Development of fragment-based inhibitors of the bacterial deacetylase LpxC as novel antibiotics

多重耐药革兰氏阴性菌数量的不断增加对人类健康和福祉构成了紧迫的威胁。因此，迫切需要具有迄今为止尚未开发的作用机制的新型抗生素。作为脂多糖的疏水膜锚的脂质A对于革兰氏阴性菌的生长和存活是必需的，抑制其生物合成代表了用于开发对革兰氏阴性菌具有选择性的抗生素的有前景的策略。脱乙酰酶LpxC催化脂质A生物合成的第一个关键步骤，可以被验证为抗菌药物靶标。在拟议的项目中，小分子LpxC抑制剂将使用创新的基于片段的方法与基于结构的方法相结合来开发。将进行针对LpxC的基于NMR的片段筛选，以鉴定与迄今未被占据的LpxC UDP结合口袋结合的片段。包含自然启发片段的片段文库将在衍生自已知以及新型抑制剂的探针存在下进行筛选。为了产生有效的酶抑制剂，将使用来自实验NMR数据的结构信息的知识，如配体间NOE和分子对接研究，以最有益的方式合并鉴定的结构。所设想的合并化合物将采用现有技术的发散合成以立体控制的方式制备。除了苄氧基乙酰异羟肟酸为基础的LpxC抑制剂，新的3，4-二取代的吗啉衍生物是没有药代动力学不利的异羟肟酸部分将被合成。将阐述所有合成的LpxC抑制剂的结构-活性关系，并通过分子对接研究进行合理化。在随后的优化步骤中，化合物的抑制活性、它们对各种革兰氏阴性菌的抗菌谱以及它们的代谢稳定性将得到改善。为了保证该项目的可行性，进行了初步的实验。蛋白质纯化，酶法测定，第一LpxC抑制剂的合成，生物活性，interligand-NOE NMR实验，和对接研究与已知的和设计的抑制剂已经成功地进行。

The constantly increasing number of multidrug-resistant Gramnegative bacteria poses a pressing threat to human health and welfare. Therefore, novel antibiotics possessing so far unexploited mechanisms of action are urgently required. As lipid A, the hydrophobic membrane anchor of lipopolysaccharides, is essential for growth and viability of Gram-negative bacteria, the inhibition of its biosynthesis represents a promising strategy for the development of antibiotics being selective for Gram-negative germs. The deacetylase LpxC catalyzes the first committed step of lipid A biosynthesis and could be validated as an antibacterial drug target. In the proposed project, small molecule LpxC inhibitors shall be developed using innovative fragment-based methods combined with structure-based approaches. NMR-based fragment screening against LpxC will be performed to identify fragments binding to the so far unoccupied UDP-binding pocket of LpxC. Fragment libraries comprising nature-inspired fragments will be screened in the presence of probes being derived from known as well as novel inhibitors. To generate potent enzyme inhibitors, the identified structures will be merged in the most beneficial way using the knowledge of the structural information derived from experimental NMR data such as Interligand NOEs and molecular docking studies. The envisaged merged compounds will be prepared in a stereocontrolled manner employing state of the art divergent syntheses. Besides benzyloxyacetohydroxamic acid-based LpxC inhibitors, novel 3,4-disubstituted morpholine derivatives being devoid of the pharmacokinetically unfavorable hydroxamate moiety will be synthesized. Structure-activity relationships will be elaborated for all of the synthesized LpxC inhibitors and rationalized by molecular docking studies. In subsequent optimization steps, the inhibitory activity of the compounds, their antibacterial spectrum against various Gram-negative bacteria as well as their metabolic stability will be improved. Preliminary experiments have been performed to ensure the feasibility of the project. Protein purification, enzymatic assays, first LpxC inhibitors synthesis, biological activities, interligand-NOE NMR experiments, and docking studies with known and designed inhibitors have been carried out successfully.