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

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- 具有抗药性的细菌感染。美国每年约有150万名患者在医院获得 感染(HAI)，死亡率约为6%。近70%的引起人类免疫缺陷的细菌至少对 一种常用的抗生素。天然产物(NPs)及其衍生物是许多 药物进展，占过去50年FDA批准的小分子药物的50%左右 包括许多重要的抗生素。对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，即表达水平太低而不能用传统的抗菌筛选试验检测到，或者不表达 完全没有表达出来。Warp Drive Bio(WDB)已经对来自中国的135,000多株放线菌基因组进行了测序 来源多样，我们的专有基因组数据库包含约350万次生代谢物 基因簇。重要的是，我们数据库中约75%的聚类族尚未在文献中报道。 这为发现结构和机械上新颖的NPs提供了一个前所未有的机会。 我们的目标是发现和开发新型β-内酰胺类药物，作为具有改良的广谱抗生素 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 代表了一种经过临床验证的类别，广泛用于抗菌治疗，具有良好的广谱活性 和良好的安全状况。不幸的是，多重耐药的出现严重限制了临床 目前的BL抗生素，作为单一药物或与β-内酰胺酶抑制剂联合使用的有效性。Wdb是 部署我们专有的天然产品基因组挖掘平台，发现新的BL抗生素。这 Platform提供了一种新的范式，其中药物发现是在基因组水平上启动的，允许快速 鉴定在传统的基于活动的发现中可能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 编码新的BL分子的基因组数据库。挖掘沉默生物合成基因的潜力 在簇的基础上，我们开发了系统地替换启动子的方法，诱导复合表达。 簇被转移到工程异源表达宿主中，以产生新的分子 以小型化的高通量平板格式分离的AS提取物用于生物测定，我们利用质量 用光谱分析技术快速鉴定新的感兴趣的BL的质量，以便于快速纯化 进一步的刻画。我们将应用我们的基因组挖掘平台来工程、表达、纯化和 在这项第一阶段的概念验证研究中，表征来自10个新的BL生物合成簇的产品。

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.