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: 7334001
• 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/7334001
• 关键词: Modulation; Protein; Cell; Functions; Heparin

A highly active heparin-mimetic HIV-1 entry inhibitor in vitro and free of anti-thrombin toxicity (PK II, SOLIS, is an outcome of pre-clinical studies generated in this project in the 1990s by our basic research findings and knowledge of the molecular and chemical glycobiology of the heparin/heparan sulfate class of polysaccharides (H/HS) and how its structural diversity provides ability to modulate functions of diverse proteins in normal and disease processes. [E.g., cell growth, secretion, development, blood coagulation, and infections by virus and other human pathogens.]. Due to the diversity of sequences within H/HS chains, libraries of unique H/HS oligoS have not been readily obtainenable for research. Pk II was isolated from a heparin-mimetic pharmaceutical comprised of a mixture of sulfated oligoxylans (S-oligoS). We devised enlarged procedures to enable the clinical preparation of PK II (SOLIS) and developed a macro combinational type strategy to obtain a library of heparin-mimetic S-oligoS for researchers. [HD 01315-01-08-10]. The latter enabled us to show that the in vitro inhibitions of HIV-1 cytotoxicity and syncytium-formation by PK II were governed by a degree of discrete structural specificities, as was its lack of anti-thrombin coagulation inhibitory activity, two essential indicators of usefulness for further drug development. SOLIS, A POTENTIAL HIV-1 BINDING/FUSION INHIBITOR: HIV-infection in Americans is reaching one million; morbidity and mortality of AIDS worldwide (estimated 50 million cases and16 million deaths since the 1980s) are increasing. There is no cure nor fully effective vaccine, despite global efforts for almost two decades. Constant daily treatments of AIDS patients with multiple anti-retroviral drugs has been successful in developed countries for long term control of viral load and deadly effects, but with high cost in toxicity, sensitivity, and mutation to multi-drug resistant strains (found in adults and children), which increasingly limit this armamentarium. FDA has approved 26 drugs; 16 vs genomic replication (reverse transcriptase), 9 vs protease, and one is vs entry (a peptide that prevents viral fusion to the CD4 cell membrane by blocking changes in conformations of the fusion subunit of the viral envelope protein. gp41). Broad consensus counsels that inhibitors vs viral components that have other functions in HIV-1 attack are critically needed. We are completing a clinical preparation of SOLIS for a small Phase I safety trial. SOLIS would be an adjunct drug vs binding and entry of virus into target cells (e.g., it inhibits CD4 cell adherence to gp120-coated t.c. plates and prevents syncytium- forming fusion of HIV-1 (gp41) with CD4 cell membrane. (See summary of our results on biological characteristics of SOLIS below). Heparin has long been known to modulate conformations of proteins and peptides. From our studies in this