Ecological and Chemical Studies on the Gq-protein Inhibitor FR900359

The cyclic depsipeptide FR900359 (FR), isolated from the tropical plant Ardisia crenata, displays a strong and selective inhibition of Gq proteins, making it an indispensable pharmacological tool to study Gq-related processes, as well as a promising drug candidate. Gq inhibition is a novel mode of action for defense chemicals and crucial for the ecological function of FR, as corroborated by in vivo experiments on mice, affinity to insect Gq proteins and insect toxicity studies. The uncultured endosymbiont of A. crenata, 'Candidatus Burkholderia crenata' was sequenced, revealing the FR nonribosomal peptide synthetase (frs) gene cluster. In this study we provide a detailed model of FR biosynthesis, supported by in vitro enzymatic and bioinformatic studies. Finally, expression of the frs genes in E. coli led to heterologous FR production in a cultivable, bacterial host for the first time, paving the way for a biotechnological production of FR independent from time- and work-intensive plant cultivation, harvesting and extraction. Direct targeting of intracellular Gα subunits is a challenging task in pursuit of chemical tools for pharmacological studies and for developing novel therapeutic approaches. We isolated four new FR analogs (1-4) from A. crenata and elucidated their structures by NMR spectroscopic data and MS-based molecular networking followed by in-depth LCMS2 analysis. Next, we analyzed all currently known inhibitors of Gq protein including YM-254890, FR900359, above mentioned novel FR-derivatives from A. crenata, and synthetic cyclic peptides to devise a strategy for the elucidation of characteristics that determine interaction with Gq. Using 2D NMR spectroscopy and molecular docking we identified unique features in the macrocycles that govern specific binding to and inhibition of Gq. While all novel compounds were devoid of effects on Gi and Gs proteins, no inhibitor surpassed biological activity of FR or YM. This raises the question of whether nature has optimized these depsipeptides for specific inhibition of Gq. Thus, rather than attempting to enhance Gq activity of newly synthesized inhibitors, future synthetic efforts on FR/YM-analogs should target Gα subunits other than Gq. Additionally, FR was detected from leaves of five other Ardisia species, among them the non-nodulated A. lucida as well as from a soil bacterium, implicating a much broader distribution of FR as originally anticipated. Furthermore the first reported fluorescent FR analogs were synthesized, biologically evaluated and applied to study the mechanism of cellular uptake of FR

Design, synthesis and evaluation of fluorescent CB2 cannabinoid receptor ligands

Cannabis has been used as a medicinal and natural product for thousands of years. Whether it has been used to make rope or paper, or been used to treat pain or depression, cannabis has always had a place in human civilisation. With the isolation of the psychoactive compounds responsible for cannabis’ effects, the discovery of two human cannabinoid receptors and an expanding knowledge of the therapeutic uses of cannabis, interest in the development of novel cannabinoids grew. The CB2 cannabinoid receptor has gained particular attention, as the often unwanted central and psychoactive effects of cannabinoids has been attributed to the CB1 cannabinoid receptor. Development of CB2 receptor selective ligands offers treatment opportunities in many areas, but most especially for pain, multiple sclerosis and immunomodulation. The preparation of fluorescently labelled ligands for a variety of receptors has improved compound screening techniques, as well as allowing use as biomolecular probes for aiding our understanding of the receptor in situ. The aim of this work is to design, synthesise and evaluate novel fluorescently labelled cannabinoids, with a particular interest in CB2 selective compounds. Focusing on the CB2 receptor selective alkylindole JWH-015, targeted substitutions were made to its naphthyl ring to identify sites that might be suitable for fluorophore attachment. With a site chosen, a series of fluorescent JWH-015 analogues was synthesised and evaluated for their CB2 receptor binding affinities. Though none of the evaluated compounds showed sufficient binding affinity for them to be used as biomolecular tools, the structure activity relationships gained suggested that improved design of fluorescent JWH-015 analogues in future could lead to the first ever active fluorescent cannabinoid

Design, synthesis and evaluation of fluorescent CB2 cannabinoid receptor ligands

Cannabis has been used as a medicinal and natural product for thousands of years. Whether it has been used to make rope or paper, or been used to treat pain or depression, cannabis has always had a place in human civilisation. With the isolation of the psychoactive compounds responsible for cannabis’ effects, the discovery of two human cannabinoid receptors and an expanding knowledge of the therapeutic uses of cannabis, interest in the development of novel cannabinoids grew. The CB2 cannabinoid receptor has gained particular attention, as the often unwanted central and psychoactive effects of cannabinoids has been attributed to the CB1 cannabinoid receptor. Development of CB2 receptor selective ligands offers treatment opportunities in many areas, but most especially for pain, multiple sclerosis and immunomodulation. The preparation of fluorescently labelled ligands for a variety of receptors has improved compound screening techniques, as well as allowing use as biomolecular probes for aiding our understanding of the receptor in situ. The aim of this work is to design, synthesise and evaluate novel fluorescently labelled cannabinoids, with a particular interest in CB2 selective compounds. Focusing on the CB2 receptor selective alkylindole JWH-015, targeted substitutions were made to its naphthyl ring to identify sites that might be suitable for fluorophore attachment. With a site chosen, a series of fluorescent JWH-015 analogues was synthesised and evaluated for their CB2 receptor binding affinities. Though none of the evaluated compounds showed sufficient binding affinity for them to be used as biomolecular tools, the structure activity relationships gained suggested that improved design of fluorescent JWH-015 analogues in future could lead to the first ever active fluorescent cannabinoid

