The Uptake of Sporopollenin Exine Capsules and Associated Bioavailability of Adsorbed Oestradiol in Selected Aquatic Invertebrates.

Lycopodium clavatum sporopollenin exine capsules (SpECs) are known to both adsorb and absorb chemicals. The aim of the present work was to determine whether oestradiol (E2) is 'bioavailable' to bioindicator species, either pre-adsorbed to, or in the presence of, SpECs. SpEC uptake was confirmed for Daphnia magna and Dreissena bugensis. E2 levels varied among treatments for Caenorhabditis elegans though there was no relationship to SpEC load. E2 was not detected in D. bugensis tissues. Expression changes of general stress and E2-specific genes were measured. For C. elegans, NHR-14 expression suggested that SpECs modulate E2 impacts, but not general health responses. For D. magna, SpECs alone and with E2 changed Vtg1 and general stress responses. For D. bugensis, SpECS were taken up but no E2 or change in gene expression was detected after exposure to E2 and/or SpECs. The present study is the first to investigate SpECs and bound chemical dynamics.

Role of Whole-Genome Sequencing in Characterizing the Mechanism of Action of para-Aminosalicylic Acid and Its Resistance.

para-Aminosalicylic acid (PAS) remains one of the drugs of last resort for the treatment of tuberculosis, but its mechanism of action is still not completely understood. The main aim of this project was to identify new potential mechanisms of action and resistance to PAS by performing whole-genome sequencing on PAS-resistant laboratory mutants. A new variant in the folC gene was identified, as well as some other mutations that require further study.

Interconvertible geometric isomers of Plasmodium falciparum dihydroorotate dehydrogenase inhibitors exhibit multiple binding modes.

Two new tricyclic ß-aminoacrylate derivatives (2e and 3e) have been found to be inhibitors of Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH) with Ki 0.037 and 0.15µM respectively. 1H and 13C NMR spectroscopic data show that these compounds undergo ready cis-trans isomerisation at room temperature in polar solvents. In silico docking studies indicate that for both molecules there is neither conformation nor double bond configuration which bind preferentially to PfDHODH. This flexibility is favourable for inhibitors of this channel that require extensive positioning to reach their binding site.

Synthesis of a Graphene-Encapsulated Fe3C/Fe Catalyst Supported on Sporopollenin Exine Capsules and Its Use for the Reverse Water-Gas Shift Reaction.

Bioderived materials have emerged as sustainable catalyst supports for several heterogeneous reactions owing to their naturally occurring hierarchal pore size distribution, high surface area, and thermal and chemical stability. We utilize sporopollenin exine capsules (SpECs), a carbon-rich byproduct of pollen grains, composed primarily of polymerized and cross-linked lipids, to synthesize carbon-encapsulated iron nanoparticles via evaporative precipitation and pyrolytic treatments. The composition and morphology of the macroparticles were influenced by the precursor iron acetate concentration. Most significantly, the formation of crystalline phases (Fe3C, α-Fe, and graphite) detected via X-ray diffraction spectroscopy showed a critical dependence on iron loading. Significantly, the characteristic morphology and structure of the SpECs were largely preserved after high-temperature pyrolysis. Analysis of Brunauer-Emmett-Teller surface area, the D and G bands from Raman spectroscopy, and the relative ratio of the C=C to C-C bonding from high-resolution X-ray photoelectron spectroscopy suggests that porosity, surface area, and degree of graphitization were easily tuned by varying the Fe loading. A mechanism for the formation of crystalline phases and meso-porosity during the pyrolysis process is also proposed. SpEC-Fe10% proved to be highly active and selective for the reverse water-gas shift reaction at high temperatures (>600 °C).

Spore exines increase vitamin D clinical bioavailability by mucoadhesion and bile triggered release.

Sporopollenin exine capsules (SpECs) are microcapsules derived from the outer shells (exines) of plant spore and pollen grains. This work reports the first clinical study on healthy volunteers to show enhanced bioavailability of vitamin D encapsulated in SpECs from Lycopodium clavatum L. spore grains vs vitamin D alone, and the first evidence (in vitro, ex vivo and in vivo) of mechanisms to account for the enhancement and release of the active in the small intestine. Evidence for mucoadhesion of the SpECs contributing to the mechanism of the enhancement is based on: (i) release profile over time of vitamin D in a double blind cross-over human study showing significant release in the small intestine; (ii) in vivo particle counting data in rat showing preferred retention of SpECs vs synthetic beads; (iii) ex vivo99mTc labelling and counting data using rat small intestine sections showing preferred retention of SpECs vs synthetic beads; (iv) in vitro mucoadhesion data. Triggered release by bile in the small intestine was shown in vitro using solid state NMR and HPLC.

