NMR uncovers direct interaction between human NEDD4-1 and p34SEI-1.

PTEN, an important tumor suppressor and a key regulator of the PI3K/AKT signaling pathway, is often deleted/mutated in different types of cancer. The E3 ubiquitin ligase NEDD4-1 catalyzes the polyubiquitination of PTEN, thereby acting as a negative regulator of PTEN. Stability of NEDD4-1, in turn, is tightly controlled by a 34 kDa oncoprotein, p34SEI-1 and it regulates PTEN degradation and activates PI3K/AKT pathway, resulting in cancer metastasis. p34SEI-1 affects not only the expression of NEDD4-1 during transcription and translation but also the subcellular localization of PTEN. This emphasizes the need to understand, at molecular level, the interaction between NEDD4-1 and p34SEI-1. A recent study showed that NEDD4-1 interacts with p34SEI-1 via its WWI domain. However, a detailed interaction for molecular level is yet unknown. We report that the WW1 domain of NEDD4-1 recognizes the SERTA domain containing the proline rich region (PRR motif) in p34SEI-1. TALOS analysis based on NMR data confirms three conserved ß-sheets in NEDD4-1 WW1 and the central ß-sheet of NEDD4-1 WW1 plays a role for protein stability by the backbone dynamics experiments. NMR titration data revealed the binding site for p34SEI-1 with NEDD4-1. Our data will provide insights into the molecular mechanism of NEDD4-1 and p34SEI-1 interaction, which will be directly used for drug design which inhibits the molecular interaction involved in different cancer signaling.

In planta biocatalysis screen of P450s identifies 8-methoxypsoralen as a substrate for the CYP82C subfamily, yielding original chemical structures.

An in vivo plant screen that allows for the analysis of exogenously applied substrates against transgenic Arabidopsis lines overexpressing individual cytochrome P450s has been developed. By deploying this screen with a subset of 91 P450s, we have identified an original substrate for members of the CYP82C subfamily. The therapeutic compound 8-methoxypsoralen was hydroxylated by plants overexpressing CYP82C2 or CYP82C4, forming 5-hydroxy-8-methoxypsoralen. Additionally, plants further modified this product to create a glycosylated compound, likely the compound 5-O-beta-D-glucopyranosyl-8-methoxypsoralen. The discovery of adducts of therapeutic compounds demonstrates the potential of this biocatalysis screening approach to create compounds that may be of pharmacological value. Additionally, this platform provides a means to expand the general knowledge base of P450 enzyme/substrate combinations and may provide valuable tools for a vast array of biocatalytic and bioremediation processes.

Enhancement of alkaloid production in opium and California poppy by transactivation using heterologous regulatory factors.

Genes encoding regulatory factors isolated from Arabidopsis, soybean and corn have been screened to identify those that modulate the expression of genes encoding for enzymes involved in the biosynthesis of morphinan alkaloids in opium poppy (Papaver somniferum) and benzophenanthridine alkaloids in California poppy (Eschscholzia californica). In opium poppy, the over-expression of selected regulatory factors increased the levels of PsCOR (codeinone reductase), Ps4'OMT (S-adenosyl-l-methionine:3'-hydroxy-N-methylcoclaurine 4'-O-methyltransferase) and Ps6OMT [(R,S)-norcoclaurine 6-O-methyltransferase] transcripts by 10- to more than 100-fold. These transcriptional activations translated into an enhancement of alkaloid production in opium poppy of up to at least 10-fold. In California poppy, the transactivation effect of regulatory factor WRKY1 resulted in an increase of up to 60-fold in the level of EcCYP80B1 [(S)-N-methylcoclaurine 3'-hydroxylase] and EcBBE (berberine bridge enzyme) transcripts. As a result, the accumulations of selected alkaloid intermediates were enhanced up to 30-fold. The transactivation effects of other regulatory factors led to the accumulation of the same intermediates. These regulatory factors also led to the production of new alkaloids in California poppy callus culture.

Expression of human nuclear receptors in plants for the discovery of plant-derived ligands.

