A protein phosphatase 2A (PP2A) inhibition assay using a recombinant enzyme for rapid detection of microcystins

Worldwide blooms of toxic cyanobacteria (blue-green algae) commonly occur in freshwater, often in drinking water sources, necessitating routine monitoring of water quality. Microcystin-LR and related cyanobacterial toxins strongly inhibit protein phosphatase 2A (PP2A) and are therefore assayable by measuring the extent of PP2A inhibition. In this study, we evaluated the suitability of the catalytic subunit of recombinant PP2A (rPP2Ac) expressed with a baculovirus system for use in a microplate microcystin assay. Five microcystin analogs, microcystin-LR, -RR, -YR, -LF, and -LW, and nodularin strongly inhibited rPP2Ac activity with IC(50) values of 0.048, 0.072, 0.147, 0.096, 0.114, and 0.54 nM, respectively. Microcystin-LR in a water sample could be assayed from 0.005 to 5 ng/ml. The assay could detect the toxin at a far lower level than required by the World Health Organization for regulation of microcystin-LR or its equivalent (1 microg/L). Pretreatment or concentration of water samples with low toxin concentrations was not necessary. The microplate assay using rPP2Ac was more sensitive than an enzyme-linked immunosorbent assay (ELISA) method and a cytotoxicity assay. The genetically engineered rPP2Ac was more stable than a commercially available dimeric enzyme, producing accurate and reproducible results. Our results confirm that the rPP2Ac we prepared is an excellent tool for detecting and quantifying microcystins in water.     

The effect of structural variation in 21 microcystins on their inhibition of PP2A and the effect of replacing cys269 with glycine

Microcystins (MCs) are a group of cyclic heptapeptide hepatotoxins produced by Microcystis and several other genera of cyanobacteria. The representative MC, MC-LR, strongly inhibits protein phosphatase 2A (PP2A), while the inhibitory potencies of at least 60 MC analogs characterized from bloom samples and cultured strains have not been fully elucidated. In this study, we determined the IC₅₀ values for 21 MC analogs for inhibiting the recombinant PP2A catalytic subunit (rPP2Ac). Of the 21 MC analogs, MC-LR was the strongest inhibitor of rPP2Ac. Comparison of the IC₅₀ values indicates that demethylation of the amino acids at positions 3 or 7 leads to a greater reduction in activity than the substitution of l-amino acids at positions 2 and 4. To obtain further insight into the MC-PP2A interaction, we substituted cysteine at position 269 in PP2Ac with glycine. The mutant PP2Ac (C269G) was comparable to the wild-type PP2Ac in the hydrolysis of p-NPP, but was more resistant to MCs as indicated by the greater IC₅₀ values. Our results indicate that cys269 in PP2Ac and N-methyldehydroalanine (Mdha) at position 7 in MCs play important roles in the enzyme-inhibitor interaction. We also determined the LC₅₀ values of the MCs for cytotoxicity assay. Our results indicate that there is a weak correlation between the cytotoxicity and PP2A inhibiting activities of the MCs. The MCs and rPP2Ac used in this study were of high purity and the IC₅₀ values were determined under the same experimental conditions, ensuring the quality of the data. The IC₅₀ values are of practical importance because they enable the precise conversion of the amounts of various MCs detected using instrumental methods to MC-LR equivalents.     

Single laboratory validation of a ready-to-use phosphatase inhibition assay for detection of okadaic acid toxins

A phosphatase inhibition assay for detection of okadaic acid (OA) toxins in shellfish, OkaTest, was single laboratory validated according to international recognized guidelines (AOAC, EURACHEM). Special emphasis was placed on the ruggedness of the method and stability of the components. All reagents were stable for more than 6 months and the method was highly robust under normal laboratory conditions. The limit of detection and quantification were 44 and 56 μg/kg, respectively; both below the European legal limit of 160 μg/kg. The repeatability was evaluated with 2 naturally contaminated samples. The relative standard deviation (RSD) calculated was 1.4% at a level of 276 μg/kg and 3.9% at 124 μg/kg. Intermediate precision was estimated by testing 10 different samples (mussel and scallop) on three different days and ranged between 2.4 and 9.5%. The IC(50) values of the phosphatase used in this assay were determined for OA (1.2 nM), DTX-1 (1.6 nM) and DTX-2 (1.2 nM). The accuracy of the method was estimated by recovery testing for OA (mussel, 78-101%; king scallop, 98-114%), DTX-1 (king scallop, 79-102%) and DTX-2 (king scallop, 93%). Finally, the method was qualitatively compared to the mouse bioassay and LC-MS/MS.     

