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: 6840716
• 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/6840716
• 关键词: antiAIDS agent; antimalarial agents; bacterial polysaccharides; biomimetics; carbohydrate structure; chemical models; chemical structure function; combinatorial chemistry; conformation; drug design /synthesis /production; heparan sulfate; heparin; infrared spectrometry; nuclear magnetic resonance spectroscopy; oligosaccharides

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, multifunctional capacity. This capacity is exerted through specific binding to, and thus modulation of 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 infections by virus and other classes of human pathogens). Established and/or newly devised biological, biochemical, and physical methods are utilized. [HD 01315-01-08]. Due to the complex diversity of oligoS sequences within H/HS chains, libraries of unique H/HS structures have not been prepared in quantities needed for extended research. This project has engaged several approaches to study structural requirements for the functions ascribed to H/HS, in particular its capacity to inhibit HIV-1: 1) CHARACTERIZATION AND PREPARATION OF A NEW CLASS OF HIV-1 INHIBITOR: We previously developed a H/HS-mimetic library by low pressure liquid chromatography fractionation of a H/HS- mimetic sulfated xylan pharmaceutical which mimicks most of the known biological actions of H. 24 sulfated oligosaccharide (S-oligoS) Components (Cps) were obtained. Cps were tested against in vitro anticoagulant and anti-HIV-1 assays [HD 01315-01-06]. Findings showed that a degree of structural specificity governed both heparin-mimetic capacities of the pharmaceutical. This led to the preparation and characterization of a highly active anti-HIV-1 agent which was essentially free of anti-thrombin toxicity (CpF-PkII). An enlarged method for producing sufficient CpF-PkII for potential Phase I testing was developed [HD 01315-04-05] and a clinical preparation is underway utilizing 40 grams of starting pharmaceutical [HD 01315-05-08]. Currently, about 50% is processed to the second or first stage purification. Two modifications were developed for faster processing: An on-line detector monitors the chromatographic separation, eliminating post-column analyses; high reproducibility among the required series of columns now allows analyses of fewer fractions to identify product (sharper cut). We expect with staff to complete the preparation of 3-3.5 grams of CpF-PkII in FY 04, and then to prepare the requirements for Phase I testing. This putative adjunct drug would provide an additional mechanism of inhibition of HIV since its mode of action in vitro prevents the binding and entry of virus into target cells and the cell-cell spreading of the infection. Our basic research studies [HD 01315-03-06] with staff will resume and include: Protein or protein regions (from literature and data banks on HIV and CD4 membrane proteins) will be tested as putative ligands for CpF-PkII by modifying the gel shift analysis for heparin oligoS-protein binding (Rosenberg, R. D. and coworkers) and using a fluorescent derivative to mark the migration of S-oligoS in the gel. Further characterization of structure-function relation of CpF-PkII and other S-oligoS by capillary HPLC/mass spectrometry will be examined. 2) DEVELOPMENT OF H/HS-MIMETIC LIBRARY of S-OLIGOS: The heparin-mimetic S-oligoS are now characterized by physical,chemical,and various functional properties and may be used to identify and examine structural properties of the functionality of S-oligoS and H/HS in the inhibition of other diseases or disorders. We successfully utilized the library to examine the anti-malaria parasite capacities of H/HS [See HD008733-02-03]. Cps also were sent to colleagues for their comparison studies on the inhibition of tumor metastasis by H. Preliminary results were positive and additional Cps will be sent for further comparison. 3) ELUCIDATION OF THE S-OLIGOS STUCTURE. Structural features of the library have been studied by biochemical, chemical, and spectroscopic (FTIR, NMR, and dye-coupling) analyses and by titration. We discovered early that the heparin-mimetic structures differed markedly from the then generally accepted model(a sulfated polyxylose with sparse glucuronic acid (GlcA) branches, one to 10 xyloses (Xyl) on average). The S-oligoS Cps contained about I GlcA to 3 Xyl and thus were largely a tetrasaccharide motif, one alpha 1,2-linked GlcA-branch per beta-1,4-linked trixylyl sequence [HD 01315-01-05]. Such motifs would display sulfated GlcA-Xyl- groupings spaced along the chain by only two xylose rings and each GlcA could assume a helical rotation of 180 degrees. Subsequent studies further diminished apparent dissimilarities between the macrostructures of H and the S-oligoS. FTIR, proton NMR and heteronuclear two-dimensional NMR proton-13C-correlation spectroscopy (analyzed by the heteronuclear multi-dimensional quantum coherences (HMQC) method) on CpF-PkII revealed that some sugar moieties were in an alternative chair conformation instead of the expected normal chair form [HD 01313-05-08]. Only one of the two sugars was seen to assume this form, and the doublet signals appeared to arise from only one sulfated carbon position. This would be consistent with a mono- sulfated Xyl which contains a GlcA branch. As with H, such sugars would have a shortening effect on the chain backbone. This finding points out an additional parameter for possible diversity in the functional structures of these heparin-mimetics as well as that of H/HS. The data taken together led us to propose a structure for CpF-PkII, (4-O Me D- GlcA-alpha 1,2 beta 1,4 D-xylyltrisaccharide) dodecaglucuronyl-oligoxylose, having several moieties of either Xyl or less likely GlcA in the alternative chair conformation. Recent FTIR data demonstrated that alternative chair characteristics are also displayed by other S-oligoS. Furthermore, the relative ratio of such alternate ring forms to the normal chair differed among Cps with different functions and mass [HD 01313-05-08]. Current 1H-13C heteronuclear NMR spectroscopy of the low mass sporozoite inhibitor (Cp 11) did not reveal doublet peaks which signal these alternative forms. This data is consistent with the low ratio of alternate forms found by FTIR analysis of Cp 12 in the low mass region. The notion that sugar conformations may be a governing factor in structural specificity of the protein-binding sugar sequences of H/HS may have a general significance, e.g., in strategies for synthesis of oligoS drugs and the design of synthetic oligoS-protective antigens. Structure-function studies including FTIR and 1H-13C correlation NMR spectroscopy on further purified inhibitors will continue.