Plants have the potential to produce a wide array of secondary metabolites that have utility as drugs to treat human diseases. To tap this potential, functional human nuclear receptors have been expressed in plants to create in planta screening assays as a tool to discover natural product ligands. Assays have been designed and validated using 3 nuclear receptors: the estrogen receptor (ER), the androgen receptor (AR), and the heterodimeric retinoid X receptor-alpha plus thyroid hormone receptor-beta (RXRA/THRB). Nuclear receptor-reporter constructs have been expressed in plants to detect the presence of natural ligands that are produced de novo in several plant species during different stages of development, in various tissues, and in response to different stress elicitors. Screening experiments with ER, AR, and RXRA/THRB have been conducted, leading to the identification of plant sources of natural product ligands of human nuclear receptors. This in planta screen has led to the identification of previously unreported ER ligands, providing evidence of the complementary value of this approach to current in vitro high-throughput screening assays.

Misassigned natural products and their revised structures.

Natural products are a major pipeline for drug development and are responsible for more than 50 % of drugs on the market. NMR is a fundamental and powerful tool for the structure determination of natural products. It is essential to provide unambiguous chemical structure information on natural products in drug development research, including the structure-activity relationship, derivatization and pharmacokinetic/pharmacodynamic studies. Advancement of NMR instruments has made it possible to deal with nanomole-scale natural products for structure elucidation, but misinterpretation of NMR spectra still occurs. We review 21 natural products with revised chemical structures and the methods used for those revisions.

Cyclolignans from Scyphocephalium ochocoa via high-throughput natural product chemistry methods.

Two 2,7'-cyclolignans, ocholignans A and B, were obtained as mass-limited samples from Scyphocephalium ochocoa via high-throughput natural products chemistry methods. The rapid structure elucidation of each compound was primarily facilitated by NMR data acquisition using a capillary-scale NMR probe, CapNMR probe. Ocholignan A was found to possess significant in vitro antibacterial activity against Gram-positive bacteria methicillin-resistant Staphylococcus aureus ATCC 33591 and S. aureus 78-13607A with a MIC of 16 microg/mL, respectively.

CD-200 induces apoptosis and inhibits Bcr-Abl signaling in imatinib-resistant chronic myeloid leukemia with T315I mutation.

Chronic myeloid leukemia (CML) is characterized by a constitutively active Bcr-Abl tyrosine kinase. Although Imatinib has been proven to be an effective drug against CML, its resistance has been observed with disease relapse due to T315I predominant point mutation. Liriodendron tulipifera L., one of the fastest growing hardwood tree species, exerts antioxidant activity and anti-inflammatory effects. However, its anticancer effect has been minimally reported. In this study, we extracted CD-200 from Liriodendron tulipifera L. and investigated its effect on cell survival or apoptosis in CML cells with Bcr-Abl/T315I (BaF3/T315I) as well as wild-type Bcr-Abl (BaF3/WT). CD-200 inhibited cell proliferation in the BaF3/WT cells, and also in the BaF3/T315I cells with Imatinib resistance. Moreover, it strongly inhibited Bcr-Abl signaling pathways in a dose-dependent manner. Also, it significantly increased the sub-G1 phase and the expression of cleaved PARP and caspase-3, as well as the TUNEL-positive apoptotic cells. In addition, we observed that CD-200 induced apoptosis with a loss of mitochondrial membrane potential by decreasing the expression of Mcl-1 and survivin. Furthermore, CD-200 showed a significant inhibition in tumor growth, compared to Imatinib in BaF3/T315I mouse xenograft models. Taken together, our study demonstrates that CD-200 exhibits apoptosis induction and anti-proliferative effect by blocking the Bcr-Abl signaling pathways in the Bcr-Abl/T315I with resistance to Imatinib. We suggest that CD-200 may be a natural product to target Bcr-Abl and overcome Imatinib resistance in CML patients.

Development of a solid-phase binding assay and identification of nonpeptide ligands for the FynB Src homology 2 domain.

