Identification of a 3-Alkylpyridinium Compound from the Red Sea Sponge Amphimedon chloros with In Vitro Inhibitory Activity against the West Nile Virus NS3 Protease.

Viruses are underrepresented as targets in pharmacological screening efforts, given the difficulties of devising suitable cell-based and biochemical assays. In this study we found that a pre-fractionated organic extract of the Red Sea sponge Amphimedon chloros was able to inhibit the West Nile Virus NS3 protease (WNV NS3). Using liquid chromatography⁻mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR) spectroscopy, the identity of the bioactive compound was determined as a 3-alkylpyridinium with m/z = 190.16. Diffusion Ordered Spectroscopy (DOSY) NMR and NMR relaxation rate analysis suggest that the bioactive compound forms oligomers of up to 35 kDa. We observed that at 9.4 μg/mL there was up to 40⁻70% inhibitory activity on WNV NS3 protease in orthogonal biochemical assays for solid phase extracts (SPE) of A. chloros. However, the LC-MS purified fragment was effective at inhibiting the protease up to 95% at an approximate amount of 2 µg/mL with negligible cytotoxicity to HeLa cells based on a High-Content Screening (HCS) cytological profiling strategy. To date, 3-alkylpyridinium type natural products have not been reported to show antiviral activity since the first characterization of halitoxin, or 3-alkylpyridinium, in 1978. This study provides the first account of a 3-alkylpyridinium complex that exhibits a proposed antiviral activity by inhibiting the NS3 protease. We suggest that the here-described compound can be further modified to increase its stability and tested in a cell-based assay to explore its full potential as a potential novel antiviral capable of inhibiting WNV replication

Gas-phase fragmentation studies of a series of biotin derivatives and of a core oligosaccharide by tandem mass spectrometry

The gas-phase fragmentation of a series of commercially available biotinyl reagents have been evaluated by electrospay ionization mass spectrometry (ESI-MS) and collision induced dissociation tandem mass spectrometry (CID-MS/MS) analyses using a QqTOF-MS/MS hybrid instrument. In general it has been observed that the CID-MS/MS fragmentation routes of the five precursor protonated molecules obtained from the biotin linkers I-5 afforded a series of product ions formed essentially by similar routes. The genesis and structural identities of all the product ions obtained from the biotin linkers I-5 have been assigned. All of the exact mass assignments of the protonated molecules and the product ions were verified by conducting separate CID-MS/MS analysis of the deuterium labeled precursor ions. -- The ESI-QqTOF-MS structural eluciadation of the core oligosaccharide of Aeromonas hydrophila (chemotype II) lipopolysaccharide has been investigated and it was demonstrated that it contained a 4-O-linked posphorylated group Kdo residue which was glycosylated by the remaining outer core oligosaccharide through its O-5 position. After, releasing the core oligosaccharide from the native LPS with acid, the phosphorylated Kdo residue eliminated phosphoric acid to produce a core oligosaccharide containing a mixture of diastereomeric 4,8- and 4,7-anhydro-α-keto acids and an open olefinic Kdo residue. The characteristic glycone sequence was elucidated by CID-MS/MS of the protonated molecule of the native core oligosaccharide. In addition, the analysis of the Hakamori permethylated core oligosaccharide was carried out by ESI-QqTOF-MS and MALDI-TOF-MS analyses. The presence of more than nine isobaric isomers of this core was noticed. The collision-induced dissociation analysis (CID-MS/MS) of the various protonated permethylated core oligosaccharide molecules showed a similar and diagnostic fragmentation pattern. -- To confirm these obtained results, the permethylation of the core oligosaccharide SJ-48R has been performed with a different methylation method (Ciucanu & Kerek method). It was realized that the extra minor satellite signals obtained in the ESI-QqTOF-MS and MALDI-TOF-MS analyses were DMSO stable covalent addition products, which have occurred by a Michael addition on the 4,8-Kdo exocyclic double bond and on the C-3-C-4 double bond of the olefinic open-chain Kdo residue. To our knowledge, this is the first time that DMSO adducts have been observed in the gas phase, and they have never before been reported

Analysis of some pharmaceuticals in municipal wastewater of Almadinah Almunawarah

