Therapeutic targeting of malarial placental cytoadherence

 描述(申请人提供)：孕期感染恶性疟原虫导致胎盘性疟疾(PM)。仅在非洲，每年就有约2500万妇女面临患多发性硬化症的风险，这种疾病导致约10万名婴儿和数万名产妇死亡。低出生体重在PM中很常见，这些新生儿非常容易死于随后的疟疾或其他感染。虽然流行区的人在成年后会获得对疟疾的保护性免疫，但妇女在怀孕期间容易感染严重疟疾。这是由于一种抗原性独特的恶性疟原虫表型在胎盘中的粘连。因此，从未怀孕的女性对这种寄生虫表型没有免疫力。在感染恶性疟原虫的第一次和第二次怀孕妇女中，附着的寄生虫被选择在胎盘中快速生长，使感染的红细胞(RBC)在胎盘中积累到高密度。这会导致炎症和胎盘功能障碍，导致PM预防或治疗PM的有效治疗是必不可少的，但目前还没有可用的方法。这种寄生虫输出一种名为VAR2CSA的变种黏附蛋白，它是恶性疟原虫红细胞膜蛋白1家族的成员，显示在红细胞表面。VA2CSA的胞外结构域介导IRBCs与胎盘CSPG受体的软骨素4-硫酸酯(C4S)部分的12聚体基序的结合。在怀孕期间获得抑制性抗VAR2CSA抗体的妇女对PM具有抵抗力，并生下健康的婴儿，这表明阻断IRBC黏附是治疗PM的合适策略然而，VAR2CSA是一种高度多态的疫苗，因此，获得可行的疫苗需要基于保守的C4S结合位点的知识进行基本设计。另一种方法是开发黏附阻断抑制剂。由于VAR2CSA在IRBC表面，即使是高极性的分子也可以接触到，这被预测为非常有前途的策略。因此，这项建议的目标是：(1)化学合成在特定位置含有两个硫酸盐残基的C4S12-MERS和一个不同大小和硫酸盐化模式的软骨素硫酸盐寡糖文库。这将使我们能够确定最佳结合的寡糖以及参与VAR2CSA-C4S结合的关键相互作用；(Ii)利用我们已经开发的新颖且灵敏的基于C4S 12聚体的荧光各向异性分析，我们将筛选药物和糖胺聚糖模拟库，以通过高通量筛选来识别阻断VAR2CSA-C4S结合的小分子抑制剂，并通过细胞实验验证这些抑制剂的有效性；(Iii)表征VAR2CSA中的C4S结合位点以确定相互作用的氨基酸残基。这些结果有望为开发治疗多发性硬化症的疗法提供必要的关键信息长期目标是开发可用于PM治疗的小分子抑制剂。

 DESCRIPTION (provided by applicant): Plasmodium falciparum infection during pregnancy results in placental malaria (PM). In Africa alone, ~25 million women yearly are at risk of developing PM, which causes ~100,000 infant and tens of thousand maternal deaths. Low birth weight is common in PM and these newborns are highly vulnerable to death from subsequent malaria or other infections. Although people in endemic areas will have acquired protective immunity against malaria by their adulthood, women are susceptible to severe malaria during pregnancy. This is due to the adherence in the placenta of an antigenically distinctive P. falciparum phenotype. So women who have never been pregnant have no immunity to this parasite phenotype. In their first and second pregnancies women infected with P. falciparum, the adherent parasites are selected in the placenta, grow rapidly, allowing infected red blood cells (RBCs) to accumulate to high density in the placenta. This results in inflammation and placental dysfunction, causing PM. An effective treatment to prevent or treat PM is essential, but is not currently available. The parasite exports a variant adhesive protein called VAR2CSA, a member of Plasmodium falciparum erythrocyte membrane protein 1 family, that is displayed on the erythrocyte surface. The ectodomain of VA2CSA mediates the binding of IRBCs to 12-mer motifs of the chondroitin 4-sulfate (C4S) moieties of the CSPG receptor in the placenta. Women who have acquired inhibitory anti-VAR2CSA antibodies during prior pregnancies are resistant to PM and deliver healthy babies, indicating that blocking of IRBC adherence is a suitable strategy to treat PM. However, VAR2CSA is a highly polymorphic and so, obtaining a viable vaccine requires rationale design based on the knowledge of conserved C4S-binding site. An alternative approach is to develop adherence-blocking inhibitors. Since VAR2CSA is on the IRBC surface and is accessible for even highly polar molecules, this is predicted to be highly promising strategy. Accordingly, the goals of this proposal are to: (i) chemically synthesize the C4S 12-mers containing two sulfate residues at specific positions and a library of chondroitin sulfate oligosaccharides of different size and sulfation patterns. This will enable us to identify the optimally binding oligosaccharide and critical interactions involved in VAR2CSA-C4S binding; (ii) Using the novel and sensitive C4S 12-mer-based fluorescence anisotropy assay that we have already developed, we will screen drug and glycosaminoglycan mimetic libraries to identify, by high-throughput screening, small molecule inhibitors that block VAR2CSA-C4S binding and validate the inhibitors by a cell-based assay (iii) characterize the C4S-binding sites in VAR2CSA to identify the interacting amino acid residues. The results are expected to provide critical information necessary for developing therapeutics for PM. The long-term objective is to develop small molecule inhibitors that can be used for PM treatment.