Bypassing the proline/thiazoline requirement of the macrocyclase PatG.

Biocatalysis is a fast developing field in which an enzyme's natural capabilities are harnessed or engineered for synthetic chemistry. The enzyme PatG is an extremely promiscuous macrocyclase enzyme tolerating both non-natural amino acids and non-amino acids within the substrate. It does, however, require a proline or thiazoline at the C-terminal position of the core peptide which means the final product must contain this group. Here, we show guided by structural insight we have identified two synthetic routes, triazole and a double cysteine, that circumvent this requirement. With the triazole, we show PatGmac can macrocyclise substrates that do not contain any amino acids in the final product.

The Streptomyces metabolite anhydroexfoliamycin ameliorates hallmarks of Alzheimer's disease in vitro and in vivo.

Anhydroexfoliamycin (1) and undecylprodigiosin (2) have been previously described as neuroprotective molecules against oxidative stress in neurons. Since oxidative stress is strongly correlated with neurodegenerative diseases, we have evaluated their effects over the principal hallmarks of Alzheimer's disease (AD). Both compounds were tested in vitro in two different neuroblastoma cellular models, one for amyloid precursor protein metabolism studies (BE(2)-M17) and another one specific for taupathology in AD (SH-SY5Y-TMHT441). Amyloid-beta (Aß) levels, ß-secretase (BACE1) activity, tau phosphorylation, extracellular signal-regulated kinase (ERK) and glycogen synthase kinase-3beta (GSK3ß) expression were analyzed and while undecylprodigiosin (2) produced poor results, anhydroexfoliamycin (1) strongly inhibited GSK3ß, reducing tau phosphorylation in vitro (0.1 µM). A competitive assay of anhydroexfoliamycin (1) and the specific c-Jun N-terminal kinase (JNK) inhibitor, SP600125, showed that the reduction of the phosphorylated tau levels is mediated by the JNK pathway in SH-SY5Y-TMHT441 cells. Thus, this compound was tested in vivo by intraperitoneal administration in 3xTg-AD mice, confirming the positive results registered in the in vitro assays. This work presents anhydroexfoliamycin (1) as a promising candidate for further studies in drug development against neurodegenerative diseases.

Bromoalkaloids protect primary cortical neurons from induced oxidative stress.

Bromoalkaloids are secondary metabolites with a demonstrated high activity in several therapeutic areas. In this research, we probe the neuroprotective and antioxidant activities of hymenialdisine and hymenin. Both structures were tested in an oxidative stress cellular model, consisting of cortical neurons that are incubated with the oxidative stress inducer hydrogen peroxide and the tested compound. Several oxidation biomarkers were analyzed, and the results of the oxidative stress induced neurons in the presence and absence of bromoalkaloids were compared. Both compounds demonstrated significant neuroprotective ability under stress conditions at low nanomolar concentrations, with hymenialdisine highlighted for demonstrating a more complete protection. Also, the activity of hymenialdisine and hymenin was studied in the nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant response element (ARE) pathway, and, for the first time, these halogenated metabolites are described as Nrf2 inducers, reinforcing the antioxidant capacity observed and therefore opening a new path of investigation. These results, added to the previously described effect of this compound family in negatively modulating several kinases and proinflammatory cytokines, position hymenialdisine and hymenin as good candidates for the development of new drugs for neurodegenerative diseases.

Bioactividad de los extractos y aislamiento de los lignanos de las semillas de Centaurea dealbata / Bioactivity of the extracts and the isolation of lignans from the seeds of Centaurea dealbata

