Self-association of okadaic acid: structural and pharmacological significance.

Okadaic acid (OA) has been an invaluable pharmacological tool in the study of cellular signaling. The great affinity of this polyether for its targets together with its high specificity to inhibit certain protein phosphatases enables the differential study of these proteins. Crystallographic structures of protein phosphatases in complex with OA show a 1:1 protein to toxin ratio. Nevertheless, it has been found that OA is able to self-associate under certain conditions although very little is known about the importance of this phenomenon. Here we review the available knowledge on the latter topic and we report on the existence of an unusual self-associated tetrameric form. The structure of these oligomers is proposed based on spectroscopic data and molecular modeling calculations.

Identification of 19-epi-okadaic acid, a new diarrhetic shellfish poisoning toxin, by liquid chromatography with mass spectrometry detection.

Okadaic acid (1) (OA) and its congeners are mainly responsible for diarrhetic shellfish poisoning (DSP) syndrome. The presence of several OA derivatives have already been confirmed in Prorocentrum and Dinophysis spp. In this paper, we report on the detection and identification of a new DSP toxin, the OA isomer 19-epi-okadaic acid (2) (19-epi-OA), isolated from cultures of Prorocentrum belizeanum, by determining its retention time (RT) and fragmentation pattern using liquid chromatography coupled with mass spectrometry (LC-MS/MS).

On the Mechanism of Action of Dragmacidins I and J, Two New Representatives of a New Class of Protein Phosphatase 1 and 2A Inhibitors.

Two new brominated bis(indole) alkaloids, dragmacidins I (1) and J (2), showing low micromolar cytostatic activity, along with three known congeners were isolated from the Tanzanian sponge Dragmacidon sp. and their structures determined by the analysis of their NMR and MS data. From the study of their mechanism of action, it can be concluded that the mitotic arrest at metaphase in treated tumor cells, mediated by inhibition of PP1 and/or PP2A phosphatases is involved in the observed antiproliferative activity. Differences in their bioactivities were rationalized, and a plausible binding mode is proposed on the basis of computational simulations.

19-epi-okadaic acid, a novel protein phosphatase inhibitor with enhanced selectivity.

A new protein phosphatase inhibitor, 19-epi-okadaic acid, was isolated from the marine dinoflagellate Prorocentrum belizeanum. Its structure and conformation in solution has been determined, and important differences were found when compared with the lead compound okadaic acid. The new metabolite showed nanomolar activities, and its selectivity for PP2A versus PP1 surpasses that shown by okadaic acid 10-fold, making it one of the most selective inhibitors of this class.

Self-assembly of okadaic acid as a pathway to the cell.

The polyether toxin okadaic acid (OA) inhibits several protein serine/threonine phosphatases that play central roles in the regulation of many essential cellular processes. The use of scanning tunneling microscopy (STM) shows that dimerization of such toxins is crucial to understand the mechanism of toxin transport across model membranes.

Identification and characterization of DTX-5c and 7-hydroxymethyl-2-methylene-octa-4,7-dienyl okadaate from Prorocentrum belizeanum cultures by LC-MS.

The main toxins produced by the dinoflagellate Prorocentrum belizeanum, DTX5c and 7-hydroxymethyl-2methylene-octa-4,7 dienyl okadaate, were studied by liquid chromatography-coupled with ion trap mass spectrometry (LC-MS/MS). Their retention times (RTs) and fragmentation patterns were established, in particular those of DTX5c, as there is a lack of data about these water soluble OA sulphated derivatives. As an application of the LC-MS methodology, a sample of the toxic P. belizeanum culture was analysed for these metabolites. Both metabolites were detected in cells as majority compounds, whereas levels of these compounds in the culture media were undetectable.

Characterisation of okadaic acid related toxins by liquid chromatography coupled with mass spectrometry.

In the Diarrhetic Shellfish Poisoning (DSP) phenomena, the parent toxins, namely okadaic acid (OA) and/or dinophysistoxin-2 (DTX2), are predominantly found esterified. Therefore, a toxicity assessment of a sample can only be performed after an alkaline hydrolysis step in order to recover the parent molecules in their free form. The presence of several OA diol esters has already been confirmed in Prorocentrum lima and Prorocentrum belizeanum cultures. This paper reports on the analysis of OA diol esters using liquid chromatography coupled with mass spectrometry (LC-MS/MS), and establishes a method for their detection and identification based upon their retention times (RT) and the fragmentation patterns of their mass spectra.

Cytotoxic Anomoian B and Aplyzanzine B, New Bromotyrosine Alkaloids from Indonesian Sponges.

