Inactivation of phospholipase A2 by manoalide. Localization of the manoalide binding site on bee venom phospholipase A2

The marine natural product manoalide (MLD), a potent inhibitor of phospholipases, completely inactivates bee venom phospholipase A2 (PLA2) by an irreversible mechanism. It has been proposed [K. B. Glaser and R. S. Jacobs, Biochem. Pharmac. 36, 2079 (1987)] that the reaction of MLD with PLA2 may involve the selective reactivity of MLD to a peptide sequence, possibly a Lys-X-X-Lys peptide. Localization of the MLD binding site on bee venom PLA2 demonstrated that upon MLD modification of bee venom PLA2 the only change in amino acid content was an apparent loss of Lys, corresponding to approximately three of the eleven Lys residues present. Selective chemical modification of Lys residues with [14C]maleic anhydride demonstrated that all eleven Lys residues on bee venom PLA2 were accessible to this reagent (11.6 mol maleyl group incorporated/mol of PLA2). Pretreatment of PLA2 with MLD (less than 0.7% residual activity) resulted in a molar ratio of 8.7, also consistent with the loss of three Lys residues upon modification by MLD. Reverse phase high performance liquid chromatography (RP-HPLC) of the cyanogen bromide (CNBr) digestion product of MLD-treated PLA2 produced three peaks (A280). The second peak showed the most intense absorbance at 434 nm. This material corresponded to residues 81-128, as determined by gas-phase microsequence analysis. Sequencing failure was observed at Lys-88 in the MLD-treated fragment. The control carboxymethylated-PLA2 fragment corresponding to residues 81-128 sequenced beyond Lys-88 without significant change in the expected yield. These data suggest that Lys-88 may correspond to one of the three MLD-modified Lys residues. The minor absorbance at 434 nm of the CNBr fragments containing residues 42-80 and 1-36 as compared to the fragment of residues 81-128 suggests that the major MLD binding fragment residues in residues 81-128.     

Inactivation of human pleural fluid phospholipase A2 by dioscin

The natural product, spirostanol glycoside dioscin, was shown to directly inactivate human pleural fluid phospholipase A₂ (PLA₂). Inactivation was dose, and time dependent. The IC₅₀ was estimated at 18 μM and virtually complete inactivation of the enzyme occurred at 50 μM. Using Michaelis-Menten kinetics, dioscin inactivated the enzyme by a competitive inhibitory manner, the apparentKi value was 6.9×10^?4M. Reversibility was studied directly by dialysis method, the inhibition was reversible. Addition of excess Ca²⁺ concentration up to 8 mM did not antagonize the inhibitory activity of dioscin. Inactivation of several kinds of PLA₂ by dioscin, showed a broad range of PLA₂ specificity. These data suggest that inactivation of PLA₂ by dioscin is due to interaction with the active site of PLA₂ and may be a useful adjunct in the theraphy of inflammatory diseases.     

Molecular pharmacology of manoalide. Inactivation of bee venom phospholipase A2

The marine natural product manoalide (MLD) was shown to directly inactivate bee venom phospholipase A2 (PLA2). Inactivation was pH dependent (maximum inactivation occurred at pH 8.0), time dependent and concentration dependent. The IC50 was estimated at 0.05 microM and virtually complete inactivation of the enzyme occurred at 4.0 microM. The time-dependent loss of PLA2 activity suggested that inactivation does not follow typical Michaelis-Menten kinetics. Reversibility was studied directly by dilution and dialysis; both methods were ineffective in dissociating the MLD-PLA2 complex. A kinetic plot of initial velocity (v) versus [PLA2] supported our hypothesis that MLD apparently inactivates bee venom PLA2 by an irreversible mechanism.     

