Heparin/heparan Sulfate Mimetics: Modulators Of Malaria

肝素/硫酸乙酰肝素模拟物：疟疾调节剂

• 批准号: 6840722
• 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/6840722
• 关键词: Plasmodium; antimalarial agents; biomimetics; drug design /synthesis /production; heparan sulfate; heparin; intracellular parasitism; liver cells; malaria; microorganism culture; parasite infection mechanism; parasitic liver disorder

This project elucidates heparin- and heparan sulfate (H/HS)-mimetic actions of sulfated xylan oligosaccharides (S-oligoS) in malarial invasion and pathogenicity. H/HS and S-oligoS appear to modulate the parasites at various infectious stages and in their sequelae: 1) As an hepatocyte receptor for malarial sporozoite invasion through binding to the circumsporozoite membrane protein; 2) as a possible erythrocyte (RBC) receptor in the invasion of RBC by newly released merozoites, as well as involved in the complex rosetting reactions between parasitized-RBC (pRBC) and normal RBC; and 3) by sequestration of p-RBC to the microvasculature in cerebral and severe malaria. The molecular basis underlying these functional reactions of H/HS remain to be clarified. Exogenous heparin was shown by others to inhibit the parasite invasion of hepatocytes and RBC and to dissolve rosettes in vitro. It also rapidly dissolved rosettes in vivo. This clinical treatment for cerebral malaria, however, had to be abandoned because of bleeding toxicity. Utilizing our library of H/HS-mimetic S-oligoS and a macro combinatorial strategy to examine the degree of specificity in the putative H/HS hepatocyte receptor function in vitro , we had demonstrated that the potencies of the inhibition of liver stage sporozoite development were differentially displayed among eight S-oligoS Components (Cp). [Two Cp (av. mass region 7200 and 3600) exhibited concentration dependent, high inhibitory capacity (ID50 est. 1 uM), while four other Cp (broad av. mass range 2300 - 6600 lacked anti-sporozoite capacity]. These data indicated that this inhibition was governed by a degree of molecular specificity. In contrast, in the in vitro assay of invasion of RBC, high potency (IC50=0.14-0.27uM) H/HS-mimetic S-oligoS against malaria parasites appeared to be limited to the high mass region (av. 8600 and 13,400) of the library; these would have relatively high anti-thrombin capacity and aPTT values. Moderate inhibitory potency (IC50=0.54-0.69uM), however, was exhibited by S-oligoS in the intermediate av mass range of 5800-4700. [details in HD01315-07,08; HD008733-02] These studies revealed that a small S-oligoS (approx. a dodecamer, av. mass 4700), which would be devoid of anti-thrombin capacity and close to a low level of 1.5 in the aPTT test, might be an approach to treatment of malaria in adults and children. It is noteworthy that our high potency anti-HIV-1 agent, CpF-Pk II, which was devoid of capacity to inhibit sporozoite invasion, had moderate potency against malaria parasite invasion of RBC. Such capacities may be found to provide a dual usefulness in treating populations subject to both diseases. Research in this project was largely inactive this year, but will resume next year with renewed lab staff. Studies this year focused on development of enlarged and further devoluted preparations of the two anti-malaria sporozoite S-oligoS and the S-oligoS region moderately inhibitory against RBC invasion (av. mass = 5800). We now have 200-500 mg of first stage preparations ready for second stage purification. Our basic research will resume, continuing in three approaches. The first is to repeat and extend through collaboration the anti-sporozoite and anti-RBC invasion assays of the putative receptor-mimetic S-oligoS. The second is to identify and elucidate the S-oligoS ligands, using a modified gel-shift analysis for heparin oligoS-protein binding. This would reveal possible specific binding of S-oligoS to, and isolation of, their protein partners in the given membranes, e.g., fluorescent receptor (monodansylated S-oligoS) would mark possible shifts in gel migration upon its binding to specific protein sequences identified in the data banks on malarial proteins of the sporozoite membrane. The third approach in the basic research on our H/HS-mimetic library is to examine the potential specificity and usefulness of the S-oligoS against cerebral malaria in children and adults by means of in vitro assays on their capacity to rapidly dissolve the pathogenic rosettes of pRB.

该项目阐明了硫酸盐二糖（S-橄榄酸）在疟疾侵袭和致病性中的硫酸肝素和乙par（H/HS）的模拟作用。 H/HS和S-Oligos似乎在各种感染阶段和后遗症中调节寄生虫：1）作为肝细胞受体，作为肝孢子虫浸入型肝细胞受体，通过与外孢子菌膜膜蛋白的结合。 2）作为新释放的Merozoites侵袭RBC的可能红细胞（RBC）受体，并参与了寄生的RBC（PRBC）和正常RBC之间的复杂玫瑰质反应； 3）通过将P-RBC固定在脑和严重疟疾中的微脉管系统中。 H/HS的这些功能反应的分子基础仍有待阐明。其他人证明了外源肝素，以抑制肝细胞和RBC的寄生虫入侵，并在体外溶解玫瑰花结。它还在体内迅速溶解了玫瑰花结。然而，由于出血毒性，这种对脑疟疾的临床治疗必须放弃。利用我们的H/HS模拟S-Oligos库和宏观组合策略来检查假定的H/HS肝细胞受体功能在体外的特异性程度，我们证明了抑制肝脏阶段孢子岩发育的能力在8 s-Oligos Components中差异化。 [两个CP（公平质量区域7200和3600）表现出浓度依赖性的，高抑制能力（ID50EST。1UM），而其他四个CP（宽公寓质量范围2300-6600缺乏抗杀菌仪的能力）。这些数据表明，这种抑制作用受到分子特异性的影响，相反。相反。相反。 （IC50 = 0.14-0.27UM）H/HS模拟的S-Oligos针对疟疾的寄生虫似乎限制在图书馆的高质量区域（AV。8600和13,400）。 5800-4700 [HD01315-07,08中的细节；值得注意的是，我们的高效力抗HIV-1药物CPF-PK II缺乏抑制孢子虫入侵的能力，对疟疾寄生虫入侵RBC具有适度的效力。可能发现这种能力可以在治疗受这两种疾病的人群中提供双重实用性。 今年对该项目的研究在很大程度上是不活动的，但明年将与新的实验室工作人员一起恢复。今年的研究重点是开发两种抗马拉里亚孢子岩S-橄榄岩和S-Oligos区域的扩大和进一步散布的制剂，对RBC入侵中等抑制作用（AV。Mass= 5800）。现在，我们准备了200-500毫克的第一阶段准备工作，准备进行第二阶段净化。我们的基础研究将恢复，继续采用三种方法。首先是通过合作重复并扩展了推定受体模仿S-Oligos的抗孢子岩和抗RBC入侵测定法。第二个是使用改良的凝胶递移分析来识别和阐明S-橄榄蛋白寡聚蛋白的结合。这将揭示S-Oligos与给定膜中其蛋白质伴侣的蛋白质伴侣的特异性结合，例如荧光受体（单二烷基化的S-橄榄岩）将标志着凝胶迁移的可能变化，其与孢子菌孢子酸酯症状症状的疟疾蛋白质中鉴定在数据库中鉴定的特定蛋白质序列的结合。关于我们的H/HS模拟图书馆的基础研究的第三种方法是检查S-Oligos对儿童和成人在儿童和成人中的潜在特异性和实用性，并通过其体外测定的能力来快速溶解 PRB的致病玫瑰花结。