连翘酯苷A与弹性蛋白酶相互作用的研究

连翘的主要成分之一连翘酯苷A(FSA)有抗炎活性,对弹性蛋白酶(PPE)有抑制作用。本文通过光谱法以及分子对接模拟法研究了FSA与PPE的相互作用。结果表明,FSA对PPE之间存在π-阳离子、π-π共轭、氢键和疏水作用力等多种作用,能够形成稳定的1∶1的复合物,使得FSA能够抑制PPE的活性,具有抗炎作用。     

来源于天然产物的基质金属蛋白酶（MMPs）抑制剂

基质金属蛋白酶（MMPs）参与一系列重大疾病的病理过程，基质金属蛋白酶抑制剂具有广阔的药用前景。本文概述了基质金属蛋白酶抑制剂的研究历史和最新的研究理念。重点回顾总结了天然产物中基质金属蛋白酶的活性抑制成分和对基质金属蛋白酶转录表达抑制的天然产物成分以及这些化合物的抗癌效果。     

快速高分离度液相色谱/质谱方法筛选组织蛋白酶B的抑制剂

1引言组织蛋白酶B(CB)是溶酶体内半胱氨酸蛋白水解酶,属于木瓜蛋白酶家族,其特异性底物为Z-Arg-Arg-7-氨基-4-甲基香豆素[1],CB催化底物肽键断裂生成7-氨基-4-甲基香豆素。近年来的研究表明,CB在肿瘤的发展中起作用的一类关键酶[2],CB通过多种机制促进血管形成,从而促进肿瘤生长,同时降解基底膜主要成分以及激活胶原酶、尿激酶等其它蛋白酶发生协同作用,使肿瘤易侵袭、转移和扩散。在肿瘤组织中还发现CB与CB抑制剂平衡作用也参与肿瘤的发     

利用多中心合成方法同步合成含N-取代甘氨酸的类肽化合物

N-取代甘氨酸聚合体是一类新的聚合体化合物, 同多肽相比, N-取代甘氨酸聚合体具有抗酶解稳定性, 同时使用市售的伯胺作为构建单元(building block)大大地扩展了天然多肽分子结构的多样性, 本文报道了利用多中心多肽合成方法合成了九个含N-取代甘氨酸聚合体多肽杂合休, 由于此类聚合体具有抗酶的能力, 因此可用于蛋白酶抑制剂的研究。     

慈菇蛋白酶抑制剂的抑制特性及其活性中心的探讨

本文采用亲和层析分离纯化了结晶慈菇蛋白酶抑制剂A和B,抑制剂A和B均为双头多功能蛋白酶抑制剂,抑制剂A能同时等当量抑制胰蛋白酶和胰凝乳蛋白酶,对激肽释放酶的抑制作用较弱,抑制剂B能当量抑制2克分子的胰蛋白酶,对激肽释放酶的抑制活力高于抑制剂A,但对胰凝乳蛋白酶的抑制作用远比抑制剂A弱。化学修饰以及胰蛋白酶与胰凝乳蛋白酶对抑制剂A的竞争性结合表明:抑制剂A和B的两个活性中心均为Lys和Arg残基,其中Lys活性中心专一抑制胰蛋白酶,而由Arg活性中心构成的活性区域则表现为多功能,能抑制多种蛋白酶,从抑制剂A和B的结构特征推测,两活性中心应分别为Lys-Ser(44—45)及Arg-Tyr-Lys(76—78),在抑制剂A中还存在一疏水性残基参与对胰凝乳蛋白酶的抑制,此残基位于由Arg活性中心所构成的活性区域中。     

肽的酶促合成及其反应机制

先后以α-胰凝乳蛋白酶和木爪酶作催化剂以逐级缩合方式合成了[色]~4亮-脑啡肽酰酶(TyGlyGlyTrpLeuNH_2),在合成中,对酶的专一性,有机溶剂的选择和合成条件等进行试探,比较了固相酶促法和液相酶促法,α-胰凝乳蛋白酶和木爪酶连接肽键中,以N-酰基氨基酸酯作为供体比相应的游离酸好,如用嗜热菌蛋白酶只能以N-酰基氨基酸为羧基组份,初步的体外实验说明合成的[色]~4-亮脑啡肽酰酶与天然亮-脑啡肽有类     

