Therapeutic targeting of malarial placental cytoadherence

 描述（由申请方提供）：妊娠期间恶性疟原虫感染导致胎盘疟疾（PM）。仅在非洲，每年约有2500万妇女面临患PM的风险，这导致约10万婴儿和数万孕产妇死亡。PM中的低出生体重很常见，这些新生儿非常容易因随后的疟疾或其他感染而死亡。虽然疟疾流行地区的人到成年时已经获得了对疟疾的保护性免疫力，但妇女在怀孕期间很容易感染严重的疟疾。这是由于抗原性独特的恶性疟原虫表型粘附在胎盘中。所以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，粘附的寄生虫在胎盘中被选择，迅速生长，使感染的红细胞（RBC）在胎盘中积累到高密度。这导致炎症和胎盘功能障碍，引起PM。预防或治疗PM的有效治疗是必不可少的，但目前还没有。该寄生虫输出称为VAR 2CSA的变体粘附蛋白，其是恶性疟原虫红细胞膜蛋白1家族的成员，其展示在红细胞表面上。VA 2CSA的胞外域介导IRBC与胎盘中CSPG受体的4-硫酸软骨素（C4 S）部分的12聚体基序的结合。在先前怀孕期间获得抑制性抗VAR 2CSA抗体的妇女对PM具有抗性并分娩健康婴儿，这表明阻断IRBC粘附是治疗PM的合适策略。然而，VAR 2CSA是高度多态性的，因此，获得可行的疫苗需要基于保守的C4 S结合位点的知识的合理设计。另一种方法是开发粘附阻断抑制剂。由于VAR 2CSA在IRBC表面上并且甚至对于高极性分子也是可接近的，因此预测这是非常有前途的策略。因此，该提议的目标是：（i）化学合成在特定位置含有两个硫酸酯残基的C4 S 12-mer和不同大小和硫酸化模式的硫酸软骨素寡糖文库。这将使我们能够鉴定最佳结合寡糖和参与VAR 2CSA-C4 S结合的关键相互作用;（ii）使用我们已经开发的基于C4 S 12-mer的新型和灵敏的荧光各向异性测定，我们将筛选药物和糖胺聚糖模拟文库，以通过高通量筛选来鉴定，阻断VAR 2CSA-C4 S结合的小分子抑制剂，并通过基于细胞的测定验证所述抑制剂（iii）表征VAR 2CSA中的C4 S结合位点以鉴定相互作用的氨基酸残基。这些结果有望为PM治疗药物的开发提供关键信息。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.