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（Oligos）结构的高度多样性与其平行，多功能能力有关。这种能力是通过特异性结合到正常和疾病过程中的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人类病原体的其他类别的感染中的多细胞反应）来施加的。使用了建立和/或新设计的生物学，生化和物理方法。 [HD 01315-01-08]。由于H/HS链中的寡核序列的复杂多样性，尚未以扩展研究所需的数量制备独特的H/HS结构的库。该项目已聘用了几种研究归因于H/HS功能的结构要求的方法，特别是其抑制HIV-1：1）表征和制备了新型的HIV-1抑制剂的表征和准备：我们先前通过低压液体色谱分离了H/H/Mimimigetate XylAcientation XylAcy Xylace Xylacy Xylacy himimical of Himimatial himimikical of hs-hs-bimetic库，该单位是H.获得了寡糖（S-Oligos）成分（CPS）。针对体外抗凝剂和抗HIV-1分析测试了CP [HD 01315-01-06]。研究结果表明，一定程度的结构特异性控制了药物的肝素模拟能力。这导致了高度活跃的抗HIV-1药物的制备和表征，该药物基本上没有抗凝血酶毒性（CPF-PKII）。开发了一种用于产生足够的CPF-PKII用于潜在I期测试的方法[HD 01315-04-05]，并利用40克启动药物[HD 01315-05-08]正在进行临床准备工作。目前，将约50％的人处理到第二阶段或第一阶段的净化。开发了两次修改以进行更快的处理：在线检测器监测色谱分离，消除了柱后分析；现在所需的一系列列之间的高可重复性可以分析较少的分数以识别产品（剪切）。我们希望工作人员在04财年完成3-3.5克CPF-PKII的准备，然后准备I期测试的要求。这种推定的辅助药物将提供抑制HIV的附加机制，因为其体外作用方式可防止病毒进入靶细胞和感染的细胞细胞扩散。我们与员工的基础研究[HD 01315-03-06]将恢复并包括：蛋白质或蛋白质区域（来自HIV和CD4膜蛋白的文献和数据库）将作为CPF-PKII的假定配体进行测试，以通过修改Heparin Oligos oligos ofortiv and a anderg anderg和R. D. R. D. R. D. R. D. R.在凝胶中标记S-Oligos的迁移。将检查CPF-PKII和其他S-Oligos通过毛细管HPLC/质谱的进一步表征。 2）S-Oligos的H/HS模仿文库的发展：肝素模拟S-Oligos现在以物理，化学和各种功能特性为特征，可用于识别和检查S-Oligos和H/HS功能的结构特性。我们成功地利用了图书馆来检查H/HS的抗马拉里亚寄生虫能力[见HD008733-02-03]。 CP还被送往同事，进行了H.初步结果抑制肿瘤转移的比较研究，并将额外的CPS发送以进行进一步比较。 3）阐明S-Oligos稳定性。图书馆的结构特征已通过生化，化学和光谱（FTIR，NMR和染料偶联）分析以及滴定研究。我们很早就发现，肝素模拟结构与当时普遍接受的模型（具有稀疏的葡萄糖酸（GLCA）分支的硫酸化聚xylose，平均为1到10木糖（Xyl））。 S-Oligos CP包含大约I GLCA至3 Xyl，因此在很大程度上是四糖基序，每个Beta-1,4-1-4-链接的Trixylyl序列[HD 01315-01-05]一个Alpha 1,2连接的GLCA分支[HD 01315-01-05]。这样的基序将仅通过两个木糖环显示沿链间隔的硫酸GLCA-Xyl-组，每个GLCA可以假设螺旋旋转180度。随后的研究进一步减少了H和S-Oligos的宏观结构之间的明显差异。 FTIR，质子NMR和异核二维NMR PROTON-13C相关光谱（通过CPF-PKII通过异核多维量子量子相干（HMQC）方法分析）在CPF-PKII上进行了一些替代椅子的椅子，而不是预期的正常椅子，而不是预期的正常椅子[HD-01313-01313-01313-01313-013。只有两种糖中只有一种假设这种形式，而双线信号似乎仅来自一个硫酸化的碳位置。这将与包含GLCA分支的单硫化Xyl一致。与H一样，此类糖会对链链链产生缩短作用。这一发现指出了这些肝素摄影和H/HS功能结构中可能多样性的附加参数。数据一起导致我们提出了CPF-PKII的结构（4-O ME D-GLCA-ALPHA 1,2β1,4D-二甘露三糖）十二核葡萄糖磺酰基 - 乙酰糖基因，具有几个Xyl或更少的Xyl或更少的GLCA，或者是更可能的GLCA。最近的FTIR数据表明，其他S-Oligos也显示了替代椅子的特征。此外，这种交替环形式与正常椅的相对比在具有不同功能和质量的CP之间有所不同[HD 01313-05-08]。低质量孢子岩抑制剂（CP 11）的当前1H-13C异核NMR光谱法没有揭示双峰峰，这些峰标志着这些替代形式。该数据与低质量区域中CP 12分析发现的替代形式的低比率一致。糖构象可能是H/HS蛋白结合糖序列的结构特异性的一个概念可能具有一般意义，例如，在合成寡核体药物的策略和合成寡核酸保护抗原的设计中。在进一步纯化的抑制剂上，包括FTIR和1H-13C相关性NMR光谱在内的结构功能研究将继续进行。