Method for detecting classes of microcystins by combination of protein phosphatase inhibition assay and ELISA: comparison with LC-MS.

Depending on the class of microcystin the protein phosphatase inhibition assay shows different sensitivities to different classes of toxin. We have determined that the IC50 values obtained from dose-response curves for the inhibition of the enzyme by micro-cystin LR, nodularin, YR, and RR were 2.2, 1.8, 9 and 175 nM, respectively. When equimolar amounts of these toxins were determined by the ELISA assay with microcystin LR as the standard, the assay showed equivalence in toxin responses. However, when the toxins were determined by the protein phosphatase inhibition assay using microcystin LR as the standard, the ratios of the values determined by PP-2A to ELISA decreased in the order: nodularin (2.23) microcystin LR (1.1)> microcystin YR (0.63)> microcystin RR (0.06). When the ratios for each standard were plotted against the IC50 values, the log-log plot was negative linear, and the lowest value for the IC50 corresponded with the lowest ratio. The differential sensitivity of the PP-2A assay to the various standards was used to establish an indicative toxicity ranking (ITR) where a ranking of 1 (the highest) was assigned to ratios of > or = 0.8 or greater, and 3 (the lowest) to values < or = 0.2. The three ranking classes corresponded to toxin equivalence represented by the four standards. The new method allows not only the determination of microcystin toxins in terms of stoichiometry (ELISA) but also in terms of indicative toxicity. The method can be performed using the same instrument (e.g. multiwell fluorimeter with absorbance capability) and offers an advantage to methods presently used to determine microcystins (e.g. ELISA or LC-MS). The former has the propensity to overestimate toxicity because it measures equivalence to microcystin LR and is a stoichiometric measurement and the latter has the disadvantage in that relatively few of the microcystins that occur naturally are available as standards. The new method was applied to the analysis of sample from lakes and streams from temperate locations and to extracts of cyanobacterial mats from ponds and streams in cold temperature locations.     

Decrease in toxicity of microcystins LA and LR in drinking water by ozonation

Unchlorinated treated waters from two Australian reservoirs were spiked with microcystin-LA and -LR extracted from a toxic scum of Microcystis aeruginosa. The two waters had considerably different water quality and therefore ozone demands. The spiked sample waters were ozonated using the batch method of ozonation at a range of doses and the samples were analysed for toxins using high-performance liquid chromatography (HPLC). The toxin content of the samples was also determined using a protein phosphatase type 2A inhibition assay (PP2A) and toxicity via the standard mouse bioassay. The HPLC results correlated well with the PP2A results and toxicity tests for both waters. A loss of both toxins and toxicity was observed with increasing ozone dose, resulting in a complete loss of toxicity for both waters once an ozone residual had been achieved. At this ozone residual no toxin was detected using HPLC. The results indicate that microcystins are not transformed into toxic by-products.     

蛋白质酪氨酸磷酸酯酶1B(PTP1B)抑制剂研究进展

蛋白质酪氨酸磷酸酯酶1B(PTP1B)在胰岛素信号传导过程中起负调节作用,是治疗糖尿病和肥胖症的潜在靶点.本文按作用位点对近年来研究的PTP1B抑制剂进行综述,分析了N-草酰胺苯甲酸类化合物、水杨酸类化合物、取代苯乙酮及肉桂酸类化合物、氨基磺酸类化合物、拟肽类物质、噻吩及苯并噻吩类化合物、苯并呋喃磺酰胺类化合物和哒嗪类化合物八大类抑制剂的结构及药理活性.     

Inhibition of nuclear protein phosphatase activity in mouse hepatocytes by the cyanobacterial toxin microcystin-LR.

