Modulation Of Protein And Cell Functions By Heparin/hepa

肝素/hepa 对蛋白质和细胞功能的调节

• 批准号: 6840709
• 负责人: AUDREY L STONE
• 金额: --
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6840709
• 关键词: antiAIDS agent; anticoagulants; antiviral agents; bacterial polysaccharides; bioassay; biomimetics; carbohydrate structure; chemical models; chemical structure function; drug design /synthesis /production; gel mobility shift assay; heparan sulfate; heparin; human immunodeficiency virus 1; infrared spectrometry; nuclear magnetic resonance spectroscopy; oligosaccharides; protein structure function

This project elucidates the fundamental molecular design of the heparin/heparan sulfate class of biological regulatory polysaccharides (H/HS) and studies how the high degree of diversity of its sulfated oligosaccharide (oligoS) structures relates to its parallel, multi-functional capacity. This capacity is exerted through specific binding to, and thus modulating the functional activity of, many different protein partners in normal and disease processes (e.g., cell growth, secretion, multi-cell reactions in development, blood coagulation, physiological stability, and in viral and other infections). Established and/or newly devised biological, biochemical, and physical methods are utilized. [HD 01315-01-07] Previously: To reveal and study putative unique structures required for specific H/HS functions, and focusing on its anti-viral capacities against HIV-1, we devised a structurally simpler, model H/HS system because despite intensive biochemical research to prepare libraries of such H/HS structures, these are difficult to generate (especially in sufficient quantities for extended research needs) due to the complex diversity of oligoS sequences within H/HS. A model H/HS library of functionally different Components was prepared by low pressure liquid chromatography of a multifunctional heparin-mimetic pharmaceutical which is comprised of a mixture of chemically sulfated xylan oligosaccharides (S-oligoS). [HD 01315-01-06]. Briefly, Components were tested against five in vitro anticoagulant and antiviral target assays. This study showed that each functional capacity was governed by a degree of structural specificity as would be expected for H/HS. This established the potential usefulness of our model system and a potent anti-HIV-1 agent, CpF-PkII, devoid of anti-thrombin activity was isolated and characterized. An enlarged method suitable for producing sufficient CpF-PkII for potential Phase I testing was developed and is underway. About 30 percent is prepared. In addition, the above macro combinatorial strategy using the S-oligoS library was successfully extended to elucidate the anti-malaria parasite capacities attributed to H/HS [See HD 008733-01]. Structural features of the S-oligoS are studied by analytical, chemical, sugar and spectroscopic-FTIR, NMR, and dye-coupling analysis, and by titration. First we discovered that the GlcA to Xyl ratio in the S-oligoS was closer to 1:3 (sugar and proton NMR analyses), indicating that there is a tetrasaccharide motif (beta-1,4-linked trixyloside with one alpha 1,2-linked branch) in the H/HS mimetic structure, rather than that of a beta xylan chain with sparse (1:10) GlcA branches as previously assumed and accepted. [HD 01315-01-05] In addition, from FTIR, proton NMR and 13C heteronuclear correlation spectroscopy data on CpF-PkII and other Components we discovered that functional structures in our S-oligoS library contained sugar moieties in an alternative conformation as well as those in the expected normal chair form. Moreover, the relative proportions of axial sulfate