胰酶的小肽类抑制剂的研究

利用噬菌体表面展示15肽库技术对胰酶抑制剂进行了3轮特异性的筛选.从中得到18个不同的肽序列,与胰酶天然抑制剂活性部位比较,对抑制剂的活性序列进行了分析.根据分析结果合成了1个9肽,其抑制常数为89±10μmol/L.     

类蛋白反应修饰海地瓜酶解物及ACE抑制肽的制备

采用类蛋白反应修饰海地瓜体壁蛋白酶解物,制得修饰肽的血管紧张素转换酶(ACE)抑制活性与原酶解液的活性相比显著提高;模拟消化实验结果表明,修饰肽与胃肠蛋白酶作用后可增强其降压活性;细胞毒性实验结果表明,样品浓度在低于10 mg/mL时对犬肾细胞(MDCK)无毒性,且低浓度可以促进细胞生长.采用超滤、Sephadex G-15凝胶柱层析和RP-HPLC等分离技术纯化修饰产物,得到高活性ACE抑制肽(IC50值为3.67μmol/L),序列结构为NQNFVQYTTNT.紫外光谱分析结果表明,修饰肽与ACE结合可引起吸光度变化.用SYBYL软件对抑制肽与ACE活性位点进行模拟对接,发现该抑制肽能与ACE很好地结合.     

中国毛虾流感病毒神经氨酸酶抑制活性肽的研究

以中国毛虾为原料, 以抑制流感病毒神经氨酸酶(NA)活性为初筛指标, 通过控制酶切位点制备了具有抑制NA活性的酶解液. 利用凝胶层析和高效液相色谱等技术分离纯化出高活性的抑制肽, 其IC₅₀ 值为96.1 μmol/L.经串联质谱测定该抑制肽序列为EISYIHAEAYRRGELK, 紫外光谱分析结果证明该抑制肽能与NA结合.基于反向对接, 应用SYBYL软件模拟抑制肽与NA活性区域结合, 确定了抑制肽与NA的结合位点. 细胞毒性实验测得该抑制肽对细胞的最大无毒浓度(TC₀)为1.26 mg/mL.在红细胞凝集实验中, 随着抑制肽浓度增大, 病毒的凝集价显著降低, 证明抑制肽的抗病毒作用具有多靶点.     

高活性燕麦蛋白源ACE抑制肽的制备、纯化及结构鉴定

利用胰蛋白酶水解燕麦蛋白制备了高血管紧张素转化酶(Angiotensin I-Converting Enzyme, ACE)抑制活性的燕麦蛋白酶解物, 分别采用离子交换色谱、凝胶过滤色谱和反相高效液相色谱等分离手段从酶解物中分离出一种新的强活性ACE抑制肽, 其IC50值为77.3 μmol/L; 通过基质辅助激光解析电离飞行时间串联质谱对其进行结构鉴定, 其氨基酸序列为Glu-Gly-Gly-Tyr-Arg.     

反相液相色谱-串联质谱法鉴定油菜蜂花粉中的蛋白质及活性肽

基于鸟枪法蛋白质组学分析方法,使用反相液相色谱-串联质谱（RPLC-MS/MS）系统分析油菜蜂花粉蛋白质的胰蛋白酶酶解产物,结合数据库检索,共鉴定到353条肽段。鉴定到的肽段所归属的蛋白质中有239个蛋白质可检索到其分子生物学功能,主要功能为结合活性、酶活性、运输活性、抑制活性等。根据血管紧张素转化酶（ACE）抑制肽活性与多肽构效之间的关系,从鉴定到的肽段中筛选并适当修饰后得到5条可能具有ACE抑制活性的肽段,化学合成肽段后进行了活性验证。结果表明5条肽段均具有良好的活性,其中肽段AELDIVLALF和LAVNLIPFP表现出较高的ACE抑制活性,半数抑制浓度（IC₅₀）分别为（10.65±0.50）μmol/L和（23.66±1.08）μmol/L。该方法速度快,成本低,大大缩短了鉴定周期,达到了高通量筛选生物活性肽的目的。     

