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147396-10-9,Endotoxin Inhibitor,H2N-Lys-Thr-Lys-Cys-Lys-Phe-Leu-Lys-Lys-Cys-COOH(Disulfide Bridge:Cys4-Cys10),H2N-KTKCKFLKKC-OH(Disulfide Bridge:C4-C10),杭州专肽生物的产品

内毒素抑制剂、Endotoxin Inhibitor

内毒素抑制剂与肽类抗生素和内毒素抑制剂多粘菌素B结构相似,可作为抗生素治疗的佐剂。

编号:181317

CAS号:147396-10-9

单字母:H2N-KTKCKFLKKC-OH(Disulfide Bridge:C4-C10)

纠错
  • 编号:181317
    中文名称:内毒素抑制剂、Endotoxin Inhibitor
    英文名:Endotoxin Inhibitor
    CAS号:147396-10-9
    单字母:H2N-KTKCKFLKKC-OH(Disulfide Bridge:C4-C10)
    三字母:H2N-Lys-Thr-Lys-Cys-Lys-Phe-Leu-Lys-Lys-Cys-OH(Disulfide Bridge:Cys4-Cys10)
    氨基酸个数:10
    分子式:C55H97N15O12S2
    平均分子量:1224.58
    精确分子量:1223.69
    等电点(PI):-
    pH=7.0时的净电荷数:7.91
    平均亲水性:0.83
    疏水性值:-0.86
    外观与性状:白色粉末状固体
    消光系数:-
    来源:人工化学合成,仅限科学研究使用,不得用于人体。
    纯度:95%、98%
    盐体系:可选TFA、HAc、HCl
    储存条件:负80℃至负20℃
    标签:二硫键环肽    抑制剂相关肽(Inhibitor Peptide)   

  • 环肽KTKCKFLKKC与内毒素脂多糖(LPS)的毒性成分脂质a形成强复合物,Ka=0.56·10⁷ M⁻¹.它在体外对LPS解毒,并在体内防止LPS诱导的细胞因子释放和致死。此外,它既没有显示抗生素活性也没有毒性,因此可能用于预防和治疗LPS介导的疾病。KTKCKFLKKC与肽类抗生素和内毒素抑制剂多粘菌素B结构相似,可作为抗生素治疗的佐剂。

    The cyclic peptide KTKCKFLKKC has ben shown to form a strong complex with lipid A, the toxic component of the endotoxin lipopolysaccharide (LPS) with Ka = 0.56 · 10⁷ M⁻¹. It detoxifies LPS in vitro and prevents LPS-induced cytokine release and lethality in vivo. Furthermore, it shows neither antibiotic activity nor toxicity and might therefore be useful in the prophylaxis and treatment of LPS-mediated diseases. KTKCKFLKKC bears a structural resemblance to the peptide antibiotic and endotoxin inhibitor polymyxin B. It could be of use as an adjuvant for antibiotic therapy.

    内毒素抑制剂与肽类抗生素和内毒素抑制剂多粘菌素B结构相似,可作为抗生素治疗的佐剂。

    Endotoxin inhibitor bears a structural resemblance to the peptide antibiotic and endotoxin inhibitor polymyxin B. It could be of use as an adjuvant for antibiotic therapy.

    二硫键广泛存在与蛋白结构中,对稳定蛋白结构具有非常重要的意义,二硫键一般是通过序列中的2个Cys的巯基,经氧化形成。
     

    形成二硫键的方法很多:空气氧化法,DMSO氧化法,过氧化氢氧化法等。
     

    二硫键的合成过程,  可以通过Ellman检测以及HPLC检测方法对其反应进程进行监测。  
       

    如果多肽中只含有1对Cys,那二硫键的形成是简单的。多肽经固相或液相合成,然后在pH8-9的溶液中进行氧化。      
     

    当需要形成2对或2对以上的二硫键时,合成过程则相对复杂。尽管二硫键的形成通常是在合成方案的最后阶段完成,但有时引入预先形成的二硫化物是有利于连合或延长肽链的。通常采用的巯基保护基有trt, Acm, Mmt, tBu, Bzl, Mob, Tmob等多种基团。我们分别列出两种以2-Cl树脂和Rink树脂为载体合成的多肽上多对二硫键形成路线:
     

    二硫键反应条件选择    
     

     二硫键即为蛋白质或多肽分子中两个不同位点Cys的巯基(-SH)被氧化形成的S-S共价键。 一条肽链上不同位置的氨基酸之间形成的二硫键,可以将肽链折叠成特定的空间结构。多肽分 子通常分子量较大,空间结构复杂,结构中形成二硫键时要求两个半胱氨酸在空间距离上接近。 此外,多肽结构中还原态的巯基化学性质活泼,容易发生其他的副反应,而且肽链上其他侧链 也可能会发生一系列修饰,因此,肽链进行修饰所选取的氧化剂和氧化条件是反应的关键因素, 反应机理也比较复杂,既可能是自由基反应,也可能是离子反应。      