groups that would be associated with the alternate ring form differed among Components with different functional specificities. [HD 01313-05] Since H/HS also contains axial sulfates on sugar moieties in an alternate chair form, this finding indicated a structural basis for the heparin-mimetic capacity of S-oligoS for the first time and highlighted that such sugar conformations may be important in function and specificity of H/HS. [HD 01315-03-04] Current: Some experiments from above were completed. We continue to characterize and expand the library of S-oligoS structures and are establishing it among colleagues studying diverse normal/pathogenic systems as a valid mimic of the H/HS structures which have the various functions ascribed to H/HS. Progress was significant in: 1) We reported this research in four presentations on the expanded library and our applications of the library to examine H/HS function. Results from a series of 12 highly characterized S-oligoS (of 24 studied) against six targets demonstrated that this library contained discrete structures which exhibited differential specificities against the targets and that the potent inhibitors could be identified. Moreover, Components are well characterized as to functional, physical and chemical properties and could now be used to elucidate the properties of the structure of the functional molecules of H/HS in the inhibition of other diseases or disorders (e.g., whether an inhibitor could be prepared devoid of anti-thrombin toxicity). From NMR, FTIR and mass studies, we also proposed that the structure of CpF-PkII is a (4-O Me D- glucuuronyl-alpha 1,2 beta 1,4 D-ylyltrisaccharide) dodecaglucuronyl-oligoxylose containiing multiple xylyl moieties in an alternative chair conformation. Such sugars have a shortening effect on the chain backbone which resembles that in heparin. The positions and nature of the alternative moieties remain to be elucidated. [Stone AL Differential structure-function relations in a family of heparin-mimetic sulfated glucuronyloligoxyloses in vitro: Malaria parasite, AIDS virus and blood coagulation inhibitors Gordon Research Conference on Proteoglycans, July 2002] 2) The first extended library samples were prepared and sent to Dr. Lubor Borsig (U. of Zurich) for his studies on heparin inhibition of tumor metastasis. 3) The potential importance of Cp11 as an agent against malaria sporozoites is a significant new finding in our studies. This H/HS mimetic has ideal characteristics for safe clinical application (See HD 008733-02) and could be produced by methods analogous to those developed for isolation of CpF-PkII. Cp 3 (highly active against parasite invasion of erythrocytes) and Cp 11 were prepared and analyses are on going for further structural characterization. 4) Heteronuclear two-dimensional NMR proton-13C-correlation spectroscopy using an inverted probe in a 600 MHz spectrometer at 60 degrees and analysis by the heteronuclear multi-dimensional quantum coherences (HMQC) method conclusively demonstrated the presence of alternative chair conformations. A hydrogen atom bonded to a sugar carbon that occurs in two distinct conformations would exhibit two complex interactions with that carbon. The NMR correlation spectrum of CpF-PkII displayed two pairs of 13C-satellite signals about 71 ppm on the carbon spectrum, showing unambiguously that at least one type of sugar moiety existed in two conformations, which confirmed the finding from FTIR. Detailed analysis of the correlation spectrum of Pk-II and FTIR spectroscopy of numerous Components now indicate that axial-like sulfates were present on sugar rings with alternative chair conformations in most S-oligoS and that in the case of PkII the axial sulfate appears to occur on carbon 3. The ratio of these forms among additional Components studied also varied. Samples of an anti-thrombin and the antimalaria S-oligoS have been prepared for correlation NMR spectroscopy and analyses are ongoing. 