Inhibition of phosphoinositide-specific phospholipase C by manoalide

Manoalide is a novel sesterterpenoid which has previously been shown to be a potent inhibitor of venom phospholipases A2. To determine whether manoalide inhibited other phospholipases, the sensitivity of phosphoinsitide-specific phospholipase C (PI-PLC) to inactivation by manoalide was examined using crude cytosolic PI-PLC and a PI-PLC purified to homogeneity from guinea pig uterus cytosol (PI-PLC I). Manoalide inhibited both cytosolic and purified PI-PLC I in a concentration-dependent fashion, exhibiting an IC50 of 3-6 microM. Inactivation of PI-PLC I was calcium- and pH-dependent, with greater inactivation occurring at alkaline pH. Manoalide inhibited hydrolysis of all three phosphoinositides by purified PI-PLC I. The substrate kinetics of PI-PLC I suggest that manoalide does not inhibit purified PI-PLC I by simple competitive or noncompetitive inhibition. Enzyme activity was not recovered after dialysis of manoalide-treated PI-PLC I, indicating that inactivation of PI-PLC I was irreversible. To determine whether manoalide inhibited PI-PLC in cells, the effects of manoalide on norepinephrine (NE)-stimulated phosphoinositide hydrolysis and calcium mobilization were investigated in a smooth muscle-like cell line, DDT1MF-2. Manoalide inhibited NE-induced inositol 1,4,5-trisphosphate and inositol 1-phosphate formation in a concentration-dependent manner. The IC50 for inhibition of inositol 1-phosphate formation was 1.5 microM. Manoalide also inhibited NE-induced calcium transients in DDT1MF-2 cells, exhibiting an IC50 of 2 microM. These data suggest that inhibition of PI-PLC may account, in part, for the anti-inflammatory actions of manoalide.     

Biochemical characterization of soluble phospholipase A2 from rheumatoid synovial fluid

Radiolabeled E. coli, Phosphatidylethanolamine (PE) and Phosphatidylcholine (PC), were used to characterize the phospholipase A2 (PLA2) activity in synovial fluid (SF) from rheumatoid arthritis (RA) patients. Cell-free fractions of SF contain a PLA2 enzyme that preferentially releases [14C]oleic acid from E. coli, requires calcium and is optimally active at neutral pH. Purified PE, but not PC is also readily degraded by the soluble enzyme. A cell-associated PLA2 present in sonicates of SF mononuclear cells and neutrophils preferentially releases [3H]AA from E. coli. These studies suggest the presence of at least two different enzymes with activity of PLA2 in rheumatoid SF.     

Inhibition of phospholipase A2 activities and some inflammatory responses by the marine product ircinin

The marine product ircinin has been tested for its effects on secretory and cytosolic phospholipase A₂ (PLA₂) activities in vitro as well as for inhibition of cellular functions in human neutrophils and inflammatory responses in mice. Ircinin inhibited Naja naja venom, human synovial recombinant, bee venom and zymosan-injected rat air pouch PLA₂ with IC₅₀ values in the M range, similar to those of the known inhibitor scalaradial. On the other hand, ircinin was less active on cytosolic PLA₂ from human monocytes and decreased potently the release of LTB₄ in human neutrophils. This marine product affected weakly human neutrophil functions like superoxide generation and degranulation. In the zymosaninjected rat air pouch ircinin inhibited in vivo the activity of PLA₂ present in exudates and reduced dose-dependently myeloperoxidase levels, whereas cell migration was inhibited only at the highest dose tested. This compound exerted a potent anti-oedematous effect after topical application in the mouse ear oedema test. Ircinin is a new inhibitor of PLA₂ activity and our results suggest a potential role for this marine product as an inhibitor of inflammatory processes.     

The inhibition of release of endothelium-derived relaxant factor by manoalide,a potent inhibitor of phospholipase A2.

1 The inhibitory action of manoalide on vascular relaxation was characterized. Manoalide was a potent inhibitor of endothelium-dependent relaxations in the isolated aorta of the rabbit. Responses to acetylcholine (ACh), A23187 and substance P were reduced by manoalide in a dose-dependent manner whilst those to nitroglycerin were unaffected. 2 Repeated washing of manoalide-treated tissues did not restore the relaxant response to ACh, indicating an irreversible action of manoalide. Scanning electron microscopic studies revealed that the endothelium remained intact on manoalide-treated tissues. 3 Rabbit aortae from which the endothelium had been removed relaxed in response to perfusion with ACh when delivered via an upstream endothelium-bearing tissue, indicating release of an endothelium-derived relaxant factor (EDRF). Incubation of the tissue without endothelium with manoalide (100 nM; 30 min) or inclusion of manoalide in the superfusate at a point just distal to the endothelium bearing tissue did not reduce the relaxant potency of EDRF. 4 Contractile responses of the guinea-pig isolated ileum to ACh were not affected by manoalide and, furthermore, binding of [3H]-quinuclidinyl benzilate to striatal membranes was not reduced by manoalide except at very high concentrations. 5 Manoalide therefore appears to inhibit vascular relaxation with a selectivity directed towards that mediated by EDRF. A direct antagonism of neither cholinoceptors nor EDRF receptors occurs and it is suggested that manoalide acts at a site within the endothelium to inhibit the synthesis and/or release of EDRF. Based upon these and previous data the possibility that EDRF is lipid-like or controlled by an arachidonic acid metabolite must continue to be considered.     

