The coupled reaction catalyzed by EchB and EchC lead to the formation of the common 2',3',5'-trihydroxy-benzene core in echosides biosynthesis.

p-Terphenyls represent a unique family of aromatic natural products generated by nonribosomal peptide synthetase-like (NRPS-like) enzyme. After formation of p-terphenyl skeleton, tailoring modifications will give rise to structural diversity and various biological activities. Here we demonstrated a two-enzyme (EchB, a short-chain dehydrogenase/reductase (SDR), and EchC, a nuclear transport factor 2 (NTF2)-like dehydratase) participated transformation from dihydroxybenzoquinone core to 2',3',5'-trihydroxy-benzene in the biosynthesis of echosides. Beginning with polyporic acid as substrate, successive steps of reduction-dehydration-reduction cascade catalyzed by EchB-EchC-EchB were concluded after in vivo gene disruption and in vitro bioassay experiments. These findings demonstrated a conserved synthesis pathway of 2',3',5'-trihydroxy-p-terphenyls in bacteria, such as Actinomycetes and Burkholderia. The parallel pathway in fungi has yet to be explored.

Terphenyl derivatives and terpenoids from a wheat-born mold Aspergillus candidus.

A new p-terphenyl derivative aspergicandidusin A (1), a new cleistanthane diterpenoid 6-deoxyaspergiloid C (13), and 12 known compounds (2-12, and 14) were isolated from the mold Aspergillus candidus. The structures of the new compounds were elucidated by spectral analysis of NMR and MS data. The absolute configuration of C-1 in 13 was determined via the circular dichroism data of the [Rh2(OCOCF3)4] complex. Compounds 2-8 and 11 showed moderate inhibitory activity against K562 cell lines with the IC50 value in the range from 17.9 to 46.3 µM. Compound 13 exhibited moderate cytotoxicity against HepG2 cells with the IC50 value of 47.7 µM. Compounds 11 and 12 exhibited moderate activity against the growth of S. aureus with MIC value of 6.25 µM, respectively.

Vialinin A and thelephantin G, potent inhibitors of tumor necrosis factor-α production, inhibit sentrin/SUMO-specific protease 1 enzymatic activity.

Several p-terphenyl compounds have been isolated from the edible Chinese mushroom Thelephora vialis. Vialinin A, a p-terphenyl compound, strongly inhibits tumor necrosis factor-α production and release. Vialinin A inhibits the enzymatic activity of ubiquitin-specific peptidase 5, one of the target molecules in RBL-2H3 cells. Here we examined the inhibitory effect of p-terphenyl compounds, including vialinin A, against sentrin/SUMO-specific protease 1 (SENP1) enzymatic activity. The half maximal inhibitory concentration values of vialinin A and thelephantin G against full-length SENP1 were 1.64±0.23µM and 2.48±0.02µM, respectively. These findings suggest that p-terphenyl compounds are potent SENP1 inhibitors.

Site-specific incorporation of a fluorescent terphenyl unnatural amino acid.

The site-specific incorporation of unnatural amino acids into proteins has a wide range of biological implications. Of particular interest is the incorporation of fluorescent probes as a mechanism to track protein function, transport, and folding. Thus, the development of a novel system for the incorporation of new fluorescent unnatural amino acids has significant utility. Specifically, we have elucidated an aminoacyl-tRNA synthetase capable of recognizing a terphenyl UAA derivative, and charging a cognate tRNA with this amino acid for protein incorporation. Moreover, we have successfully incorporated this fluorescent UAA into GFP at several key residues, demonstrating a novel means to modulate fluorescence within the protein.

New metabolites from endolichenic fungus Pleosporales sp.

Eight new metabolites were obtained from the culture of an endolichenic fungus, Pleosporales sp. Their structures were determined as three terphenyl derivatives, cucurbitarins A-C (1-3, resp.), two structurally related compounds, cucurbitarins D and E (4 and 5, resp.), two benzocoumarins, 3,10-dihydroxy-4,8-dimethoxy-6-methylbenzocoumarin (6) and 3,8,10-trihydroxy-4-methoxy-6-methylbenzocoumarin (7), as well as one cyclohexenone, (5R)-5-hydroxy-2,3-dimethylcyclohex-2-en-1-one (8), based on the spectroscopic data.

Identification and catalytic characterization of a nonribosomal peptide synthetase-like (NRPS-like) enzyme involved in the biosynthesis of echosides from Streptomyces sp. LZ35.

