Identification of small molecule probes for dissecting the roles of sorting platform components within the type III secretion system

鉴定小分子探针，用于剖析 III 型分泌系统中分选平台组件的作用

• 批准号: 9806976
• 负责人: WILLIAM D. PICKING
• 金额: $ 22.73万
• 依托单位: UNIVERSITY OF KANSAS LAWRENCE
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-06-01 至 2021-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9806976
• 关键词: ATP phosphohydrolase; Anti-Infective Agents; Architecture; Bacteria; Binding; Binding Sites; Biology; Bordetella pertussis; Cell physiology; Cells; Chemicals; Clinical; Communication; Complex; Crystallization; Cytoplasmic Tail; Diarrhea; Distant; Dysentery; Ensure; Eukaryotic Cell; Foundations; Gram-Negative Bacteria; Heart; Homologous Protein; Human; Impairment; In Situ; In Vitro; Investigation; Kansas; Laboratories; Laboratory Study; Libraries; Ligand Binding; Mechanics; Membrane; Needles; Nosocomial Infections; Pertussis; Pilot Projects; Plague; Positioning Attribute; Proteins; Pseudomonas; Pseudomonas aeruginosa; Publishing; Recombinants; Reporting; Resolution; Role; Salmonella; Shigella; Site; Sorting - Cell Movement; Structural Protein; Structure; Structure-Activity Relationship; System; Testing; Therapeutic Agents; Time; Type III Secretion System Pathway; Typhoid Fever; Universities; Virulence; Virulence Factors; Work; Yersinia pestis; antimicrobial; base; cell envelope; experimental study; high risk; high throughput screening; human pathogen; inhibitor/antagonist; kinetosome; nanomachine; novel; pathogenic bacteria; protein protein interaction; protein structure; response; scaffold; screening; small molecule; small molecule inhibitor; small molecule libraries; stoichiometry; synergism; tool

SUMMARY Our laboratory studies the bacterial type III secretion system (T3SS) which is a sophisticated nanomachine that permits communication with eukaryotic cells to subvert normal cellular functions. T3SSs are essential virulence factors for a number of biomedically important human pathogens including Yersinia pestis (plague), Shigella spp. (dysentery), Salmonella spp. (diarrhea/enteric fever), Bordetella pertussis (whooping cough), and Pseudomonas aeruginosa (opportunistic/nosocomial infections). While these systems are diverse with regard to the effector proteins they deliver, the T3SS apparatus (T3SA) is well-conserved with respect to overall structure and architecture. The T3SA is comprised of: 1) a cytoplasmic sorting platform that recognizes secretion substrates and powers secretion; 2) a rigid basal body spanning the entire cell envelope; and 3) an exposed needle with tip complex that delivers cargo into host cells. The protein components of the sorting platform have been identified, but little was known about how they assemble until we published the first description of the in situ Shigella T3SA sorting platform. Since then, we have determined the structural details and dynamics of this platform, including the essential protein-protein contacts. Based on this background information, we hypothesize that key interfaces within the sorting platform can be targeted to block type III secretion in Shigella. Furthermore, we can extrapolate our findings to identify inhibitors of homologous interactions within distantly related T3SA sorting platforms such as that form P. aeruginosa. Here, we will target the interaction between inner membrane-imbedded MxiG (via its cytoplasmic domain, MxiGc) and its newly identified binding partner, MxiK, in Shigella. The homologous pair from P. aeruginosa (PscDc and PscK, respectively) will also be targeted. The specific aims of this pilot project application are to: 1) Complete high-throughput screens of extended chemical libraries to identify small molecules that interfere with the interaction of MxiGc (PscDc) with MxiK (PscK); and 2) Validate hits for inhibitors of the MxiG-MxiK and PscD-PscK interactions by performing ligand binding experiments and determining their effects on Shigella and Pseudomonas T3SS-related virulence functions. This high-risk, high-payoff potential of this exploratory project will employ specialized core laboratories in high-throughput screening, computational chemical biology and protein structure determination at the University of Kansas to generate a synergy that will allow an unprecedented level of understanding of the mechanism by which the T3SA sorting platform operates.

概括 我们的实验室研究是细菌III型分泌系统（T3SS），这是一种精致的纳米机械 这允许与真核细胞的通信颠覆正常的细胞功能。 T3SS是必不可少的 许多生物医学重要的人类病原体（包括耶尔森氏菌（Pestis）（瘟疫））的毒力因子， Shigella spp。 （痢疾），沙门氏菌属。 （腹泻/肠发烧），百日咳（百日咳）和 铜绿假单胞菌（机会性/医院感染）。尽管这些系统在 他们传递的效应蛋白，T3SS设备（T3SA）相对于整体结构得到了很好的保存 和建筑。 T3SA由：1）识别分泌的细胞质分类平台 底物和力量分泌； 2）横跨整个细胞包膜的刚体； 3）暴露 带有尖端复合物的针头，将货物输送到宿主细胞中。排序平台的蛋白质成分具有 已经确定了，但对它们如何组装知之甚少，直到我们出版了第一个描述 SITU SHIGELLA T3SA分类平台。从那以后，我们确定了结构的细节和动态 平台，包括必需的蛋白质 - 蛋白质接触。基于此背景信息，我们 假设分类平台内的关键接口可以针对以阻止III类型的分泌 志贺氏菌。此外，我们可以推断我们的发现以识别同源相互作用的抑制剂 在远距离相关的T3SA排序平台中，例如铜绿假单胞菌。在这里，我们将针对 内膜成膜的MXIG（通过其细胞质结构域MXIGC）及其新鉴定的相互作用 装订合作伙伴MXIK在Shigella中。来自铜绿假单胞菌（分别PSCDC和PSCK）的同源对将 也是针对的。该试点项目申请的具体目的是：1）完整的高通量屏幕 扩展的化学文库以识别干扰MXIGC（PSCDC）相互作用的小分子 MXIK（PSCK）; 2）通过执行MXIG-MXIK和PSCD-PSCK的抑制剂验证命中 配体结合实验并确定其对志贺氏菌和假单胞菌T3SS相关毒力的影响 功能。这个探索性项目的高风险，高付的潜力将采用专业的核心实验室 在高通量筛选中，计算化学生物学和蛋白质结构在 堪萨斯大学产生一种协同作用，该协同作用将使人们对 T3SA排序平台运行的机制。