    反应条件有多种选择,比如空气氧化,DMSO氧化等温和的氧化过程,也可以采用H2O2,I2, 汞盐等激烈的反应条件。
     

    空气氧化法: 空气氧化法形成二硫键是多肽合成中最经典的方法,通常是将巯基处于还原态的多肽溶于水中,在近中性或弱碱性条件下(PH值6.5-10),反应24小时以上。为了降低分子之间二硫键形成的可能,该方法通常需要在低浓度条件下进行。
     

    碘氧化法:将多肽溶于25%的甲醇水溶液或30%的醋酸水溶液中,逐滴滴加10-15mol/L的碘进行氧化,反应15-40min。当肽链中含有对碘比较敏感的Tyr、Trp、Met和His的残基时,氧化条件要控制的更精确,氧化完后,立即加入维生素C或硫代硫酸钠除去过量的碘。 当序列中有两对或多对二硫键需要成环时,通常有两种情况:
     

    自然随机成环:       序列中的Cys之间随机成环,与一对二硫键成环条件相似;
     

    定点成环:       定点成环即序列中的Cys按照设计要求形成二硫键,反应过程相对复杂。在 固相合成多肽之前,需要提前设计几对二硫键形成的顺序和方法路线,选择不同的侧链 巯基保护基,利用其性质差异,分步氧化形成两对或多对二硫键。       通常采用的巯基保护 基有trt, Acm, Mmt, tBu, Bzl, Mob, Tmob等多种基团。