area, inhibition of the conformational changes in gp41 which are required for fusion progression, is a possible mechanism of action of SOLIS and if so would be relatively insensitive to virus mutation. The laboratory is in a unique position to test this idea given our library of S-oligoS. Pk II, however, also inhibited cytopathic effects of HIV-1, suggesting that such S-oligoS might interfere with other critical elements of its attack in addition to binding/entry. SUMMARY OF PROPERTIES OF PK II (SOLIS): TEST SYSTEMS: S-oligoS libraries; preparation samplings FUNCTIONS: cytopathology protection; syncytium/fusion inhibition; inhibition of binding between gp120 and CD4 cell; anticoagulation capacity vs thrombin, endotoxin content. MEASUREMENTS : cell-killing (formazan), EC50 ug/ml equals 0.2-0.3 (See note.); cell fusion count, EC50 ug/ml equals 0.05-0.1 (See note.); CD4-cell adherence to gp120-coated plates; Heparin Units/mg equals less than 0.05-0.3 (Rosenberg assay); FDA assayy (LAL), sampling during preps, less than 0.06 eu. MEASUREMENTS respectively with FUNCTIONS. OUTCOMES: differential capacity, positive protection vs.cytopathology; differential potency, highly active vs virus (gp41)-cell fusion; differential capacity, concentration-dependent prevention of cell (CD4) binding to gp120; differential potency, essentially negligable anti-thrombin toxicity; conforms to FDA laboratory monitoring level of pyrogen exclusion. Note: NIH laboratory data; this may be 7-10 times higher in some commercial labs. IN VIVO Efficacy of SOLIS will be tested in a SCID mouse model through the NIAID DAID program, if applicable. AN FDA LICENSED US heparin-mimetic, similar to the European made starting source of SOLIS (PK II) is currently investigated as an alternate and found to be suitable in preparative aspects. Second stage product, however, remain to be studied in further detail. Negotiations are underway to continue cooperation in a CDA with the pharmaceutical company and ultimately to obtain additional starting product for subsequent trials. VIRUS LIGANDS: Viral proteins that enable HIV to attack, survive, and replicate in target human cells have been elucidated in the field and are available for in vitro study. We have proposed to identify and isolate putative H/HS ligand(s) under conditions of HIV-1 tissue culture infection in the presence of a bifunctional SOLIS probe, and to achieve chemical bonding by flash illumination to endogenous viral/cell protein structures during virus attack. After cell membranes are purified, isolated proteins would be analyzed for fluorescent probe by gel electrophoresis. The probe would be synthesized by our method: synthesizing a bifunctional hydrazine derivative and subsequent reaction with the reducing aldehyde terminus of the inhibitor. Such ligand(s) might be protective antigens and/or a means of obtaining endogenous H/HS receptors. STRUCTURE-FUNCTION (SF): The mixture of S-oligoS remaining after chemical sulfation of the native xylan was shown previously to unexpectedly comprise a family of oligomers that increased in