TRPA1 is activated by direct addition of cysteine residues to the N-hydroxysuccinyl esters of acrylic and cinnamic acids.

The nociceptor TRPA1 is thought to be activated through covalent modification of specific cysteine residues on the N terminal of the channel. The precise mechanism of covalent modification with unsaturated carbonyl-containing compounds is unclear, therefore by examining a range of compounds which can undergo both conjugate and/or direct addition reactions we sought to further elucidate the mechanism(s) whereby TRPA1 can be activated by covalent modification. Calcium signalling was used to determine the mechanism of activation of TRPA1 expressed in HEK293 cells with a series of related compounds which were capable of either direct and/or conjugate addition processes. These results were confirmed using physiological recordings with isolated vagus nerve preparations. We found negligible channel activation with chemicals which could only react with cysteine residues via conjugate addition such as acrylamide, acrylic acid, and cinnamic acid. Compounds able to react via either conjugate or direct addition, such as acrolein, methyl vinyl ketone, mesityl oxide, acrylic acid NHS ester, cinnamaldehyde and cinnamic acid NHS ester, activated TRPA1 in a concentration dependent manner as did compounds only capable of direct addition, namely propionic acid NHS ester and hydrocinnamic acid NHS ester. These compounds failed to activate TRPV1 expressed in HEK293 cells or mock transfected HEK293 cells. For molecules capable of direct or conjugate additions, the results suggest for the first time that TRPA1 may be activated preferentially by direct addition of the thiol group of TRPA1 cysteines to the agonist carbonyl carbon of α,ß-unsaturated carbonyl-containing compounds.

Synthesis and analysis of small molecules to restrain the function of tissue factor within tumour cells.

Introduction: The restriction of prolyl-protein cis/trans isomerase 1 (Pin1) activity has been shown to prevent the release of tissue factor (TF) leading to the accumulation of the latter protein within the cell. This study tested the ability of novel small molecules to inhibit Pin1, suppress TF activity and release, and induce cellular apoptosis. Methods: Four compounds were designed and synthesised based on modification of 5-(p-methoxyphenyl)-2-methylfuran-3-carbonyl amide and the outcome on MDA-MB-231 and primary cells examined. These compounds contained 3-(2-naphthyl)-D-alanine (4a), D-tryptophan (4b), D-phenylalanine (4c), and D-tyrosine (4d) at the amino-termini. Results: Treatment of cells with compound 4b and 4d reduced the cell-surface TF activity after 60 min on MDA-MB-231 cells. Incubation with compound 4d also reduced TF antigen on the cell surface and its incorporation into microvesicles, while compounds 4a and 4b significantly increased TF release. None of the four compounds significantly altered the total amount of TF antigen or TF mRNA expression. Compound 4b and 4d also suppressed the binding of Pin1 to TF-cytoplasmic domain peptide. However, compound 4d reduced while compound 4b increased the Pin1 isomerase activity. Finally, treatment with compound 4b and 4d reduced the cell numbers, increased nuclear localisation of p53, Bax protein and bax mRNA expression and induced cellular apoptosis in MDA-MB-231 but not primary endothelial cells. Conclusions: In conclusion, we have identified small molecules to regulate the function of TF within cells. Two of these compounds may prove to be beneficial in moderating TF function specifically and restrain TF-mediated tumour growth without detrimental outcomes on normal vascular cells.

Recent advances in drug discovery for diabetic kidney disease.

