O-Glycan Synthesis: Potential Targets for Intervention in Cryptosporidiosis

O-聚糖合成：干预隐孢子虫病的潜在目标

• 批准号: 8496716
• 负责人: Honorine D Ward
• 金额: $ 22.42万
• 依托单位: TUFTS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-07-01 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8496716
• 关键词: AIDS therapy; Acetylgalactosamine; Acquired Immunodeficiency Syndrome; Affinity; Alcohol or Other Drugs use; Antibodies; Binding; Biological Assay; Cell Communication; Cells; Collaborations; Cryptosporidiosis; Cryptosporidium; Cryptosporidium parvum; Data; Development; Disease; Emerging Communicable Diseases; Enzyme Inhibitor Drugs; Enzyme Inhibitors; Enzymes; Epithelial Cells; Funding; Future; Genes; Genome; Glycobiology; Glycoproteins; Goals; Immune response; Immunocompromised Host; In Vitro; Infection; Intervention; Intestines; Invaded; Lectin; Link; Mediating; Molecular; Monoclonal Antibodies; Mucins; New England; Parasites; Parasitology; Patients; Peptides; Pharmaceutical Chemistry; Pharmaceutical Preparations; Play; Polysaccharides; Proteins; Publishing; Recombinants; Resources; Role; Surface; System; Technology; Testing; Therapeutic; Toxoplasma gondii; Transferase; United States National Institutes of Health; Universities; base; biodefense; effective therapy; enzyme activity; high throughput screening; inhibitor/antagonist; novel; polypeptide; ppGalNAc-T; screening; small molecule; small molecule libraries; sugar

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.

描述（由申请人提供）： 机会性顶复形寄生虫隐孢子虫（Crypto）在世界范围内引起可对AIDS患者造成破坏性的生殖道疾病。然而，目前，对于艾滋病相关的隐孢子虫病，还没有有效的、特异性的治疗方法。该项目的长期目标是通过靶向合成O-聚糖的酶来开发艾滋病患者隐孢子虫病的治疗方法，O-聚糖是寄生虫附着和侵入肠上皮细胞的能力不可或缺的。我们先前的和初步的研究强烈表明，O-聚糖在介导感染和诱导免疫应答中起重要作用，并暗示合成它们的酶（UDP-N-乙酰基-D-半乳糖胺：多肽N-乙酰基半乳糖胺转移酶或ppGalNAc-Ts）可以作为免疫功能低下患者，特别是AIDS患者中隐孢子虫病干预的靶点。然而，很少，如果有的话，是知道这些酶在密码。我们的中心假设是，催化关键粘蛋白型糖蛋白上的0-聚糖合成的ppGalNAc-Ts对于Crypto感染是必需的。具体目的是阐明ppGalNAc-Ts在Crypto感染中的功能作用，使用通过与新英格兰生物防御和新兴传染病卓越区域中心（NERCE-BEID）的国家小分子筛选和药物化学（NSRB）核心合作的小分子文库的高通量筛选（HTS）鉴定的抑制剂。将筛选最有效的酶活性抑制剂，以确定其特异性阻断隐O-聚糖合成的能力。然后将测试这些抑制剂在体外抑制Crypto感染的能力。在该项目完成时，我们预计已经表达了所有四种Cp GalNAc T的酶活性重组形式，并鉴定了在体外阻断酶活性、O-聚糖合成和感染的小分子抑制剂。这些研究将确定O-聚糖和催化其合成的酶在Crypto-host细胞相互作用中的作用，并显着推进该领域。该项目的结果将为未来关于特定糖蛋白上的Crypto ppGalNAc-Ts和O聚糖的研究提供信息，这些糖蛋白对于介导寄生虫附着和入侵宿主细胞至关重要。小分子抑制剂，阻断隐ppGalNAc T酶的活性，O-聚糖的合成和感染在体外可以开发为潜在的治疗艾滋病相关的隐孢子虫病在未来的研究。

项目成果

Molecular cloning, expression, and characterization of UDP N-acetyl-α-d-galactosamine: Polypeptide N-acetylgalactosaminyltransferase 4 from Cryptosporidium parvum.

UDP N-乙酰-α-d-半乳糖胺的分子克隆、表达和表征：来自小隐孢子虫的多肽 N-乙酰半乳糖胺基转移酶 4。

• DOI: 10.1016/j.molbiopara.2018.03.002
• 发表时间: 2018
• 期刊: Molecular and biochemical parasitology
• 影响因子: 1.5
• 作者: DeCiccoRePass,MariaA;Bhat,Najma;Heimburg-Molinaro,Jamie;Bunnell,Stephen;Cummings,RichardD;Ward,HonorineD
• 通讯作者: Ward,HonorineD