La Centaurea dealbata Willd. (familia: Asteraceae) pertenece al género Centaurea, que comprende unas 500 especies. Para evaluar la actividad antioxidante y la toxicidad general de los extractos de n-hexano, diclorometano (DCM) y metanol (MeOH) de las semillas de C. dealbata se han utilizado, respectivamente, el ensayo DPPH y el ensayo de letalidad de gambas en salmuera. Tanto el extracto de DCM como el de MeOH presentaron niveles signifi cativos de actividad antioxidante, con valores de RC50 de 6,8 x 10-2 y 4,7 x 10-2 mg/mL, respectivamente. Ninguno de los extractos presentó una toxicidad general signifi cativa (LD50 = >1000 mg/mL). Se observó que los tres principales componentes bioactivos del extracto de MeOH fueron los lignanos arctigenina, arctiina y matairesinosida. Las estructuras de estos lignanos se dilucidaron mediante análisis espectroscópicos exhaustivos y comparación directa con los datos respectivos publicados. Éste es el primer informe sobre la ocurrencia de arctiina y matairesinol en C. dealbata. También se presenta la distribución de estos lignanos dentro del género Centaurea

Centaurea dealbata Willd. (Family: Asteraceae) belongs to the big genus Centaurea that comprises ca. 500 species. The n-hexane, dichloromethane (DCM) and methanol (MeOH) extracts of the seeds of C. dealbata have been assessed for antioxidant activity and general toxicity using, respectively, the DPPH assay, and the brine shrimp lethality assay. Both the DCM and the MeOH extract showed signifi cant levels of antioxidant activities with an RC50 value 6.8 x 10-2 and 4.7 x 10-2 mg/mL, respectively. None of the extracts exhibited any signifi cant general toxicity (LD50 = >1000 mg/mL). Three major bioactive components of the MeOH extract were found to be the lignans, arctigenin, arctiin and matairesinoside. The structures of these lignans were elucidated by comprehensive spectroscopic analyses, and also by direct comparison with the respective published data. This is the fi rst report on the occurrence of arctiin and matairesionl in C. dealbata. The distribution of these lignans within the genus Centaurea has also been presented

Comparative studies on biological activities of Prunus padus and P. spinosa.

Comparative studies on the antibacterial and free radical scavenging activities of the n-hexane, dichloromethane and methanol extracts of the seeds of Prunus padus and P. spinosa have been carried out. General toxicity of these extracts has also been determined.

Irreversible and reversible pore formation by polymeric alkylpyridinium salts (poly-APS) from the sponge Reniera sarai.

1. In this study, we investigated the electrophysiological actions of a high molecular weight fraction, predominantly containing two polymeric 1,3-alkylpyridinium salts (poly-APS) of 5.5 and approximately 19 kDa isolated from the marine sponge Reniera sarai. The biological properties of poly-APS are of particular interest because this preparation may be used to deliver macromolecules into the intracellular environment without producing long-term damage to cells. Poly-APS (50-0.05 micro g ml(-1)) was applied to cultured dorsal root ganglion neurones or HEK 293 cells and changes in cell membrane properties were measured using whole-cell patch-clamp recording and fura-2 Ca(2+) imaging. 2. Poly-APS (50 micro g ml(-1)) evoked irreversible depolarisations in membrane potential and reductions in input resistance. However, doses of 5 micro g ml(-1) and less produced reversible effects on these cell membrane characteristics and on Ca(2+) permeability. 3. At 0.05 micro g ml(-1), poly-APS could robust transient increases in Ca(2+) permeability without damaging the neurones or subsequently attenuating Ca(2+) entry through voltage-activated channels. 4. Bathing cells in NaCl-based extracellular medium containing 1.5 mM zinc attenuated the irreversible and reversible effects of poly-APS on membrane properties (membrane potential, input resistance and whole-cell currents). In both DRG neurones and HEK 293 cells, zinc attenuated Ca(2+) entry evoked by poly-APS. These effects of zinc were only observed if zinc was continually present during poly-APS application. However, zinc failed to attenuate the actions of poly-APS if it was applied after the sponge toxin preparation had evoked changes in membrane properties. 5. In conclusion, the pore-forming preparation poly-APS can have dose-dependent interactions with cell membranes and at low doses these can be reversible. Additionally, the interactions between poly-APS and cell membranes could be attenuated by zinc.

Bioactivity of secoiridoid glycosides from Centaurium erythraea.

