靶向庚糖转移酶的新型抗耐药革兰氏阴性菌药物研究

Bacterial infection is one of the greatest potential threat in the public health, and multidrug resistance is becoming commonplace in the nosocomial setting. Specifically, infections due to multidrug resistant, gram-negative pathogens are responsible for high mortality rates and may leave few effective antimicrobial options with few antibiotics available for treatment, and even fewer drugs in the antibiotic pipeline. Research and development of new drugs that target a novel pathway with new mechanisms is another key approach for the treatment bacterial infection. Lipopolysaccharide (LPS), also known as endotoxins, is a key component of the outer membrane of Gram-negative bacteria. It is of crucial importance to Gram-negative bacteria, which die if it is mutated or removed. LPS induces a strong response from normal host immune systems. The mortality of many infectious diseases is closely related to the amount of circulating LPS endotoxins found in patient sera. Heptose is a key component in LPS. Gram-negative bacteria that lack heptose display the deep-rough phenotype and show a reduction in outer membrane protein content, an increased sensitivity towards detergents or hydrophobic antibiotics, and are much more susceptible to phagocytosis by macrophages. Therefore, the heptosyltransferases implied in LPS biosynthesis represent attractive targets, the inhibition of which could attenuate the virulence of diverse bacterial strains. In our previous study，we systematically studied the LPS biosynthetic pathway and the heptosyltransferase-I (WaaC) assay, and found that some molecular probes had a good inhibition potency for WaaC. In this study, we will rationally design and synthesize a novel series of donor-substrate-based heptosyltransferase-I inhibitors on the basis of the scaffold of two probes and the WaaC crystal structure. A unique library targeting to WaaC will be constructed by efficient chemical synthesis to explore the structure-active relationship for the inhibition of WaaC. We expect to find 1-2 leading compounds with a strong inhibition of WaaC activity for the development of new drug for the treatment of multidrug resistant, gram-negative pathogens.

耐药细菌感染是当前公共卫生安全的潜在威胁之一，尤其多药耐药的革兰氏阴性菌(G- 菌)引起的院内感染致死率很高，临床上对多药耐药感染面临无药可用的巨大风险。针对新靶标研发作用机制新颖的抗菌药物，是应对耐药菌感染的关键。脂多糖(LPS)又称内毒素，是维持G-菌正常结构和生存的基本组分，也是主要的毒力因子。庚糖是细菌LPS的特异单糖，抑制G-菌庚糖转移酶可能成为抗菌药物研发的重要靶标。在前期研究中，我们建立了庚糖转移酶I (WaaC)抑制剂的高效特异性筛选方法，并发现了若干具有一定抑制作用的探针分子。本申请拟在前期研究的基础上，依据WaaC晶体结构，通过合理药物设计和定向高效化学合成，构建靶向WaaC的小分子化合物库，然后进行WaaC活性筛选，并对活性化合物进行体外抗菌增敏活性评价及动物保护试验，力图发现1-2个靶向WaaC的抗G-菌药物先导化合物，为研发新型抗耐药G-菌药物打下基础。

Heptosyltransferase-I (WaaC) 是抗毒力研究机制中的重要靶标，发现全新作用机制的活性化合物可以有效克服细菌耐药的蔓延。本项目是基于庚糖转移酶-I作为靶点，设计合成了不同类型的WaaC小分子抑制剂。主要研究内容包括：第一部分就是优化WaaC活性测试方法，建立其酶活测试方法并用于小分子化合物的活性筛选。第二部分主要集中在小分子抑制剂的设计和合成。第三部分是抑制WaaC活性评价及抗菌活性研究。通过对本项目的研究工作，构建了基于WaaC靶点研究的技术平台，设计合成了84个新型含有腺苷复杂结构的小分子化合物库，发现了2个活性较好的衍生物，酶活水平小于 10 μM（IC50 = 6.0 -5.7 μM），显著提高了其酶活抑制效果，正在进行初步的抗毒力活性评价。首次探索了WaaC天然底物的构效关系研究，优化和开发了一套全新的双磷酸腺苷庚糖衍生物合成方法，该方法具有较好底物适应性，尤其对于含有叠氮类型的点击化学化学基团具有较好的兼容性，通过该方法可以高效合成了全新的类天然腺苷双磷酸底物和WaaC天然底物，对后续的机制研究提供了新型的工具分子，尤其是在脂多糖LPS新型叠氮化学修饰分子的生物合成提供新的可能。此外，发现了一类新型抑菌剂-羰基硫脲类衍生物，抗菌活性水平（最小抑菌浓度MIC）在0.78 μg/mL-0.1 μg/mL，可能是WaaC潜在抑制剂，开展了其抗菌作用机制研究，发现该类化合物显示出较好的抑菌活性（不是杀菌剂），能够在较低浓度快速抑制细菌增殖，值得进行深入的研究工作。