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：通过功能不同的非加性四级结构的相互转换进行变构调节最近描述了必需酶胆色素原合酶（PBGS）的组装。这 PBGS 观察到的寡聚重排与 MWC 或 Koshland 变构模型不同；因此，术语“吗啡因”被引入来描述变构的这种结构基础。小分子可以稳定活性或非活性四级结构组装，本文称为形态锁，是建议用作治疗剂。非活性四级结构组装的稳定性胆色素原合酶（PBGS）被提议作为一类新型抗生素的基础。虽然 PBGS 催化四吡咯辅因子（例如血红素）生物合成的第一个常见步骤，是对于人类和大多数人类病原体都是必需的，假定的系统发育存在广泛的变异形态锁结合位点。这使我们能够以 PBGS 为目标进行药物开发。有四个具体目标。目标 1 是应用计算机方法（蛋白质结构建模和对接）来发现形态锁将捕获人类病原体中 PBGS 的无活性四级结构组装体铜绿假单胞菌、霍乱弧菌、弓形虫和恶性疟原虫。对接后标准将用于选择购买的化合物。目标 2 是对购买的产品进行体外测试化合物作为目标病原体的 PBGS 抑制剂。使用天然凝胶位移分析和广泛的动力学标准，购买的化合物将经过仔细和严格的评估，以选择起作用的抑制剂通过低聚物稳定机制和物种选择性。目标 3 是体内测试使用人类细胞培养系统和标准微生物测定法选择形态锁。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。为进一步探究选择形态锁的结构/功能，我们将合成和评估结构类似物。拟议的方法已被用来发现第一个形态锁，形态锁-1，它抑制植物中的 PBGS 通过捕获不活跃的六聚体组装来进行物种选择性时尚。如果建议的吗啡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。