    定义
    酶是用于生化反应的非常有效的催化剂。它们通过提供较低活化能的替代反应途径来加快反应速度。酶作用于底物并产生产物。一些物质降低或什至停止酶的催化活性被称为抑制剂。
    发现
    1965年,Umezawa H分析了微生物产生的酶抑制剂,并分离出了抑制亮肽素和抗痛药的胰蛋白酶和木瓜蛋白酶,乳糜蛋白酶抑制的胰凝乳蛋白酶,胃蛋白酶抑制素抑制胃蛋白酶,泛磷酰胺抑制唾液酸酶,乌藤酮抑制酪氨酸羟化酶,多巴汀抑制多巴胺3-羟硫基嘧啶和多巴胺3-羟色胺酶酪氨酸羟化酶和多巴胺J3-羟化酶。最近,一种替代方法已应用于预测新的抑制剂:合理的药物设计使用酶活性位点的三维结构来预测哪些分子可能是抑制剂1。已经开发了用于识别酶抑制剂的基于计算机的方法,例如分子力学和分子对接。
    结构特征
    已经确定了许多抑制剂的晶体结构。已经确定了三种与凝血酶复合的高效且选择性的低分子量刚性肽醛醛抑制剂的晶体结构。这三种抑制剂全部在P3位置具有一个新的内酰胺部分,而对胰蛋白酶选择性最高的两种抑制剂在P1位置具有一个与S1特异性位点结合的胍基哌啶基。凝血酶的抑制动力学从慢到快变化,而对于胰蛋白酶,抑制的动力学在所有情况下都快。根据两步机理2中稳定过渡态络合物的缓慢形成来检验动力学。
    埃米尔•菲舍尔(Emil Fischer)在1894年提出,酶和底物都具有特定的互补几何形状,彼此恰好契合。这称为“锁和钥匙”模型3。丹尼尔·科什兰(Daniel Koshland)提出了诱导拟合模型,其中底物和酶是相当灵活的结构,当底物与酶4相互作用时,活性位点通过与底物的相互作用不断重塑。
    在众多生物活性肽的成熟过程中,需要由其谷氨酰胺(或谷氨酰胺)前体形成N末端焦谷氨酸(pGlu)。游离形式并与底物和三种咪唑衍生抑制剂结合的人QC的结构揭示了类似于两个锌外肽酶的α/β支架,但有多个插入和缺失,特别是在活性位点区域。几种活性位点突变酶的结构分析为针对QC相关疾病5的抑制剂的合理设计提供了结构基础。
    作用方式
    酶是催化化学反应的蛋白质。酶与底物相互作用并将其转化为产物。抑制剂的结合可以阻止底物进入酶的活性位点和/或阻止酶催化其反应。抑制剂的种类繁多,包括:非特异性,不可逆,可逆-竞争性和非竞争性。可逆抑制剂 以非共价相互作用(例如疏水相互作用,氢键和离子键)与酶结合。非特异性抑制方法包括最终使酶的蛋白质部分变性并因此不可逆的任何物理或化学变化。特定抑制剂 对单一酶发挥作用。大多数毒药通过特异性抑制酶发挥作用。竞争性抑制剂是任何与底物的化学结构和分子几何结构非常相似的化合物。抑制剂可以在活性位点与酶相互作用,但是没有反应发生。非竞争性抑制剂是与酶相互作用但通常不在活性位点相互作用的物质。非竞争性抑制剂的净作用是改变酶的形状,从而改变活性位点,从而使底物不再能与酶相互作用而产生反应。非竞争性抑制剂通常是可逆的。不可逆抑制剂与酶形成牢固的共价键。这些抑制剂可以在活性位点附近或附近起作用。
    功能
    工业应用中, 酶在商业上被广泛使用,例如在洗涤剂,食品和酿造工业中。蛋白酶用于“生物”洗衣粉中,以加速蛋白质在诸如血液和鸡蛋等污渍中的分解。商业上使用酶的问题包括:它们是水溶性的,这使得它们难以回收,并且一些产物可以抑制酶的活性(反馈抑制)。
    药物分子,许多药物分子都是酶抑制剂,药用酶抑制剂通常以其特异性和效力为特征。高度的特异性和效力表明该药物具有较少的副作用和较低的毒性。酶抑制剂在自然界中发现,并且也作为药理学和生物化学的一部分进行设计和生产6。
    天然毒物 通常是酶抑制剂,已进化为保护植物或动物免受天敌的侵害。这些天然毒素包括一些已知最剧毒的化合物。
    神经气体( 例如二异丙基氟磷酸酯(DFP))通过与丝氨酸的羟基反应生成酯,从而抑制了乙酰胆碱酯酶的活性位点。
    参考
    1、Scapin G (2006). Structural biology and drug discovery. Curr. Pharm. Des.,      12(17):2087–2097.
    2、Krishnan R, Zhang E, Hakansson K, Arni RK, Tulinsky A, Lim-Wilby MS, Levy OE, Semple JE, Brunck TK (1998). Highly selective mechanism-based thrombin inhibitors:  structures of thrombin and trypsin inhibited with rigid peptidyl aldehydes. Biochemistry, 37 (35):12094-12103.
    3、Fischer E (1894). Einfluss der configuration auf die wirkung der enzyme. Ber. Dt. Chem. Ges., 27:2985–2993.
    4、Koshland DE (1958). Application of a theory of enzyme specificity to protein synthesis. PNAS., 44 (2):98–104.
    5、Huang KF, Liu YL, Cheng WJ, Ko TP, Wang AH (2005). Crystal structures of human glutaminyl cyclase, an enzyme responsible for protein N-terminal pyroglutamate formation. PNAS., 102(37):13117-13122.
    6、Holmes CF, Maynes JT, Perreault KR, Dawson JF, James MN (2002). Molecular enzymology underlying regulation of protein phosphatase-1 by natural toxins. Curr Med Chem., 9(22):1981-1989.

     

    Definition
    Enzymes are very efficient catalysts for biochemical reactions. They speed up reactions by providing an alternative reaction pathway of lower activation energy. Enzyme acts on substrate and gives rise to a product. Some substances reduce or even stop the catalytic activities of enzymes are called inhibitors.

    Discovery
    In 1965, Umezawa H analysed enzyme inhibitors produced by microorganisms and isolated leupeptin and antipain inhibiting trypsin and papain, chymostatin inhibiting chymotrypsin, pepstatin inhibiting pepsin, panosialin inhibiting sialidases, oudenone inhibiting tyrosine hydroxylase, dopastin inhibiting dopamine 3-hydroxylase, aquayamycin and chrothiomycin inhibiting tyrosine hydroxylase and dopamine J3-hydroxylase . Recently, an alternative approach has been applied to predict new inhibitors: rational drug design uses the three-dimensional structure of an enzyme's active site to predict which molecules might be inhibitors 1. Computer-based methods for identifying inhibitor for an enzyme have been developed, such as molecular mechanics and molecular docking.

    Structural Characteristics
    The crystal structures of many inhibitors have been determined. The crystal structures of three highly potent and selective low-molecular weight rigid peptidyl aldehyde inhibitors complexed with thrombin have been determined. All the three inhibitors have a novel lactam moiety at the P3 position, while the two with greatest trypsin selectivity have a guanidinopiperidyl group at the P1 position that binds in the S1 specificity site. The kinetics of inhibition vary from slow to fast with thrombin and are fast in all cases with trypsin. The kinetics are examined in terms of the slow formation of a stable transition-state complex in a two-step mechanism 2.