Two new bromotyrosine derivatives, anomoian B (1) and aplyzanzine B (2), were isolated, respectively, from the organic extracts of a Verongida sponge belonging to the Hexadella genus and from a two-sponge association (Jaspis sp. and Bubaris sp.), both collected off the coast of Indonesia. The planar structure of 1 and 2 was determined by 1D and 2D NMR experiments and by high-resolution mass spectrometry, while their absolute stereochemistry was assigned by comparison with optical rotation values of known bromotyrosines and by chemical degradation. Both compounds showed moderate antiproliferative activity against a panel of different cancer cell lines. Their cytotoxic activity is facilitated through the induction of apoptosis, which is mediated neither by the generation of reactive oxygen species nor by the inhibition of histone deacetylases in these cell lines.

DTX5c, a new OA sulphate ester derivative from cultures of Prorocentrum belizeanum.

Prorocentrum belizeanum is a dinoflagellate known for its okadaic acid (OA) and dinophysitoxins (DTXs) production, both OA and DTX are polyether toxins of the Diarrhetic Shellfish Poisoning (DSP) group. We have recently published the isolation of a new diol-ester of okadaic acid from cultures of P. belizeanum. On this occasion we present a new sulphated water-soluble derivative, DTX-5c, isolated from this microalga, whose structure was established on the basis of its spectroscopical data.

Novel Lobophorins Inhibit Oral Cancer Cell Growth and Induce Atf4- and Chop-Dependent Cell Death in Murine Fibroblasts.

As part of the International Cooperative Biodiversity Groups (ICBG) Program, we were interested in identifying biologically active unfolded protein response (UPR) inducing compounds from marine microorganisms isolated from Costa Rican biota. With this aim in mind we have now generated more than 33,000 unique prefractionated natural product extracts from marine and terrestrial organisms that have been submitted to the Center of Chemical Genomics (CCG) at the University of Michigan for high throughput screening (HTS). An effective complementary cell-based assay to identify novel modulators of UPR signaling was used for screening extracts. Active fractions were iteratively subjected to reverse-phase HPLC chromatographic analysis, and together with lobophorin A, B, E, and F (1-4), three new lobophorin congeners, designated as CR1 (5), CR2 (6), and CR3 (7) were isolated. Herein, we report that secondary assays revealed that the new lobophorins induced UPR-associated gene expression, inhibited oral squamous cell carcinoma cell growth, and led to UPR-dependent cell death in murine embryonic fibroblast (MEF) cells.

High-throughput screen of natural product extracts in a yeast model of polyglutamine proteotoxicity.

Proteins with expanded polyglutamine (polyQ) segments cause a number of fatal neurodegenerative disorders, including Huntington's disease (HD). Previous high-throughput screens in cellular and biochemical models of HD have revealed compounds that mitigate polyQ aggregation and proteotoxicity, providing insight into the mechanisms of disease and leads for potential therapeutics. However, the structural diversity of natural products has not yet been fully mobilized toward these goals. Here, we have screened a collection of ~11 000 natural product extracts for the ability to recover the slow growth of ΔProQ103-expressing yeast cells in 384-well plates (Z' ~ 0.7, CV ~ 8%). This screen identified actinomycin D as a strong inhibitor of polyQ aggregation and proteotoxicity at nanomolar concentrations (~50-500 ng/mL). We found that a low dose of actinomycin D increased the levels of the heat-shock proteins Hsp104, Hsp70 and Hsp26 and enhanced binding of Hsp70 to the polyQ in yeast. Actinomycin also suppressed aggregation of polyQ in mammalian cells, suggesting a conserved mechanism. These results establish natural products as a rich source of compounds with interesting mechanisms of action against polyQ disorders.

Comparative toxicological study of the novel protein phosphatase inhibitor 19-Epi-okadaic acid in primary cultures of rat cerebellar cells.

Okadaic acid (OKA) and analogues are frequent contaminants of coastal waters and seafood. Structure analysis of the isolated OKA analogue 19-epi-OKA showed important conformation differences expected to result in lower protein phosphatase (PP) inhibitory potencies than OKA. However, 19-epi-OKA and OKA inhibitory activities versus PP2A were unexpectedly found to be virtually equipotent. To investigate the toxicological relevance of these findings, we tested the effects of 19-epi-OKA on cultured cerebellar cells and compared them with those of OKA and its isomer dinophysistoxin-2. 19-epi-OKA caused degeneration of neurites and neuronal death with much lower potency than its congeners. The concentration of 19-epi-OKA that reduced after 24h the maximum neuronal survival (EC5024) by 50% was ~300nM compared with ~2nM and ~8nM for OKA and dinophysistoxin-2, respectively. Exposure to 19-epi-OKA resulted also in less toxicity for cultured glial cells (EC5024,19-epi-OKA ~ 600nM; EC5024,OKA ~ 20nM). 19-epi-OKA induced apoptotic condensation and fragmentation of chromatin, activation of caspases, and activation of ERK1/2 MAP kinases, features previously reported for OKA and dinophysistoxin-2. Also, differential sensitivity to 19-epi-OKA was observed between neuronal and glial cells, a specific characteristic shared by OKA and dinophysistoxin-2 but not by other toxins. Our results are consistent with 19-epi-OKA being included among the group of toxins of OKA and derivatives and support the suitability of cellular bioassays for the detection of these compounds.