As part of our on-going search for bioactive compounds from Scottish plants, two secoiridoid glycosides, swertiamarin and sweroside, have been isolated from the aerial parts of Centaurium erythraea Rafn (Family: Gentianaceae) by reversed-phase preparative HPLC coupled with a photo-diode-array detector. The structures of these compounds were elucidated unambiguously by UV, FABMS and extensive 1D and 2D NMR spectroscopic analyses and also by comparing experimental data with literature data. Antibacterial, free radical scavenging activities and general toxicity of these glycosides have been assessed. Both compounds inhibited the growth of Bacillus cereus, Bacillus subtilis, Citrobacter freundii and Escherichia coli. While swertiamarin was also active against Proteus mirabilis and Serratia marcescens, sweroside inhibited the growth of Staphylococcus epidermidis. Swertiamarin and sweroside exhibited significant general toxicity in brine shrimp lethality bioassay and the LD50 values were 8.0 microg/ml and 34 microg/ml, respectively, whereas that of the positive control podophyllotoxin, a well known cytotoxic lignan, was 2.79 microg/ml. Chemotaxonomic implications of these compounds in the family Gentianaceae have also been discussed briefly.

Biological activity of Euonymus europaeus.

The n-hexane, dichloromethane and methanol extracts of the seeds of Euonymus europaeus have been screened for antibacterial and free radical scavenging activity. General toxicity (brine shrimp lethality assay) of these extracts has also been assessed.

Biological activity of Glechoma hederacea.

The n-hexane, dichloromethane and methanol extracts of the aerial parts of Glechoma hederacea have been screened for antibacterial and free radical scavenging activity. General toxicity (brine shrimp lethality assay) of these extracts has also been assessed.

Novel activities of saliva from the octopus Eledone cirrhosa (Mollusca; Cephalopoda).

Haemolytic activity against mammalian erythrocytes was detected in very low concentrations of saliva from the octopus Eledone cirrhosa. It was not caused by any previously identified component of this saliva. The activity was slightly inhibited by trypsin and more drastically by chymotrypsin. Locust bioassay was used to test HPLC fractions isolated from E. cirrhosa saliva. An LD(50) of 378.4 microg/g locust weight was calculated for whole saliva injected into the desert locust Schistocerca gregaria. This study was the first record of haemolytic activity and toxicity to insects in cephalopod saliva. Both haemolytic and insecticidal fractions could be separated using reversed phase HPLC.

Psammaplin A, a chitinase inhibitor isolated from the Fijian marine sponge Aplysinella rhax.

Several brominated tyrosine derived compounds, psammaplins A (1), K (2) and L (3) as well as bisaprasin (4) were isolated from the Fijian marine sponge Aplysinella rhax during a bioassay guided isolation protocol. Their structures were determined using NMR and MS techniques. Psammaplin A was found to moderately inhibit chitinase B from Serratia marcescens, the mode of inhibition being non-competitive. Crystallographic studies suggest that a disordered psammaplin A molecule is bound near the active site. Interestingly, psammaplin A was found to be a potent antifungal agent.

Indole alkaloids from the seeds of Centaurea cyanus (Asteraceae).

Preparative RP-HPLC analysis of a methanol extract of the seeds of Centaurea cyanus afforded four indole alkaloids: moschamine, cis-moschamine, centcyamine and cis-centcyamine, the latter two being new natural products. Structures of these compounds were elucidated by comprehensive spectroscopic analyses. General toxicity of the isolates was determined by Brine Shrimp Lethality bioassay.

Stelliferin riboside, a triterpene monosaccharide isolated from the Fijian sponge Geodia globostellifera.

A triterpene monosaccharide, stelliferin riboside (1), was isolated from the Fijian marine sponge Geodia globostellifera. In addition, stellettins A and B (2, 3) were also isolated. This is the first report of a stelliferin from this species and the first example of a saccharide derivative of a stelliferin.

Analysis of the structure and electrophysiological actions of halitoxins: 1,3 alkyl-pyridinium salts from Callyspongia ridleyi.

