Small molecule inhibitors of lytic transglycosylase to potentiate beta-lactam antibiotics

抗生素抗药性细菌的迅速出现是一个重大的全球健康威胁。这促使人们有必要重新审视 关键的抗菌药物靶点，如肽聚糖(PG)层。PG合成机器的目标是 β-内酰胺类抗生素，可抑制青霉素结合蛋白(PBP)，使PG链发生交联。一个主要的抵抗力量 其机制是β-内酰胺酶的表达可以降解β-内酰胺类抗生素。因此，迫切需要新的 抗生素或使细菌对β-内酰胺类抗生素重新敏感的途径。对于后者，一种方法是 开发β-内酰胺酶抑制剂；不幸的是，目前的5种抑制剂不能抑制某些关键的β-内酰胺酶， 耐药机制是通过耐药的β-内酰胺酶实现的。第二种方法是抑制PG 降解裂解转糖基酶(LT)，本应用的重点。 抑制LTS或基因敲除LTs已被证明可以恢复β-内酰胺的疗效 许多严重病原体中的抗生素，包括大肠杆菌、脑膜炎奈瑟菌、假单胞菌 铜绿假单胞菌、产气肠杆菌、鲍曼不动杆菌、幽门螺杆菌和空肠弯曲杆菌。 这种β-内酰胺增强涉及两种可能的机制，根据病原体的不同，或者 两者都有贡献。在第一种机制中，PBP和LT的抑制都导致了长的非交联型PG 导致细胞壁膨胀的链条，削弱细胞壁。在第二种机制中，LT活动产生 二糖PG产物，当循环到细胞质时，在某些情况下增加β-内酰胺酶的表达 病原体。尽管有这些令人信服的观察结果，但目前只有一种有希望的LT抑制剂，即Bulgecin A； 然而，这种天然产物碳水化合物为基础的抑制剂是非常具有挑战性的药物化学工作。AS 因此，Bulgecin A不太容易将抑制研究推进到动物研究和 更远一点。该应用程序建议通过开发新的LT抑制剂来克服这一关键障碍 支架(S)通过有偏见的(目标1)和无偏见的基于片段的方法(目标2)而不同于Bulgecin A。 目的1：鉴定新的保留牛油菌素A关键N-乙酰基的抑制物片段。含N-乙酰基 化合物将通过对接进行选择或设计；其LT结合和抑制将通过以下方式进行探测 生物物理技术、蛋白质结晶学和酶分析。化合物将被测试与 多个已知结合Bulgecin A的LT，它们对结晶学(大肠杆菌、铜绿假单胞菌和 空肠弯曲菌)，以鉴定至少一个与一个LT结合的片段作为优化的新起点。 目的2：为了鉴定与LT活性部位结合的非乙酰基片段，我们将筛选非乙酰基 目标1中针对LTS的偏向片段的结合和抑制。这样的片段可以与N-乙酰基结合 装订口袋或与相邻口袋相连。化合物将从SP3片段文库和In中获得 对更大的图书馆进行电子筛选。来自AIMS 1和2的点击将被修改/增长/链接，以改善绑定和 抑制一种或多种LT，并对β-内酰胺类抗生素的增强作用进行微生物学测试。

The rapid emergence of antibiotic-resistant bacteria is a major global health threat. This spurs the need to revisit key antibacterial drug targets such as the peptidoglycan (PG) layer. The PG synthesis machinery is targeted by β-lactam antibiotics that inhibit penicillin-binding proteins (PBP) which crosslink PG strands. A main resistance mechanism is the expression of β-lactamases that can degrade β-lactams. There is thus a critical need for new antibiotics or for avenues to re-sensitize bacteria to β-lactam antibiotics. For the latter, one approach is developing β-lactamase inhibitors; unfortunately, the 5 current inhibitors do not inhibit certain key β-lactamases, and there are resistance mechanisms via inhibitor-resistant β-lactamases. A second approach is to inhibit PG degrading lytic transglycosylases (LT), the focus of this application. Inhibition of LTs or knocking out LTs genetically has been shown to restore the efficacy of β-lactam antibiotics in many serious pathogens including Escherichia coli, Neisseria meningitides, Pseudomonas aeruginosa, Enterobacter aerogenes, Acinetobacter baumannii, Helicobacter pylori, and Campylobacter jejuni. This β-lactam potentiation involves two possible mechanisms of which, depending on the pathogen, either or both contribute. In the first mechanism, the inhibition of both PBP and LT leads to long non-cross-linked PG strands that cause cell wall bulges, weakening the cell wall. In the second mechanism, LT activity generates disaccharide PG product that, when recycled to the cytoplasm, increases β-lactamase expression in certain pathogens. Despite these compelling observations, there is only one promising LT inhibitor known, bulgecin A; however, this natural product carbohydrate-based inhibitor is very challenging for medicinal chemistry efforts. As a result, bulgecin A has not been very amenable to advancing inhibition studies towards animal studies and beyond. This application proposes to overcome this key roadblock by developing new LT inhibitors with scaffold(s) different from bulgecin A via biased (Aim 1) and non-biased fragment-based approaches (Aim 2). Aim 1: To identify new inhibitor fragments that retain bulgecin A's key N-acetyl group. N-acetyl containing compounds will be selected or designed aided by docking; their LT binding and inhibition will be probed by biophysical techniques, protein crystallography, and enzymatic assays. Compounds will be tested against multiple LTs known to bind bulgecin A, and which are amenable to crystallography (E. coli, P. aeruginosa, and C. jejuni) in order to identify at least one fragment binding to one LT as a novel starting point for optimization. Aim 2: To identify non-acetyl containing fragments that bind to the active site of LT, we will screen non- biased fragments against LTs for binding and inhibition as in Aim 1. Such fragments could bind to the N-acetyl binding pocket or to the adjacent pockets. Compounds will be obtained from an sp3 fragment library and from in silico screening of larger libraries. Hits from Aims 1 and 2 will be modified/grown/linked to improve binding and inhibition of one or more of the LTs and also tested microbiologically for the potentiation of β-lactam antibiotics.