Echosides, isolated from Streptomyces sp. LZ35, represent a class of para-terphenyl natural products that display DNA topoisomerase I and IIα inhibitory activities. By analyzing the genome draft of strain LZ35, the ech gene cluster was identified to be responsible for the biosynthesis of echosides, which was further confirmed by gene disruption and HPLC analysis. Meanwhile, the biosynthetic pathway for echosides was proposed. Furthermore, the echA-gene, encoding a tri-domain nonribosomal peptide synthetase (NRPS)-like enzyme, was identified as a polyporic acid synthetase and biochemically characterized in vitro. This is the first study to our knowledge on the biochemical characterization of an Actinobacteria quinone synthetase, which accepts phenylpyruvic acid as a native substrate. Therefore, our results may help investigate the function of other NRPS-like enzymes in Actinobacteria.

Structural elucidation and synthesis of vialinin C, a new inhibitor of TNF-α production.

A new inhibitor of TNF-α production (IC50=0.89 µM) named vialinin C (1) was isolated from dry fruiting bodies of an edible Chinese mushroom, Thelephora vialis. The structure of 1 was determined by high-resolution MS, NMR spectroscopic analysis, and confirmed by synthesis. Synthesis of ganbajunin B (5) obtained from the same origin was also described.

Ubiquitin-specific peptidase 5, a target molecule of vialinin A, is a key molecule of TNF-α production in RBL-2H3 cells.

Tumor necrosis factor alpha (TNF-α), a central mediator of the inflammatory response, is released from basophilic cells and other cells in response to a variety of proinflammatory stimuli. Vialinin A is a potent inhibitor of TNF-α production and is released from RBL-2H3 cells. Ubiquitin-specific peptidase 5 (USP5), a deubiquitinating enzyme, was identified as a target molecule of vialinin A and its enzymatic activity was inhibited by vialinin A. Here we report production of TNF-α is decreased in USP5 siRNA-knockdown RBL-2H3 cells, compared with control cells. The finding of the present study strongly suggests that USP5 is one of the essential molecules for the production of TNF-α in RBL-2H3.

Vialinin A is a ubiquitin-specific peptidase inhibitor.

Vialinin A, a small compound isolated from the Chinese mushroom Thelephora vialis, exhibits more effective anti-inflammatory activity than the widely used immunosuppressive drug tacrolimus (FK506). Here, we show that ubiquitin-specific peptidase 5/isopeptidase T (USP5/IsoT) is a target molecule of vialinin A, identified by using a beads-probe method. Vialinin A inhibited the peptidase activity of USP5/IsoT and also inhibited the enzymatic activities of USP4 among deubiquitinating enzymes tested. Although USPs are a member of thiol protease family, vialinin A exhibited no inhibitions for other thiol proteases, such as calpain and cathepsin.

p-Terphenyls from fungus Paxillus curtisii chelate irons: a proposed role of p-terphenyls in fungus.

Diverse p-terphenyl compounds, named curtisians, have been isolated from the fungus Paxillus curtisii, and degradation of wood by this fungus is thought to be progressed by iron chelation of p-terphenyl curtisians. In this study, the iron chelation ability of p-terphenyls has been proved by chrome azurol S (CAS) assay, reducing power, and UV-visible spectroscopic analyses. The catechol moiety of p-terphenyl is an essential factor for the potent iron chelation ability, and thus deacylated curtisian with a tetrahydroxyl moiety in the central ring of p-terphenyl is more effective than acylated curtisians.

Synthesis of tetrahydroxybiphenyls and tetrahydroxyterphenyls and their evaluation as amyloid-ß aggregation inhibitors.

3,3',4,4'-Tetrahydroxybiphenyl and three isomeric 3,3″,4,4″-tetrahydroxyterphenyls with varying geometries around the central phenyl ring have been synthesized and evaluated for their in vitro activity against aggregation of Alzheimer's amyloid-ß peptide (Aß). Results from Congo red spectral-shift assays reveal that all four compounds successfully inhibit association of Aß monomers. For the tetrahydroxyterphenyls, efficacy varies with linker geometry: the ortho-arrangement affords the most successful inhibition and the para-geometry the least, perhaps due to differing abilities of these compounds to bind Aß. Of the four small molecules studied, 3,3',4,4'-tetrahydroxybiphenyl is the most effective inhibitor, reducing Aß aggregation by 50% when present in stoichiometric concentrations.

Determination of different putative allosteric binding pockets at the lutropin receptor by using diverse drug-like low molecular weight ligands.