Rheumatoid arthritis synovial fluid phospholipase A2 activating protein (PLAP) stimulates human neutrophil degranulation and superoxide ion production

Rheumatoid arthritis is characterized by excessive eicosanoid production, and phospholipase enzymes are the rate limiting step in eicosanoid synthesis. We have shown previously that cells from patients with rheumatoid arthritis express enhanced phospholipase A2 enzyme activities. Recently, we have isolated a phospholipase A2 activating protein termed PLAP from rheumatoid synovial fluid. This novel human protein shares biochemical and antigenic similarities with melittin, a bee venom phospholipase activating protein. Because melittin has been shown to induce neutrophil degranulation and superoxide formation, and because exuberent release of lysosomal enzymes and superoxide have been implicated in the pathogenesis of rheumatoid arthritis, we examined the role of PLAP on inducing these neutrophil functions.     

Modulation of acute and chronic inflammatory processes by cacospongionolide B, a novel inhibitor of human synovial phospholipase A2

1. Cacospongionolide B is a novel marine metabolite isolated from the sponge Fasciospongia cavernosa. In in vitro studies, this compound inhibited phospholipase A2 (PLA2), showing selectivity for secretory PLA2 (sPLA2) versus cytosolic PLA2 (cPLA2), and its potency on the human synovial enzyme (group II) was similar to that of manoalide. 2. This activity was confirmed in vivo in the 8 h zymosan-injected rat air pouch, on the secretory enzyme accumulating in the pouch exudate. Cacospongionolide B, that is bioavailable when is given orally, reduced the elevated levels of sPLA2 present in paw homogenates of rats with adjuvant arthritis. 3. This marine metabolite showed topical anti-inflammatory activity on the mouse ear oedema induced by 12-O-tetradecanoylphorbol acetate (TPA) and decreased carrageenin paw oedema in mice after oral administration of 5, 10 or 20 mg kg(-1). 4. In the mouse air pouch injected with zymosan, cacospongionolide B administered into the pouch, induced a dose-dependent reduction in the levels of eicosanoids and tumour necrosis factor alpha (TNFalpha) in the exudates 4 h after the stimulus. It also had a weak effect on cell migration. 5. The inflammatory response of adjuvant arthritis was reduced by cacospongionolide B, which did not significantly affect eicosanoid levels in serum, paw or stomach homogenates and did not induce toxic effects. 6 Cacospongionolide B is a new inhibitor of sPLA2 in vitro and in vivo, with anti-inflammatory properties in acute and chronic inflammation. This marine metabolite was active after oral administration and able to modify TNFalpha levels, and may offer an interesting approach in the search for new anti-inflammatory agents.     

Mechanism of inhibition of a poxvirus topoisomerase by the marine natural product sansalvamide A.

At present no antiviral agents are available for treatment of infection by the pathogenic poxvirus molluscum contagiosum virus (MCV). Here we report the identification and characterization of an inhibitor active against the virus-encoded type-1 topoisomerase, an enzyme likely to be required for MCV replication. We screened a library of marine extracts and natural products from microorganisms using MCV topoisomerase assays in vitro. The cyclic depsipeptide sansalvamide A was found to inhibit topoisomerase-catalyzed DNA relaxation. Sansalvamide A was inactive against two other DNA-modifying enzymes tested as a counterscreen. Assays of discrete steps in the topoisomerase reaction cycle revealed that sansalvamide A inhibited DNA binding and thereby covalent complex formation, but not resealing of a DNA nick in a preformed covalent complex. Sansalvamide A also inhibits DNA binding by the isolated catalytic domain, thereby specifying the part of the protein sensitive to sansalvamide A. These data specify the mechanism by which sansalvamide A inhibits MCV topoisomerase. Cyclic depsipeptides related to sansalvamide A represent a potentially promising chemical family for development of anti-MCV agents.     

