Modulation Of Protein And Cell Functions By Heparin/heparan Sulfate And Mimetics

肝素/硫酸乙酰肝素及其模拟物对蛋白质和细胞功能的调节

• 批准号: 7594173
• 负责人: AUDREY L STONE
• 金额: $ 72.25万
• 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/7594173
• 关键词: Accounting; Adherence; Affect; Aliquot; American; Anti-HIV Agents; Anticoagulants; Anticoagulation; Antigens; Automation; Binding; Biological; Biological Assay; Biological Markers; Biological Process; Biopolymers; Blood; Blood capillaries; Blood coagulation; Blood drug level result; CD4 Positive T Lymphocytes; CYP1A1 gene; Calculi; Cell fusion; Cell membrane; Cell physiology; Cells; Chemicals; Chemistry; Child; Class; Clinical; Clinical Markers; Clinical Research; Color; Combined Modality Therapy; Condition; Consensus; Counseling; Cytopathology; Developed Countries; Developing Countries; Development; Disease; Dose; Drug Formulations; Endopeptidases; Endotoxins; Equipment; European; Exhibits; Family; Fluorescent Probes; Formazans; German population; Giant Cells; Glycobiology; Glycoconjugates; Goals; Growth; HIV; HIV Envelope Protein gp120; HIV Infections; HIV-1; HMQC; Health Sciences; Heparin; Heparitin Sulfate; Human; Immunity; Immunologics; In Vitro; Incidence; Individual; Infection; Inorganic Sulfates; Investigation; Journals; Knowledge; Laboratories; Libraries; Licensing; Ligands; Maintenance; Malaria; Mass Spectrum Analysis; Measurement; Medical; Membrane Fusion; Membrane Proteins; Methods; Modeling; Modification; Molecular; Molecular Conformation; Multi-Drug Resistance; NMR Spectroscopy; Nature; Numbers; Oligonucleotides; Oligosaccharides; Optics; Oral; Oral Administration; Patients; Pentosan Sulfuric Polyester; Peptide Hydrolases; Personal Satisfaction; Persons; Pharmaceutical Preparations; Pharmacologic Substance; Phase; Polysaccharides; Preparation; Procedures; Process; Protein Conformation; Proteins; Protons; Public Health; Publishing; Range; Relative (related person); Reporting; Reproducibility; Research; Reverse Transcriptase Inhibitors; Role; Safety; Sampling; Source; Specificity; Spectroscopy, Fourier Transform Infrared; Staging; Structure; System; Techniques; Thinking; Thrombin; Time; Toxic effect; United States Food and Drug Administration; Unspecified or Sulfate Ion Sulfates; Vaccines; Viral; Viral Load result; Viral Proteins; Virus; Virus Diseases; Virus Inhibitors; Work; Xylans; abstracting; base; capillary; cell growth; cell killing; chemical bond; combinatorial; cytotoxicity; density; dosage; drug development; inhibitor/antagonist; mimetics; novel; pathogen; polypeptide; prevent; protein structure; receptor; sugar; sulfation; tissue culture; two-dimensional

Heparin and heparan sulfates (H/HS) are a class of highly sulfated glycoamino-polysaccharide biopolymers found on the membranes of cells in the body. They have a large diversity in sugar modifications and in sulfate density (aninonic density) along the polysaccharide chains. This structural diversity provides H/HS with a sufficiently large number of unique sequences to account for its known ability to modulate precisely the functions of many diverse proteins and biological systems 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, however, libraries of the unique H/HS oligoS have not been readily obtainable for research. We based a strategy to obtain a macro combinatorial type of heparin-mimetic family (library) on the structure-function model for anticoagulant heparin (Rosenberg and coworkers), and selected a chemically sulfated natural xylan German phamaceutical comprised of S-oligoS that mimicked heparin in almost all its known biological actions, at similar uM concentrations, to obtain a pilot heparin-mimetic family of S-OligoS HD 01315-01-08-10. This enabled us to show that the in vitro inhibitions of HIV-1 cytotoxicity and syncytium-formation were each governed by a degree of structural specificity as well as separability from the anticoagulant S-oligoS of the pharmaceutical, two essential indicators of their usefulness for further drug development AL Stone, et al 1998 Structure-function relations of heparin-mimetic sulfated xylan oligosaccharides: inhibition of Human Immunodeiciency Virus-1 in vitro Glycoconjugate Journal 15:697-712. We devised enlarged procedures to enable the clinical preparation of a highly active HIV-1 virus fusion inhibitor free of anti-thrombin toxicity (PK II, SOLIS). SOLIS, POTENTIAL HIV-1 VIRAL ENTRY INHIBITOR: With HIV-infection in Americans at appx. one million and 50 times that worldwide, no cure or fully effective vaccine, the successful long term control of viral load and deadly effects in developed countries threatened by toxicity and multi-drug resistant strains, and most drugs approved by the FDA being protease or reverse transcriptase inhibitors with one inhibitor of virus entry, broad consensus counsels that inhibitors against viral components which have other functions in HIV-1 attack are critically needed. We are working on completing a clinical preparation of SOLIS for formulation in a small Phase I i.v.-administration safety trial. SOLIS would be an adjunct drug selected from the family library as an inhibitor of binding and entry of virus into target cells (e.g., it inhibits CD4 cell adherence to gp120-coated t.c. plates (binding assay) and is highly active against syncytium-formation of HIV-1 (gp41) with CD4 cell membrane (fusion assay). Heparin has long been known to affect conformation of proteins as a means of modulating their biological function. Thus, inhibition of the conformational changes in gp41, which are thought to be required for fusion progression, by SOLIS is a possible mechanism of action. SUMMARY OF PROPERTIES OF PK II: IC 50 vs cell-killing (formazan assay) 0.20-0.3 ug/ml; -------- anticoagulation vs thrombin ----- less than 0.05-0.3 HU/mg IC 50 vs vs virus-cell fusion 0.05-0.1 ug/ml;---------endotoxin content FDA LAL Assay ---less than 0.06 eu (IC 50 may be 7-10 x greater in a commercial lab) ---;--------inhib. adherence CD4 cell to gp120-- concentration-dependent inhib. c It has been known for many years that S-oligoS are very poorly absorbed by oral administration. Clinical studies of others using the sulfated xylan pharmaceutical were flawed by use of indirect means to assess blood levels of the drug during or after i.v. administraation, absent an available direct assay. Our strategy for the preparation of a biomarker is expected to enable a direct blood assay and accurate assessment of dosages of SOLIS relative to in vitro IC 50. I.v. administration of S-oligoS requires relatively large doses per person. An FDA licensed heparin-mimetic, similar to the European made starting source of SOLIS (PK II) is under our investigation as a possible ready source and was reported to be suitable in first stage preparative aspects last year. This product undergoes an additional