The lutropin/choriogonadotrophin receptor (LHCGR) is a family A G protein-coupled receptor (GPCR) which binds the endogenous hormone-ligands at the large extracellular domain. In contrast, several drug-like low-molecular-weight ligands (LMWs) have been reported to interact allosterically within the seven transmembrane domain (7TMD) of the LHCGR. Here, we were interested to study the putative allosteric LHCGR binding region with focus on the determination of two pockets for LMW ligands. A library of compounds was screened for their ability to modify the binding of an allosteric radiolabeled LMW agonist [³H]Org 43553. Further experimental and computational studies revealed that the putative binding pocket for a newly identified allosteric enhancer (LUF5419) and a previously described allosteric inhibitor (LUF5771) are overlapping and that this site is different from the Org 43553 binding site. The present study showed that these compounds are useful tools to reveal details on different allosteric binding sites located within the 7TMD of the LHCGR.

Binding affinity of triphenyl acrylonitriles to estrogen receptors: quantitative structure-activity relationships.

AIM: The quantitative structure-activity relationship approach was applied to understand the relative binding affinity of triphenyl acrylonitriles to estrogen receptors. MATERIAL AND METHODS: A sample of previously studied triphenyl acrylonitriles was divided into training (18 compounds) and test sets (7 compounds) using a stratified random approach. The molecular descriptor family on vertices cutting (MDFV) approach was used in order to translate the structural information into descriptors. The relationship between binding activity and structural descriptors was identified using the multiple linear regression procedure. RESULTS: An optimal three-parameter equation with a determination coefficient of 0.9580 and a cross-validation leave-one-out parameter of 0.9408 was identified. The optimal model was assessed on a test set and a determination coefficient of 0.9004 was obtained. The MDFV model proved not to be significantly different from the previously reported model in terms of goodness-of-fit. In terms of information criteria (Akaike's, Bayesian, Amemiya, and Hannan-Quinn) and Kubinyi function, the MDFV model proved to perform better than the previously reported model. CONCLUSION: The optimal MDFV model was able to explain approximately 96% of the total variance in the estrogenic binding relative affinity of triphenyl acrylonitriles and to have estimation and prediction abilities. Although there were no significant differences in terms of goodness-of-fit, the MDFV model proved to exhibit better information parameters compared to the previously reported model using the same number of molecular descriptors.

Rational quantitative structure-activity relationship (RQSAR) screen for PXR and CAR isoform-specific nuclear receptor ligands.

Constitutive androstane receptor (CAR) and pregnane X receptor (PXR) are closely related orphan nuclear receptor proteins that share several ligands and target overlapping sets of genes involved in homeostasis and all phases of drug metabolism. CAR and PXR are involved in the development of certain diseases, including diabetes, metabolic syndrome and obesity. Ligand screens for these receptors so far have typically focused on steroid hormone analogs with pharmacophore-based approaches, only to find relatively few new hits. Multiple CAR isoforms have been detected in human liver, with the most abundant being the constitutively active reference, CAR1, and the ligand-dependent isoform CAR3. It has been assumed that any compound that binds CAR1 should also activate CAR3, and so CAR3 can be used as a ligand-activated surrogate for CAR1 studies. The possibility of CAR3-specific ligands has not, so far, been addressed. To investigate the differences between CAR1, CAR3 and PXR, and to look for more CAR ligands that may be of use in quantitative structure-activity relationship (QSAR) studies, we performed a luciferase transactivation assay screen of 60 mostly non-steroid compounds. Known active compounds with different core chemistries were chosen as starting points and structural variants were rationally selected for screening. Distinct differences in agonist versus inverse agonist/antagonist effects were seen in 49 compounds that had some ligand effect on at least one receptor and 18 that had effects on all three receptors; eight were CAR1 ligands only, three were CAR3 only ligands and four affected PXR only. This work provides evidence for new CAR ligands, some of which have CAR3-specific effects, and provides observational data on CAR and PXR ligands with which to inform in silico strategies. Compounds that demonstrated unique activity on any one receptor are potentially valuable diagnostic tools for the investigation of in vivo molecular targets.

Accumulation of lipophilic dications by mitochondria and cells.

Lipophilic monocations can pass through phospholipid bilayers and accumulate in negatively-charged compartments such as the mitochondrial matrix, driven by the membrane potential. This property is used to visualize mitochondria, to deliver therapeutic molecules to mitochondria and to measure the membrane potential. In theory, lipophilic dications have a number of advantages over monocations for these tasks, as the double charge should lead to a far greater and more selective uptake by mitochondria, increasing their therapeutic potential. However, the double charge might also limit the movement of lipophilic dications through phospholipid bilayers and little is known about their interaction with mitochondria. To see whether lipophilic dications could be taken up by mitochondria and cells, we made a series of bistriphenylphosphonium cations comprising two triphenylphosphonium moieties linked by a 2-, 4-, 5-, 6- or 10-carbon methylene bridge. The 5-, 6- and 10-carbon dications were taken up by energized mitochondria, whereas the 2- and 4-carbon dications were not. The accumulation of the dication was greater than that of the monocation methyltriphenylphosphonium. However, the uptake of dications was only described by the Nernst equation at low levels of accumulation, and beyond a threshold membrane potential of 90-100 mV there was negligible increase in dication uptake. Interestingly, the 5- and 6-carbon dications were not accumulated by cells, due to lack of permeation through the plasma membrane. These findings indicate that conjugating compounds to dications offers only a minor increase over monocations in delivery to mitochondria. Instead, this suggests that it may be possible to form dications within mitochondria that then remain within the cell.

