吡咯烷类胞液型磷脂酶A2抑制剂的比较分子力场分析

胞液型磷脂酶A2能引发关节炎，针对胞液型磷脂酶A2的抑制剂有可能成为治疗关节炎的特效药，因此引起了广泛的关注.文章对于吡咯烷类胞液型磷脂酶A2抑制剂进行了三维定量构效关系研究，利用比较分子力场分析构建了该类分子的定量构效关系，得到三维等值线图，为胞液型磷脂酶A2抑制剂的进一步改造提供了有益的启示.     

磷脂酶A2及其复合物的分子动力学研究

本文运用分子动力学方法对磷脂酶A2的自由态以及有机小分子形成的复合物进行了研究．通过模型构建分子动力学模拟得到了磷脂酶A2与配体结合的模型，与磷脂酶A2的自由态相比，其口袋更为宽松，组成口袋的残基的结构趋于稳定，但催化残基的柔性变大．研究结果为药物分子设计提供了有用的信息．     

磷脂酶A2吲哚类抑制剂的结构和活性关系

应用一种新的预测酶-配体复合物亲和性的方法来研究磷脂酶A2蚓哚类抑制剂的结构-活性关系．磷脂酶A2-抑制剂复合物的三维结构是用分子模构的方法搭建的，复合物的亲和性由程序SCORE计算．共分析了12个抑制剂分子．计算所得的亲和性和实验测定值吻合得很好，明显优于用CoMFA方法分析同一系列化合物所给出的结果．通过分析SCORE的输出结果，识别出了蚓哚类抑制剂和磷脂酶A2相互作用的关键位点，为设计新型的磷脂酶A2抑制剂提供了指导．     

基于结构设计合成新型喹啉类分泌型PLA2抑制剂

磷脂酶A2(PLA2)在很多人类疾病的病理研究中起重要作用,是药物化学研究所的热点之一。因此,发展新型的PLA2抑制剂对生物有机研究和临床应用均有重要意义。我们设计合成分泌型PLA2的非底物类似物以寻找新型PLA2抑制剂。本文基于结构设计并合成了喹啉－4－乙酰胺作为PLA2抑制剂,目标化合物结构经1^HNMR,IR,MS和元素分析确认,初步活性检测显示该类化合物具有较好体外活性及动物活性。     

稀土及其配合物对蛇毒磷脂酶A_2活性的影响

分别研究了三价稀土离子 (La3 + ,Eu3 + ,Dy3 + ,Yb3 + )、二乙三胺五乙酸及其衍生物二乙三胺五乙酸 -双二甲酰胺 ,二乙三胺五乙酸 -双 (异烟肼 )与稀土离子的配合物以及Tb -谷氨酰胺配合物对蛇毒磷脂酶A2 活性的影响 .浓度低于 <3μmol/L的稀土离子可以激活磷脂酶A2 ,浓度大于 5 μmol/L后稀土离子对酶活性表现出抑制作用 ;外源Ca2 + 离子的加入可以缓解稀土离子对酶活性的抑制作用 ,表明稀土离子和钙离子是竞争性地结合在酶的活性部位 ;稀土离子和二乙三胺五乙酸及其衍生物的配合物对酶活性没有明显影响 ;Tb -谷氨酰胺在浓度大于 10 μmol/L后开始抑制酶的活性     

Ca2+离子对PLA2结构和功能的影响

从旅顺产白眉蝮蛇(Glyodius blomhoffi Brevicaudaus,GBB)蛇毒(GBBSV)中首次纯化得到了电泳纯和质谱纯磷脂酶A2(GBB-PLA2). 采用电泳、质谱、等离子体发射光谱、紫外、荧光光谱测试技术对GBB-PLA2进行了系统表征. HPLC-ESI-MS法测得的精确分子量为13 965.07 amu,HPLC-ESI-MS/MS法鉴定GBB-PLA2为江浙蝮蛇和尖吻蝮蛇蛇毒PLA2的同源性蛋白,分别得到4个和1个同源性肽段. 凝胶过滤、质谱和SDS-PAGE测试技术结果表明,GBB-PLA2以稳定的单体形式存在. 电感耦合等离子体发射光谱分析法(ICP-AES)测得每1个磷脂酶A2含有1个Ca2+离子,Ca2+是PLA2活性所必需的. Ca2+的除去使PLA2活性降低90%,外加Ca2+明显加速PLA2水解底物DPPC的速度. Ca2+起稳定PLA2结构的作用,可使GBB-PLA2的热变性温度提高3 ℃.     