purification step to obtain a whiter color. Two small preparations of components analogous to SOLIS are now completed and one exhibits inhibitory capacity in the formazan assay (cytopathology protection) similar to that of Pk II. Reproducibility of the chromatographic processes were not fully satisfactory, however. VIRUS LIGANDS: Viral proteins that enable HIV to attack, survive, and replicate in target human cells are available for in vitro study. We have proposed, when research staff would be available, 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 to endogenous viral/cell protein structures during virus attack. After cell membranes are purified, isolated membrane proteins would be analyzed for fluorescent probe. Such ligand(s) might be protective antigens and/or a means of obtaining endogenous H/HS receptors. STUDIES ON COMBINATION THERAPY: We are preparing Cp11 samples to elucidate 1.)whether the addition of increasing amounts of Cp11 (relatively inactive vs HIV-1) to the usual Pk II doses in the formazan cytotoxicity protection assay will effect the capacity of Pk II to protect cells, and 2.) whether constant molarity of total S-oligoS in usual dose range of Pk II, diminishing Pk II proportion, will yield the expected protection. Aliquots of Cp 8, highly active in the formazan assay, are in preparation for similar studies with Pk II if indicated. BIOMARKERS: Aliquots of Components 8 and 9 are under preparation to serve in devising the methods for obtaining a biomarker for direct measurement of i.v. administered SOLIS. Particular synthetic polypeptides will be selected for linkage to these S-OLIGOS based on our speculation of a mode of action of SOLIS, and immunologic biomarkers will be sought. STRUCTURE-FUNCTION (SF): Early on we showed that the mixture of S-oligoS remaining after chemical sulfation of the native xylan unexpectedly comprised a family of oligomers that range in mass from about 20 to less than 2 K on average. These contain decreasing numbers of a putative -D-glucuronyl-alpha 1,2 beta 1,4 D-(xylyl)3 (tetrasacchaide) motif and varying amount of axial sulfates (alterrnative chair conformation). (FTIR, HMQC heteronuclear two-dimensional NMR proton-13C correlation spectra, GlcA analysis published oral report abstracts: AL Stone and MO Longas 1999 Sulfated oligo-xylans .... : Characterization by FTIR spectroscopy FASEB J 13:A1385; AL Stone and MO Longas 1999 Structure- function relations ......: proton NMR studies Glycobiology 10:1119; MO Longas and AL Stone 2001 Structure-function relations ......: Study of alternate chair conformations by NMR spectroscopy Glycobiology 11:921-922. We are currently preparing samples of Cp II and Cp 8b for advanced FTIR spectral analysis and HMQC correlation spectra. SF underlying the above inhibitions of HIV-1 indicates that S-oligoS structure against gp120 binding to receptor(s) could contain a max of 4 motifs to retain maximum potency while that for inhibition of gp41 helical change could be 3. Capillary HPLC-mass spectroscopy will serve to analyze and model more specifically the various structures of Pk II. This information might help define structures for potential inhibitors that might have simpler chemistry.

