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

裂解性转糖基酶小分子抑制剂可增强 β-内酰胺抗生素的作用

基本信息

批准号：
10078254
负责人：
FOCCO VAN DEN AKKER
金额：
$ 21.01万
依托单位：
CASE WESTERN RESERVE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-01-01 至 2022-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10078254
关键词：
Acinetobacter baumannii Active Sites Anabolism Animals Anti-Bacterial Agents Antibiotics Antimicrobial Resistance Bacteria Bacterial Antibiotic Resistance Bacterial Proteins Binding Biological Biological Assay Campylobacter jejuni Carbohydrates Cell Death Cell Wall Cells Chemicals Chemosensitization Crystallography Cytolysis Cytoplasm Disaccharides Docking Drug Targeting Enterobacter cloacae Enzyme Inhibitor Drugs Enzymes Escherichia coli Glucosamine Helicobacter pylori Klebsiella aerogenes Knock-out Label Libraries Link Lytic Mechanics Microbiology Monobactams Multiple Bacterial Drug Resistance Natural Products Neisseria Neisseria gonorrhoeae Penicillin-Binding Proteins Peptidoglycan Pharmaceutical Chemistry Proteins Pseudomonas aeruginosa Pyrrolidines Resistance Shapes Structure Testing bacterial resistance base beta-Lactamase beta-Lactams biophysical techniques crosslink design global health glycosyltransferase improved in silico inhibitor/antagonist novel novel therapeutics pathogen pathogenic bacteria protein crosslink resistance mechanism scaffold screening small molecule inhibitor tool

项目摘要

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.

抗药性细菌的迅速出现是一个重大的全球健康威胁。这促使我们有必要重新审视关键的抗菌药物靶点，如肽聚糖（PG）层。PG合成机制的目标是抑制交联PG链的青霉素结合蛋白（PBP）的β-内酰胺抗生素。主要抵抗力量其作用机制是表达能降解β-内酰胺类的β-内酰胺酶。因此，迫切需要新的抗生素或用于使细菌对β-内酰胺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，并且存在通过耐药β-内酰胺酶的耐药机制。第二种方法是抑制PG 降解裂解性转糖基酶（LT），这是本申请的焦点。抑制LT或基因敲除LT已被证明可恢复β-内酰胺的疗效抗生素在许多严重的病原体，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-内酰胺增强作用涉及两种可能的机制，根据病原体的不同，两者都有贡献。在第一种机制中，PBP和LT的抑制导致长的非交联PG 导致细胞壁隆起，削弱细胞壁的链。在第二种机制中，LT活动产生二糖PG产物，当再循环到细胞质时，在某些细胞中增加β-内酰胺酶表达，病原体尽管有这些令人信服的观察结果，只有一个有前途的LT抑制剂已知，bulgecin A; 然而，这种天然产物基于碳水化合物的抑制剂对于药物化学研究是非常具有挑战性的。作为因此，bulgecin A不太适合将抑制研究推进到动物研究，超越。本申请提出通过开发新的LT抑制剂来克服这一关键障碍，通过基于偏倚（Aim 1）和基于非偏倚片段的方法（Aim 2），获得不同于bulgecin A的支架。目的1：鉴定保留球蛋白A关键N-乙酰基的新抑制剂片段。含N-乙酰基将通过对接辅助选择或设计化合物;将通过以下方法探测其LT结合和抑制：生物物理技术、蛋白质晶体学和酶测定。将对化合物进行测试，已知结合bulgecin A的多种LT，并且其服从晶体学（E.大肠杆菌，铜绿假单胞菌，和 C. jejuni），以鉴定与一种LT结合的至少一个片段作为优化的新起点。目的2：为了鉴定与LT活性位点结合的不含乙酰基的片段，我们将筛选非乙酰基的片段。如目的1所述，针对LT的偏向片段用于结合和抑制。这样的片段可以结合到N-乙酰基上，装订口袋或相邻口袋。化合物将从sp3片段文库获得，并从更大文库的计算机筛选。来自目标1和2的命中将被修改/生长/链接以改善结合，对一种或多种LT的抑制作用，并且还对β-内酰胺抗生素的增强作用进行了微生物测试。

项目成果

期刊论文数量（2）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Turnover Chemistry and Structural Characterization of the Cj0843c Lytic Transglycosylase of Campylobacter jejuni.

DOI：
10.1021/acs.biochem.1c00027
发表时间：
2021-04-13
期刊：
BIOCHEMISTRY
影响因子：
2.9
作者：
Kumar, Vijay;Mathure, Snigdha A.;Lee, Mijoon;Boorman, Jacob;Zeng, Ximin;Lin, Jun;Hesek, Dusan;Lastochkin, Elena;Mobashery, Shahriar;van den Akker, Focco
通讯作者：
van den Akker, Focco

Exploring the inhibition of the soluble lytic transglycosylase Cj0843c of Campylobacter jejuni via targeting different sites with different scaffolds.