MODULATION OF PROTEIN AND CELL FUNCTIONS BY HEPARIN/HEPARAN SULFATES

肝素/硫酸乙酰肝素对蛋白质和细胞功能的调节

• 批准号: 6432564
• 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/6432564
• 关键词: antiAIDS agent; anticoagulants; antiviral agents; chemical models; chemical structure function; heparan sulfate; heparin; human immunodeficiency virus 1; human tissue; nuclear magnetic resonance spectroscopy; virus infection mechanism

The project elucidates the fundamental molecular design of heparins and heparan sulfates (H/HS) and studies structure- function relations of their specific modulations of diverse normal and diseased protein and cell membrane systems, using biological, biochemical, and physical approaches. We examine H/HS as a modulator of viral infectivity, and isolate and study Components (S-oligoS) of a heparin-mimetic, sulfated xylan drug which, as does heparin, inhibits the infectivity of HIV-1 in vitro: 1) We had revealed differential functional potencies and physical properties of the S-oligoS Components, which led to our isolation and identification of a minimal-sized, potent anti-HIV-1 S-oligoS, CpF and CpF-PkII [HD 01315-01-03]. Moreover, CpF is separated from antithrombin activity which is associated with different structurally specific Components. Upscaled procedures were explored for use in obtaining a CpF-PkII drug against AIDS in humans [HD 01315-04-05]. Our optimized procedures are reliably reproducible and continue to be labor intensive, with ~ 8 % yield. Multiple preparations to obtain gram quantities of CpF-PkII for a Phase I trial remain a primary focus and continue in cooperation with the drug manufacturer with whom we have negotiated a confidential agreement (30% of goal are now completed through final step). 2) Structure-function relations are studied by FTIR, exciton-absorption and NMR spectroscopy. Proton NMR and sugar analysis support our proposal of a tetrasaccharide grouping in heparin-mimetic structures (a beta 1,4-linked trixyloside containing an alpha 1,2 D-GlcA branch) [HD01315-01-03,05]. FTIR of CpF-PkII revealed sugars in the alternate as well as normal chair forms (axial (ax) as well as expected equatorial (eq) sulfates) [HD01315-03-04], and these structures are displayed differentially among Components. Structures containing axial sulfates may be of significance in functional specificity: a high mass, antithrombin Component (4B) was associated with 1.7-fold greater proportion of such alternate chair conformations than PK-II [HD01313-05] and we find by FTIR that anticaoagulant, anti-HIV-1 Component 4A displays an ax-to-eq sulfate ratio of 3.1 compared with 0.11 for that of Component 12B which is inactive against thrombin and low in anti-HIV-1 capacity. From proton NMR spectra of PkII, signals at 4.83, 4.93, 4.6 and 5.18 ppm suggest alternate chair forms. A 13C-proton correlation study of PkII designed to elucidate the sugar(s) and carbon atom(s) associated with ax sufate groups is in progress. 3) The molecular mechanism underlying the inhibition of HIV-1 by S-oligoS and H/HS are investigated: Fluorescent photoactive-crosslinking CpF-PkI-probes were synthesized [HD 01315-05]. Studies to mprove % derivatization by direct nucleophilic addition to the aldehyde using aminonaptholsulfonic acid hydrazide continue. These unique probes will assist in studies to identify and isolate a putative cell membrane S-oligoS receptor involved in viral infectivity. 4) Heparin-mimetic inhibition of the malarial parasite by Components in vitro is examined: Components are prepared and currently assayed for specificity.

该项目阐明了使用生物学，生化和物理方法，阐明了肝素和硫酸盐（H/HS）（H/HS）的基本分子设计（H/HS），并研究了其特定正常和患病蛋白质和细胞膜系统的特定调制的结构功能关系。 We examine H/HS as a modulator of viral infectivity, and isolate and study Components (S-oligoS) of a heparin-mimetic, sulfated xylan drug which, as does heparin, inhibits the infectivity of HIV-1 in vitro: 1) We had revealed differential functional potencies and physical properties of the S-oligoS Components, which led to our isolation and identification of a minimal-sized,有效的抗HIV-1 S-Oligos，CPF和CPF-PKII [HD 01315-01-03]。此外，CPF与抗凝血酶活性分离，这与不同的结构特异性成分相关。探索了用于获得针对人类艾滋病的CPF-PKII药物的上升程序[HD 01315-04-05]。我们的优化程序可可靠地重现，并继续进行劳动密集型，产量约为8％。为I期试验获得革兰氏数量的CPF-PKII的多项准备仍然是主要重点，并继续与我们与我们协商机密协议进行谈判的药物制造商合作（目前有30％的目标已通过最后一步完成）。 2）通过FTIR，激子吸收和NMR光谱研究结构功能关系。质子NMR和糖分析支持我们对肝素模拟结构中四糖组的建议（含有α1,2D-GLCA分支的β1,4-链接的二氧甲酰胺）[HD01315-01-01-03,05]。 CPF-PKII的FTIR在交替的椅子和正常椅子形式（轴向（AX）以及预期的赤道（EQ）硫酸盐）[HD01315-03-04]中显示出糖，这些结构在组件之间差异显示。含有轴向硫酸盐的结构在功能特异性中可能具有重要意义：高质量，抗凝结成分（4b）与PK-II的比例比PK-II比1.7倍（HD01313-05）[HD01313-05]高1.7倍，而我们通过FTIR发现了抗凝蛋白的抗凝聚力，抗抗凝蛋白的速度为3.组件12b对凝血酶无活性，抗HIV-1容量低。从PKII的质子NMR光谱中，信号为4.83、4.93、4.6和5.18 ppm，提出了替代椅子的形式。 PKII的13C-蛋白质相关研究旨在阐明与AX舒服基团相关的糖原子和碳原子。 3）研究了S-Oligos和H/HS抑制HIV-1的分子机制：合成了荧光光活性 - 跨CPF-PKI-PROBES [HD 01315-05]。 使用氨基硫醇磺酸氢氮化甲酰基通过直接核仁添加到醛中直接添加醛的衍生化研究。这些独特的探针将有助于研究和分离出参与病毒感染性的推定细胞膜S-橄榄岩受体。 4）检查了体外成分对疟疾寄生虫对疟疾的抑制：制备成分并目前对特异性进行测定。