INTRODUCTION: Diabetic kidney disease (DKD) is a leading cause of end-stage renal disease (ESRD), and 40% of patients with diabetes develop DKD. Although some pathophysiological mechanisms and drug targets of DKD have been described, the effectiveness or clinical usefulness of such treatment has not been well validated. Therefore, searching for new targets and potential therapeutic candidates has become an emerging research area. AREAS COVERED: The pathophysiological mechanisms, new drug targets and potential therapeutic compounds for DKD are addressed in this review. EXPERT OPINION: Although preclinical and clinical evidence has shown some positive results for controlling DKD progression, treatment regimens have not been well developed to reduce the mortality in patients with DKD globally. Therefore, the discovery of new therapeutic targets and effective target-based drugs to achieve better and safe treatment are urgently required. Preclinical screening and clinical trials for such drugs are needed.

N-Cinnamoylanthranilates as human TRPA1 modulators: Structure-activity relationships and channel binding sites.

The transient receptor potential ankyrin 1 (TRPA1) channel is a non-selective cation channel, which detects noxious stimuli leading to pain, itch and cough. However, the mechanism(s) of channel modulation by many of the known, non-reactive modulators has not been fully elucidated. N-Cinnamoylanthranilic acid derivatives (CADs) contain structural elements from the TRPA1 modulators cinnamaldehyde and flufenamic acid, so it was hypothesized that specific modulators could be found amongst them and more could be learnt about modulation of TRPA1 with these compounds. A series of CADs was therefore screened for agonism and antagonism in HEK293 cells stably transfected with WT-human (h)TRPA1, or C621A, F909A or F944A mutant hTRPA1. Derivatives with electron-withdrawing and/or electron-donating substituents were found to possess different activities. CADs with inductive electron-withdrawing groups were agonists with desensitising effects, and CADs with electron-donating groups were either partial agonists or antagonists. Site-directed mutagenesis revealed that the CADs do not undergo conjugate addition reaction with TRPA1, and that F944 is a key residue involved in the non-covalent modulation of TRPA1 by CADs, as well as many other structurally distinct non-reactive TRPA1 ligands already reported.

Design and synthesis of potent inhibitors of the malaria parasite dihydroorotate dehydrogenase.

Pyrimidine biosynthesis presents an attractive drug target in malaria parasites due to the absence of a pyrimidine salvage pathway. A set of compounds designed to inhibit the Plasmodium falciparum pyrimidine biosynthetic enzyme dihydroorotate dehydrogenase (PfDHODH) was synthesized. PfDHODH-specific inhibitors with low nanomolar binding affinities were identified that bind in the N-terminal hydrophobic channel of dihydroorotate dehydrogenase, the presumed site of ubiquinone binding during oxidation of dihydroorotate to orotate. These compounds also prevented growth of cultured parasites at low micromolar concentrations. Models that suggest the mode of inhibitor binding is based on shape complementarity, matching hydrophobic regions of inhibitor and enzyme, and interaction of inhibitors with amino acid residues F188, H185, and R265 are supported by mutagenesis data. These results further highlight PfDHODH as a promising new target for chemotherapeutic intervention in prevention of malaria and provide better understanding of the factors that determine specificity over human dihydroorotate dehydrogenase.

Protein free microcapsules obtained from plant spores as a model for drug delivery: ibuprofen encapsulation, release and taste masking.

Sporopollenin exine capsules (SEC) extracted from Lycopodium clavatum spores were shown to encapsulate ibuprofen as a drug model, with 97 ± 1% efficiency as measured by recovery of the loaded drug and absence of the drug on the SEC surface by scanning electron microscopy (SEM). The encapsulated ibuprofen was shown to be unchanged from its bulk crystalline form by solid state NMR, FTIR and XRD. Essential for drug delivery applications, SEC were shown to be non-toxic to human endothelial cells and free of allergenic protein epitopes by MALDI-TOF-MS and ESI-QqToF-MS. Potential application for targeted release into the intestinal region of the gastrointestinal tract (GIT) was demonstrated by 88 ± 1% of the drug being retained in simulated gastric fluid (SGF) after 45 minutes and 85 ± 2% being released after 5 min in buffer (PBS; pH 7.4). The SEC were shown to provide significant taste masking of encapsulated ibuprofen in a double blind trial with 10 human volunteers.

Pharmacological comparison of novel synthetic fenamate analogues with econazole and 2-APB on the inhibition of TRPM2 channels.

