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

耐多药革兰氏阴性细菌的数量不断增加，对人类的健康和福利构成了紧迫的威胁。因此，迫切需要具有迄今尚未开发的作用机制的新型抗生素。脂类A是脂多糖的疏水性膜锚，对革兰氏阴性菌的生长和生存是必不可少的，因此抑制其生物合成是开发对革兰氏阴性菌具有选择性的抗生素的一种有前途的策略。脱乙酰酶LpxC催化脂质A生物合成的第一步，可作为抗菌药物的靶点。在拟议的项目中，将使用创新的基于片段的方法和基于结构的方法相结合来开发小分子LpxC抑制剂。将进行基于核磁共振的针对LpxC的片段筛选，以鉴定与LpxC迄今未被占据的UDP结合口袋结合的片段。包含受自然启发的片段的片段文库将在存在来自已知和新的抑制剂的探针的情况下进行筛选。为了产生有效的酶抑制剂，将利用从实验核磁共振数据中获得的结构信息，如配体间NOES和分子对接研究，以最有益的方式合并鉴定的结构。设想的合并化合物将以立体控制的方式使用最先进的发散合成来制备。除了基于苯甲氧基乙酰异羟肟酸的LpxC抑制剂外，还将合成新的3，4-二取代吗啉衍生物，这些衍生物不含药动学上不利的异羟甲酸酯部分。将详细阐述所有合成的LpxC抑制剂的结构-活性关系，并通过分子对接研究使其合理化。在后续的优化步骤中，这些化合物的抑制活性、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都将得到提高。为确保该项目的可行性，已进行了初步试验。已经成功地进行了蛋白质纯化、酶分析、第一批LpxC抑制剂的合成、生物活性、配体间NOE核磁共振实验以及与已知和设计的抑制剂的对接研究。

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.