新型氨基嘧啶环戊并[b]吲哚衍生物的合成及抗肿瘤活性

3-吲哚丙酸为原料，经环化、N-甲基化、Claisen-Schmidt缩合、环加成反应得到新型氨基嘧啶环戊并[b]吲哚衍生物。经¹HNMR、¹³CNMR以及HR-MS（ESI）表征。采用细胞增殖抑制实验法（MTT法）测定6a~6j对人肝癌（HepG-2）、人子宫颈癌（Hela）和人鼻咽癌（CNE-1）瘤株的体外抑制活性。6b对这些肿瘤细胞的抑制活性最好，其IC50值分别为大于50 μmol/L（HepG-2）、15.2 μmol/L（Hela）和14.8 μmol/L（CNE-1），但均弱于顺铂。      

快速高分离度液相色谱-质谱联用方法筛选Tau蛋白激酶2抑制剂

建立了测定Tau 蛋白激酶2(Tau protein kinase 2, TPK2)催化反应产物磷酸化十肽(PKpTPKKAKKL)的快速高分离度液相色谱-质谱方法(RRLC/MS)用于筛选TPK2抑制剂. 反应溶液总体积为50 μL, 将终浓度为20 nmol/L的TPK2与终浓度为5 μmol/L的底物十肽(PKTPKKAKKL)于30 ℃反应30 min, 用等体积乙腈终止反应并加入终浓度为1 μmol/L的磷酸化十肽(PKpTPKKAKKV)作为内标, 采用Agilent SB-C₁₈色谱柱, 以乙腈-水为流动相梯度洗脱分离后, 用RRLC/MS进行定性及定量分析. 该方法可通过精确定量一定时间内的酶促反应产物来反映酶活性被抑制的程度, 作为TPK2抑制剂的筛选方法, 本方法具有简单、 灵敏、 快速的优点, 能够避免光谱法筛选可能产生的假阳性结果, 可用于TPK2的先导化合物的高通量筛选.     

具有谷胱甘肽过氧化物酶(GPx)活力的含硒四肽(SecRGD)

以精氨酸-甘氨酸-天冬氨酸(RGD)序列为基础, 在N-端引入硒代半胱氨酸(Sec)设计了SecRGD序列模拟谷胱甘肽过氧化物酶(GPx), 利用Fmoc固相合成法合成了SecRGD. 采用ESI-MS质谱和氢化物原子荧光光谱法对硒肽进行表征, 采用酶偶联法进行GPx活力测定和酶动力学分析, 用噻唑蓝(MTT)比色法评价了硒肽的抗氧化效果. 结果表明, 该硒肽的存在形式为SecRGD的二聚体. 该硒肽具有GPx活力, 其催化谷胱甘肽(GSH)还原H2O2的GPx活力为5.54 U/μmol, 高于经典的GPx模拟物Ebselen. 稳态动力学分析结果表明, 该硒肽的催化机制为乒乓机制. 该硒肽具有分子量小, 易溶于水, 毒性低及可有效保护Vero细胞免受氧化损伤的优点, 具有作为抗氧化药物的应用前景.     

新型喹唑啉衍生物的设计、合成及体外抗肿瘤活性

为了寻找高效、毒性小的抗肿瘤药物,设计并合成了一系列新型的喹唑啉衍生物,其结构经¹H NMR、 ¹³C NMR、 IR、 MS（ESI）和元素分析表征。采用细胞增殖抑制实验法（MTT法）测定了化合物3a～10c对人盲肠腺癌细胞（HCT-8）、人肝癌细胞（HepG2）、人肺癌细胞（A549） 和人高转移性肝癌细胞（MHCC-97H）的体外抑制活性。结果表明：4-位为氨基苯甲基丙二酸二甲酯时（3a~6c）,化合物的肿瘤抑制作用比其相应水解产物（7a~10c）更强;氨基在苯甲基丙二酸二甲酯间位时,抑制活性明显优于对位取代。其中,5c对HCT-8、 A549及MHCC-97H的抑制活性较显著,IC₅₀值分别为13.613 μmol/L, 13.578 μmol/L和25.804 μmol/L。      