一种钙/钙调蛋白依赖性的蛇毒磷脂酶A_2

分别研究了钙离子和三价稀土离子对白眉蝮蛇 (Agkistrodon blomhoffii Ussurensis)蛇毒磷脂酶 A2(PLA2 )活性的影响以及钙调蛋白对它的激活作用 .实验结果表明 ,PLA2 的活性对钙离子表现出依赖性 ,钙调蛋白能够激活该蛇毒 PLA2 ,钙调蛋白的拮抗剂三氟甲基吩噻嗪 (Trifluoperazine)能够完全抑制它对 PLA2的激活作用 .三价稀土离子 La3 、Eu3 、Dy3 、Yb3 对该 PLA2 的活性表现出抑制作用 ,其中离子半径较大的La3 和 Eu3 对酶活的抑制程度要小于半径较小的 Dy3 和 Yb3 .     

靶向流感病毒进入(细胞)抑制剂研究进展

目前临床上抗流感病毒感染的药物主要有M2离子通道抑制剂及神经氨酸酶抑制剂两大类.随着流行性感冒、季节性流感、高致病性禽流感的爆发以及耐药流感病毒株的出现,使得对具有新作用机制的抗流感药物的需求更加紧迫.近年来,随着流感病毒进入细胞分子机制研究的深入,为以"病毒进入细胞为靶"的抗流感抑制剂的研究带来了契机.本文在介绍流感病毒进入细胞分子机制的基础上,重点介绍了靶向流感病毒进入的抑制剂及其作用机制的最新研究进展.     

O-GlcNAcase抑制剂

O-连接的N-乙酰葡糖胺糖基化修饰 (O-GlcNAcylation) 是一种存在于蛋白质Ser/Thr上的翻译后修饰。与磷酸化相似,它参与细胞内的信号传递,并与神经退行性疾病、Ⅱ型糖尿病、癌症等许多疾病的发病机理密切相关。O-连接的N-乙酰葡糖胺水解酶 (O-GlcNAcase, OGA) 是生物体内唯一水解蛋白质O-GlcNAc修饰的糖苷酶。因此,研究高效、专一的OGA小分子抑制剂是调节细胞中蛋白质O-GlcNAc水平的有效策略,利于阿尔茨海默病等相关神经退行性疾病新型药物的开发。结合本实验室对OGA抑制剂的研究,本文介绍了OGA的结构、催化机理及目前OGA抑制剂的研究进展,讨论了各种抑制剂的构效关系,并对OGA抑制剂的研究前景进行了展望。     

新型小分子抗凝血药物的合成、作用机制及构效关系研究进展

以达比加群酯为代表的Ⅱa因子抑制剂和以利伐沙班为代表的Xa因子抑制剂在抗凝血药物中起到了重要作用。本文综述了已上市的新型小分子抗凝血药物的的合成、作用机制及其构效关系,并对其进行了深入分析,同时对此类药物的发展趋势和前景进行了展望。     

A Biomimetic Nanoparticle to “Lure and Kill” Phospholipase A2

Inhibition of phospholipase A2 (PLA2) has long been considered for treating various diseases associated with an elevated PLA2 activity. However, safe and effective PLA2 inhibitors remain unavailable. Herein, we report a biomimetic nanoparticle design that enables a “lure and kill” mechanism designed for PLA2 inhibition (denoted “L&K-NP”). The L&K-NPs are made of polymeric cores wrapped with modified red blood cell membrane with two inserted key components: melittin and oleyloxyethyl phosphorylcholine (OOPC). Melittin acts as a PLA2 attractant that works together with the membrane lipids to “lure” in-coming PLA2 for attack. Meanwhile, OOPC acts as inhibitor that “kills” PLA2 upon enzymatic attack. Both compounds are integrated into the L&K-NP structure, which voids toxicity associated with free molecules. In the study, L&K-NPs effectively inhibit PLA2-induced hemolysis. In mice administered with a lethal dose of venomous PLA2, L&K-NPs also inhibit hemolysis and confer a significant survival benefit. Furthermore, L&K-NPs show no obvious toxicity in mice. and the design provides a platform technology for a safe and effective anti-PLA2 approach.     

