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.

摘要 在2015年，我们报道了四环素破坏酶（TDases）的发现，这是一个黄素酶家族， 通过酶降解使四环素（泰特）抗生素失活，将其与经典的机械- 对泰特的抵抗。自那份报告以来，我们已将已知TDases的库扩展到>100个功能性TDases 鉴定了酶，报道了许多TDases的晶体结构，并提出了一类小分子 抑制剂来对抗这些酶。TDases现在被广泛认为是一种临床相关的耐药机制， 主义。这项提议的中心动机是为了更好地理解分子机制，进化 的起源和结构特征，以便合理设计更好的诊断和抑制剂， 随着TDases继续传播并成为一种广泛的原因， 发病率和死亡率。我们的合作努力产生了有影响力的科学成果，我们是理想的 有能力实现我们三个独立但互补的具体目标：1）阐明 通过TDases的泰特失活，2）理解TDases在遗传和群体水平上的进化，以及3） 开发TDases的抑制剂和诊断剂。第一个目标将测试多样化的子系统的假设 策略结合模式和FAD辅因子方向决定了泰特氧化和reg的原子位置。 优化TDases的催化循环。我们建议，我们可以关联观察到的酶降解 使用X射线晶体学，光亲和交联，酶动力学， 和同位素标记研究与各种TDases和底物。第二个目标是检查序列 黄素单加氧酶向泰特失活演变的决定因素以及选择性的ad- TDases在表达不同机制的细菌群体中提供的Vantage 泰特抵抗。我们将通过迭代的基于序列的预测和表型分析来识别新的酶。 验证，使用饱和诱变和DNA改组鉴定活性所需的结构元件，和 使用高通量报告基因测定来检查TDases的群体水平适应性优势。第三 目的是确定脱水四环素（aTC）类似物是否可以通过以下方式优化以抑制TDases： 控制配体结合模式和显色Tets是否可以作为诊断剂， 病原体中的TDase表达。我们将使用我们开发的稳健的半合成方法进行修饰 的泰特和aTC支架，并研究所产生的新化合物与严格的生化分析，X射线 晶体学和表型全细胞研究。这项研究意义重大，因为抗生素可以重新... 耐药是一种公共卫生危机，降解所有已知四环素类药物的TDases广泛分布于不同的地区， 环境细菌和病原细菌。这项研究是有影响力的，因为它结合了基金- 随着协同治疗和诊断技术的发展，对酶的进化和机制的认识也在不断加深。 具有减轻酶促泰特失活所造成的新威胁的潜力的治疗剂。