Microcystin-LR (MCLR) is a cyanobacterial hepatotoxin and protein phosphatase inhibitor that contaminates water reservoirs worldwide. MCLR localizes to the cytosol of hepatocytes, however, immunohistochemical studies indicate that it accumulates in the nucleus. MCLR toxicosis is associated with decreased hepatic protein phosphatase activity, but effects in nuclear protein phosphatase activity have not been investigated. Balb/c mice were given lethal (100 microg/kg) or sublethal (12, 23 and 45 microg/kg) i.p. doses of MCLR and hepatic nuclear extracts were analyzed for protein phosphatase 1 and 2A activity. There was profound inhibition of nuclear protein phosphatase activity within 50 min of lethal dosing, however an inhibition was not detected with sublethal doses. MCLR immunohistochemistry revealed widespread lobular staining in the lethal group and centrilobular staining in the sublethal groups. At the cellular level there was nuclear and cytoplasmic staining of equal intensity. As an indicator of nuclear protein phosphatase activity, the phosphorylation of p53, a nuclear phosphoprotein and known substrate for protein phosphatases 1 and 2A, was evaluated. Balb/c mice were treated with sublethal doses of MCLR or saline vehicle after induction of hepatic p53 by the DNA damaging agent diethylnitrosamine (DEN). P53 was immunoprecipitated and probed with phosphoserine specific antibodies by Western blotting. There was greater phosphoserine reactivity of p53 protein in animals treated with MCLR relative to saline treated controls, consistent with increased phosphorylation of serine sites. It is concluded that an interaction of this toxin with nuclear protein phosphatases occurs within 50 min of lethal dosing, which leads to a profound inhibition of enzymatic activity. Even sublethal doses of MCLR that do not result in significant inhibition of activity in bulk nuclei, result in detectable changes in phosphorylation of p53.     

Bacterial degradation of microcystin toxins in drinking water eliminates their toxicity.

Microcystin-LR and -LA were readily biodegraded by a bacterium, Sphingpoyxis sp. LH21, in a treated reservoir water. Detection of the microcystins was conducted using high-performance liquid chromatography (HPLC), protein phosphatase 2A (PP2A) inhibition assay and a cell-based cytotoxicity assay. The HPLC results correlated well with the two assays. The decrease in cytotoxicity, coupled with the associated decrease in microcystin concentrations, indicated that no cytotoxic by-products were being generated, highlighting the applicability of biodegradation as a feasible treatment option for effective microcystin removal.     

榅桲果实及种子不同提取部位的蛋白酪氨酸磷酸酶1B抑制活性筛选

目的探讨榅桲果实和种子的甲醇提取物及其不同溶剂提取部位对蛋白酪氨酸磷酸酶1B(PTP1B)的抑制作用,筛选抗糖尿病活性部位。方法利用PTP1B体外筛选系统,对榅桲果实和种子不同提取部位进行细致筛选,确定其降糖活性部位。结果榅桲果实和种子各部位均有不同程度抑制PTP1B活性的作用,其中榅桲种子乙酸乙酯部位、榅桲果实二氯甲烷部位和榅桲果实水部位的体积分数为50%的乙醇溶液洗脱部位对PTP1B抑制作用强,其半数抑制质量浓度(IC50)分别为0.844,0.423和0.395mg·L-1,初步推测榅桲果实及种子提取物具有抗2型糖尿病的潜力。结论榅桲种子乙酸乙酯部位、榅桲果实二氯甲烷部位和榅桲果实水部位的体积分数为50%的乙醇溶液洗脱部位是主要的抗糖尿病活性部位。     

Involvement of mitogen-activated protein kinases and protein kinase C in cadmium-induced prostaglandin E2 production in primary mouse osteoblastic cells

We previously reported that cadmium (Cd) induced prostaglandin E₂ (PGE₂) biosynthesis through the activation of cytosolic phospholipase A₂ (cPLA₂) and induction of cyclooxygenase 2 (COX-2) in primary mouse osteoblastic cells. In the present study, we further investigated the mechanism of PGE₂ production by Cd focusing on the main mitogen-activated protein kinase (MAPK) subfamilies that mediate prostaglandin synthesis, extracellular signal-regulated kinase (ERK1/2 MAPK), c-jun-amino-terminal kinase (JNK MAPK) and p38 MAPK, and protein kinase C (PKC) which is activated by Cd in several kinds of cells. Cd at 2 μM and above stimulated PGE₂ production in osteoblastic cells and its production was inhibited by the kinase-specific inhibitors PD98059, SB203580, curcumin, and calphostin C. Calphostin C also inhibited the production of PGE₂ by phorbol 12-myristate 13-acetate (PMA), which is a potent activator of PKC. PD98059 inhibited PGE₂ production stimulated by PMA as well as Cd, indicating that activation of PKC by ERK1/2 MAPK was necessary for Cd-stimulated PGE₂ production. Moreover, Cd stimulated the phosphorylation of these three MAPKs, and inhibition of the phosphorylation of ERK1/2 MAPK by calphostin C was also observed. On the other hand, Cd was found to phosphorylate cPLA₂ and the phosphorylation was inhibited by PD98059, indicating that cPLA₂ was activated by Cd through ERK1/2 MAPK and released arachidonic acid (AA), a substrate of COX-2, from membranous phospholipids. From these results, it was suggested that activation of each of the ERK1/2, p38, and JNK MAPK cascades in addition to that of PKC and cPLA₂ played an important role in the Cd-stimulated biosynthesis of PGE₂ in mouse osteoblastic cells.     