海洋天然产物brevianamide K的全合成

摘 要：目的 对海洋曲霉菌的次级代谢产物brevianamide K进行全合成研究,旨在解决其天然来源有限的问题,进而为其生物活性、作用机制的进一步研究以及同家族其他化合物的合成奠定基础。方法 以N-Pht-甘氨酸为起始原料,通过酰化、去保护、胺酯交换、乙酰基保护反应得到羟醛缩合反应的1个片段。以吲哚为起始原料,通过氯代、取代、Vilsmeier-Haack反应、保护基保护得到另1个片段。2个片段发生羟醛缩合反应后脱除保护基完成目标分子的全合成。结果 从商业可得化合物出发,通过最长线性7步反应,以9%的总收率完成brevianamide K的首次全合成,为后续活性研究和同家族其他化合物的合成奠定了基础。关键词：brevianamide K;吲哚二酮哌嗪生物碱;羟醛缩合     

Inhibition of crotoxin phospholipase A(2) activity by manoalide associated with inactivation of crotoxin toxicity and dissociation of the heterodimeric neurotoxic complex

Crotoxin (CACB complex) is a convulsant heterodimeric neurotoxic phospholipase A(2) (PLA(2)). The role of phospholipid hydrolysis in its epileptogenic properties remains unresolved. We, thus, studied the effect of manoalide (MLD), a PLA(2) inhibitor, on the toxin catalytic activity and its central and peripheral toxicity. Incubation of crotoxin with MLD fully and irreversibly inactivated its enzymatic activity. Interestingly, crotoxin also lost its central neurotoxicity after intracerebroventricular injection and peripheral toxicity after intravenous administration. MLD-treated crotoxin prevented the high affinity binding of [125I]-radiolabeled crotoxin on rat cortex synaptic plasma membranes. Further analysis of MLD-treated crotoxin by non-denaturing PAGE and surface plasmon resonance indicated that the crotoxin complex was dissociated after MLD treatment. Although the loss of MLD-treated crotoxin peripheral neurotoxicity could not be attributed to this dissociation, the presence of free CA subunit might explain the observed competition in binding experiments. In conclusion, the dissociation of the crotoxin complex by MLD, as demonstrated in this study, did not permit to specify the role of the enzymatic activity in crotoxin epileptogenic properties. Other approaches would be required to resolve this question.     

Inhibition by manoalide of fMLP-stimulated elastase release from human neutrophils

Incubation of human polymorphonuclear leukocytes (PMNLs) with the chemotactic factor N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) resulted in a concentration-dependent release of the neutral protease elastase. This response was inhibited by pretreatment of the PMNLs with manoalide (IC50 approximately 0.08 microM). To understand the mechanism of this inhibition, we examined the effect of manoalide on the signal-transduction pathway believed to mediate fMLP stimulation. We observed in fura-2 loaded cells that pretreatment with manoalide blocked fMLP-induced increases in cytosolic free-calcium (IC50 approximately 0.15 microM). However, manoalide had no effect on inositol 1,4,5-trisphosphate (IP3) production at concentrations which completely inhibited the Ca2+ signal. Furthermore, manoalide was approximately 50-fold less potent as an inhibitor of phospholipase C activity in membrane preparations of PMNLs than as an inhibitor of calcium mobilization in whole cells. These data indicate that manoalide can block stimulation of human PMNLs through inhibition of Ca2+ mobilization, but that this occurs at a site beyond phospholipase C activation and inositol phosphate turnover.     

Diffusion of oxyphenbutazone into synovial fluid,synovial tissue,joint cartilage and cerebrospinal fluid

Summary The diffusion of oxyphenbutazone into synovial and cerebrospinal fluids and synovium and joint cartilage was investigated in 25 patients receiving short-term treatment. In the synovial fluid, the mean oxyphenbutazone concentration, was 57.1±13.4% of the plasma level, due to its excellent diffusion into the joint cavity. In synovial tissue, the oxyphenbutazone level was higher in patients with severe inflammation than in those with no or little inflammation. Penetration into joint cartilage is less than into synovial tissue. In cerebrospinal fluid the concentration was close to the level of free plasma oxyphenbutazone. The findings show increased diffusion of oxyphenbutazone towards its site of action in inflammation.     

