Structure-activity relationship within a series of okadaic acid derivatives

Nishiwaki, Shinji; Fujiki, Hirota; Suganuma, Masami; Furuya-Suguri, Hiroko; Matsushima, Rie; Iida, Yukari; Ojika, Makoto; Yamada, Kiyoyuki; Uemura, Daisuke; Yasumoto, Takeshi; Schmitz, Francis J.; Sügimura, Takashi

doi:10.1093/carcin/11.10.1837

Cited by 134 publications

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“…2). In our structure, the acid motif in OA accepts a hydrogen bond from the hydroxyl group of Tyr-272 (20). Other hydrogen bonding interactions occur between Arg-96 and the C-2 hydroxyl of the inhibitor and between Arg-221 and the C-24 hydroxyl group of OA.…”

Section: Resultsmentioning

confidence: 93%

“…The importance of the hydrogen bond between Tyr-272 and the acid group of OA is exemplified by the observation that esterification or removal of the acidic moiety in OA results in elimination of its inhibitory activity and by the fact that mutation of Tyr-272 to Phe results in a 50-fold increase in the K i value (20,22). Despite the intimate interaction of the C-2 hydroxyl group of OA with the Arg-96 side chain, removal of the hydroxyl group results in only a 7-fold increase in the K i value (24).…”

Section: Discussionmentioning

confidence: 99%

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Crystal Structure of the Tumor-promoter Okadaic Acid Bound to Protein Phosphatase-1

Maynes¹,

Bateman²,

Cherney³

et al. 2001

Journal of Biological Chemistry

154

143

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Protein phosphatase-1 (PP1) plays a key role in dephosphorylation in numerous biological processes such as glycogen metabolism, cell cycle regulation, smooth muscle contraction, and protein synthesis. Microorganisms produce a variety of inhibitors of PP1, which include the microcystin class of inhibitors and okadaic acid, the latter being the major cause of diarrhetic shellfish poisoning and a powerful tumor promoter. We have determined the crystal structure of the molecular complex of okadaic acid bound to PP1 to a resolution of 1.9 Å. This structure reveals that the acid binds in a hydrophobic groove adjacent to the active site of the protein and interacts with basic residues within the active site. Okadaic acid exhibits a cyclic structure, which is maintained via an intramolecular hydrogen bond. This is reminiscent of other macrocyclic protein phosphatase inhibitors. The inhibitor-bound enzyme shows very little conformational change when compared with two other PP1 structures, except in the inhibitor-sensitive ␤12-␤13 loop region. The selectivity of okadaic acid for protein phosphatases-1 and -2A but not PP-2B (calcineurin) may be reassessed in light of this study.The phosphorylation and dephosphorylation of proteins is vital to the regulation of many cellular pathways and processes. Two classes of enzymes in the cell that catalyze cellular dephosphorylation activity are tyrosine phosphatases and serine/threonine phosphatases (1). Classification of serine/threonine phosphatases can be subdivided into four categories: protein phosphatase-1 (PP1), 1 -2A (PP2A), -2B(PP2B) and -2C (PP2C) (2). The first three of these categories comprise what is known as the PPP family of protein phosphatases since they contain extensive sequence similarity in their catalytic domains and little or no sequence homology to PP2C or to tyrosine phosphatases. There are several natural toxin inhibitors of the PPP family of enzymes. These include microcystins, calyculins, tautomycin and okadaic acid (OA) ( Fig. 1) (1).OA is a tumor-promoting C 38 polyether fatty acid produced by marine dinoflagellates (1, 3-6). OA contains acidic and hydrophobic moieties and is cyclic (via an intramolecular hydrogen bond) (6). This toxin can accumulate in filter-feeding organisms and is the principle cause of diarrhetic shellfish poisoning worldwide (4).There have been many biochemical and modeling studies on the inhibition of the PPP family of phosphatases by the natural toxins, but the lone crystal structure is of microcystin-LR (MCLR) bound to PP1 (␣ isoform) (8). Here we describe the crystal structure of OA bound to the recombinant catalytic subunit of PP1 (␥ isoform). EXPERIMENTAL PROCEDURESCrystallization-The catalytic subunit of protein phosphatase-1 ␥ isoform was purified as described previously (9, 10). OA was purified from Prorocentrum lima (9, 10). Crystals were obtained by the hanging drop vapor diffusion method at room temperature. The enzyme and inhibitor were mixed in a 1:2 molar ratio with the concentration of protein being ϳ0.4 mM. The P...