Titration-based screening for evaluation of natural product extracts: identification of an aspulvinone family of luciferase inhibitors.

The chemical diversity of nature has tremendous potential for the discovery of molecular probes and medicinal agents. However, sensitivity of HTS assays to interfering components of crude extracts derived from plants, and macro- and microorganisms has curtailed their use in lead discovery. Here, we describe a process for leveraging the concentration-response curves obtained from quantitative HTS to improve the initial selection of "actives" from a library of partially fractionated natural product extracts derived from marine actinomycetes and fungi. By using pharmacological activity, the first-pass CRC paradigm improves the probability that labor-intensive subsequent steps of reculturing, extraction, and bioassay-guided isolation of active component(s) target the most promising strains and growth conditions. We illustrate how this process identified a family of fungal metabolites as potent inhibitors of firefly luciferase, subsequently resolved in molecular detail by X-ray crystallography.

Complementary cell-based high-throughput screens identify novel modulators of the unfolded protein response.

Despite advances toward understanding the prevention and treatment of many cancers, patients who suffer from oral squamous cell carcinoma (OSCC) confront a survival rate that has remained unimproved for more than 2 decades, indicating our ability to treat them pharmacologically has reached a plateau. In an ongoing effort to improve the clinical outlook for this disease, we previously reported that an essential component of the mechanism by which the proteasome inhibitor bortezomib (PS-341, Velcade) induced apoptosis in OSCC required the activation of a terminal unfolded protein response (UPR). Predicated on these studies, the authors hypothesized that high-throughput screening (HTS) of large diverse chemical libraries might identify more potent or selective small-molecule activators of the apoptotic arm of the UPR to control or kill OSCC. They have developed complementary cell-based assays using stably transfected CHO-K1 cell lines that individually assess the PERK/eIF2α/CHOP (apoptotic) or the IRE1/XBP1 (adaptive) UPR subpathways. An 66 K compound collection was screened at the University of Michigan Center for Chemical Genomics that included a unique library of prefractionated natural product extracts. The mycotoxin methoxycitrinin was isolated from a natural extract and found to selectively activate the CHOP-luciferase reporter at 80 µM. A series of citrinin derivatives was isolated from these extracts, including a unique congener that has not been previously described. In an effort to identify more potent compounds, the authors examined the ability of citrinin and the structurally related mycotoxins ochratoxin A and patulin to activate the UPR. Strikingly, it was found that patulin at 2.5 to 10 µM induced a terminal UPR in a panel of OSCC cells that was characterized by an increase in CHOP, GADD34, and ATF3 gene expression and XBP1 splicing. A luminescent caspase assay and the induction of several BH3-only genes indicated that patulin could induce apoptosis in OSCC cells. These data support the use of this complementary HTS strategy to identify novel modulators of UPR signaling and tumor cell death.

Belizeanic acid: a potent protein phosphatase 1 inhibitor belonging to the okadaic acid class, with an unusual skeleton.

Belizeanic acid (BA), a novel metabolite belonging to the okadaic acid class of protein phosphatase inhibitors, was isolated from artificial cultures of the dinoflagellate Prorocentrum belizeanum. The structure and conformational behaviour of BA was elucidated by a combination of NMR spectroscopy and conformational analysis. The isolation of this metabolite, which possesses a simplified version of the okadaic acid skeleton, supports the biogenetic pathway previously reported for this class of compounds. BA showed potent inhibitory activity against protein phosphatase 1 (PP1) within the nM range. A plausible model for the interaction of BA with the PP1 binding pocket was derived from computational docking studies.

New targets in diarrhetic shellfish poisoning control.

The toxic profile of dinoflagellates varies even among identical species, raising an interesting question about the mechanism of toxin regulation and production. In consequence, it also poses a considerable problem in their control methods. In this paper, we report on the isolation and structural elucidation of several new ester derivatives of okadaic acid (OA) from artificial cultures of the genus Prorocentrum. These new compounds enlarge the range of target molecules that must be considered in the monitoring programs.