We have chemically characterized a preparation of halitoxins, (1,3 alkyl-pyridinium salts) isolated from the marine sponge Callyspongia ridleyi. At concentrations of 50 and 5 microg/ml the halitoxin preparation caused irreversible membrane potential depolarization, decreased input resistance and inhibited evoked action potentials when applied to cultured dorsal root ganglion neurones. Under whole cell voltage clamp the halitoxins produced an increase in cation conductance that was attenuated by replacing sodium with N-methyl-d-glucamine. Fura-2 fluorescence ratiometric calcium imaging was used to directly measure calcium flux into neurones after exposure to halitoxins. Calcium influx, evoked by the halitoxins, persisted when the neurones were bathed in medium containing the voltage-activated calcium channel antagonists cadmium and nickel. Experiments on undifferentiated F-11 cells showed little or no calcium influx in response to depolarizing concentrations of potassium and indicated that halitoxins evoked massive calcium influx in the absence of voltage-activated calcium channels. The halitoxins also produced transient increases in intracellular calcium when F-11 cells were bathed in calcium-free medium suggesting that the toxins could release calcium from intracellular stores. The pore-forming action of the halitoxins was identified when the toxins were applied to artificial lipid bilayers composed of phosphatidylcholine and cholesterol. Halitoxins evoked channel-like activity in the lipid bilayers, with estimated unitary conductances of between 145pS and 2280pS, possibly indicating that distinct channels could be produced by the different components in the preparation of halitoxins.

Glutathione-dependent conversion of N-ethylmaleimide to the maleamic acid by Escherichia coli: an intracellular detoxification process.

The electrophile N-ethylmaleimide (NEM) elicits rapid K(+) efflux from Escherichia coli cells consequent upon reaction with cytoplasmic glutathione to form an adduct, N-ethylsuccinimido-S-glutathione (ESG) that is a strong activator of the KefB and KefC glutathione-gated K(+) efflux systems. The fate of the ESG has not previously been investigated. In this report we demonstrate that NEM and N-phenylmaleimide (NPM) are rapidly detoxified by E. coli. The detoxification occurs through the formation of the glutathione adduct of NEM or NPM, followed by the hydrolysis of the imide bond after which N-substituted maleamic acids are released. N-ethylmaleamic acid is not toxic to E. coli cells even at high concentrations. The glutathione adducts are not released from cells, and this allows glutathione to be recycled in the cytoplasm. The detoxification is independent of new protein synthesis and NAD(+)-dependent dehydrogenase activity and entirely dependent upon glutathione. The time course of the detoxification of low concentrations of NEM parallels the transient activation of the KefB and KefC glutathione-gated K(+) efflux systems.

A bioactive secosterol with an unusual A- and B-ring oxygenation pattern isolated from an Indonesian soft coral Lobophytum sp.

A secosterol with a gorgosterol side chain and an unusual oxygenation pattern on the A and B rings was isolated from an Indonesian soft coral Lobophytum sp. The A and B rings of the reported compound 1 have hydroxyl groups at C-3 and C-7 and an epoxide ring at C-5-C-6. The structures of 1 was solved by 2D NMR methods and by chemical shift analogy to the known secogorgosterol 2. Compound 1 was found to have activity against human ovarian tumor and human leukemia cell lines.

Using scalarane sesterterpenes to examine a sponge taxonomic anomaly.

A parallel study was conducted on two Indo-Pacific foliose sponges. The first specimen contains 3-hydroxy-20,22-dimethyl-20-deoxoscalarin (2), while the second contains 3-oxo-20,22-dimethyl-20-dioxoscalarin 8 (3). The physical properties as well as X-ray results confirming the structure and stereochemical features of these compounds are presented first. The difficulty we encountered in the taxonomic identification of these species is also discussed. One of our specimens is identical to material considered by different taxonomists as either Phyllospongia vermicularis or Dysidea vermicularis. The other is identified as Carteriospongia sp. We outline that the parallel chemistry of these two specimens suggests that they are closely related taxonomically.

The cyclorenierins, sesquiterpenoid quinols from the sponge Haliclona sp. collected in Vanuatu.

Two sesquiterpene quinols, cyclorenierins A [1] and B [2], which are closely related to panicein A2 [3] and renierin A [4], were isolated as an inseparable mixture from the marine sponge Haliclona sp. collected in Vanuatu. Structures were proposed using nmr shift analogies to 3 and 4 and confirmed with 2D nmr data.