Low Activity Oligomers of Porphobilinogen Synthase as Antibiotic Targets

作为抗生素靶标的胆色素原合酶的低活性寡聚物

• 批准号: 7935543
• 负责人: EILEEN K JAFFE
• 金额: $ 43.63万
• 依托单位: RESEARCH INST OF FOX CHASE CAN CTR
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-23 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7935543
• 关键词: Active Sites; Allosteric Regulation; Anabolism; Antibiotics; Binding; Binding Sites; Biological Assay; Biological Models; Cancer Patient; Cell Culture System; Cell Culture Techniques; Cessation of life; Childhood; Collaborations; Complex; Computer Simulation; Core Facility; DNA Sequence Rearrangement; Developing Countries; Development; Disease; Docking; Encephalitis; Enzymes; Equilibrium; Gelshift Analysis; Heme; Homo; Hospitals; Human; Immune; In Vitro; Individual; Infection; Kinetics; Knowledge; Laboratories; Libraries; Malaria; Methods; Microbe; Modeling; Morphologic artifacts; National Institute of Allergy and Infectious Disease; Organism; Parasites; Phylogenetic Analysis; Plants; Plasmodium falciparum; Porphobilinogen Synthase; Positioning Attribute; Postdoctoral Fellow; Preparation; Process; Protein Isoforms; Proteins; Pseudomonas aeruginosa; Relative (related person); Resources; Roentgen Rays; Scheme; Structure; Surface; Technology; Testing; Tetrapyrroles; Therapeutic Agents; Toxoplasma gondii; Variant; Vibrio cholerae; Virulence; Work; X-Ray Crystallography; abstracting; analog; antimicrobial drug; base; cofactor; comparative; cystic fibrosis patients; drug development; drug discovery; enzyme activity; expression cloning; in vitro testing; in vivo; inhibitor/antagonist; non-drug; pathogen; protein oligomer; protein structure; small molecule; virtual

Abstract: Allosteric regulation through the interconversion of functionally distinct non-additive quaternary structure assemblies has recently been described for the essential enzyme porphobilinogen synthase (PBGS). The oligomeric rearrangements seen with PBGS are different from the MWC or Koshland models for allostery; thus, the term “morpheein” has been introduced to describe this structural basis for allostery. Small molecules that can stabilize either an active or inactive quaternary structure assembly, herein called morphlocks, are proposed to function as therapeutic agents. Stabilization of an inactive quaternary structure assembly of porphobilinogen synthase (PBGS) is proposed as the basis for a new class of antibiotic agents. Although PBGS, which catalyzes the first common step in the biosynthesis of the tetrapyrrole cofactors (e.g. heme), is essential in both humans and most human pathogens, there is extensive phylogenetic variation in the putative morphlock binding sites. This allows us to target PBGS for drug development. There are four Specific Aims. Aim 1 is the application of in silico methods (protein structure modeling and docking) to the discovery of morphlocks that will trap inactive quaternary structure assemblies of PBGS from the human pathogens Pseudomonas aeruginosa, Vibrio cholera, Toxoplasma gondii, and Plasmodium falciparum. Post-docking criteria will be used to select compounds for purchase. Aim 2 is the in vitro testing of the purchased compounds as inhibitors of PBGS from the targeted pathogens. Using native gel shift analysis and extensive kinetic criteria, purchased compounds will be carefully and critically evaluated to select inhibitors that function through the oligomer stabilization mechanism and with species selectivity. Aim 3 is the in vivo testing of chosen morphlocks using a human cell culture system and standard microbiological assays. In Aim 4 we will use X-ray crystallography to evaluate the structures of the morphlock-PBGS complexes. To further probe the structure/function of select morphlocks, we will synthesize and evaluate structural analogs. The proposed methods have been used to discover the first morphlock, morphlock-1, which inhibits a plant PBGS in a species selective fashion by trapping the inactive hexameric assembly. Should the proposed morpheein mechanism for allosteric regulation be applicable to other medically important proteins, the proposed work will serve as a roadmap for harnessing this allosteric mechanism for drug discovery.

摘要： 通过功能不同的非加性四级结构的相互转换的别构调节 最近已经描述了必需酶胆色素原合酶（PBGS）的组装。的 用PBGS观察到的寡聚重排与变构的MWC或Koshland模型不同;因此， 引入术语吗啡因来描述变构的这种结构基础。的小分子 可以稳定活性或非活性四级结构组装体，本文称为吗啉嵌段， 建议用作治疗剂。的无活性四级结构组装体的稳定化 胆色素原合酶（PBGS）被提议作为一类新的抗生素剂的基础。虽然 PBGS催化四吡咯辅因子（例如血红素）生物合成中的第一个共同步骤， 在人类和大多数人类病原体中都是必需的，在假定的病原体中存在广泛的系统发育变异。 morphlock结合位点。这使我们能够针对PBGS进行药物开发。有四个具体目标。 目的1是应用计算机模拟方法（蛋白质结构建模和对接）发现 将捕获来自人类病原体的PBGS的无活性四级结构组装体的morphlock 铜绿假单胞菌、霍乱弧菌、刚地弓形虫和恶性疟原虫。后入坞 将使用标准来选择购买的化合物。目标2是对购买的 作为来自靶向病原体的PBGS的抑制剂的化合物。使用天然凝胶位移分析和广泛的 动力学标准，购买的化合物将进行仔细和严格的评估，以选择抑制剂的功能， 通过低聚物稳定化机制和物种选择性。目的3是体内测试 使用人细胞培养系统和标准微生物测定法选择吗啉。在目标4中， 使用X射线晶体学来评估吗啉-PBGS复合物的结构。为了进一步探索 结构/功能的选择，我们将合成和评估结构类似物。拟议 方法已经被用来发现第一个morphlock，morphlock-1，它抑制植物PBGS， 通过捕获无活性的六聚体组装的物种选择性方式。建议中的莫菲因 变构调节机制适用于其他医学上重要的蛋白质，拟议的工作将 作为利用这种变构机制进行药物发现的路线图。