mass by additions which included a putative -D-glucuronyl-alpha 1,2 beta 1,4 D-(xylyl)3 motif. The SoligoS range from about 20 to less than 2 K on the average in mass, contain more or less sugars in alternate chair conformations, and a large proportion of GlcA lacking an 4-Ome substituent (FTIR, HMQC heteronuclear two-dimensional NMR proton-13C correlation spectra, GlcA analysis). Recent consideration of the SF underlying the above inhibitions of HIV-1 indicates that S-oligoS action against gp120 binding to the cell receptor(s) requires a minimum of 4 motifs to retain maximum potency. The requirement differs for the inhibition of gp41 helical change where the max number of motifs is 3. Capillary HPLC-mass spectroscopy will be used shortly to analyze and model more specifically the various structures of Pk II and other S-oligoS. This information might help define structures for potential inhibitors that might have simpler chemistry.

高度活跃的肝素模拟HIV-1进入体外抑制剂并且没有抗脑组毒性（PK II，SOLIS，SOLIS，是我们1990年代在该项目中产生的临床前研究的结果，我们的基础研究结果和对分子和化学性化学的甲基硫酸盐/Heparan sulfate suplations and-heparan sulfate and/hs/hss soldations and/hs/hshs的糖基化（H/hs）的知识（正常和疾病过程中的蛋白质。寡素（S-Oligos）。 [HD 01315-01-08-10]。后者使我们能够表明，PK II对HIV-1细胞毒性和合成形式的体外抑制受到一定程度的离散结构特异性的控制，因为它缺乏抗凝血酶凝血抑制活性，这是两个对进一步药物开发的有用性的基本指标。 Solis是一种潜在的HIV-1结合/融合抑制剂：美国人的HIV感染已达到100万；全球艾滋病的发病率和死亡率（1980年代以来估计有5000万例和1600万例死亡）正在增加。尽管近二十年来，尽管全球努力，但仍无法治愈或完全有效的疫苗。多种抗逆转录病毒药物的艾滋病患者的持续日常治疗在发达国家已经成功地控制了病毒载荷和致命作用，但毒性，敏感性和对多药耐药菌株的敏感性和突变成本很高（在成人和儿童中发现），这越来越多地限制了该臂臂。 FDA批准了26种药物； 16 vs基因组复制（逆转录酶），9 vs蛋白酶，一个是与输入相比（一种通过阻止病毒包膜蛋白融合亚基的构象变化来防止病毒融合到CD4细胞膜的肽。迫切需要在HIV-1攻击中具有其他功能的病毒成分的广泛共识。我们正在完成一项小型I期安全试验的SOLIS的临床准备。 SOLIS将是一种辅助药物与病毒进入靶细胞的结合和进入（例如，它抑制CD4细胞依从性，与GP120涂层的T.C.平板并预防HIV-1（GP41）（GP41）与CD4细胞膜的合成融合（GP41）（gp41）与CD4细胞膜的综合性综合性的综合量。从我们在这一领域的研究中，融合进展所需的构象变化是溶液的一种可能的机理，如果这是对Sy-Oligos的cytig siv，则可能是对病毒突变的独特位置。除了结合/进入外，其攻击的关键要素。 PK II（SOLIS）的性质摘要： 测试系统：S-Oligos库；准备采样 功能：细胞病理学保护；合成/融合抑制；抑制结合 在GP120和CD4细胞之间；抗凝能力与凝血酶，内毒素含量。 测量值：杀伤细胞（甲状腺），EC50 ug/ml等于0.2-0.3（请参阅注释）；细胞融合计数，EC50 ug/ml等于0.05-0.1（请参阅注释）； CD4细胞遵守GP120涂层板；肝素单位/毫克等于不到0.05-0.3（Rosenberg分析）； FDA Assayy（LAL），预备过程中的采样，小于0.06 EU。分别使用功能进行测量。 结果：差异能力，积极保护与周期病理学；差异效力，高活性与病毒（GP41） - 细胞融合；差异能力，浓度依赖性预防细胞（CD4）与GP120结合；差异效力，本质上可以忽略不可抗脑栓的毒性；符合FDA实验室监测水平的热原排除水平。注意：NIH实验室数据；在某些商业实验室中，这可能是7-10倍。 如果适用，将通过NIAID DAID程序在SCID鼠标模型中测试SOLIS的体内功效。目前研究了FDA许可的美国肝素模仿，类似于欧洲制造的Solis（PK II）的起始来源（PK II），作为替代方案，发现适合制备方面。但是，第二阶段的产品仍有详细研究。正在进行谈判，以继续与制药公司在CDA中进行合作，并最终获得随后试验的其他起始产品。病毒配体：使艾滋病毒能够在靶标细胞中攻击，生存和复制的病毒蛋白在该田间已阐明，并且可以进行体外研究。我们已经提议在存在双功能的Solis探针的情况下在HIV-1组织培养感染的条件下鉴定和分离推定的H/HS配体，并在病毒攻击过程中通过闪光照明对内源性病毒/细胞蛋白结构实现化学键合。纯化细胞膜后，将通过凝胶电泳分析分离的蛋白质中的荧光探针。该探针将通过我们的方法合成：合成双功能氢氮衍生物和随后与抑制剂还原醛末端的反应。这种配体可能是保护性抗原和/或获得内源性H/HS受体的方法。结构功能（SF）：天然Xylan化学硫化后剩余的S-Oligos的混合物先前已显示出意外的构成一系列低聚物家族，其质量增加了质量，其中包括推定的-D-葡萄糖醛醛-Alpha 1,2 beta 1,2 beta 1,4 d-（Xylyl）3个基序。 Soligos的质量平均值的范围从大约20至2 K范围，替代椅子构象中包含或多或少的糖，并且很大一部分GLCA缺乏4-OME取代基（FTIR，FTIR，HMQC，HMQC异核二维NMR NMR Proton-13c相关光谱，GLCA分析）。最近考虑了上述HIV-1抑制作用的SF，这表明S-Oligos对GP120与细胞受体结合的作用至少需要4个基序才能保留最大的效力。抑制GP41螺旋变化的要求有所不同，其中最大基序数为3。毛细管HPLC-Mass光谱法将很快使用，以更具体地分析和模拟PK II和其他S-Oligos的各种结构。这些信息可能有助于定义可能具有更简单化学的潜在抑制剂的结构。