摘要

尽管广泛使用小分子抗微生物联合疗法，但在美国，抗生素耐药性感染每年杀死大约23,000人并且每年花费20,000,000,000美元。抗生素组合通常具有累加效应：组合的功效匹配单独使用时每种抗生素的效力总和。当组合的功效大于附加功效时，小分子也可以协同作用。然而，协同组合很少见，历史上难以确定。协同配对的高通量鉴定受到潜在组合规模的限制：1000个小分子的适度收集涉及100万个配对组合。在这里，我们描述了一种高通量快速鉴定协同小分子对的方法，overlap2方法（O2M）。 O2M从化学基因数据库中提取模式，这些数据集是在一系列突变体在数百个不同小分子存在的情况下生长时产生的，从而产生由突变集合中每个小分子诱导的精确表型。当用已知的协同分子处理时，显示相同表型的突变体的鉴定使我们能够确定也协同起作用的另外的分子组合。作为概念的证据，我们关注与抗生素甲氧苄氨嘧啶和磺胺甲四唑的组合，这是抗尿路感染的标准疗法，直到广泛的抵抗力降低疗效。使用O2M，我们筛选了一个包含2,000个小分子的文库，并确定了几个与抗生素甲氧苄氨嘧啶和/或磺胺甲四唑协同作用的文库。这些协同相互作用中最有效的是抗病毒药物叠氮胸苷（AZT）。然后我们证明理解小分子协同相互作用的分子机制可以合理设计旁路抗药性的其他组合。甲氧苄啶和磺胺甲四唑都是叶酸生物合成抑制剂。我们发现这种活性破坏了核苷酸稳态，它在AZT存在下阻止DNA复制。基于这些数据，我们显示其他通过其他机制（羟基脲和氟尿苷）破坏核苷酸稳态的小分子也与AZT协同作用。这些新的组合抑制耐甲氧苄氨嘧啶临床大肠杆菌和肺炎克雷伯菌分离株的生长和毒力，表明它们可能能够迅速进入临床使用。总之，我们提出了一种通用的方法来筛选新的协同组合，识别导致协同作用的特定机制，并使用机械知识合理设计绕过耐药性的新组合。

Antibiotic-resistant infections kill approximately 23,000 people and cost $20,000,000,000 each year in the United States alone despite the widespread use of small-molecule antimicrobial combination therapy. Antibiotic combinations typically have an additive effect: the efficacy of the combination matches the sum of the efficacies of each antibiotic when used alone. Small molecules can also act synergistically when the efficacy of the combination is greater than the additive efficacy. However, synergistic combinations are rare and have been historically difficult to identify. High-throughput identification of synergistic pairs is limited by the scale of potential combinations: a modest collection of 1,000 small molecules involves 1 million pairwise combinations. Here, we describe a high-throughput method for rapid identification of synergistic small-molecule pairs, the overlap2 method (O2M). O2M extracts patterns from chemical-genetic datasets, which are created when a collection of mutants is grown in the presence of hundreds of different small molecules, producing a precise set of phenotypes induced by each small molecule across the mutant set. The identification of mutants that show the same phenotype when treated with known synergistic molecules allows us to pinpoint additional molecule combinations that also act synergistically. As a proof of concept, we focus on combinations with the antibiotics trimethoprim and sulfamethizole, which had been standard treatment against urinary tract infections until widespread resistance decreased efficacy. Using O2M, we screened a library of 2,000 small molecules and identified several that synergize with the antibiotic trimethoprim and/or sulfamethizole. The most potent of these synergistic interactions is with the antiviral drug azidothymidine (AZT). We then demonstrate that understanding the molecular mechanism underlying small-molecule synergistic interactions allows the rational design of additional combinations that bypass drug resistance. Trimethoprim and sulfamethizole are both folate biosynthesis inhibitors. We find that this activity disrupts nucleotide homeostasis, which blocks DNA replication in the presence of AZT. Building on these data, we show that other small molecules that disrupt nucleotide homeostasis through other mechanisms (hydroxyurea and floxuridine) also act synergistically with AZT. These novel combinations inhibit the growth and virulence of trimethoprim-resistant clinical Escherichia coliand Klebsiella pneumoniae isolates, suggesting that they may be able to be rapidly advanced into clinical use. In sum, we present a generalizable method to screen for novel synergistic combinations, to identify particular mechanisms resulting in synergy, and to use the mechanistic knowledge to rationally design new combinations that bypass drug resistance.

http://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.2001644