DEVELOPING KINASE INHIBITORS TO COUNTERACT PARASITE VIRULENCE

开发激酶抑制剂来对抗寄生虫毒力

• 批准号: 7644748
• 负责人: L. David Sibley
• 金额: $ 37.93万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-06-01 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7644748
• 关键词: Acute; Adult; Alleles; Animal Model; Antibodies; Biochemical; Bioinformatics; Biological Assay; Cells; Chemicals; Chemotherapy-Oncologic Procedure; Chronic; Databases; Disease; Family; Gel; Genes; Genetic; Genotype; Grant; HIV Infections; Homology Modeling; Human; Immune system; Immunocompromised Host; In Vitro; Infant; Infection; Infection prevention; Lead; Libraries; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Mental disorders; Modeling; Molecular; Molecular Epidemiology; Molecular Models; Monitor; Mus; Ocular Toxoplasmosis; Organ Transplantation; Parasites; Peptides; Phosphoproteins; Phosphorylation; Phosphotransferases; Pro-Q aerosol foam; Process; Protein-Serine-Threonine Kinases; Proteins; Proteomics; Research Design; Risk Factors; Screening procedure; Staining method; Stains; Structure; Substrate Specificity; Testing; Therapeutic Intervention; Time; Toxoplasma; Toxoplasma gondii; Toxoplasmosis; Triad Acrylic Resin; Virulence; Virulence Factors; base; chemotherapy; companion animal; cytotoxic; epidemiology study; foodborne pathogen; high throughput screening; improved; in utero; in vivo; inhibitor/antagonist; insight; kinase inhibitor; member; molecular modeling; mouse model; prevent; public health relevance; rhoptry; secretory protein; tool; trait

DESCRIPTION (provided by applicant): Toxoplasma gondii is a widespread protozoan parasite of wild, domestic, and companion animals that also commonly infects humans. Severe infections are normally only found in immunocompromised patients, including HIV infection, cancer chemotherapy, organ transplant, or infants infected in utero. However, emerging studies indicate that severe ocular toxoplasmosis can also occur in healthy adults, and that chronic infection is an underlying risk factor for some forms of psychiatric disease. Toxoplasma isolates differ dramatically in their virulence in animal models and also in human infections. Recent findings reveal that the major virulence determinants of this parasite are secretory proteins derived from the rhoptries (ROPs), which are injected to the host cell at the time of invasion. Many ROPs contain a conserved serine / threonine kinase domain and while most appear not to be active, several contain a conserved catalytic triad and have been shown to function as kinases. In preliminary studies, we have developed biochemical assays to examine the ability of ROP kinases to phosphorylate a variety of substrates in vitro. In the proposed studies, proteomic, biochemical, mass spectrometry, and bioinformatic analyses will be used to identify substrates of ROP kinases in infected host cells. These studies will provide insight into the mechanism of enhanced virulence conferred by ROP kinases. In vitro kinase assays will be further optimized to provide high-throughput screening for specific inhibitors of ROP kinases. We will also will solve the crystal structure of key members of the ROP kinase family and use molecular modeling to examine specific interactions with inhibitors. Compounds discovered though this process will be analyzed for their potential to prevent infection in vivo using the mouse model. These studies will help define the substrate specificity and inhibitor profile of an important class of parasite virulence factors and may lead to improved therapeutic intervention against toxoplasmosis. PUBLIC HEALTH RELEVANCE: Toxoplasma gondii is an important food borne pathogen of humans that causes disease in those with weakened immune systems. Our studies are designed to identify new chemical inhibitors that counteract the parasite's major virulence factors. Such inhibitors may lead to improved therapeutic intervention against severe infections caused by this parasite.

描述（由申请人提供）：弓形虫弓形虫是一种普遍感染人类的​​野生，家庭和伴侣动物的广泛的原生动物寄生虫。严重感染通常仅在免疫功能低下的患者中发现，包括HIV感染，癌症化疗，器官移植或子宫内感染的婴儿。然而，新兴的研究表明，健康成年人也可能发生严重的眼毒，并且慢性感染是某些形式的精神病患者的潜在危险因素。弓形虫分离株在动物模型和人类感染中的毒力差异很大。最近的发现表明，该寄生虫的主要毒力决定因素是衍生自rhoptries（ROPS）的分泌蛋白，在入侵时被注入宿主细胞。许多ROP包含保守的丝氨酸 /苏氨酸激酶结构域，虽然大多数似乎不活跃，但其中一些包含保守的催化三合会，并且已显示起到激酶的作用。在初步研究中，我们开发了生化测定，以检查ROP激酶在体外磷酸化的各种底物的能力。在拟议的研究中，将使用蛋白质组学，生化，质谱法和生化分析来鉴定感染宿主细胞中ROP激酶的底物。这些研究将洞悉ROP激酶赋予的增强毒力的机制。体外激酶测定将进一步优化，以提供针对ROP激酶特异性抑制剂的高通量筛选。我们还将解决ROP激酶家族的关键成员的晶体结构，并使用分子建模来检查与抑制剂的特定相互作用。发现该过程的化合物将被分析，以防止使用鼠标模型在体内感染的潜力。这些研究将有助于定义一类重要的寄生虫毒力因子的底物特异性和抑制剂概况，并可能导致改善针对弓形虫病的治疗干预措施。公共卫生相关性：弓形虫弓形虫是人类的重要食物传播病原体，可导致免疫系统疲软的患者。我们的研究旨在确定应对寄生虫主要毒力因素的新化学抑制剂。这种抑制剂可能会导致对这种寄生虫引起的严重感染的治疗干预。