Protein phosphorylation profile and adduct formation in liver and kidney of microcystin-LR-treated mice.

Microcystins are cyclic peptide toxins implicated in several livestock and human deaths. The toxicity of microcystins has been attributed to the highly specific inhibition of serine/theronine protein phosphatases-1 and 2A. Reversible protein phosphorylation is an essential regulatory mechanism in many cellular processes. We aimed to investigate the protein phosphatase inhibition, profile of phosphorylated proteins of serine and threonine residues and microcystin-protein phosphatase adduct in vivo after microcystin-LR exposure by intraperitoneal route in mice. At 1 LD(50), there was significant inhibition of protein phosphatases 1 and 2A activity in liver after 30-120 min exposure but there was no effect in kidney. At 0.5 LD(50) there was no inhibition of protein phosphatase activity in both liver and kidney. Similarly, time-dependent phosphorylation of serine and threonine residues was observed at 1 LD(50). Microcystin-LR-protein phosphatase adduct was time and dose dependent in liver. At 0.5 LD(50) the adduct could be detected at 1 and 3 days post-exposure. No adduct could be detected in kidney.     

Oatp-associated uptake and toxicity of microcystins in primary murine whole brain cells

Microcystins (MCs) are naturally occurring cyclic heptapeptides that exhibit hepato-, nephro- and possibly neurotoxic effects in mammals. Organic anion transporting polypeptides (rodent Oatp/human OATP) appear to be specifically required for active uptake of MCs into hepatocytes and kidney epithelial cells. Based on symptoms of neurotoxicity in MC-intoxicated patients and the presence of Oatp/OATP at the blood-brain-barrier (BBB) and blood-cerebrospinal-fluid-barrier (BCFB) it is hypothesized that MCs can be transported across the BBB/BCFB in an Oatp/OATP-dependent manner and can induce toxicity in brain cells via inhibition of protein phosphatase (PP). To test these hypotheses, the presence of murine Oatp (mOatp) in primary murine whole brain cells (mWBC) was investigated at the mRNA and protein level. MC transport was tested by exposing mWBCs to three different MC-congeners (MC-LR, -LW, -LF) with/without co-incubation with the OATP/Oatp-substrates taurocholate (TC) and bromosulfophthalein (BSP). Uptake of MCs and cytotoxicity was demonstrated via MC-Western blot analysis, immunocytochemistry, cell viability and PP inhibition assays.All MC congeners bound covalently and inhibited mWBC PP. MC-LF was the most cytotoxic congener followed by -LW and -LR. The lowest toxin concentration significantly reducing mWBC viability after 48 h exposure was 400 nM (MC-LF). Uptake of MCs into mWBCs was inhibited via co-incubation with excess TC (50 and 500 μM) and BSP (50 μM). MC-Western blot analysis demonstrated a concentration-dependent accumulation of MCs. In conclusion, the in vitro data support the assumed MC-congener-dependent uptake in a mOatp-associated manner and cytotoxicity of MCs in primary murine whole brain cells.     

Differential modulation of agonist and antagonist structure activity relations for rat TRPV1 by cyclosporin A and other protein phosphatase inhibitors

The transient receptor potential V1 channel (vanilloid receptor, TRPV1) represents a promising therapeutic target for inflammatory pain and other conditions involving C-fiber sensory afferent neurons. Sensitivity of TRPV1 is known to be subject to modulation by numerous signaling pathways, in particular by phosphorylation, and we wished to determine whether TRPV1 structure activity relations could be differentially affected. We demonstrate here that the structure activity relations of TRPV1, as determined by ⁴⁵Ca² uptake, were substantially altered by treatment of the cells with cyclosporin A, an inhibitor of protein phosphatase 2B. Whereas the potency of resiniferatoxin for stimulation of ⁴⁵Ca² was not altered by cyclosporin A treatment, the potencies of some other agonists were increased up to 8-fold. Among the antagonists examined, potencies were reduced to a lesser extent, ranging from 1- to 2.5-fold. Finally, the efficacy of partial agonists was increased. In contrast to cyclosporin A, okadaic acid, an inhibitor of protein phosphatases 1 and 2A, had little effect on agonist potencies, and calyculin A, an inhibitor of protein phosphatases 1 and 2A but with somewhat different selectivity from that of okadaic acid, caused changes in structure activity relations distinct from those induced by cyclosporin A. Because phosphatase activity differentially modulates the structure activity relations of TRPV1 agonists and antagonists, our findings predict that it may be possible to design agonists and antagonists selective for TRPV1 in a specific regulatory environment. A further implication is that it may be desirable to tailor screening approaches for drug discovery to reflect the desired regulatory state of the targeted TRPV1.     

