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

基本信息

批准号：
8069778
负责人：
EILEEN K JAFFE
金额：
$ 1.07万
依托单位：
RESEARCH INST OF FOX CHASE CAN CTR
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-09-23 至 2010-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8069778
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

项目摘要

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.

抽象的：通过功能上不同的非腺体结构的互连来调节变构调节最近已经描述了基本卟啉原合酶（PBG）的基本酶。这使用PBG看到的寡聚重排不同于MWC或Koshland模型的变构模型。因此，引入了morpheein一词来描述变构的结构基础。小分子可以在此称为morphlocks中稳定活性或不活跃的四级结构组件提议充当治疗剂。稳定非活性四级结构组装提出了卟啉原合酶（PBG）作为新类抗生素剂的基础。虽然 PBG是催化四吡咯辅助因子（例如血红素）的生物合成中的第一个常见步骤在人类和大多数人类病原体中必不可少的，假定的系统发育差异很大 Morphlock结合位点。这使我们能够针对PBG进行药物开发。有四个具体目标。 AIM 1是在硅方法中的应用（蛋白质结构建模和对接）将捕获人类病原体PBG的非活性四级结构组件的形态锁铜绿假单胞菌，弧菌霍乱，弓形虫弓形虫和恶性疟原虫。船后标准将用于选择购买化合物。 AIM 2是购买的体外测试从靶向病原体的PBG抑制剂作为抑制剂。使用本地凝胶移位分析和广泛动力学标准，购买的化合物将经过仔细和严格的评估，以选择功能的抑制剂通过低聚物稳定机制和物种选择性。 AIM 3是体内测试使用人类细胞培养系统和标准的微生物学测定法选择了形态锁。在目标4中，我们将使用X射线晶体学评估Morphlock-PBGS复合物的结构。进一步探测精选的morphlocks的结构/功能，我们将合成和评估结构类似物。提议方法已用于发现第一个morphlock，morphlock-1，该morphlock-1抑制了植物PBG 物种选择性时尚，通过捕获无活跃的六聚体组件。应该拟议的形成蛋白变构调节的机制适用于其他医学上重要的蛋白质，拟议的工作将用作利用这种变构的药物发现的路线图。