Connecting structure and fitness landscapes to overcome antibiotic resistance

连接结构和健身景观以克服抗生素耐药性

基本信息

批准号：
10679332
负责人：
Christian Bernard Macdonald
金额：
$ 6.95万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-09-13 至 2025-09-12
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10679332
关键词：
Acetylation Amino Acids Antibiotic Resistance Antibiotics Area Binding Biochemistry Biological Assay Biophysics Chemosensitization Clinical Collaborations Complex Coupled Coyotes Crystallography Development Drug Design Drug resistance Enterococcus faecium Environment Enzymatic Biochemistry Escherichia coli Event Evolution Failure Family Future Genes Genetic Recombination Goals Grant Heterogeneity Homologous Gene In Vitro Laboratories Libraries Link Longevity Maps Measurement Measures Mediating Mentorship Microbe Modeling Molecular Molecular Conformation Mutation Nature Outcome Pathway interactions Phenotype Phylogenetic Analysis Play Positioning Attribute Process Property Proteins Research Resistance Resources Ribosomes Role Sampling Scanning Sequence Analysis Streptogramins Streptomyces Structural Biochemistry Structure Substrate Specificity Techniques Temperature Universities Variant Work analog antimicrobial biophysical model biophysical properties combinatorial enzyme activity fitness gain of function global health improved insertion/deletion mutation insight microbial mutant mutation screening novel novel strategies pathogen permissiveness predictive modeling preempt protein function protein structure resistance gene resistance mechanism response screening training opportunity

项目摘要

Project summary - Connecting structure and fitness landscapes to overcome antibiotic resistance Antibiotic resistance is a pressing, multifaceted challenge. Pathogen evolution is outstripping the supply of new compounds and analogues, threatening a global health crisis. New approaches to understand adaptation are clearly necessary, but this is a difficult problem. The mechanisms of resistance are often unknown, as well as the overall combination of changes that produce overall microbial fitness changes. Technical developments in high-throughput biochemistry have allowed massive variant libraries to be assayed, which opens the door to constructing predictive models of resistance, but these have until our recent work ignored complex mutations, specifically insertions and deletions, which play a massive role by producing major changes to underlying fitness landscapes with small mutations. To study how, we will combine experimental evolution, deep mutational scanning, and multitemperature crystallography to produce an integrated model for how insertions and deletions permit rapid changes to protein function. We will use the Streptogramin A family as our model antibiotic. These are ribosome-targeting compounds produced by Streptomyces. Resistance occurs through Vat proteins, which specifically inactivate Streptogramin A (SA) via acetylation. A collaboration with the Seiple lab at UCSF has led to a modular synthesis of SA that allows variants to be simply produced, as well as several novel compounds with demonstrated reduced acetylation in vitro. We will determine how adaptation to these novel compounds proceeds, and how indels within a key variable substrate-binding loop modulate it. In my first aim, I will use experimental evolution to uncover how Vat proteins adapt to streptogramins, and how the addition of insertions and deletions within this loop change the adaptive potential. In my second aim, I will conduct high-throughput stability measurements to determine the mechanistic basis for resistance changes, and then use deep mutational scanning to measure the mutational accessibility and biophysical basis for an evolved adaptive trajectory. In my third aim, I will use cryo- and multitemperature crystallography to determine the static and dynamic basis for indel-potentiated changes to substrate specificity towards new SAs. The completion of these aims will produce an integrated picture of microbial adaptation and essential new insight into how complex but understudied mutations radically shift the adaptive potential of genes. The cross- disciplinary nature of the project will expose me to a number of techniques and approaches that will provide me with excellent training opportunities under the mentorship of field-defining experts. The new frameworks and techniques I will develop will help establish my scientific maturity as I pursue my goal of an independent research position studying the mechanistic basis of molecular protein evolution at a university.

项目摘要 - 连接结构和健身景观以克服抗生素耐药性抗生素耐药性是一种紧迫的多方面挑战。病原体进化超越了新的供应化合物和类似物，威胁着全球健康危机。了解适应的新方法是显然是必要的，但这是一个困难的问题。抵抗的机制通常是未知的，以及变化的总体组合产生了整体微生物适应性的变化。技术发展高通量生物化学允许测定大量的变体库，这打开了通往的大门构建抗性的预测模型，但是直到我们最近的工作忽略了复杂突变，特别是插入和删除，通过对基础产生重大变化来发挥巨大作用适应性景观，具有小突变。为了研究如何，我们将结合实验进化，深突变扫描和多振体晶体学，以产生插入方式的集成模型缺失允许快速改变蛋白质功能。我们将使用链球菌作为家族作为模型抗生素。这些是核糖体靶向化合物由链霉菌产生。电阻通过增值税蛋白发生，该蛋白特别活化通过乙酰化A（SA）链球素A（SA）。与UCSF的Seiple Lab的合作导致了模块化 SA的合成可以简单地产生变体，以及几种具有的新型化合物显示体外乙酰化降低。我们将确定如何适应这些新型化合物进行，以及如何在关键变量基材结合环中进行置换。在我的第一个目的中，我将使用实验进化来揭示增值税蛋白如何适应链链球菌，以及如何适应在此循环中添加插入和缺失改变了自适应电位。在我的第二个目标中，我会进行高通量稳定性测量以确定电阻变化的机理基础，然后使用深层突变扫描来测量突变的可及性和生物物理基础进化的自适应轨迹。在我的第三个目标中，我将使用冷冻和多振体晶体学来确定对新SAS的底物特异性变化的静态和动态基础。这些目标的完成将产生微生物适应和必不可少的新图片深入了解复杂但研究的突变如何从根本上转移基因的适应潜力。交叉该项目的纪律性质将使我了解许多能够为我提供的技术和方法在现场定义专家的指导下，具有出色的培训机会。新框架和我将开发的技术将有助于建立我的科学成熟度，因为我追求独立的目标研究位置研究了大学分子蛋白进化的机理基础。