5) This finding, that sugar conformations may be a governing parameter in structural specificity of the protein-binding sugar sequences of H/HS, may also have far reaching important implications in the areas of development of strategies for synthesis of oligoS drugs and in the design of synthetic oligoS-protective antigens. The above studies will be continued and include initiation of testing of putative proteins as partners for CpF-PkII, Cp11 and Cp3 by modification of the gel shift analysis for heparin oligoS-protein binding. Preparation of CpF-PkII will be resumed when required staff arrives. [See HD 008733-02 for other references.]

该项目阐明了肝素/肝素硫酸盐类别的生物调节多糖（H/HS）的基本分子设计，并研究其硫酸寡糖（寡糖）结构的高度多样性与其平行，多函数的能力相关。这种能力是通过特异性结合与正常和疾病过程中许多不同蛋白质伴侣的功能活性（例如细胞生长，分泌，发育中的多细胞反应，血液凝结，生理稳定性以及病毒和其他感染中的多细胞反应）来调节功能活性的。使用了建立和/或新设计的生物学，生化和物理方法。 [HD 01315-01-07]先前：要揭示和研究特定H/HS功能所需的假定独特结构，并专注于其针对HIV-1的抗病毒能力，我们设计了一个更简单的HS系统，模型的H/HS系统，因为尽管需要进行大量的生物化学量，但这些量很难构成这些复杂性，尤其是在这些复杂的研究中，尤其是尤其是HS结构（尤其是生成生成的库） H/HS内的寡核序列的多样性。功能上不同组件的模型H/HS库是通过多功能肝素模拟药物的低压液相色谱制备的，该药物由化学硫酸化的Xylan寡糖（S-Oligos）组成。 [HD 01315-01-06]。简而言之，针对五个体外抗凝剂和抗病毒靶测定进行了测试。这项研究表明，每个功能能力都受到H/HS预期的结构特异性的影响。这确立了我们的模型系统和有效的抗HIV-1药物CPF-PKII的潜在有用性，该抗凝血酶活性没有抗凝血酶活性。开发了一种用于生产足够的CPF-PKII用于潜在I期测试的扩大方法，并且正在进行中。准备大约30％。此外，成功扩展了使用S-Oligos库的上述宏观组合策略，以阐明归因于H/HS的抗马拉里亚寄生虫能力[见HD 008733-01]。通过分析，化学，糖和光谱ftir，NMR和染料偶联分析以及滴定，研究了S-橄榄的结构特征。 First we discovered that the GlcA to Xyl ratio in the S-oligoS was closer to 1:3 (sugar and proton NMR analyses), indicating that there is a tetrasaccharide motif (beta-1,4-linked trixyloside with one alpha 1,2-linked branch) in the H/HS mimetic structure, rather than that of a beta xylan chain with sparse (1:10) GlcA branches as previously假设并接受。 [HD 01315-01-05]此外，来自FTIR，Proton NMR和13C异核相关光谱光谱数据以及其他组件以及其他组件，我们发现我们S-Oligos中的功能结构在我们的S-Oligos库中包含了替代构造中的糖部分，并在预期的正常椅子上均包含这些构造。此外，在具有不同功能特异性的组件之间，与替代环形式相关的轴向硫酸盐基团的相对比例有所不同。 [HD 01313-05]由于H/HS还以替代椅子形式的糖部分上含有轴向硫酸盐，因此该发现表明S-Oligos的肝素模拟能力的结构性基础首次表明，这种糖构型在H/HS的功能和特定性中可能很重要。 [HD 01315-03-04] 电流：上面的一些实验完成了。我们继续表征和扩展S-Oligos结构的库，并在研究各种正常/致病系统的同事中建立它，作为H/HS结构的有效模拟，这些HS结构具有各种功能归因于H/HS。进度在以下方面很大：1）我们在有关扩展的库以及图书馆的应用中检查H/HS功能的四个演讲中报告了这项研究。来自六个目标的一系列12个高度特征的S-Oligos（共24个研究）的结果表明，该文库包含离散的结构，这些结构表现出差异化的特异性针对靶标，并且可以鉴定出有效的抑制剂。此外，组件在功能，物理和化学特性方面具有很好的特征，现在可以用来阐明H/HS功能分子在抑制其他疾病或疾病中的功能分子结构的性质（例如，是否可以制备抑制剂，没有抗Thrombin毒性）。根据NMR，FTIR和大规模研究，我们还提出了CPF-PKII的结构是（4-O Me D-葡萄糖酮-Alpha 1,2β1,4d-糖酸糖）dodecagagulonyl- ol-磺酰基含量），含有多个Xylyl moieties，含有多个Xylyl Moieties。这种糖对肝素中的链链链具有缩短作用。替代部分的位置和性质仍有待阐明。 [在体外的肝素模拟硫酸葡萄糖lo-核苷家族中的石材差异结构连接关系：疟疾寄生虫，艾滋病病毒和血液凝结抑制戈登蛋白聚糖研究会议，2002年7月，2002年7月] 2）转移。 3）在我们的研究中，CP11作为针对疟疾孢子虫的药物的潜在重要性是一个重要的新发现。该H/HS模拟物具有用于安全临床应用的理想特征（请参见HD 008733-02），可以通过类似于用于隔离CPF-PKII的方法来产生。制备了CP 3（对红细胞的寄生虫侵袭高度活跃）和CP 11，并进行了分析，以进行进一步的结构表征。 4）异核二维NMR质子13C相关光谱在60度下使用600 MHz光谱仪中的倒探针，并通过异核多核量子对焦（HMQC）进行分析，最终证明了替代椅构象的存在。与糖碳结合到两个不同构象中的糖碳的氢原子将与该碳表现出两种复杂的相互作用。 CPF-PKII的NMR相关光谱在碳光谱上显示了两对13C-卫星信号，大约71 ppm，明确地表明，两种构型中至少存在一种类型的糖部分，这证实了FTIR的发现。对许多成分的PK-II和FTIR光谱的相关光谱的详细分析现在表明，在大多数S-Oligos中，具有替代椅子构象的糖环存在轴向样硫酸盐，并且在PKII的情况下，在PKII的情况下，轴向硫酸盐硫酸盐似乎在碳3上似乎发生在这些组合中。抗凝血酶和抗疟疾s-橄榄蛋白的样品已经制备用于相关性NMR光谱，并且正在进行分析。 5）这一发现，糖构象可能是H/HS的蛋白质结合糖序列的结构特异性的一个管理参数，这对合成寡寡做药物的策略的发展和Synthetic Oligos-Oligos-Oligos-Oligos-protective Antigens的设计也可能具有重要意义。上述研究将继续进行，并包括通过修改肝素寡聚蛋白结合的凝胶移位分析来启动推定蛋白作为CPF-PKII，CP11和CP3的伴侣的测试。当需要员工到达时，将恢复CPF-PKII的准备。 [有关其他参考，请参见HD 008733-02。]