Synthesis, activity, and molecular modeling studies of novel human aldose reductase inhibitors based on a marine natural product

Aldose reductase (ALR2) has been implicated in the etiology of diabetic complications, including blindness. Because of the limited number of currently available drugs for the prevention of these long-term complications, the discovery of new ALR2 inhibitors appears highly desirable. In this study, a polybrominated diphenyl ether (1) naturally occurring in a marine sponge was found to inhibit recombinant human ALR2 with an IC(50) of 6.4 microM. A series of polyhalogenated analogues that were synthesized and tested in vitro to explore the structure-activity relationships displayed various degrees of inhibitory activity. The most active compounds were also capable of preventing sorbitol accumulation inside human retinal cells. In this cell-based assay, the most potent synthesized analogue (16) showed a 17-fold increase in inhibitory activity compared to that of sorbinil (IC(50) = 0.24 vs 4 microM). A molecular representation of human ALR2 in complex with the natural product was built using homology modeling, automated docking, and energy refinement methods. AMBER parameters for the halogen atoms were derived and calibrated using condensed phase molecular dynamics simulations of fluorobenzene, chlorobenzene, and bromobenzene. Inhibitor binding is proposed to cause a conformational change similar to that recently reported for zenarestat. A free energy perturbation thermodynamic cycle allowed us to assess the importance of a crucial bromine atom that distinguishes the active lead compound from a much less active close natural analogue. Remarkably, the spatial location of this bromine atom is equivalent to that occupied by the only bromine atom present in zenarestat.     

HPLC determination of adenosine in human synovial fluid

A high-performance liquid chromatographic method has been developed for the quantitative determination of adenosine in human synovial fluid. The method is simple, rapid and, overall, selective. No interference with the components of the biological matrix was observed in these chromatographic conditions. An ODS (250×4.6 mm) 5 μm column was used with an isocratic elution of a phosphate buffer–acetonitrile mobile phase. Detection was carried out on a UV detector at 260 nm. Calibration curve was found to be linear in the 0.7–70 μg ml⁻¹ range. Linear regression analysis of the data demonstrates the efficacy of the method in terms of precision and accuracy. The precision of this method, calculated as the relative standard deviation (RSD) of the recoveries (1.57–2.21%), was excellent. The limits of quantitation (LOQ) and detection (LOD) were respectively 0.7 and 0.2 μg ml⁻¹. The method was applied to some samples of synovial effusion from patients affected by rheumatoid arthritis. The concentrations of adenosine which were found were included in the range of the calibration curve.     

海洋天然产物Polycarpaurines C的合成及其对新型冠状病毒SARS-CoV-2的抑制作用

摘 要：目的：合成海洋天然产物,含硫海洋生物碱Polycarpaurines C,并测试其对SARS-CoV-2的抑制作用。方法：将4-乙酰基苯硼酸为原料经过过氧化氢氧化、甲基化、溴化、引入甲胺基、与2-乙基异硫脲氢溴酸盐缩合、与S2Cl2氧化偶联等反应,经过7步合成Polycarpaurines C,并对其测试对于SARS-CoV-2的抑制作用,利用分子对接技术测试Polycarpaurines C与Mpro的结合。结果：合成了目标天然产物Polycarpaurines C,并对其测试了对SARS-CoV-2的抑制作用,结果显示Polycarpaurine C对SARS-CoV-2具有不错的抑制作用,测得IC50为13.76 μM,但是与阳性药还有一定的差距,可作为药物进一步开发的模板骨架。     

Selective inhibition of cytokinesis in sea urchin embryos by the marine natural product pseudopterolide.

Low concentrations (1-10 microM) of the marine natural product pseudopterolide inhibited cytokinesis and induced formation of multinucleate cells in fertilized Strongylocentrotus purpuratus embryos. As determined by immunofluorescence microscopy using fluorescent stains for actin filaments, microtubules, and chromosomes, pseudopterolide inhibited cytokinesis selectively by disrupting the contractile ring, whereas spindle microtubule organization and mitotic chromosome segregation to opposite spindle poles were unimpaired. At somewhat higher concentrations (16-20 microM), pseudopterolide induced formation of microtubule spiral asters, which are believed to be caused by rotation of the cytoplasm relative to the cell cortex. The effects of pseudopterolide on cytokinesis were cell-cycle dependent. The actions of pseudopterolide in fertilized sea urchin embryos were strikingly similar to the actions of another marine natural product, stypoldione, a structurally unrelated orthoquinone that reacts covalently with the sulfhydryl groups of glutathione, beta-mercaptoethanol, cysteine, and a number of proteins [Mol. Pharmacol. 35:635-642 (1989)]. In the present study, pseudopterolide was also found to react with sulfhydryl groups of glutathione, beta-mercaptoethanol, and cysteine. The results indicate that the cellular target for pseudopterolide, like the target for stypoldione, may be an especially sensitive sulfhydryl-containing protein involved in the formation or function of the contractile ring.