肝素和肝素硫酸盐（H/HS）是一类高度硫化的乙酰糖氨基 - 果糖糖酸生物聚合物，这些生物聚合物在体内的细胞膜上发现。它们的糖修饰和沿多糖链的硫酸盐密度（Aninonic密度）具有较大的多样性。这种结构多样性为H/HS提供了足够数量的独特序列，以说明其已知的能力，可以精确调节许多不同蛋白质和生物系统在正常和疾病过程中的功能。例如，病毒和其他人类病原体的细胞生长，分泌，发育，血液凝结以及感染。然而，由于H/HS链中的序列多样性，尚未轻松获得独特的H/HS寡聚物的库。 We based a strategy to obtain a macro combinatorial type of heparin-mimetic family (library) on the structure-function model for anticoagulant heparin (Rosenberg and coworkers), and selected a chemically sulfated natural xylan German phamaceutical comprised of S-oligoS that mimicked heparin in almost all its known biological actions, at similar uM concentrations, to obtain a pilot heparin-mimetic family of S-Oligos HD 01315-01-08-10。 This enabled us to show that the in vitro inhibitions of HIV-1 cytotoxicity and syncytium-formation were each governed by a degree of structural specificity as well as separability from the anticoagulant S-oligoS of the pharmaceutical, two essential indicators of their usefulness for further drug development AL Stone, et al 1998 Structure-function relations of heparin-mimetic sulfated xylan oligosaccharides: inhibition of Human免疫性病毒-1体外糖缀合物杂志15：697-712。 我们设计了扩大的程序，以使高度活性的HIV-1病毒融合抑制剂无抗凝血酶毒性（PK II，SOLIS）进行临床制备。 SOLIS，潜在的HIV-1病毒进入抑制剂：Appx美国人的HIV感染。全球一百万到50倍，无法治愈或完全有效的疫苗，在受毒性和多药抗性菌株受到威胁的发达国家中的长期长期控制和致命的影响，以及大多数由FDA批准的药物是蛋白酶或逆转录酶抑制剂，具有一种抗衡性的群体，该抑制剂具有侵害性群体的抑制作用，该抑制剂具有其他与群体的抑制作用，该动作具有其他对群体的影响，该动力是对群众的侵害。需要。 我们正在努力完成在小型I.V.-Administration安全试验中制定SOLIS的临床准备。 SOLIS将是一种从家庭文库中选择的辅助药物作为病毒和进入靶细胞的抑制剂（例如，它抑制CD4细胞对GP120涂层的T.C.平板（结合分析）的不符合性，并且高度活跃，并且对HIV-1（gp41）的cd4细胞膜膜构型（GP41）具有flosemations Assay（fypections Ass）。因此，调节其生物学功能。 PK II的性质摘要： IC 50 vs杀细胞（甲贡测定）0.20-0.3 ug/ml; ---------抗凝凝血蛋白-------------------------------------------- IC 50与病毒细胞融合0.05-0.1 ug/mL; ----------------------------------------------------------------------------》 （在商业实验室中，IC 50可能大7-10 x）---; --------抑制作用。遵守CD4细胞对GP120-浓度依赖性抑制。 c 多年来，S-Oligos被口服给药非常糟糕。使用硫酸化Xylan药物的其他人的临床研究通过使用间接手段在静脉注射期间或之后评估药物的血液水平存在缺陷。管理，没有可用的直接测定。 我们准备生物标志物的制备策略有望直接血液测定法，并准确评估SOLIS相对于体外IC 50。 S-Oligos的给药需要每人相对较大的剂量。 FDA许可的肝素模仿，类似于欧洲制造的Solis（PK II），我们正在研究作为可能的现成来源，据报道，去年在第一阶段的准备方面适合使用。 该产品经历了额外的纯化步骤，以获得白色的颜色。 现在已经完成了类似于SOLIS的两种小型组件制剂，其中一种与PK II相似的甲贡测定法（细胞病理保护）表现出抑制能力。但是，色谱过程的可重复性并不完全令人满意。 病毒配体：使HIV能够在靶标人细胞中攻击，生存和复制的病毒蛋白可用于体外研究。 我们提出，在可用研究人员的情况下，在HIV-1组织培养物感染的条件下，在患有双功能的Solis探针的情况下，识别和分离了假定的H/HS配体，并在病毒攻击过程中实现化学键合成内源性病毒/细胞蛋白结构。 纯化细胞膜后，将分析分离的膜蛋白中的荧光探针。 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。 STUDIES ON COMBINATION THERAPY: We are preparing Cp11 samples to elucidate 1.)whether the addition of increasing amounts of Cp11 (relatively inactive vs HIV-1) to the usual Pk II doses in the formazan cytotoxicity protection assay will effect the capacity of Pk II to protect cells, and 2.) whether constant molarity of total S-oligoS in usual dose range of Pk II, diminishing Pk II proportion, will yield预期的保护。 CP 8的等分试样在甲半氮测定中高度活跃，正在准备与PK II相似的研究。 生物标志物：组件8和9的等分试样正在准备工作，以设计用于获得静脉内直接测量的生物标志物的方法。管理的索利斯。根据我们对Solis的作用方式，将选择特定的合成多肽与这些S-Oligos链接，并寻求免疫生物标志物。 结构功能（SF）：早期，我们表明，天然Xylan化学硫酸化的S-Oligos的混合物出乎意料地组成了一个质量家族，质量的含量为质量，平均约为20至2 K。 这些含有减少的假定-D-葡萄糖醛醛-Alpha 1,2β1,4d-（Xylyl）3（四链肌）基序和不同量的轴向硫酸盐（替代椅子构象）。 (FTIR, HMQC heteronuclear two-dimensional NMR proton-13C correlation spectra, GlcA analysis published oral report abstracts: AL Stone and MO Longas 1999 Sulfated oligo-xylans .... : Characterization by FTIR spectroscopy FASEB J 13:A1385; AL Stone and MO Longas 1999 Structure- function relations ......: proton NMR studies Glycobiology 10：1119; Mo Longas和Al Stone 2001结构 - 功能关系……：NMR光谱的替代椅子构型11：921-922我们目前正在准备CP II和CP 8B的样品。与受体的结合可以包含4个基序以保持最大效能，而抑制GP41螺旋变化的效力可能为3。毛细管HPLC-MAS光谱学将有助于分析和模拟PK II的各种结构，以帮助定义潜在的抑制结构。