Differential effects of tautomycetin and its derivatives on protein phosphatase inhibition, immunosuppressive function and antitumor activity

In the present work, we studied the structure-activity relationship (SAR) of tautomycetin (TMC) and its derivatives. Further, we demonstrated the correlation between the immunosuppressive fuction, anticancer activity and protein phosphatase type 1 (PP1) inhibition of TMC and its derivatives. We have prepared some TMC derivatives via combinatorial biosynthesis, isolation from fermentation broth or chemical degradation of TMC. We found that the immunosuppressive activity was correlated with anticancer activity for TMC and its analog compounds, indicating that TMC may home at the same targets for its immunosuppressive and anticancer activities. Interestingly, TMC-F1, TMC-D1 and TMC-D2 all retained significant, albeit reduced PP1 inhibitory activity compared to TMC. However, only TMC-D2 showed immunosuppressive and anticancer activities in studies carried out in cell lines. Moreover, TMC-Chain did not show any significant inhibitory activity towards PP1 but showed strong growth inhibitory effect. This observation implicates that the maleic anhydride moiety of TMC is critical for its phosphatase inhibitory activity whereas the C1-C18 moiety of TMC is essential for the inhibition of tumor cell proliferation. Furthermore, we measured in vivo phosphatase activities of PP1 in MCF-7 cell extracts treated with TMC and its related compounds, and the results indicate that the cytotoxicity of TMC doesn't correlate with its in vivo PP1 inhibition activity. Taken together, our study suggests that the immunosuppressive and anticancer activities of TMC are not due to the inhibition of PP1. Our results provide a novel insight for the elucidation of the underlying molecular mechanisms of TMC's important biological functions.     

PP2A Inhibition Assay Using Recombinant Enzyme for Rapid Detection of Okadaic Acid and Its Analogs in Shellfish

Okadaic acid and its analogs (OAs) responsible for diarrhetic shellfish poisoning (DSP) strongly inhibit protein phosphatase 2A (PP2A) and thus are quantifiable by measuring the extent of the enzyme inhibition. In this study, we evaluated the suitability of the catalytic subunit of recombinant human PP2A (rhPP2Ac) for use in a microplate OA assay. OA, dinophysistoxin-1(DTX1), and hydrolyzate of 7-O-palmitoyl-OA strongly inhibited rhPP2Ac activity with IC(50) values of 0.095, 0.104, and 0.135 nM, respectively. The limits of detection and quantitation for OA in the digestive gland of scallops and mussels were 0.0348 μg/g and 0.0611 μg/g respectively, and, when converted to the whole meat basis, are well below the regulation level proposed by EU (0.16 μg/g whole meat). A good correlation with LC-MS data was demonstrated, the correlation coefficient being 0.996 with the regression slope of 1.097.     

Comparison of protein phosphatase inhibitory activity and apparent toxicity of microcystins and related compounds.

Two metabolites of microcystin-LR glutathione conjugate and, microcystin-cysteine conjugate, as well as microcystin-RR (MCRR) are less toxic than microcystin-LR (MCLR). In the present study, we investigated why these compounds are weakly toxic in comparison with MCLR, as the reason is still unknown and no systematic study has so far been carried out for a clarification of this issue. Although they showed almost the same inhibitory activity against protein phosphatases 1 and 2A as MCLR in vitro, the apparent toxicity of these three compounds by intratracheal administration to mice decreased to about 1/12 the level of MCLR at 100microg/kg. An immunostaining study showed that these conjugates at a sublethal dose of 200microg/kg were prominently observed in the intestine and kidney, whereas effective accumulation and bleeding were not found in the liver in spite of the larger dosage. As an explanation for these results, there may be two possibilities. First, the transport system to the liver might not function well, and second, transported toxins may be effectively eliminated by an appropriate system such as the GS-X (ATP-dependent glutathione S-conjugate exported) pump. It was concluded that the inhibitory activity against protein phosphatases is not always related to the apparent LD(50) level, and that the appearance of toxicity by microcystins depends on the balance between accumulation and metabolism in the liver.     

