O-Glycan Synthesis: Potential Targets for Intervention in Cryptosporidiosis

描述(由申请人提供)： 机会性顶端复合体寄生虫隐孢子虫在世界范围内引起腹泻疾病，对艾滋病患者来说可能是毁灭性的。然而，目前还没有针对艾滋病相关隐孢子虫病的有效、特异的治疗方法。该项目的长期目标是通过靶向合成O-糖链的酶来开发治疗艾滋病患者隐孢子虫病的药物，O-糖链是寄生虫附着和入侵肠道上皮细胞能力所不可或缺的。我们以前的研究和初步研究表明，O-葡聚糖在介导感染和诱导免疫应答中发挥着重要作用，并暗示合成O-葡聚糖的酶(UDP-N-乙酰-D-氨基半乳糖胺：多肽N-乙酰氨基半乳糖转移酶或ppGalNAc-ts)可能成为干预免疫低下者，特别是艾滋病患者隐孢子虫病的靶点。然而，如果对Crypto中的这些酶有任何了解的话，那就是非常少。我们的中心假设是，ppGalNAc-ts催化关键的粘蛋白类糖蛋白上O-糖链的合成，是隐形病毒感染所必需的。其具体目的是利用小分子文库高通量筛选(HTS)确定的抑制剂，与新英格兰地区生物防御和新出现传染病卓越中心(NERCE-BEID)的国家小分子筛选和药物化学(NSRB)核心合作，阐明ppGalNAc-ts在隐蔽感染中的功能作用。将筛选出最有效的酶活性抑制剂，以确定它们是否能够特异性地阻断O-隐聚糖的合成。然后将测试这些抑制剂在体外抑制Crypto感染的能力。在这个项目完成时，我们预计已经表达了所有四种具有酶活性的重组形式的CP GalNAc ts，并已经确定了能够阻断酶活性、O-糖链合成和体外感染的小分子抑制剂。这些研究将确定O-葡聚糖及其催化合成的酶在隐形细胞-宿主细胞相互作用中的作用，并显着推动该领域的发展。该项目的结果将为未来对特定糖蛋白上的隐含ppGalNAc-ts和O糖链的研究提供信息，这些糖蛋白对于介导寄生虫对宿主细胞的附着和入侵至关重要。小分子抑制剂可以在体外阻断Crypto ppGalNAc T酶的活性、O-糖链的合成和体外感染，在未来的研究中可能成为治疗艾滋病相关隐孢子虫病的潜在药物。 公共卫生相关性： 隐孢子虫是一种寄生虫，可引起艾滋病患者的腹泻疾病，目前尚无具体有效的治疗方法。这种寄生虫利用其表面的糖分子感染宿主细胞。该项目的目标是确定利用抑制这些糖分子的物质来制造这些糖分子的酶的作用，以便开发治疗这种寄生虫引起的艾滋病患者疾病的新药。

DESCRIPTION (provided by applicant): The opportunistic apicomplexan parasite Cryptosporidium (Crypto) causes diarrheal disease worldwide which can be devastating in AIDS patients. However, currently, there is no effective, specific therapy for AIDS- associated cryptosporidiosis. The long term goal of this project is to develop therapeutics for cryptosporidiosis in AIDS patients by targeting enzymes that synthesize O-glycans that are integral to the ability of the parasite to attach to and invade intestinal epithelial cells. Our previous and preliminary studies strongly suggest that O- glycans play a significant role in mediating infection and inducing immune responses and imply that the enzymes (UDP-N-acetyl-D-galactosamine: polypeptide N-acetylgalactosaminyl transferases or ppGalNAc-Ts) which synthesize them may serve as targets for intervention for cryptosporidiosis in the immunocompromised, particularly AIDS patients. However, very little, if anything is known about these enzymes in Crypto. Our central hypothesis is that ppGalNAc-Ts that catalyze the synthesis of O-glycans on key mucin-type glycoproteins are essential for Crypto infection. The specific aim is to elucidate the functional role of ppGalNAc-Ts in Crypto infection using inhibitor identified by high throughput screening (HTS) of small molecule libraries in collaboration with National Small Molecule Screening and Medicinal Chemistry (NSRB) Core at the New the England Regional Center of Excellence for Biodefense and Emerging Infectious Diseases (NERCE-BEID). The most potent inhibitors of enzyme activity will be screened for their ability to specifically block synthesis of Crypto O-glycans. These inhibitors will then be tested for their ability to inhibit Crypto infection in vitro. At the completion of this project we expect to have expressed enzymatically active, recombinant forms of all four Cp GalNAc Ts and to have identified small molecule inhibitors which block enzyme activity, O-glycan synthesis and infection in vitro. These studies will establish the role of O-glycans and the enzymes that catalyze their synthesis in Crypto-host cell interactions and significantly advance the field. Results from this project will inform future studies on Crypto ppGalNAc-Ts and O glycans on specific glycoproteins that are critical for mediating attachment to and invasion of host cells by the parasite. Small molecule inhibitors which block Crypto ppGalNAc T enzyme activity, O-glycan synthesis and infection in vitro can be developed as potential therapeutics for AIDS-associated cryptosporidiosis in future studies. PUBLIC HEALTH RELEVANCE: Cryptosporidium is a parasite which causes diarrheal illness in AIDS patients for which there is no specific effective treatment. This parasite uses sugar molecules on its surface to infect host cells. The goal of this project is to determine the role of the enzymes that make these sugar molecules using substances that inhibit them, in order to develop new drugs for treatment of disease caused by this parasite in AIDS patients.