Heparin/heparan Sulfate/Mimetics Modulators Of Malaria

肝素/硫酸乙酰肝素/疟疾模拟调节剂

• 批准号: 6541337
• 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/6541337
• 关键词: Plasmodium; antimalarial agents; biomimetics; heparan sulfate; heparin; intracellular parasitism; liver cells; malaria; microorganism culture; microorganism disease chemotherapy; parasitic liver disorder

This project elucidates the actions of heparin and heparan sulfate (H/HS) and heparin-mimetic sulfated xylan oligosacchrides in malarial infection and pathogenicity. Heparan sulfate proteoglycan is believed to be an hepatocyte receptor for malarial sporozoite invasion through the binding of the circumsporozoite membrane protein (Nussenzweig & coworkers,1993). In addition, erythocyte membrane H/HS might be a receptor for the invasion of normal RBC by newly released merozoites, and it is involved as a receptor in the complex reactions of sequestration of parasitized RBC to the microvasculature in severe malaria. Heparin has been shown to inhibit the parasite invasion of hepatocytes and RBC in vitro, and it also rapidly disolves rosettes in vitro and in vivo. Heparin was tried as a treatment for cerebral malaria but was abandoned because of bleeding toxicity. The molecular details of these functional reactions of H/HS remain to be clarified and applied. This year, to study whether there is a degree of structural specificity in the H/HS hepatocyte receptor function, we conducted a preliminary study of the capacity of eight S-oligoS, range of concentration 2.5 ug - 25 ug, to inhibit hepatocyte invasion by malaria sporozoites in vitro, following our macro combinatorial strategy [HD 01315-07]. The S-oligoS Components were representative of the size and anionic distribution range of our complete series. The target test was an Inhibition of Liver Stage Development Assay (ILSDA) using Plasmodium yoelii (J. Sacci, 2001). Briefly, sporozoites are prepared from the heads and thorax of infected mosquitoes by the Ozaki method. ~24h murine hepatocyte monolayer cultures in multi-well plates are infected and incubated for 3 h, washed and incubated for 48 h. Schizonts are visualized by immunoflourescent staining and counted under an epifluorescent microscope to measure the percent inhibition. Results showed that only two of the 8 S-oligoS exhibited significantly high and concentration-dependent capacity to inhibit the schizont development . One Component was relatively small, ~ 3500 in mass (~ a dodecamer which would be devoid of anti-thrombin capacity). The other Component was ~ 7000 in mass (~22 saccharides). Complete dose-response curves in the ILSDA assays were not measured in this study, but an estimate of the IC50s for these S-oligoS was ~1 uM. A similar prliminary study was conducted on the capacity of these S-oligoS, concentration 0.5 ng - 5000 ng, to inhibit the invasion of erythrocytes by plasmodium parasite clones D6 and W2. Only one Component displayed inhibitory capacity at these low concentrations. This S-oligoS was ~12000 in mass and exhibited an IC50 against W2 comparable to that of chloroquine (0.26 nM vs 0.41 nM, resp.) but less active against D6 (0.21 nM vs 0.01 nM). The mefloquine standard was ~10 fold more potent in both clones (W2, 0.26 nM vs 0.01 nM; D6, 0.17 nM vs 0.02 nM). These assays for the IC50s will be repeated at a higher concentration range. We express the relative activities on the basis of molarity rather than ug because the mass of the S-oligoS varies and all are higher than that of the standards. The above results will be reported at the November Glycobiology meeting and the abstract published: AL Stone "Structure-Function Relations of heparin-mimetic sulfated oligoxylans in the inhibition of the invasion of hepatocytes by malaria parasites in vitro" Glycobiology Vol. 11. 2001. The above data indicate that S-oligoS may have differential capacities to inhibit sporozoite invasion of hepatocytes. Thus, further study of possible structural specificity in this H/HS function is warranted. We will repeat the study using complete dose-response curves. In addition,. a new approach utilizing sensitive binding techniques and varuius potential protein partners will be employed to discriminate between inhibitory and inactive S-oligoS. Such studies would have immediate application to research on the H/HS family.

该项目阐明了肝素和硫酸乙酰肝素（H/HS）和肝素模拟硫酸二甲烷寡素在疟疾感染和致病性中的作用。硫酸乙酰肝素蛋白聚糖被认为是肝孢子虫浸入疟疾的肝细胞受体，它通过割孢子虫膜蛋白的结合（Nussenzweig＆Coolorkers，1993）。此外，红细胞膜H/HS可能是新释放的蛋白酶侵袭正常RBC的受体，并且它作为一种受体参与了寄生虫的复杂反应，寄生RBC对严重疟疾中微腔的隔离反应。肝素已显示可在体外抑制肝细胞和RBC的寄生虫侵袭，并且还迅速在体外和体内迅速溶解玫瑰花结。肝素被试用为脑疟疾的治疗方法，但由于出血毒性而被废弃。 H/HS的这些功能反应的分子细节仍有待澄清和应用。今年，为了研究H/HS肝细胞受体功能是否存在一定程度的结构特异性，我们对八个S-Oligos的能力进行了初步研究，浓度范围为2.5 ug-25 ug，以抑制型疟疾孢子虫在体内抑制型肝脏侵入性肝癌的肝脏侵袭，遵循我们的宏观组合策略[H/HS HS-25 ug-25 ug-25 ug-25 UG。 S-Oligos组件代表了我们完整系列的大小和阴离子分布范围。目标检测是使用疟原虫YOELII对肝期发育分析（ILSDA）的抑制（J. Sacci，2001）。简而言之，通过Ozaki方法，从感染蚊子的头部和胸部制备了孢子菌。 〜24H多孔板中的鼠肝细胞单层培养物被感染并孵育3小时，洗涤并孵育48小时。通过免疫染色可视化精神分裂症，并在表含量显微镜下计数以测量抑制百分比。结果表明，在8个S-Oligos中，只有两个表现出显着较高的浓度依赖性能力来抑制Schizont发育。一个成分相对较小，质量〜3500（〜一个没有抗凝血酶容量的十二烷）。另一个成分在质量中约为7000个（〜22糖）。在本研究中未测量ILSDA测定中的完全剂量反应曲线，但是这些S-Oligos的IC50估计值约为1 UM。对这些S-橄榄族的能力（浓度为0.5 ng -5000 ng）进行了类似的临界研究，以抑制寄生虫寄生虫克隆D6和W2侵袭红细胞的侵袭。在这些低浓度下，只有一个组件显示出抑制能力。该S-Oligos的质量约为12000，与氯喹相当（0.26 nm vs 0.41 nm，rups。）表现出IC50，但针对D6（0.21 nm vs 0.01 nm）的活性较低。在两个克隆中，甲氟喹标准的有效性更高（W2，0.26 nm vs 0.01 nm; D6，0.17 nm vs 0.02 nm）。这些IC50的测定法将在较高的浓度范围内重复。我们根据摩尔性而不是UG表达相对活性，因为S-Oligos的质量变化了，并且所有这些都高于标准。上述结果将在11月的糖生物学会议上报告，并发表的摘要：“ al石头模拟硫化寡聚的寡素的结构 - 功能关系在抑制疟疾寄生虫在体外抑制肝细胞的侵袭中”。 11。2001。上述数据表明，S-Oligos可能具有差异能力来抑制子孢子侵袭肝细胞。因此，有必要进一步研究此H/HS功能中可能的结构特异性。我们将使用完整的剂量反应曲线重复研究。此外，。利用敏感结合技术和Varuius潜在蛋白质伴侣的一种新方法将采用抑制性和不活跃的S-Oligos区分。此类研究将立即应用H/HS家族。