2′,3′-乙氧甲撑基腺苷5′-硫代磷酰氨基酸酯的合成和谱学分析

核苷磷酰氨基酸酯化合物是一类倍受重视的药物 ,特别是它可作为寡聚核苷酸的类似物用于反义药物 [1] .HIV逆转录酶是治疗艾滋病的有效靶点 ,目前普遍使用的抗 HIV核苷类似物中 ,2′,3′-双脱氧核苷 (dd Ns)具有良好的疗效 [2 ] .苯氧基取代的核苷 -磷酰氨基酸酯是 HIV逆转录酶的有效抑制剂[3 ] ,其药物毒性比 dd Ns低 ,但容易在体内被核酸酶水解 .由于硫代磷酸对核酸酶具有抵抗性 ,它可以抑制核酸酶对它的水解 [4 ] ,因此我们设计合成了核苷 5′-硫代磷酰氨基酸酯化合物 ,希望开发出一种全新的 HIV逆转录酶抑制剂 .由于分子中引入氨基…     

2,6-二氯靛酚滴定法测定蜂胶中维生素C

蜂胶是一种具有重要生理功效的天然药物和保健食品[1],是蜜蜂从植物芽孢和树干处采集的树脂,混入蜜蜂上颚腺分泌物和蜂蜡等形成的一种具有芳香味的黏性胶状固形物,其化学成分复杂,含有酚、酸、醇、酯、醚、萜、烯、甾醇及多种微量元素[2]。近年来,国内外学者所做的大量研究表明:蜂胶具有治疗心血管、糖尿病、皮肤病、胃肠疾病、抗癌、增强免疫、抗菌消炎等重要的生理功效[3],已经成为保健食品研究领域的热点。随着乙醇提取法、酶解法、超临界     

组蛋白去乙酰化酶抑制剂的分子对接与构效关系研究

肿瘤是仅次于心血管疾病的第二大"杀手",肿瘤的发生与核心组蛋白的乙酰化及去乙酰化的失衡有密切的关系.这两种酶是一对功能相互拮抗的蛋白酶,分别对组蛋白氮端氨基酸残基进行乙酰化和去乙酰化调节染色质的结构,进而调控基因转录.组蛋白去乙酰酶抑制剂在体外和体内实验中均能引起乙酰化核小体组蛋白的堆积,提高p21基因的表达水平,抑制肿瘤细胞的增殖,诱导细胞分化或凋亡.研究组蛋白去乙酰化酶抑制剂和酶的分子对接可以阐明抑制剂与蛋白酶的作用模式,揭示抗癌机理.同时利用CoMFA方法研究了抑制剂结构和活性之间的关系,得到了预测能力较好的定量模型.     

α-卤代丁二酰氧肟酸类基质金属蛋白酶抑制剂的合成及活性评价

基质金属蛋白酶(MMPs)是一族Zn2+依赖的蛋白水解酶. 该族酶的过度表达与多种病理过程密切相关, 因此其抑制剂可用于这些疾病的治疗. 本文设计合成了15个α-卤代丁二酰氧肟酸类新型基质金属蛋白酶抑制剂, 经核磁共振氢谱和质谱进行了结构表征, 并以伊洛马司他(Ilomastat)为阳性对照, 分别测定了它们对基质金属蛋白酶MMP-2和MMP-9的体外抑制活性. 结果显示, 4个化合物对MMP-2的抑制活性与阳性对照相当; 5个化合物对MMP-9的抑制活性与对照药相当.     

Binding structure of elastase inhibitor scyptolin A

Natural bioactive compounds are of general interest to pharmaceutical research because they may be used as leads in drug development campaigns. Among them, scyptolin A and B from Scytonema hofmanni PCC 7110 are known to inhibit porcine pancreatic elastase, which in turn resembles the attractive drug target neutrophil elastase. The crystal structure of scyptolin A as bound to pancreatic elastase was solved at 2.8 A resolution. The inhibitor occupies the most prominent subsites S1 through S4 of the elastase and prevents a hydrolytic attack by covering the active center with its rigid ring structure. The observed binding structure may help to design potent elastase inhibitors.     

Benzimidazole tethered thioureas as a new entry to elastase inhibition and free radical scavenging: Synthesis,molecular docking,and enzyme inhibitory kinetics

The porcine pancreatic elastase inhibition and free-radical scavenging play a crucial role in age progression. All the series of 10 newly synthesized benzimidazole thioureas ( 4a-j ) were assessed for elastase inhibition and radical scavenging activity to identify the suitable anti-aging ingredient for cosmetics products. The compounds 4e , 4f , 4g , and 4h showed inhibition better than the standard, while compound 4f showed the most significant elastase inhibition with the IC₅₀ value of 1.318 ± 0.025 μM compared with oleanic acid IC₅₀ 13.451 ± 0.014 used ±1.989 and 41.563 ± 0.824, respectively, as standard. Molecular docking studies were performed and the compound 4f showed binding energy of 7.2 kcal/mol. Kinetics studies revealed inhibition of the pancreatic elastase in a competitive manner. The relative binding energy and structure activity relationship (SAR) identified compound 4f as an effective inhibitor of porcine pancreatic elastase. Compounds 4e and 4i showed remarkable free-radical scavenging activity with SC₅₀ values of 26.421.     