N端结构改造的抑咽侧体素拟肽类化合物的设计、合成及生物活性研究

抑咽侧体素(Allatostatin,AST)是一类具有抑制咽侧体合成保幼激素(JH)功能的昆虫神经肽.然而,由于天然AST易代谢失活,限制了其在害虫控制方面的应用.本研究在前期工作基础上,以高活性拟肽类化合物B1作为二级先导,采用模拟肽学方法进行结构改造,通过N端引入脲桥结构消除酶解位点,以提高抗酶解能力,共设计合成了10个目标化合物II01~II10,并对其抑制保幼激素合成离体生物活性进行了测定.结果表明,10个化合物均具有抑制保幼激素合成活性,其中,II04(IC_(50)=0.21μmol/L)抑制活性略高于一级先导天然核心五肽(IC_(50)=0.24μmol/L).虽然抑制活性比二级先导B1(IC_(50)=0.09μmol/L)略低,但简便的合成路线,使其成为具有研究潜力的先导化合物.     

血红蛋白片段的合成及生物活性

采用多肽固相合成方法, 以Wang 树脂为载体, Fmoc为N-端氨基酸保护基, HOBt-HBTU为缩合试剂, 合成了一系列血红蛋白α链的片段, 产物经RP-HPLC和质谱进行了确定. 生物活性研究结果表明, 该系列多肽具有较高的血管紧张素Ⅰ转换酶抑制活性, 但不具有α-葡萄糖苷酶抑制活性.     

血管紧张素转化酶活性抑制剂──丝素肽的分离、纯化和结构鉴定

 可溶性丝素粉末经碱性蛋白酶Alcalase水解后 ,其酶解产物对血管紧张素转化酶 (ACE)的活性有很强的抑制作用。采用凝胶过滤色谱SephadexG 15和反相高效液相色谱 (RP HPLC)对水解度为 2 0 %的酶解产物进行分离纯化 ,利用质谱鉴定其中一种ACE抑制剂是肽 ,其结构为Gly Tyr。     

Antihypertensive Effect of Angiotensin‐Converting Enzyme Inhibitory Peptide Obtained from Hen Ovotransferrin

Angiotensin I‐converting enzyme (ACE) inhibitory peptide was isolated from the hen ovotransferrin hydrolysate using chymotryptic hydrolysis by two steps of reverse‐phase high‐performance liquid chromatography. The amino sequence of this novel peptide was identified as Lys‐Val‐Arg‐Glu‐Gly‐Thr‐Thr‐Tyr that inhibited ACE activity in vitro in a concentration‐dependent manner with an effective concentration (IC₅₀) of 102.8 μM. Also, this inhibition was identified as noncompetitive using the Lineweaver‐Burk plot. Moreover, the antihypertensive action of this novel peptide was investigated by an intravenous injection into spontaneously hypertensive rats (SHR). A dose‐dependent reduction of systolic blood pressure by this peptide was observed after 40 min of treatment and it decreased the blood pressure markedly at the maximal dose (1 nmol/mL/kg). The maximal blood pressure lowering activity of this peptide was calculated as 163% of captopril (10 pmol/mL/kg) that was used as positive control. In conclusion, the obtained data suggests that Lys‐Val‐Arg‐Glu‐Gly‐Thr‐Thr‐Tyr has an ability to inhibit ACE activity and decrease the systolic blood pressure in hypertensive animals.     