A Biomimetic Nanoparticle to “Lure and Kill” Phospholipase A2

Inhibition of phospholipase A2 (PLA2) has long been considered for treating various diseases associated with an elevated PLA2 activity. However, safe and effective PLA2 inhibitors remain unavailable. Herein, we report a biomimetic nanoparticle design that enables a “lure and kill” mechanism designed for PLA2 inhibition (denoted “L&K‐NP”). The L&K‐NPs are made of polymeric cores wrapped with modified red blood cell membrane with two inserted key components: melittin and oleyloxyethyl phosphorylcholine (OOPC). Melittin acts as a PLA2 attractant that works together with the membrane lipids to “lure” in‐coming PLA2 for attack. Meanwhile, OOPC acts as inhibitor that “kills” PLA2 upon enzymatic attack. Both compounds are integrated into the L&K‐NP structure, which voids toxicity associated with free molecules. In the study, L&K‐NPs effectively inhibit PLA2‐induced hemolysis. In mice administered with a lethal dose of venomous PLA2, L&K‐NPs also inhibit hemolysis and confer a significant survival benefit. Furthermore, L&K‐NPs show no obvious toxicity in mice. and the design provides a platform technology for a safe and effective anti‐PLA2 approach.     

Crystal Structures of the Plant Phospholipase A1 Proteins Reveal a Unique Dimerization Domain

Phospholipase is an enzyme that hydrolyzes various phospholipid substrates at specific ester bonds and plays important roles such as membrane remodeling, as digestive enzymes, and the regulation of cellular mechanism. Phospholipase proteins are divided into following the four major groups according to the ester bonds they cleave off: phospholipase A1 (PLA1), phospholipase A2 (PLA2), phospholipase C (PLC), and phospholipase D (PLD). Among the four phospholipase groups, PLA1 has been less studied than the other phospholipases. Here, we report the first molecular structures of plant PLA1s: AtDSEL and CaPLA1 derived from Arabidopsis thaliana and Capsicum annuum, respectively. AtDSEL and CaPLA1 are novel PLA1s in that they form homodimers since PLAs are generally in the form of a monomer. The dimerization domain at the C-terminal of the AtDSEL and CaPLA1 makes hydrophobic interactions between each monomer, respectively. The C-terminal domain is also present in PLA1s of other plants, but not in PLAs of mammals and fungi. An activity assay of AtDSEL toward various lipid substrates demonstrates that AtDSEL is specialized for the cleavage of sn-1 acyl chains. This report reveals a new domain that exists only in plant PLA1s and suggests that the domain is essential for homodimerization.     

HPLC Assay of Phospholipase A2 Activity Using Low-Temperature Derivatization of Fatty Acids

Abstract

A modified isocratic reverse phase high-performance liquid-chromatographic (RP-HPLC) method for phospholipase (PLA) activity assay is developed. Natural lecithins and synthetic phospholipids are used as substrates, and released fatty acids are analyzed after single-phase derivatization (with p-bromophenacyl bromide) at low temperature. The procedure allows simultaneous determination of the total and specific phospholipase activity. This method was successfully applied to snake venom PLA₂ activity assay using natural (soybean and egg yolk lecithins) and synthetic (dipalmytoylphosphatydylcholine) substrates for quantitative determination of the enzyme activity.     

Mechanism of inhibition of human secretory phospholipase A2 by flavonoids: rationale for lead design

The human secretory phospholipase A2 group IIA (PLA2-IIA) is a lipolytic enzyme. Its inhibition leads to a decrease in eicosanoids levels and, thereby, to reduced inflammation. Therefore, PLA2-IIA is of high pharmacological interest in treatment of chronic diseases such as asthma and rheumatoid arthritis. Quercetin and naringenin, amongst other flavonoids, are known for their anti-inflammatory activity by modulation of enzymes of the arachidonic acid cascade. However, the mechanism by which flavonoids inhibit Phospholipase A2 (PLA2) remained unclear so far. Flavonoids are widely produced in plant tissues and, thereby, suitable targets for pharmaceutical extractions and chemical syntheses. Our work focuses on understanding the binding modes of flavonoids to PLA2, their inhibition mechanism and the rationale to modify them to obtain potent and specific inhibitors. Our computational and experimental studies focused on a set of 24 compounds including natural flavonoids and naringenin-based derivatives. Experimental results on PLA2-inhibition showed good inhibitory activity for quercetin, kaempferol, and galangin, but relatively poor for naringenin. Several naringenin derivatives were synthesized and tested for affinity and inhibitory activity improvement. 6-(1,1-dimethylallyl)naringenin revealed comparable PLA2 inhibition to quercetin-like compounds. We characterized the binding mode of these compounds and the determinants for their affinity, selectivity, and inhibitory potency. Based on our results, we suggest C(6) as the most promising position of the flavonoid scaffold to introduce chemical modifications to improve affinity, selectivity, and inhibition of PLA2-IIA by flavonoids.     

