New Approaches to Mining the Complete Actinomycete Genome to Discover Novel Beta-Lactam Antibiotics

挖掘完整放线菌基因组以发现新型β-内酰胺抗生素的新方法

• 批准号: 9409126
• 负责人: Lucy Foulston
• 金额: $ 15.46万
• 依托单位: WARP DRIVE BIO, INC
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-11 至 2018-06-01
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9409126
• 关键词: Accounting; Actinomyces Infections; Aminoglycosides; Anti-Bacterial Agents; Antibiotic Therapy; Antibiotics; Bacteria; Bacterial Infections; Bioinformatics; Biological; Biological Assay; CD69 antigen; Categories; Cell Wall; Clinical; Data; Databases; Drug resistance; Drug-sensitive; Engineering; Escherichia coli; Evaluation; FDA approved; FK506; Family; Family member; Fermentation; Gene Cluster; Genes; Genetic Engineering; Genome; Genomics; Goals; Growth; Knock-out; Lactamase; Lactams; Literature; Mass Spectrum Analysis; Medicine; Methods; Mining; Monobactams; Multi-Drug Resistance; National Institute of Allergy and Infectious Disease; Natural History; Natural Products; Nosocomial Infections; Patients; Penicillin-Binding Proteins; Peptidyltransferase; Pharmaceutical Preparations; Pharmacologic Substance; Pharmacopoeias; Phase; Production; Reporting; Research; Resistance; Risk; Safety; Sirolimus; Source; Structure; Techniques; Therapeutic; United States National Institutes of Health; antimicrobial; bacterial resistance; base; beta-Lactamase; beta-Lactams; biodefense; clinical efficacy; combat; drug discovery; improved; inhibitor/antagonist; innovation; interest; miniaturize; mortality; novel; novel strategies; pathogen; programs; promoter; scale up; screening; search engine; small molecule; synthetic biology; tool

ABSTRACT There is a critical need to discover and develop safe and effective antibiotics for the treatment of multidrug- resistant bacterial infections. Each year approximately 1.5 million patients in the US get hospital acquired infections (HAIs), with a mortality rate of ~6%. Nearly 70% of the bacteria causing HAIs are resistant to at least one commonly used antibiotic. Natural products (NPs) and their derivatives are the basis of many pharmaceutical advances, representing ~50% of FDA approved small molecule drugs over the past 5 decades including many important antibiotics. Genome mining for novel natural products is quickly replacing traditional approaches to antibiotic discovery. However approximately 90% of all actinomycete gene clusters are cryptic or silent, i.e., expressed at levels too low to detect using traditional antibacterial screening assays, or not expressed at all. Warp Drive Bio (WDB) has sequenced over 135,000 actinomycete strain genomes from diverse sources worldwide, and our proprietary genomic database contains ~3.5 million secondary metabolite gene clusters. Importantly ~75% of cluster families in our database have yet to be reported in the literature. This provides an unprecedented opportunity to discover structurally and mechanistically novel NPs. Our objective is to discover and develop novel β-lactams (BLs) as broad-spectrum antibiotics with improved therapeutic profiles aimed at combating current and emerging multidrug-resistant Gram (-)/(+) pathogens. BLs represent a clinically validated class widely used in antimicrobial therapy with good, broad-spectrum activity and favorable safety profiles. Unfortunately, the emergence of multi-drug resistance severely limits the clinical efficacy of current BL antibiotics, as single agents or in combination with β-lactamase inhibitors. WDB is deploying our proprietary natural product genome-mining platform to discover novel BL antibiotics. This platform offers a new paradigm in which drug discovery is initiated at the genome level, allowing rapid identification of novel compounds that would be otherwise overlooked in a traditional activity-based discovery paradigm. We first deploy our bioinformatics search engines to rapidly identify gene clusters within WDB's genomic database that encode novel BL molecules. To access the potential of silent biosynthetic gene clusters, we have developed methods to systematically replace promoters, inducing compound expression. Clusters are transferred into engineered heterologous expression hosts to produce novel molecules that are isolated as extracts in a miniaturized high throughput plate format for bioassay, and we utilize mass spectrometry analytics to rapidly identify the masses of novel BLs of interest to facilitate rapid purification for further characterization. We will apply our genome-mining platform to engineer, express, purify, and characterize the products from 10 novel BL biosynthetic clusters in this Phase I proof-of-concept study.

摘要 迫切需要发现和开发安全有效的抗生素来治疗多药耐药- 耐药细菌感染。美国每年约有150万患者在医院获得 感染（HAI），死亡率约为6%。近70%的引起HAI的细菌对至少 一种常用的抗生素天然产物（NPs）及其衍生物是许多生物技术的基础。 制药进展，在过去50年中，约占FDA批准的小分子药物的50% 包括许多重要的抗生素。新天然产物的基因组挖掘正在迅速取代传统的 抗生素发现的方法然而，大约90%的放线菌基因簇是隐蔽的 或沉默，即，表达水平太低，无法使用传统的抗菌筛选测定法检测， 在所有表达。Warp Drive Bio（WDB）已经对来自世界各地的135，000多个放线菌菌株基因组进行了测序。 我们的专有基因组数据库包含约350万种次级代谢产物， 基因簇重要的是，我们数据库中约75%的簇家族尚未在文献中报道。 这为发现结构和机制新颖的纳米粒子提供了前所未有的机会。 我们的目标是发现和开发新的β-内酰胺类抗生素， 旨在对抗当前和新出现的多重耐药革兰氏（-）/（+）病原体的治疗概况。BLS 代表广泛用于抗菌治疗的临床验证类别，具有良好的广谱活性 和良好的安全性。然而，多药耐药的出现严重限制了其临床应用， 作为单一药剂或与β-内酰胺酶抑制剂组合的当前BL抗生素的功效。WDB是 利用我们专有的天然产物基因组挖掘平台，发现新型BL抗生素。这 平台提供了一个新的范式，其中药物发现是在基因组水平启动，允许快速 识别在传统的基于活性的发现中会被忽视的新化合物 范例我们首先部署我们的生物信息学搜索引擎来快速识别WDB中的基因簇， 编码新BL分子的基因组数据库。利用沉默生物合成基因的潜力 簇，我们已经开发了系统地替换启动子，诱导化合物表达的方法。 将簇转移到工程化的异源表达宿主中以产生新的分子， 在用于生物测定的小型化高通量板格式中分离为提取物，并且我们利用质量 光谱分析，以快速鉴定感兴趣的新BL的质量，以促进快速纯化， 进一步表征。我们将应用我们的基因组挖掘平台来工程，表达，纯化， 在这一I期概念验证研究中，对来自10种新型BL生物合成簇的产物进行表征。