反相高效液相色谱/基质辅助激光解吸电离飞行时间质谱 分离鉴定螺蛳血管紧张素转换酶抑制肽

运用反相高效液相色谱(RP-HPLC)对酶解螺蛳腹足肌得到的血管紧张素转换酶(ACE)抑制肽进行两步分离提纯,第一步主要得到8个组分;选取其中活性最高的组分进一步分离,得到2个组分,其中活性较高组分的ACE半抑制浓度为43.5 μmol/L,基本为单一肽组分。对提纯的组分分别使用高效液相色谱/电喷雾离子质谱法(HPLC/ESI-MS)和基质辅助激光解吸电离飞行时间质谱法(MALDI-TOF MS)进行分析,同时结合氨基酸组成分析结果,最终得到的肽链一级结构为Lys-Glu-Ile-Trp(KEIW),符合已知的高活性ACE抑制肽的结构规律。经过对两种方法分析过程的比较,认为ESI-MS可以得到多方面的信息,但无法确定肽的序列;MALDI-TOF MS可以得到精确的二级质谱图(m/z精确至0.0001),从而可以得到确定的肽的序列。     

Microwave-assisted Solid-phase Synthesis,Biological Evaluation and Molecular Docking of Angiotensin I-converting Enzyme Inhibitors

Short peptides based on the tripeptides, Leu-Arg-Pro and Leu-Lys-Pro, were synthesized by microwaveassisted solid-phase synthesis method, in order to make a search for potential inhibitors for angiotensin I-converting enzyme(ACE) with minimum side effects in the treatment of hypertension. One peptide with the sequence Leu-Arg-Pro-Phe-Phe shows the strongest inhibition towards ACE with an IC50 value of 0.26 μmol/L in vitro. The study of structure-activity relationship shows that the introduction of a bulky group into the N-terminal of this series of inhibitors may enlarge steric hindrance, resulting in the poor inhibitory activity towards ACE. The inhibitory activity decreased in turn when L-Pro, D-Pro or Ac6c was at the C-terminal respectively. The binding interaction between each of these inhibitors and testicular ACE(tACE) was performed by molecular docking. The results suggest that Leu-Arg-Pro-Phe-Phe mainly occupied the S1 subsite of tACE, and made contact with tACE via seven H-bonds. It appeared that the site on the peptide that bound with tACE was influenced by the configuration of the amino acid, L- or D-form, at the C-terminal of the peptide.     

Structure-based design and optimization of antihypertensive peptides to obtain high inhibitory potency against both renin and angiotensin I-converting enzyme

The human renin–angiotensin system (RAS) plays an essential role in regulating blood pressure and systemic vascular resistance. Renin and angiotensin I-converting enzyme (ACE) are two key enzymes in RAS and have long been recognized as attractive antihypertensive targets. Here, a synthetic strategy was proposed integrating quantitative structure–activity relationship (QSAR), molecular dynamics (MD) simulation and binding free energy analysis to discover novel dual renin and ACE peptidic inhibitors. With the strategy a number of candidates were generated virtually, from which eight promising peptides were selected and synthesized for biological assay. Consequently, three peptides (RYLP, YTAWVP and YRAWVL) were successfully identified to have satisfactory inhibitory profile against both renin and ACE with IC₅₀ values of <1 mM and <10 μM, respectively. Structural analysis and energetic dissection revealed different binding modes of peptide to renin and ACE; a peptide only inserts its C-terminus into the active site of ACE, whereas the whole peptide packs tightly against renin. In addition, when limited to structural diversity it is hard to reconcile the renin and ACE inhibitory activities of short peptides such as dipeptides. These findings can be used to guide peptide optimization with improved biological activity.     

A new monoterpenoid glycoside from Myrica esculenta and the inhibition of angiotensin I-converting enzyme

One new monoterpenoid glycoside, myresculoside (1), and eleven known compounds, were isolated from methanol extract of Myrica esculenta leaves by repeated column chromatography. The effects of these compounds on angiotensin I-converting enzyme (ACE) inhibition were investigated. Compounds 3 and 4 showed the most potent ACE inhibition with rates of 29.97% and 25.63% at concentration of 100 μM, respectively. Compounds 5, 6, and 11 showed weak activity with inhibitory rates of 0.07-1.41% at concentration of 100 μM.