Time-resolved fluorescence microscopy using an improved europium chelate BHHST for the in situ detection of Cryptosporidium and Giardia.

Fluorescent immunoconjugates prepared with the europium chelate BHHCT (4,4'-bis(1'',1'',1'',2'',2'',3'',3''-heptafluoro-4'',6''-hexanedion-6''-yl)-chlorosulfo-o-terphenyl) have previously been reported as suitable labels for time-resolved fluorescence applications. BHHCT is limited by a tendency to destabilize immunoglobulins when covalently bound to the protein at moderate to high fluorophore to protein ratios (F/P). We report a new derivative of BHHCT prepared by appending a short hydrophylic tether to the chlorosulfonate activating group on BHHCT. The new derivative, BHHST (4,4'-bis-(1'',1'',1'',2'',2'',3'',3''-heptafluoro-4'',6''-hexanedion-6''-yl)sulfonylamino-propyl-ester-N-succinimide-ester-o-terphenyl), was activated to bind at the tether terminus with a succinimide leaving group that displayed less aggressive coupling activity and improved storage stability. BHHST has been used to prepare a stable and useful immunoconjugate with the anti-Cryptosporidium monoclonal antibody CRY104. The BHHST immunoconjugate provides more than a 10-fold enhancement in the signal to noise ratio (SNR) of labeled oocyst fluorescence over background when observed using TRFM techniques. An immunoconjugate was also prepared with BHHST and (goat) anti-mouse that effectively labeled Giardia cysts in situ. Detection of cysts with the TRFM was achieved with an 11-fold increase in SNR when a gate-delay of 60 micros was employed. The storage half-life of both immunoconjugates is extended more than 20-fold when compared to immunoconjugates prepared with BHHCT.

Enzymatic synthesis of oligosaccharides, alkyl and terpenyl glucosides, by recombinant Escherichia coli-expressed Pichia etchellsii beta-glucosidase II.

The biosynthetic activity of yeast Pichia etchellsii beta-glucosidase II (BglII) expressed in recombinant Escherichia coli was utilized for synthesis of cellooligosaccharides, alkyl and terpene glucosides. Cellooligosaccharides with a degree of polymerization of 3 and greater were resolved by thin-layer chromatography (TLC) using an ethyl acetate:1-propanol:2-propanol:water (8:5:1:1) solvent system followed by visualization with 0.2% naphthoresorcinol reagent. Using 2M cellobiose and 15 IU of partially purified BglII, 57 mmol/L of oligosaccharides (comprising mostly cellotriose and cellopentaose) was synthesized in 16 h. Similarly, alkyl glucosides with chain lengths from 6 to 10 carbons were synthesized and products extracted to near purity by ethyl acetate extraction. The same extraction method was employed to separate, to near purity, various monoterpenyl (nerol, geraniol, citronellol) glucosides. A reliable and simple method for separation of cellooligosaccharides using a combination of Bio-Gel P-2 gel filtration and charcoal celite adsorption chromatography was developed. The cellooligosaccharides were separated to purity as confirmed by TLC. The enzyme was among the very few that could synthesize a wide variety of glycoconjugates.

Isolation, structure, and HIV-1-integrase inhibitory activity of structurally diverse fungal metabolites.

HIV-1 integrase is a critical enzyme for replication of HIV, and its inhibition is one of the most promising new drug strategies for anti-retroviral therapy, with potentially significant advantages over existing therapies. In this report, a series of HIV-1 inhibitors isolated from the organic extract of fermentations from terrestrial fungi is described. These fungal species, belonging to a variety of genera, were collected from throughout the world following the strict guidelines of Rio Convention on Biodiversity. The polyketide- and terpenoid-derived inhibitors are represented by two naphthoquinones, a biphenyl and two triphenyls, a benzophenone, four aromatics with or without catechol units, a linear aliphatic terpenoid, a diterpenoid, and a sesterterpenoid. These compounds inhibited the coupled and strand-transfer reaction of HIV-1 integrase with an IC(50) value of 0.5-120 micro M. The bioassay-directed isolation, structure elucidation, and HIV-1 inhibitory activity of these compounds are described.