Amino acids and peptides. XXX. Synthesis of eglin c (41-49) and eglin c (60-63) and examination of their inhibitory activity towards human leukocyte elastase, cathepsin G, porcine pancreatic elastase and alpha-chymotrypsin

H-Ser-Pro-Val-Thr-Leu-Asp-Leu-Arg-Tyr-OH and H-Thr-Asn-Val-Val-OH, which correspond to the sequences 41-49 and 60-63 of eglin c, respectively, were synthesized by a conventional solution approach using the newly developed 6-chloro-2-pyridyl ester method. The inhibitory activities of the above two peptides against human leukocyte elastase, cathepsin G, porcine pancreatic elastase and alpha-chymotrypsin were examined in comparison with those of the corresponding methyl esters.     

Reactivity and selectivity in the inhibition of elastase by 3-oxo-beta-sultams and in their hydrolysis

3-oxo-beta-sultams are both beta-sultams and beta-lactams and are a novel class of time-dependent inhibitors of elastase. The inhibition involves formation of a covalent enzyme-inhibitor adduct with transient stability by acylation of the active-site serine resulting from substitution at the carbonyl centre of the 3-oxo-beta-sultam, C-N fission, and expulsion of the sulfonamide. The lead compound, N-benzyl-4,4-dimethyl-3-oxo-beta-sultam 1 is a reasonably potent inhibitor against porcine pancreatic elastase with a second-order rate constant of 768 M(-1) s(-1) at pH 6, but also possesses high chemical reactivity with a half-life for hydrolysis of only 6 mins at the same pH in water. Interestingly, the hydrolysis of 3-oxo-beta-sultams occurs at the sulfonyl centre with S-N fission and expulsion of the amide leaving group, whereas the enzyme reaction occurs at the acyl centre. Increasing selectivity between these two reactive centres was explored by examining the effect of substituents on the reactivity of 3-oxo-beta-sultam towards hydrolysis and enzyme inhibition. The inhibition activity against porcine pancreatic elastase has a much higher sensitivity to substituent variation than does the rate of alkaline hydrolysis. A difference of 2000-fold is observed in the second-order rate constants, k(i), for inhibition whereas there is only a 100-fold difference in the second-order rate constants, k(OH), for alkaline hydrolysis within the series. The higher sensitivity of enzyme inhibition to substituents than that of simple chemical reactivity indicates a significant degree of molecular recognition of the 3-oxo-beta-sultams by the enzyme.     

The efficiency of C-4 substituents in activating the beta-lactam scaffold towards serine proteases and hydroxide ion

The presence of a leaving group at C-4 of monobactams is usually considered to be a requirement for mechanism-based inhibition of human leukocyte elastase by these acylating agents. We report that second-order rate constants for the alkaline hydrolysis and elastase inactivation by N-carbamoyl monobactams are independent of the pKa of the leaving group at C-4. Indeed, the effect exerted by these substituents is purely inductive: electron-withdrawing substituents at C-4 of N-carbamoyl-3,3-diethylmonobactams increase the rate of alkaline hydrolysis and elastase inactivation, with Hammett pI values of 3.4 and 2.5, respectively, which indicate the development of a negative charge in the transition-states. The difference in magnitude between these pI values is consistent with an earlier transition-state for the enzymatic reaction when compared with that for the chemical process. These results suggest that the rate-limiting step in elastase inactivation is the formation of the tetrahedral intermediate, and that beta-lactam ring-opening is not concerted with the departure of a leaving group from C-4. Monobactam sulfones emerged as potent elastase inhibitors even when the ethyl groups at C-3, required for interaction with the primary recognition site, are absent. For one such compound, a 1 : 1 enzyme-inhibitor complex involving porcine pancreatic elastase has been examined by X-ray crystallography and shown to result from serine acylation and sulfinate departure from the beta-lactam C-4.     

Purification and partial characterization of the pancreatic proteolytic enzymes trypsin, chymotrypsin, and elastase from the chicken.

The purpose of this work was to isolate, purify and partially sequence trypsin, chymotrypsin and elastase from the chicken pancreas. The extraction of the pancreatic zymogens with 0.5 M CaCl2 at pH 7.5 for 9 h appeared to be most effective in obtaining maximum recovery of the three enzymes. The sequential Cucurbita maxima trypsin inhibitor I/bovine pancreas trypsin inhibitor/soybean trypsin inhibitor affinity chromatography gave the best result for the isolation of trypsin, chymotrypsin and elastase, respectively, from the same extract. For each proteinase, multiple form of enzymatic activity could be observed after gel electrophoresis and each form was further purified on an ion-exchange column. The N-terminal amino acid sequence of trypsin and chymotrypsin showed homologies with the bovine enzymes whereas elastase showed homologies with the porcine enzyme. The molecular mass of trypsin, chymotrypsin and elastase were estimated to be 23,500, 25,700 and 25,000, respectively, which are values close to those in mammalian species. Although some kinetic constants (Km and k(cat)/Km) appeared different from those observed in other species, the pH dependent enzymatic activities were similar to those reported in other animal species.     

