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: 6290224
• 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/6290224
• 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 the structure-function relations of their specific modulations of diverse normal and diseased protein and cell membrane systems, using various 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 identification and isolation of a minimal-sized, potent anti-HIV-1 S-oligoS, CpF (and purified CpF-PkII) [HD 01315-01-03]. Moreover, CpF is separated from antithrombin activity. This heparin-mimetic activity is associated with different structurally specific Components. Upscaled procedures (including automated medium pressure LC) are explored for use in obtaining a CpF-PkII drug against AIDS in humans. These procedures continue to be labor intensive, with ~8% yield. Multiple preparations to obtain sufficient purified CpF for a Phase I trial remain a primary focus and are continuing. A large scale preparation of CpF might be feasible in cooperation with the drug manufacturer with whom we have negotiated a confidential exchange. 2) Structure-function relations are studied by FTIR and quantitative proton NMR spectroscopy at 600 MHz. CpF-PKI and II are found by proton NMR to contain GlcA and Xyl in a ratio of 1:2.9 and 1:3.0, which supports our proposal [HD 01315-01- 03] of a tetraS grouping in heparin-mimetic structures (a b 1,4-linked trixyloside containing an a 1,2 D-GlcA branch). FTIR of CpF-PkII reveal sugars in the alternate as well as normal chair forms (axial as well as expected equatorial sulfates) [HD 01315-03]. Further purified PkII displays the same FTIR spectral pattern. From the proton NMR spectra we find Xyl in alternate to normal chair ratios of 1:9 in PkI and PkII. Alternate chair structures may be of significance in functional specificity since we now find that that a high mass, anti-thrombin Component (4B) displayed a 1.7-fold greater proportion of such alternate chair conformations (1:5.2), while having the same GlcA to Xyl ratio of 1:2.9. 3) The molecular mechanism underlying the inhibition of HIV-1 by S-oligoS and H/HS are investigated: Fluorescent photoactive- crosslinking CpF-PkI-probes are synthesized by direct nucleophilic addition to the aldehyde using aminonaptholsulfonic acid hydrazide (ANS) which we synthesized, or by formation of a glycamine of the S-oligoS to provide an amine for reaction with commercial photocrosslinking reagents. The ANS reaction gave good recovery of S-oligoS and ~25-50% derivatization, which will be improved by adjusting pH and T. These unique probes will assist in studies to identify and isolate a putative cell membrane S-oligoS receptor involved in viral infectivity.

该项目阐明了使用各种生物学，生物化学和物理方法的不同正常和患病蛋白和细胞膜系统的特定特定调制的特定调制调节的肝素和乙糖硫酸盐（H/HS）的基本分子设计。 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 identification and isolation of a minimal-sized,有效的抗HIV-1 S-Oligos，CPF（和纯化的CPF-PKII）[HD 01315-01-03]。此外，CPF与抗凝血酶活性分离。这种肝素模拟活性与不同的结构特异性成分有关。探索了探索高尺度的程序（包括自动化中型压力LC），以用于获得针对人类艾滋病的CPF-PKII药物。这些程序继续进行劳动密集型，产量约为8％。为I期试验获得足够纯化的CPF的多项准备仍然是主要重点，并且仍在继续。 CPF的大规模准备可能是与我们与我们谈判的机密交易所谈判的药物制造商合作的。 2）通过FTIR和600 MHz的定量质子NMR光谱研究结构功能关系。 Proton NMR发现CPF-PKI和II以1：2.9和1：3.0的比例包含GLCA和XYL，这支持了我们的建议[HD 01315-01-03]肝素模拟结构中的Tetras分组（A B 1,4链接的Trixylososide含有A 1,4链式的Trixylososide含有A 1,1,2 rca 1,2 d-gca）。 CPF-PKII的FTIR揭示了替代和正常椅子形式（轴向和预期赤道硫酸盐）的糖[HD 01315-03]。进一步纯化的PKII显示相同的FTIR光谱模式。从质子NMR光谱中，我们在PKI和PKII中发现xyl的正常椅子比为1：9。替代椅子结构在功能特异性中可能具有重要意义，因为我们现在发现，高质量，抗凝血酶成分（4B）显示出1.7倍的替代椅子构象（1：5.2），而具有相同的GLCA与Xyl的比率为1：2.9。 3) The molecular mechanism underlying the inhibition of HIV-1 by S-oligoS and H/HS are investigated: Fluorescent photoactive- crosslinking CpF-PkI-probes are synthesized by direct nucleophilic addition to the aldehyde using aminonaptholsulfonic acid hydrazide (ANS) which we synthesized, or by formation of a glycamine of the S-Oligos为商业光叠链链接试剂提供胺提供胺。 ANS反应可以很好地恢复S-Oligos和〜25-50％的衍生化，通过调整pH和T进行改进。这些独特的探针将有助于研究并隔离参与病毒感染性的推定细胞膜S-橄榄岩受体。