Comparison of the enzymatic and edema-producing activities of two venom phospholipase A2 enzymes.

The edema-producing activity of NNAVPLA₂, an acidic phospholipase A₂ (PLA₂) enzyme from Naja naja atra venom (NNAV), was less potent than that of TMVPLA₂ II, a basic PLA; from Trimeresurus mucrosquamatus venom (TMV). These edema-forming effects were greatly suppressed by pretreatment of rats with diphenhydramine/ methysergide or compound 48/80, which reduced the tissue content of histamine and serotonin. Heparin abolished and suppressed the paw edema caused by protamine and TMVPLA₂ II, respectively, but had no effect on the NNAVPLA₂-induced response. In isolated rat peritoneal mast cells, both PLA₂ concentration dependently induced the release of histamine and β-glueuronidase. Again, TMVPLA₂ II was more potent than NNAVPLA₂. This degranulation effect of mast cells caused by TMVPLA₂ II and protamine was inhibited by heparin, while that caused by NNAVPLA₂ was unaffected. The edema-forming and mast cell degranulation effects were greatly decreased in both PBPB-modified NNAVPLA₂ and PBPB-modified TMVPLA₂ II, in which the catalytic activity of the enzymes was completely lost. PBPB-modified TMVPLA₂ II-induced paw edema was also suppressed by heparin. Furthermore, this edematous response was totally reversed in rat pretreated with aspirin in combination with diphenhydramine and methysergide. These results suggest that the edema-forming effect of PLA₂ is probably dependent on the presence of catalytic, positive charge and pharmacological sites on its molecule.     

Inhibition of protein tyrosine phosphatase 1B by triterpenes isolated from Potentilla discolor Bge

Seven oleanene triterpenes were isolated from the roots of Potentilla discolor Bge and their structures were identified as3-oxoolean-12-en-27-oic acid (1), gypsogenic acid (2), 3α-hydroxyolean-12-en-27-oic acid (3), 3β-hydroxyolean-12-en-27-oic acid (4), aceriphyllic acid A (5), aceriphyllic acid A methyl ester (6), and oleanolic acid (7). Compounds 1–7 inhibited protein tyrosine phosphatase 1B (PTP1B) activity, with IC₅₀ values ranging from (7.5±0.5) to (22.7±0.5) μmol/L. Among the isolates, compounds 1, 2, 3 and 7 from thePotentilla discolor Bge were found to exhibit selective PTP1B inhibitory activity.     

Functional diversity of mammalian type 2C protein phosphatase isoforms: new tales from an old family

1. The Type 2C protein phosphatases (PP2C) represent a highly conserved gene family in the mammalian genome. Recent studies have revealed that PP2C isoforms possess unique patterns of tissue and subcellular distribution associated with diverse functionalities. 2. The functional importance of PP2C isoforms has been shown in a plethora of signalling networks controlling cell differentiation, proliferation, growth, survival and metabolism. However, little is known about the regulatory mechanisms of PP2C at the molecular level. It is uncertain how PP2C isoforms are recruited, activated and inactivated during signalling transduction processes. 3. In the present paper, an overview of the critical functions of individual PP2C isoforms in regulating cellular signalling events will be provided, along with our perspectives on the challenging issues to be addressed. It is clear that a better understanding of the complex biological effects elicited by specific signalling pathways involving PP2C isoforms has great potential for developing novel therapies for a variety of human diseases, including cancer, diabetes and neural disorders, as well as cardiovascular diseases.     

Design, synthesis, and biological evaluation of bromophenol derivatives as protein tyrosine phosphatase 1B inhibitors

3‐Bromo‐4,5‐bis(2,3‐dibromo‐4,5‐dihydroxybenzyl)‐1,2‐benzenediol (BDB) is a bromophenol purified from the marine red alga Rhodomela confervoides and exhibits potent protein tyrosine phosphatase 1B (PTP1B) inhibition (IC₅₀ = 1.7 µmol/L). In an effort to improve the PTP1B inhibitory activity, a series of derivatives were designed, synthesized, and evaluated in vitro. The preliminary structure–activity relationship indicated that the tricyclic scaffold and multi‐bromine atoms (four to five) attached to the aryl rings are important for PTP1B inhibition. Among these, compound 26 exhibited remarkable inhibitory activity against PTP1B with an IC₅₀ of 0.89 µmol/L, which was approximately two‐fold more potent than the initial lead compound BDB.