The chemical pollution of water resources is a major challenge facing the humanity in this century. Pharmaceuticals and personal care products (PPCPs) are a group of emerging environmental chemical pollutants distinguished by their bioactivity and high solubility. They may also cause health complications to humans and living organisms. Pharmaceuticals enter the environment, mainly via wastewater and can eventually reach the surface and ground water. Despite this, PPCPs received less attention as environmental pollutants than other chemical pollutants (e.g. heavy metals and pesticides). The purpose of this work was to investigate the presence of some of the most frequently dispensed drugs for the residents of Almadinah Almunawarah, Saudi Arabia in the municipal wastewater before and after treatment. For this purpose, wastewater samples were collected biweekly from the city's sewage treatment plant for a period of 4 months and analyzed the targeted drugs using tandem LC–MS. Out of the 19 investigated drugs, 5 pharmaceuticals have been found in concentrations greater than the limit of detection in both the influents and effluents of the sewage treatment plant. As expected, the concentrations of investigated pharmaceuticals in the wastewater were found to be low. These drugs and their average concentrations (in ng mL−1) in the influents were: acetaminophen (38.9), metformin (15.2), norfluoxetine (7.07), atenolol (2.04), and cephalexin (1.88). Meanwhile, the effluents contained slightly lower levels (in ng mL−1) than those of influents: acetaminophen (31.2), metformin (3.19), norfluoxetine (7.25), atenolol (0.545), and cephalexin (1.53). The results of this study supported by many other investigations indicate the inefficiency of current conventional wastewater treatment protocols in eliminating such a group of active and potentially hazardous pollutants from the wastewater

Extraction, Characterization, and Antioxidant Activity of Polysaccharides from Ajwa Seed and Flesh

The date palm has been cultivated in dry and hot areas of the planet for much of human history. In the Kingdom of Saudi Arabia, dates are the main crop used as a source of food. Among several species of date fruits, the Ajwa AL-Madinah date is unique, growing only in Al-Madinah geographical region. The Ajwa date is used in traditional medicine due to its abundant active components and therapeutic properties. This study investigates the structural properties and the antioxidant effects of water-soluble polysaccharides extracted from Ajwa flesh and seed. The polysaccharides were isolated by two techniques including hot water and ultrasonic extraction. After isolation and partial purification, the physicochemical properties of four samples of polysaccharides extracted from flesh and seed were studied by several techniques including FTIR, solid-state NMR, elemental analysis, and mass spectrometry. Several radical scavenging experiments were combined to study the antioxidant activity of the polysaccharide compounds. FTIR and NMR results showed a structure typical of heterogeneous polysaccharides. Mass spectrometry revealed that the polysaccharide samples were composed mainly of mannose, glucose, galactose, xylose, arabinose, galacturonic acid, and fucose. In addition, the physicochemical properties and composition of polysaccharides extracted from flesh and seed were compared. The extracted polysaccharides showed antioxidant activity, with 2, 2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging, Fe chelating ability, hydroxyl free radical scavenging ability, and 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging. These results highlight their potential to be a useful nutritional element or supplemental medication

Anti-cancer agents in Saudi Arabian herbals revealed by automated high-content imaging

<div><p>Natural products have been used for medical applications since ancient times. Commonly, natural products are structurally complex chemical compounds that efficiently interact with their biological targets, making them useful drug candidates in cancer therapy. Here, we used cell-based phenotypic profiling and image-based high-content screening to study the mode of action and potential cellular targets of plants historically used in Saudi Arabia’s traditional medicine. We compared the cytological profiles of fractions taken from <i>Juniperus phoenicea</i> (Arar), <i>Anastatica hierochuntica</i> (Kaff Maryam), and <i>Citrullus colocynthis</i> (Hanzal) with a set of reference compounds with established modes of action. Cluster analyses of the cytological profiles of the tested compounds suggested that these plants contain possible topoisomerase inhibitors that could be effective in cancer treatment. Using histone H2AX phosphorylation as a marker for DNA damage, we discovered that some of the compounds induced double-strand DNA breaks. Furthermore, chemical analysis of the active fraction isolated from <i>Juniperus phoenicea</i> revealed possible anti-cancer compounds. Our results demonstrate the usefulness of cell-based phenotypic screening of natural products to reveal their biological activities.</p></div

The cytotoxic effect of plant fractions on HeLa cells.

<p>A-B) Cells were treated with various concentrations of plant extracts for 24 h or 48 h, stained with Hoechst, and assessed using HCS. The cells exhibited cytotoxicity, which is indicator of induced apoptosis or necrosis. C) The distribution of cells during the cell cycle: G2/M, S, G0/G1 and low phases after a 24-h treatment with plant fractions. Data shown are means ± SD.</p

Plant fractions induced an apoptotic effect on HeLa cells.

<p>HeLa cells were treated with several concentrations (1.56, 3.12, 6.25, 12.5, 25, and 50 μg/ml) of plant fractions for 24 h or 48 h. A, B) Automated HCS was used to measure the activity of caspase-9 and C, D) p53. The fluorescence readout was normalized against an in-plate control. Each sample was tested in quadruplicate. Data are presented as means ± SD.</p

Assesment of the double-strand breaks in the DNA.

<p>HeLa cells were treated with different concentrations of SPE fractions to detect the expression of γ-H2AX. Data are presented as means ± SD.</p