BACKGROUND AND PURPOSE: Fenamate analogues, econazole and 2-aminoethoxydiphenyl borate (2-APB) are inhibitors of transient receptor potential melastatin 2 (TRPM2) channels and are used as research tools. However, these compounds have different chemical structures and therapeutic applications. Here we have investigated the pharmacological profile of TRPM2 channels by application of newly synthesized fenamate analogues and the existing channel blockers. EXPERIMENTAL APPROACH: Human TRPM2 channels in tetracycline-regulated pcDNA4/TO vectors were transfected into HEK293 T-REx cells and the expression was induced by tetracycline. Whole cell currents were recorded by patch-clamp techniques. Ca(2+) influx or release was monitored by fluorometry. KEY RESULTS: Flufenamic acid (FFA), mefenamic acid (MFA) and niflumic acid (NFA) concentration-dependently inhibited TRPM2 current with potency order FFA > MFA = NFA. Modification of the 2-phenylamino ring by substitution of the trifluoromethyl group in FFA with -CH(3), -F, -CF(3), -OCH(3), -OCH(2)CH(3), -COOH, and -NO(2) at various positions, reduced channel blocking potency. The conservative substitution of 3-CF(3) in FFA by -CH(3) (3-MFA), however, gave the most potent fenamate analogue with an IC(50) of 76 µM, comparable to that of FFA, but unlike FFA, had no effect on Ca(2+) release. 3-MFA and FFA inhibited the channel intracellularly. Econazole and 2-APB showed non-selectivity by altering cytosolic Ca(2+) movement. Econazole also evoked a non-selective current. CONCLUSION AND IMPLICATIONS: The fenamate analogue 3-MFA was more selective than other TRPM2 channel blockers. FFA, 2-APB and econazole should be used with caution as TRPM2 channel blockers, as these compounds can interfere with intracellular Ca(2+) movement.

Effect of non-steroidal anti-inflammatory drugs and new fenamate analogues on TRPC4 and TRPC5 channels.

Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used anti-inflammatory therapeutic agents, among which the fenamate analogues play important roles in regulating intracellular Ca²âº transient and ion channels. However, the effect of NSAIDs on TRPC4 and TRPC5 is still unknown. To understand the structure-activity of fenamate analogues on TRPC channels, we have synthesized a series of fenamate analogues and investigated their effects on TRPC4 and TRPC5 channels. Human TRPC4 and TRPC5 cDNAs in tetracycline-regulated vectors were transfected into HEK293 T-REx cells. The whole cell current and Ca²âº movement were recorded by patch clamp and calcium imaging, respectively. Flufenamic acid (FFA), mefenamic acid (MFA), niflumic acid (NFA) and diclofenac sodium (DFS) showed inhibition on TRPC4 and TRPC5 channels in a concentration-dependent manner. The potency was FFA>MFA>NFA>DFS. Modification of 2-phenylamino ring by substitution of the trifluoromethyl group in FFA with F, CH3, OCH3, OCH2CH3, COOH, and NO2 led to the changes in their channel blocking activity. However, 2-(2'-methoxy-5'-methylphenyl)aminobenzoic acid stimulated TRPC4 and TRPC5 channels. Selective COX1-3 inhibitors (aspirin, celecoxib, acetaminophen, and indomethacin) had no effect on the channels. Longer perfusion (> 5 min) with FFA (100 µM) and MFA (100 µM) caused a potentiation of TRPC4 and TRPC5 currents after their initial blocking effects that appeared to be partially mediated by the mitochondrial Ca²âº release. Our results suggest that fenamate analogues are direct modulators of TRPC4 and TRPC5 channels. The substitution pattern and conformation of the 2-phenylamino ring could alter their blocking activity, which is important for understanding fenamate pharmacology and new drug development targeting the TRPC channels.

Synthesis of brequinar analogue inhibitors of malaria parasite dihydroorotate dehydrogenase.

A series of 2-phenyl quinoline-4-carboxylic acid derivatives related to brequinar, an inhibitor of human dihydroorotate dehydrogenase (DHODH), has been prepared and evaluated as inhibitors of DHODH from the malaria parasite Plasmodium falciparum. Brequinar was essentially inactive against PfDHODH (IC(50) 880 microM) whereas several members of the series inhibited PfDHODH. Unexpectedly, replacement of the carboxylic acid required for brequinar to inhibit hDHODH was not essential in the diisopropylamides that inhibited PfDHODH.