Probing the degradation mechanism of forsythiaside A and simultaneous determination of three forsythiasides in Forsythia preparations by a single marker

Forsythiaside A is the major component of Forsythia suspensa. This study investigated the degradation mechanism of forsythiaside A. Eight degraded components including forsythiaside I, forsythiaside H, forsythiaside E, caffeic acid, suspensaside A, β‐hydroxy forsythiaside I, β‐hydroxy forsythiaside H, and β‐hydroxy forsythiaside A were identified by using ultra‐high performance liquid chromatography quadrupole time‐of‐flight mass spectrometry. Then, the quantitative analysis of multi‐components by a single‐marker was performed with ultra‐high performance liquid chromatography to simultaneously determine forsythiaside A, forsythiaside H, and forsythiaside I in Forsythia suspensa preparations. The result showed good linear relationships within 2.871–287.1, 0.231–23.1, and 0.983–98.3 μg/mL (r > 0.9998), with average recoveries of 97.7, 95.7, and 95.8% and relative standard deviations of 1.4, 2.4, and 1.8%, respectively. Using forsythiaside A as an internal reference, the relative retention values of forsythiaside H and forsythiaside I to forsythiaside A were calculated to be 0.89 and 0.61, respectively, and the relative correction factors were 0.816 and 0.799, respectively. The method for quantitative analysis of multi‐components by a single‐marker was applied to evaluate the overall quality of forsythia preparations. There was no significant difference in the measurement results of the method developed and the method of external standard.     

Evaluation of Ionic Liquids and Ionic Liquids Active Pharmaceutical Ingredients Inhibition in Elastase Enzyme Activity

Human neutrophil elastase (HNE) is used as diagnostic biomarker for inflammation/infection. In this work, 10 ionic liquids (ILs) and 11 ionic liquids active pharmaceutical ingredients (ILs-APIs) were tested to evaluate the inhibition effect on the activity of porcine pancreatic elastase enzyme, frequently employed as a model for HNE. The insertion of ionic liquids in some drugs is useful, as the insertion of ILs with inhibitory capacity will also slow down all processes in which this enzyme is involved. Therefore, a spectrophotometric method was performed to the determination of EC₅₀ values of the compounds tested. EC₅₀ values of 124 ± 4 mM to 289 ± 11 mM were obtained, with the most toxic IL for elastase being tetrabutylammonium acetate and the least toxic 1-butyl-3-methylimidazolium acetate. Moreover, sodium salicylate (raw material) presented the lower and benzethonium bistriflimide the higher EC₅₀ when compared with all the IL-APIs tested. This work provides significant information about the effect of the studied IL and IL-APIs in elastase enzyme activity.     

Molecular dynamics simulation of the 1:1 enzyme-ligand complex between porcine pancreatic elastase and acetyl-alanine-proline-alanine

A 31.5 picosecond (ps) MD calculation has been completed for the 1:1 enzyme-ligand complex between porcine pancreatic elastase (PPE) and acetyl-alanine-proline-alanine (APA). The 1:2 complex studied crystallographically at product saturation conditions precludes the study of a 1:1 complex (PPE and APA1); this objective has been achieved by computational methods described here. The acetyl group of the ligand was found to occupy two neighboring sites, one within the primary specificity site and the other out into solution. The primary change of the ligand structure is ψ₁ torsion angle being 171.5°. Supported by an interactive graphic display, the dynamical fluctuations of a smaller ligand compared with the width of the active site as PPE were observed by the MD simulation, although the complex has not been detected by any spectroscopic method.     

Study on inclusion behaviour of forsythiaside A with β-cyclodextrin

Inclusion behaviour of forsythiaside A with β-cyclodextrin (β-CD) was investigated by fluorescence spectrum, nuclear magnetic resonance (NMR) and molecular modelling. A ratio of 1:1 stoichiometry has been proposed for the inclusion complex of forsythiaside A with β-CD in aqueous media according to the continuous variation Job’s method based on the fluorescence spectroscopy data. The stability constant (K) of the inclusion complex was 669 M^?1. The pH, ionic strength and temperature of solution showed great effect on the formation of inclusion complex. The spatial configuration of complex demonstrated that the B ring of forsythiaside A might be embedded inside the lipophilic cavity of β-CD and the A ring of the forsythiaside A might be exposed outside the cavity of β-CD according to NMR spectra and molecular modelling.