Design and Synthesis of Novel Pyrazole‐based Lp‐PLA2 Inhibitors

A series of novel pyrazole‐based lipoprotein‐associated phospholipase A₂ (Lp‐PLA₂) inhibitors have been designed and synthetized by a variety of acetophenones via a 10‐step convergent approach. The synthetic approach is carefully optimized, and an unsuccessful alternative route is also discussed. The in vitro biological activity reveals that all the synthesized compounds are potent Lp‐PLA₂ inhibitors with compound 13b being the most potent one (Lp‐PLA₂, IC₅₀=1.5 nmol/L).     

The Cytotoxic Effect of Apis mellifera Venom with a Synergistic Potential of Its Two Main Components—Melittin and PLA2—On Colon Cancer HCT116 Cell Lines

Colon carcinogenesis is ranked second globally among human diseases after cardiovascular failures. Bee venom (BV) has been shown to possess in vitro anticancer effects against several types of cancer cells. The two main biopeptides of Apis mellifera BV, namely, melittin (MEL) and phospholipase A2 (PLA2), are suspected to be the biomolecules responsible for the anticancer activity. The present work aims to evaluate the cytotoxic effect of the A. mellifera venom on human colon carcinoma cells (HCT116), and to assess the synergistic effect of MEL and PLA2 on these cells. After analyzing, through high-pressure liquid chromatography, the proportions of MEL and PLA2 on BV, we have established a cell viability assay to evaluate the effect of BV, MEL, PLA2, and a mixture of MEL and PLA2 on the HCT116 cells. Results obtained showed a strong cytotoxicity effect induced by the A. mellifera venom and to a lower extent MEL or PLA2 alone. Remarkably, when MEL and PLA2 were added together, their cytotoxic effect was greatly improved, suggesting a synergistic activity on HCT116 cells. These findings confirm the cytotoxic effect of the A. mellifera venom and highlight the presence of synergistic potential activities between MEL and PLA2, possibly inducing membrane disruption of HCT116 cancer cells. Altogether, these results could serve as a basis for the development of new anticancer treatments.     

Comparison of the biological properties of several marine sponge-derived sesquiterpenoid quinones

Eight naturally occurring marine-sponge derived sesquiterpenoid quinones were evaluated as potential inhibitors of pyruvate phosphate dikinase (PPDK), a C4 plant regulatory enzyme. Of these, the hydroxyquinones ilimaquinone, ethylsmenoquinone and smenoquinone inhibited PPDK activity with IC50's (reported with 95% confidence intervals) of 285.4 (256.4-317.7), 316.2 (279.2-358.1) and 556.0 (505.9-611.0) microM, respectively, as well as being phytotoxic to the C4 plant Digitaria ciliaris. The potential anti-inflammatory activity of these compounds, using bee venom phospholipase A2 (PLA2), was also evaluated. Ethylsmenoquinone, smenospongiarine, smenospongidine and ilimaquinone inhibited PLA2 activity (% inhibition of 73.2 +/- 4.8 at 269 microM, 61.5 +/- 6.1 at 242 microM, 41.0 +/- 0.6 at 224 microM and 36.4 +/- 8.2 at 279 microM, respectively). SAR analyses indicate that a hydroxyquinone functionality and a short, hydroxide/alkoxide side-chain atC-20 is preferred for inhibition of PPDK activity, and that a larger amine side-chain at C-20 is tolerated for PLA2 inhibitory activity.     

A novel class of zinc-binding inhibitors for the phosphatidylcholine-preferring phospholipase C from Bacillus cereus

The phospholipase C (PLC) isozymes catalyze the hydrolysis of phospholipids to provide diacylglycerol (DAG) and a phosphorylated headgroup. Because DAG has been implicated in cellular signal transduction cascades in mammalian systems, there has been considerable interest in the development of inhibitors of these enzymes. Toward this end, we have discovered that the cyclic N,N'-dihydroxyureas 6-10 inhibit the phosphatidylcholine preferring PLC from Bacillus cereus (PLCBc). This class of inhibitors is believed to function by the bidentate chelation of the N,N'-dihydroxyurea array to one or more of the zinc ions at the active site of the enzyme. Because the affinities of these compounds correlate with the pKaS of the N-OH hydroxyl groups, it is apparent that one or both of the hydroxyl groups must be ionized for effective coordination to the zinc ions. It is also apparent that there may be rather strict steric requirements for these inhibitors.