The nonreceptor tyrosine kinase FynB is known to be required in the induction of long-term potentiation (LTP), a cellular mechanism for learning and memory. Ligands of the FynB SH2 domain as a possible FynB activator are, thus, of great interest. In this study, a solid-phase ligand binding assay was established to meet the screening requirement of high-throughput and ease of use, and in an attempt to find the specific ligands for the FynB SH2 domain. This assay measures the competitive inhibition of the binding of the biotinylated phosphopeptide (GGSETDDY*AEIID), derived from a binding sequence in human focal adhesion kinase, to the SH2 domain of FynB precoated as a glutathione S-transferase fusion protein on a solid-phase. Using this high-throughput screening method for SH2 ligands, a modest size of chemical library was screened, and two non-peptide compounds, 4-acetamidobenzene sulfinic acid and 1-allylpyridinium 3-sulfonate, were identified by their strong binding affinity to the FynB SH2 domain. This result demonstrates the feasibility of the developed assay in high-throughput screening. Further studies on the molecular structures of the identified SH2-binding ligands will allow presentation of specific models for ligand-domain complexes for improving the ligands and will help to develop a potential lead compound for improving LTP.

Plant cell wall polysaccharides as potential resources for the development of novel prebiotics.

Prebiotic oligosaccharides, with a degree of polymerization (DP) of mostly less than 10, exhibit diverse biological activities that contribute to human health. Currently available prebiotics are mostly derived from disaccharides and simple polysaccharides found in plants. Subtle differences in the structures of oligosaccharides can cause significant differences in their prebiotic proper-ties. Therefore, alternative substances supplying polysaccharides that have more diverse and complex structures are necessary for the development of novel oligosaccharides that have actions not present in existing prebiotics. In this review, we show that structural polysaccharides found in plant cell walls, such as xylans and pectins, are particularly potential resources supplying broadly diverse polysaccharides to produce new prebiotics.

Anti-HCV bioactivity of pseudoguaianolides from Parthenium hispitum.

Five new (1-5) and four known (6-9) C14-oxygenated 1alpha-hydroxy-11(13)-pseudoguaien-6beta,12-olides with potent inhibition of hepatitis C virus (HCV) replication were obtained from Parthenium hispitum via high-throughput natural product chemistry methods. A semipreparative HPLC system was used to purify these compounds. The miniaturization of the structure elucidation and dereplication for the mass-limited samples were performed primarily utilizing a capillary-scale NMR probe. Compounds 2-4 were found to possess in vitro anti-HCV activity in the subgenomic HCV replicon system containing luciferase reporter with significant inhibition above 90% at 2 microM concentration.

Bacterial biofilm inhibitors from Diospyros dendo.

One new (1) and four known (2-5) ursene triterpenes with potent inhibition of the formation of the bacterial biofilm Pseudomonas aeruginosa PA01 were obtained from Diospyros dendo using a high-throughput natural products chemistry procedure. These compounds were isolated as mass-limited samples. The miniaturization of the structure elucidation and dereplication was performed primarily utilizing a capillary-scale NMR probe.

Application of capillary-scale NMR for the structure determination of phytochemicals.

Employing a capillary-scale NMR probe enables the miniaturisation of structure determination and de-replication of purified natural products from plants using only 5-100 microg of material. Approximately 5 microg are required to perform one-dimensional proton and two-dimensional homonuclear (COSY and NOESY) NMR experiments; some 30 microg are needed to acquire HMQC- or HSQC-NMR spectra; ca. 75-100 microg are necessary to measure HMBC-NMR spectra; and around 200 microg of a compound are needed to perform 13C- and DEPT-NMR experiments. In order to illustrate the integration of the outputs from high-throughput natural product chemistry methods with the capabilities of the state-of-the-art CapNMR technology, the preparation of a natural product library from the extract of Penstemon centranthifolius, and the subsequent isolation, purification and structure determination of six known iridoid glycosides with 25-300 microg of material are presented.

Suaveolindole, a new mass-limited antibacterial indolosesquiterpene from Greenwayodendron suaveolens obtained via high-throughput natural products chemistry methods.