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Section: Resultsmentioning

confidence: 93%

Section: Discussionmentioning

confidence: 99%

Crystal Structure of the Tumor-promoter Okadaic Acid Bound to Protein Phosphatase-1

Maynes¹,

Bateman²,

Cherney³

et al. 2001

Journal of Biological Chemistry

154

143

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“…Methyl okadaate (MeOk) is a methyl ester derivative of the OA, artificially produced in the laboratory, but also found in shellfish, and even in naturally collected or cultured dinoflagellates from the genera Prorocentrum and Dinophysis (Hu et al, 1992;Vale and Sampayo, 1999;Suzuki et al, 2004;Suarez-Gomez et al, 2005). Usually, MeOk has been considered as an inactive molecule because of its low activity in inhibiting PPs (Nishiwaki et al, 1990;Takai et al, 1992). However, recent studies indicated that MeOk induced reorganization of the actin cytoskeleton of human neuroblastoma cells (Vilarino et al, 2008), and although the potency of MeOk was lower than that of OA, its mechanism of action was unknown.…”

Section: Introductionmentioning

confidence: 99%

The methyl ester of okadaic acid is more potent than okadaic acid in disrupting the actin cytoskeleton and metabolism of primary cultured hepatocytes

Espiña

Louzao

Cagide

et al. 2010

British J Pharmacology

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The methyl ester of okadaic acid is more potent than okadaic acid in disrupting the actin cytoskeleton and metabolism of primary cultured hepatocytesb ph_512 337.. 344 Begoña Espiña Japan Food Research Laboratories, Tama, Tokyo 206-0025, JapanBackground and purpose: Okadaic acid (OA) and microcystins (MCs) are structurally different toxins with the same mechanism of action, inhibition of serine/threonine protein phosphatases (PPs). Methyl okadaate (MeOk), a methyl ester derivative of OA, was considered almost inactive due to its weak inhibition of PP1 and PP2A. Here, we have investigated the activity and potency of MeOk in hepatic cells in comparison with that of OA and MCs. Experimental approach: We tested the effects of MeOK, OA and microcystin-leucine and arginine (MC-LR) on the metabolic rate, the actin cytoskeleton and glucose uptake in a rat hepatocyte cell line (Clone 9) and in primary cultured rat hepatocytes. PP2A was assayed to compare OA and MeOk activity. Key results: MeOk disrupted the actin cytoskeleton and depressed the metabolic rate of both types of rat hepatocytes, being six-fold less potent than OA in Clone 9 cells but nearly six-fold more potent in primary cultured hepatocytes. However, unlike OA, MeOk did not change glucose uptake in these cells, suggesting a weak inhibition of PP2A, as confirmed in direct assays of PP2A activity. Conclusions and implications:Although MeOk was originally described as a weakly bioactive molecule, it clearly depressed the metabolic rate and disrupted the cytoskeleton in primary and immortalized rat hepatocytes. Furthermore, MeOk affected primary hepatocytes at much lower concentrations than those affecting immortalized cells. These effects were unrelated to PP2A inhibition. Our results suggest the risk to public health from MeOk in foodstuffs should be re-evaluated.British Journal of Pharmacology (2010) 159, 337-344; doi:10.1111/j.1476-5381.2009 published online 15 December 2009 Keywords: actin cytoskeleton; glucose uptake kinetics; metabolic rate; methyl okadaate; microcystin; okadaic acid; phycotoxins; protein phosphatase and rat hepatocytes Abbreviations: DSP, diarrheic shellfish poisoning; MC-LR, microcystin-leucine and arginine; MeOk, methyl okadaate; OA, okadaic acid; PP1, protein phosphatase-1; PP2A, protein phosphatase-2A IntroductionMany natural toxins such as okadaic acid (OA) potently inhibit the catalytic activity of serine/threonine protein phosphatases (PPs), of which PP1 and PP2A are the most abundant in cells. Both PPs have a wide spectrum of activity being able to dephosphorylate a large number of substrates in vitro (Fernandez et al., 2002). The list of PPs inhibitors includes a structurally diverse group of compounds: polyether fatty acid substances similar to okadaic acid (OA), cyclic peptides like microcystins (MCs) and nodularins, terpenoids such as cantharidin and thyrsiferyl 23-acetate and polyketides, such as tautomycin and calyculin A (Fernandez et al., 2002). OA is the best representative of the diarrheic shellfish poisoning ...

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“…Immobilized MC-LR has been used to affinity purify PP2A [21]. However, the binding capacity (3 ,ug PP2A/l mg MC-LR) was only 0.03% of that ex-petted, probably because coupling to the matrix involved one of the MC-LR carboxyl groups that is important for binding to PP2A [22].…”

Section: Introductionmentioning

confidence: 99%

Identification of protein phosphatase 2A as the primary target for microcystin‐LR in rat liver homogenates

1994

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Structure-activity relationship within a series of okadaic acid derivatives

Cited by 134 publications

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Crystal Structure of the Tumor-promoter Okadaic Acid Bound to Protein Phosphatase-1

Crystal Structure of the Tumor-promoter Okadaic Acid Bound to Protein Phosphatase-1

The methyl ester of okadaic acid is more potent than okadaic acid in disrupting the actin cytoskeleton and metabolism of primary cultured hepatocytes

Identification of protein phosphatase 2A as the primary target for microcystin‐LR in rat liver homogenates

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