Structural, mechanistic, & evolutionary characterization of tetracycline destructases

结构性、机械性、

• 批准号: 10298624
• 负责人: Gautam Dantas
• 金额: $ 66.76万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-02-11 至 2026-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10298624
• 关键词: Address; Antibiotics; Bacteria; Base Sequence; Binding; Binding Sites; Biochemical; Biological Assay; Cells; Clinical; Color; Crystallization; DNA Shuffling; Detection; Development; Diagnostic; Disadvantaged; Distal; Elements; Enzyme Kinetics; Enzymes; Evolution; Family; Flavins; Funding; Generations; Genes; Genetic; Human; Human Microbiome; In Vitro; Infection; Intervention; Isotope Labeling; Knowledge; Ligand Binding; Maps; Mediating; Methods; Microbiology; Mission; Mixed Function Oxygenases; Modification; Molecular; Morbidity - disease rate; Motivation; Mutagenesis; Outcome; Oxides; Partner in relationship; Pharmaceutical Preparations; Phenotype; Plasmids; Population; Public Health; Reporter; Reporting; Research; Resistance; Resort; Ribosomes; Site; Soil; Structure; Testing; Tetracycline Resistance; Tetracyclines; Therapeutic; Treatment Failure; United States National Institutes of Health; Validation; Variant; X-Ray Crystallography; analog; base; clinically relevant; cofactor; combat; crosslink; design; drug resistant bacteria; experimental study; fitness; human disease; human pathogen; improved; inhibitor/antagonist; innovation; interest; markov model; mortality; mouse model; multi-drug resistant pathogen; multidisciplinary; novel; oxidation; pathogen; pathogenic bacteria; pressure; rapid detection; resistance mechanism; scaffold; small molecule; small molecule inhibitor; transmission process

ABSTRACT In 2015, we reported the discovery of the tetracycline destructases (TDases), a family of flavoenzymes capable of inactivating tetracycline (Tet) antibiotics by enzymatic degradation, distinguishing them from canonical mech- anisms of Tet resistance. Since that report we have expanded the pool of known TDases to >100 functionally identified enzymes, reported crystal structures of numerous TDases, and proposed a class of small molecule inhibitors to combat these enzymes. TDases are now widely recognized as a clinically-relevant resistance mech- anism. The central motivation for this proposal is to better understand the molecular mechanisms, evolutionary origins, and structural features of TDases in order to rationally design better diagnostics and inhibitors to restore efficacy of a vital class of antibiotics as TDases continue to disseminate and become a widespread cause of morbidity and mortality. Our collaborative effort has yielded impactful scientific results, and we are ideally equipped to carry out our three independent yet complementary specific aims: 1) Elucidate the mechanism of Tet inactivation by the TDases, 2) Understand the evolution of TDases at genetic and population levels, and 3) Develop inhibitors and diagnostic agents for TDases. The first aim will test the hypothesis that diverse sub- strate-binding modes and FAD cofactor orientations determine the atomic site of Tet oxidation and reg- ulate the catalytic cycle of the TDases. We propose that we can correlate observed enzymatic degradation products with respective binding modes using X-ray crystallography, photoaffinity crosslinking, enzyme kinetics, and isotopic labeling studies with a variety of TDases and substrates. The second aim examines the sequence determinants of flavin monooxygenase evolution toward Tet inactivation as well as the selective ad- vantage that TDases provide in the context of bacterial populations expressing different mechanisms of Tet resistance. We will identify novel enzymes through iterative sequence-based predictions and phenotypic validation, identify structural elements required for activity using saturation mutagenesis and DNA shuffling, and examine the population-level fitness advantages of TDases using high-throughput reporter assays. The third aim will determine whether anhydrotetracycline (aTC) analogs can be optimized to inhibit TDases by controlling ligand binding mode and whether chromogenic Tets can serve as diagnostic agents for TDase expression in pathogens. We will use robust semi-synthetic methods developed by us for modification of the Tet and aTC scaffolds and study the resulting novel compounds with rigorous biochemical assays, X-ray crystallography, and phenotypic whole-cell studies. The proposed research is significant because antibiotic re- sistance is a public health crisis, and TDases that degrade all known tetracyclines are widely distributed in di- verse environmental and pathogenic bacteria. The proposed research is impactful because it combines funda- mental understanding of enzyme evolution and mechanism with the development of co-therapeutic and diag- nostic agents that have the potential to mitigate the emerging threat posed by enzymatic Tet inactivation.

抽象的 2015年，我们报道了四环素破坏酶（TDases）的发现，这是一群能够 通过酶促降解使四环素（TET）抗生素灭活，将其与规范机械区分开 TET抗性的敌人。由于该报告，我们将已知TDase的池扩展到> 100个功能 鉴定出酶，报道了许多TD酶的晶体结构，并提出了一类小分子 抑制剂以对抗这些酶。现在，TDas酶被广泛认为是临床上与临床相关的抗药性机甲 anism。该建议的主要动机是更好地了解分子机制，进化 起源和TD酶的结构特征，以合理设计更好的诊断和抑制剂以恢复 随着TD酶继续传播，一系列重要类别的抗生素的功效，并成为广泛的原因 发病率和死亡率。我们的协作努力取得了影响力的科学成果，理想情况下我们是 配备了我们的三个独立但互补的特定目的：1）阐明 TD酶的TET失活，2）了解TD酶在遗传和人群水平上的演变，以及3） 开发抑制剂和TD酶的诊断剂。第一个目标将检验以下假设，即不同的子 策略结合模式和FAD辅助方向决定了TET氧化和反状的原子位点 阐明TDas酶的催化循环。我们建议我们可以将观察到的酶促降解相关联 具有相应结合模式的产品，使用X射线晶体学，光亲和力交联，酶动力学， 以及各种TD酶和底物的同位素标记研究。第二个目标检查了序列 黄素单加氧酶进化的决定因素，用于TET灭活以及选择性AD- Tdases在细菌种群中提供的有利位置，表达不同机制 TET电阻。我们将通过基于迭代序列的预测和表型来鉴定新酶 验证，使用饱和诱变和DNA改组所需的活动所需的结构元素，以及 使用高通量记者测定法检查TDase的人群级适应性优势。第三 AIM将确定是否可以优化甲非在苯乙烯（ATC）类似物以通过 控制配体结合模式以及发色质TET是否可以用作诊断剂 病原体中的TD酶表达。我们将使用我们开发的强大半合成方法进行修改 TET和ATC支架，并使用严格的生化测定，X射线研究所得的新型化合物 晶体学和表型全细胞研究。拟议的研究很重要，因为抗生素重新 SISTANCE是一种公共卫生危机，降解所有已知四环素的TD酶都广泛分布 诗歌环境和致病细菌。拟议的研究具有影响力，因为它结合了基础 对酶的进化和机制的心理理解，随着共同治疗和诊断的发展 有潜力减轻酶促灭活构成的新兴威胁的现象。