    Emil Fischer in 1894 suggested that both the enzyme and the substrate possess specific complementary geometric shapes that fit exactly into one another.This is known as "the lock and key" model 3. Daniel Koshland suggested induced fit model where substrate and enzymes are rather flexible structures, the active site is continually reshaped by interactions with the substrate as the substrate interacts with the enzyme 4.

    N-terminal pyroglutamate (pGlu) formation from its glutaminyl (or glutamyl) precursor is required in the maturation of numerous bioactive peptides. The structure of human QC in free form and bound to a substrate and three imidazole-derived inhibitors reveals an alpha/beta scaffold akin to that of two-zinc exopeptidases but with several insertions and deletions, particularly in the active-site region. The structural analyses of several active-site-mutant enzymes provide a structural basis for the rational design of inhibitors against QC-associated disorders 5.

    Mode of Action
    Enzymes are proteins that catalyze chemical reactions. Enzymes interact with substrate and convert them into products. Inhibitor binding can stop a substrate from entering the enzyme's active site and/or hinder the enzyme from catalyzing its reaction. There are a variety of types of inhibitors including: nonspecific, irreversible, reversible - competitive and noncompetitive. Reversible inhibitors bind to enzymes with non-covalent interactions like hydrophobic interactions, hydrogen bonds, and ionic bonds. Non-specific methods of inhibition include any physical or chemical changes which ultimately denature the protein portion of the enzyme and are therefore irreversible. Specific Inhibitors exert their effects upon a single enzyme. Most poisons work by specific inhibition of enzymes. A competitive inhibitor is any compound which closely resembles the chemical structure and molecular geometry of the substrate. The inhibitor may interact with the enzyme at the active site, but no reaction takes place. A noncompetitive inhibitor is a substance that interacts with the enzyme, but usually not at the active site.  The net effect of a non competitive inhibitor is to change the shape of the enzyme and thus the active site, so that the substrate can no longer interact with the enzyme to give a reaction. Non competitive inhibitors are usually reversible. Irreversible Inhibitors form strong covalent bonds with an enzyme.  These inhibitors may act at, near, or remote from the active site .

    Functions
    Industrial application, enzymes are widely used commercially, for example in the detergent, food and brewing industries. Protease enzymes are used in 'biological' washing powders to speed up the breakdown of proteins in stains like blood and egg. Problems using enzymes commercially include: they are water soluble which makes them hard to recover and some products can inhibit the enzyme activity (feedback inhibition) .

    Drug molecules, many drug molecules are enzyme inhibitors and a medicinal enzyme inhibitor is usually characterized by its specificity and its potency. A high specificity and potency suggests that a drug will have fewer side effects and less toxic. Enzyme inhibitors are found in nature and are also designed and produced as part of pharmacology and biochemistry 6.

    Natural poisons are often enzyme inhibitors that have evolved to defend a plant or animal against predators. These natural toxins include some of the most poisonous compounds known.

    Nerve gases such as diisopropylfluorophosphate (DFP) inhibit the active site of acetylcholine esterase by reacting with the hydroxyl group of serine to make an ester.

    References

    Scapin G (2006). Structural biology and drug discovery. Curr. Pharm. Des.,      12(17):2087–2097.

    Krishnan R, Zhang E, Hakansson K, Arni RK, Tulinsky A, Lim-Wilby MS, Levy OE, Semple JE, Brunck TK (1998). Highly selective mechanism-based thrombin inhibitors:  structures of thrombin and trypsin inhibited with rigid peptidyl aldehydes. Biochemistry, 37 (35):12094-12103.

    Fischer E (1894). Einfluss der configuration auf die wirkung der enzyme. Ber. Dt. Chem. Ges., 27:2985–2993.

    Koshland DE (1958). Application of a theory of enzyme specificity to protein synthesis. PNAS., 44 (2):98–104.

    Huang KF, Liu YL, Cheng WJ, Ko TP, Wang AH (2005). Crystal structures of human glutaminyl cyclase, an enzyme responsible for protein N-terminal pyroglutamate formation. PNAS., 102(37):13117-13122.

    Holmes CF, Maynes JT, Perreault KR, Dawson JF, James MN (2002). Molecular enzymology underlying regulation of protein phosphatase-1 by natural toxins. Curr Med Chem., 9(22):1981-1989.

  • DOI名称
    10.1016/s0169-328x(03)00331-0Endotoxin inhibitor blocks heat exposure-induced expression of brain cytokine mRNA in aged rats下载
    10.1007/s00268-005-7409-9Effect of intraperitoneal application of an endotoxin inhibitor on survival time in a laparoscopic model of peritonitis in rats下载
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