Utilizing high-throughput isolation, purification, and analysis methods applied to a natural products library, a new mass-limited antibacterial indolosesquiterpene, suaveolindole (1), was obtained from Greenwayodendron suaveolens. The miniaturization of the structure elucidation of 1 was performed primarily using the CapNMR probe. Compound 1 was found to possess significant in vitro antibacterial activity against the Gram-positive bacteria Bacillus subtilis (ATCC 43223), Staphylococcus aureus (ATTC 6538P), and methicillin-resistant Staphylococcus aureus (ATTC 33591), with MIC values of 4, 8, and 8 microg/mL, respectively.

Miniaturization of the structure elucidation of novel natural products--two trace antibacterial acylated caprylic alcohol glycosides from Arctostaphylos pumila.

High-throughput isolation, purification and analysis methods applied to natural products libraries from plants gave rise to the discovery of two novel acylated caprylic alcohol glycosides (1, 2) produced by Arctostaphylos pumila. The NMR spectra were acquired using the CapNMR probe and performed on mass-limited samples, which enabled us to elucidate the structures of 2,6-diacetyl-3,4-diisobutyl-1- O-octylglucopyranoside (1, 200 microg) and 2,6-diacetyl-3,4-dimethylbutyl-1- O-octylglucopyranosid (2, 70 microg). Compounds 1 and 2 exhibited antibacterial activity against Gram-positive methicillin-resistant Staphylococcus aureus with an MIC of 128 microg/mL and 64 microg/mL, respectively.

Vidalenolone, a novel phenolic metabolite from the tropical red alga Vidalia sp.

The Indonesian red alga Vidalia sp. was identified as a candidate for fractionation because its crude lipid extract showed activity in a mechanism-based anticancer assay (Fyn SH2-inhibitory activity). A chemically novel phenolic metabolite, vidalenolone, as well as two previously described and structurally simple phenols, were isolated as SH2-inactive substances. Their structures were determined by an interplay of spectroscopic methods, principally 2D NMR, and reference to literature data.

A phthalide with in vitro growth inhibitory activity from an oidiodendron strain.

A screening campaign was implemented utilizing capillary electrophoresis as a primary assay to discover binders to the cancer target Akt1 from a crude natural extract library. Fungal extracts with binding activities were characterized for biochemical inhibition of Akt1 to phosphorylate the downstream substrate protein Bad. One of the crude extracts with bioactivity selected for isolation and structure elucidation from fermentation of the fungal culture Oidiodendron sp. F01895 yielded a new trihydroxy phthalide (1). The structure of 1 was determined by a combination of 1D and 2D NMR spectroscopic data along with high-resolution mass spectrometric data. Compound 1 displays inhibition of Akt1 biochemical activity in vitro and confers growth inhibition on some cancer-derived cell lines in culture.

Identification of an endogenous ligand that activates pregnane X receptor-mediated sterol clearance.

The nuclear receptor PXR (pregnane X receptor) is a broad-specificity sensor that recognizes a wide variety of synthetic drugs and xenobiotic agents. On activation by these compounds, PXR coordinately induces a network of transporters, cytochrome P450 enzymes, and other genes that effectively clear xenobiotics from the liver and intestine. Like PXR, the majority of its target genes also possess a broad specificity for exogenous compounds. Thus, PXR is both a sensor and effector in a well integrated and generalized pathway for chemical immunity. Although it is clear that PXR responds to numerous foreign compounds, it is unclear whether it possesses an endogenous ligand. To address this issue, we noted that there is substantial overlap in the substrate specificities of PXR and its critical CYP3A target gene. This prompted us to ask whether endogenous CYP3A substrates also serve as PXR ligands. We demonstrate that 5beta-cholestane-3alpha,7alpha,12alpha-triol (triol), a cholesterol-derived CYP3A substrate, is a potent PXR agonist that effectively induces cyp3a expression in mice. This defines a critical salvage pathway that can be autoinduced to minimize triol accumulation. In contrast, triol can accumulate to very high levels in humans, and unlike mice, these people develop the severe clinical manifestations of cerebrotendinous xanthomatosis. The reason for these dramatic species differences has remained unclear. We now demonstrate that triol fails to activate human PXR or induce the CYP3A-salvage pathway. This explains why humans are more susceptible to sterol accumulation and suggests that synthetic ligands for human PXR could be used to treat cerebrotendinous xanthomatosis and other disorders of cholesterol excess.