surface free energy and water absorption rate, besides the release mechanisms driven by the kind of polymer used. Hence, it [...] Read more.
The performance of compressed tablet drug delivery systems made using polymeric materials depend on multiple factors, such as surface properties like contact angle, surface free energy and water absorption rate, besides the release mechanisms driven by the kind of polymer used. Hence, it should be possible to establish a relationship between the surface properties and the drug release kinetics. Compressed tablets with different proportions of poly(maleic acid-alt-octadecene) potassium salt (0%, 10%, 20%, 30% and 40%) were prepared. Blends of a model drug (ampicillin trihydrate) and the polymer material were analyzed by DSC. The surface properties of the tablets were determined by the sessile drop method, while the surface energy was determined using the semi-empirical Young-Dupre, Neumann and OWRK models. The release profiles were determined simulating in vitro conditions (buffer solutions pH 1.2 and pH 7.4 with ionic strength of 1.5 M at 37 °C (310.15 K)). A kinetic analysis of the dissolution profiles using different models (zero order, first order, Higuchi and Korsmeyer-Peppas) was realized. The results showed a significant effect of the proportion of polymer in both the surface properties of the tablets and the dissolution release, indicating a relationship between the kinetic and thermodynamic properties. Full article
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Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>Graphical representation of the equilibrium states described by Young to define the contact angle. (<b>A</b>) Solid surface interacting with three different liquids; (<b>B</b>) Three solid surfaces interacting with the same liquid.</p> Full article ">Figure 2
<p>SEM photographs of: (<b>A</b>) ampicillin trihydrate (600×); (<b>B</b>) PAM-18K (2250×).</p> Full article ">Figure 3
<p>Thermograms of ampicillin trihydrate with PAM-18K polymer at different proportions.</p> Full article ">Figure 4
<p>Contact angle variation between ultra-pure water and ampicillin trihydrate tablets at different PAM-18 K polymer proportions. (<b>A</b>) 0%; (<b>B</b>) 10%; (<b>C</b>) 20%; (<b>D</b>) 30%; (<b>E</b>) 40%.</p> Full article ">Figure 5
<p>Variation of <span class="html-italic">θ<sub>c</sub></span> for ampicillin trihydrate-PAM-18K tablets respect to the three test liquids.</p> Full article ">Figure 6
<p>Water absorption process on surface of tablets at different times.</p> Full article ">Figure 7
<p>Ultra-pure water absorption profiles on the solid surface of ampicillin and PAM-18K tablets versus time. ■ = 0%, ○ = 10%, □: 20%, ×: 30%, ∆: 40%.</p> Full article ">Figure 8
<p>Dissolution profiles of ampicillin trihydrate from compressed tablets containing different proportions of PAM-18K polymer in two in vitro dissolution media with ionic strength of 0.15 M: (<b>A</b>) pH 1.2; (<b>B</b>) pH 7.4.</p> Full article ">
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