Anti-angiogenic and toxicity effects of Derris trifoliata extract in zebrafish embryo

Introduction: Derris trifoliata has been traditionally used as folk for the treatment of , rheumatic joints, diarrhoea, and dysmenorrhea, and rotenoids isolated from the plant have shown to exhibit anti-cancer properties. This study aimed to assess the toxicity effects and antiangiogenic activity of extract of Derris trifoliata on zebrafish embryo model. Materials and Methods: Zebrafihs embryos were treated with aqueous extract of Derris Trifoliata to evaluate its effects on angiogenesis and zebrafish-toxicity. Angiogenic response was analyzed using whole-mount alkaline phosphatase (AP) vessel staining on 72 hours post fertilization (hpf) zebrafish embryos. Results: 1.0 mg/ml concentration was toxic to zebrafish embryos and embryos exposed to concentrations at 0.5 mg/ml and below showed some malformations. Derris trifoliata aqueous extract also showed some anti-angiogenic activity in vivo in the zebrafish embryo model wereby at high concentration inhibited vessel formation in zebrafish embryo. Conclusions: The anti-angiogenic response of extract of Derris trifoliata in zebrafish in vivo model suggest its therapeutic potential as anti-cancer agent

Drug Discovery

Natural products are a constant source of potentially active compounds for the treatment of various disorders. The Middle East and tropical regions are believed to have the richest supplies of natural products in the world. Plant derived secondary metabolites have been used by humans to treat acute infections, health disorders and chronic illness for tens of thousands of years. Only during the last 100 years have natural products been largely replaced by synthetic drugs. Estimates of 200 000 natural products in plant species have been revised upward as mass spectrometry techniques have developed. For developing countries the identification and use of endogenous medicinal plants as cures against cancers has become attractive. Books on drug discovery will play vital role in the new era of disease treatment using natural products

The new technique for accurate estimation of the spinal cord circuitry:recording reflex responses of large motor unit populations

We propose and validate a non-invasive method that enables accurate detection of the discharge times of a relatively large number of motor units during excitatory and inhibitory reflex stimulations. HDsEMG and intramuscular EMG (iEMG) were recorded from the tibialis anterior muscle during ankle dorsiflexions performed at 5%, 10%, and 20% of the maximum voluntary contraction (MVC) force, in 9 healthy subjects. The tibial nerve (inhibitory reflex) and the peroneal nerve (excitatory reflex) were stimulated with constant current stimuli. In total, 416 motor units were identified from the automatic decomposition of the HDsEMG. The iEMG was decomposed using a state-of-the-art decomposition tool and provided 84 motor units (average of two recording sites). The reflex responses of the detected motor units were analyzed using the peri-stimulus time histogram (PSTH) and the peri-stimulus frequencygram (PSF). The reflex responses of the common motor units identified concurrently from the HDsEMG and the iEMG signals showed an average disagreement (the difference between number of observed spikes in each bin relative to the mean) of 8.2±2.2% (5% MVC), 6.8±1.0% (10% MVC), and 7.5±2.2% (20% MVC), for reflex inhibition, and 6.5±4.1%, 12.0±1.8%, 13.9±2.4%, for reflex excitation. There was no significant difference between the characteristics of the reflex responses, such as latency, amplitude and duration, for the motor units identified by both techniques. Finally, reflex responses could be identified at higher force (four of the nine subjects performed contraction up to 50% MVC) using HDsEMG but not iEMG, because of the difficulty in decomposing the iEMG at high forces. In conclusion, single motor unit reflex responses can be estimated accurately and non-invasively in relatively large populations of motor units using HDsEMG. This non-invasive approach may enable a more thorough investigation of the synaptic input distribution on active motor units at various force levels

The Potential of Dietary Antioxidants

Oxidative stress causes chronic diseases such as cardiovascular disease, cancer, Alzheimer, chronic obstructive pulmonary, and neurodegenerative pathologies. Antioxidant systems defend human cells from free radicals. They act by stopping free radicals, decreasing their development, and quenching the formed ROS and RNS. The antioxidant molecules are classified into primary and secondary defense molecules. The primary antioxidant molecules (i.e., vitamins C and E, ubiquinone, and glutathione) reduce oxidation effects by moving a proton to the free radical species or electron donors, or by terminating the chain reactions The secondary antioxidants (i.e., N-acetyl cysteine and lipoic acid) act as cofactors for some enzyme systems or neutralize the production of free radicals by transition metals. This work comprises original research papers and reviews on antioxidant molecules in food, the agricultural practices that maximize their levels in plants, the potential preventive effects of selected classes of antioxidant molecules, their potential use in functional foods, and the pharmaceutical delivery systems that maximize their potential activity when used as supplements