A New Antibacterial Drug Target: Analyzing Inhibitor Binding to a Bacterial Metal

描述(由申请人提供)：几种病原菌菌株的出现突显了该项目的重要性，这些菌株对目前所有可用的抗生素都有抗药性。对抗抗生素耐药性的一种方法是在耐药细菌菌株中建立新的酶靶点，并设计和合成针对这些酶的小分子抑制剂。基于细菌遗传信息，MDAP/赖氨酸生物合成途径提供了几个潜在的抗菌靶点，这些靶点尚未被探索。由于哺乳动物中没有类似的途径，针对这一生物合成途径中的一个或多个酶的抑制剂可能只对细菌表现出选择性毒性。已有研究表明，其中一种酶的编码基因缺失对幽门螺杆菌和耻垢分枝杆菌是致命的。即使在添加赖氨酸的培养基中，幽门螺杆菌也不能生长。因此，DapE9对于细胞的生长和增殖是必不可少的。开发一种针对DAPE酶的新型抗生素的一个主要限制是缺乏X射线结晶学信息。最近，PI与Argonne国家实验室的Boguslaw Nocek博士合作，解决了致病细菌流感嗜血杆菌的单核和双核锌(II)DapE酶的2.0和2.3E分辨结构。这些结构为研究PI最近发现的几种新的、有效的DAPE抑制剂的结合提供了所需的基础，其中一些已被证明对大肠杆菌具有抗菌活性。这一建议的具体目的是：i)通过高通量筛选发现新的DAPE抑制剂，ii)分析底物/抑制剂与DapE结合的决定因素，以及iii)检测参与底物/抑制剂结合的活性部位残基。从这些研究中获得的结果将确定新的药物化学线索的优化以及对底物识别和结合的决定因素的机制洞察。这些数据将提供洞察哪些官能团是最重要的增加抑制剂结合热到DapE。最有希望发现的化合物将在体内针对几种致病菌菌株进行抗菌性能测试，为将新发现的抗菌药线索转化为临床提供了一条途径。鉴于多药耐药生物体对人类健康构成严重且日益严重的威胁，预计本提案所述研究的成功完成将为医疗保健和社会普遍福利带来好处。 与公共卫生相关：该项目的重要性因出现了几种对目前所有可用的抗生素都具有抗药性的致病细菌菌株而得到强调。为了解决这个问题，我们建议寻找新的药物化学线索进行优化，并分析底物/抑制物与流感嗜血杆菌编码的N-琥珀酰基-L，L-二氨基吩甲酸脱丁二酸酶(DAPE)结合的决定因素，该酶是细菌赖氨酸生物合成途径中的一个新的抗菌靶点。最有希望的化合物将在体内针对几种致病菌菌株进行抗菌性能测试，为将新发现的抗菌药线索转化为临床提供了一条途径。

DESCRIPTION (provided by applicant): The importance of this project is underscored by the emergence of several pathogenic bacterial strains that are resistant to all currently available antibiotics. One way to combat antibiotic resistance is to establish new enzymatic targets within resistant bacterial strains and design and synthesize small molecule inhibitors to target these enzymes. Based on bacterial genetic information, the meso-diaminopimelate (mDAP)/lysine biosynthetic pathway offers several potential anti-bacterial targets that have yet to be explored. Since there are no similar pathways in mammals, inhibitors that target one or more of the enzymes in this biosynthetic pathway will likely exhibit selective toxicity against only bacteria. It has been shown that deletion of the gene encoding for one of these enzymes, the dapE-encoded N-succinyl-L,L-diaminopimelic acid desuccinylase (DapE; EC 3.5.1.18), is lethal to Helicobacter pylori and Mycobacterium smegmatis. Even in the presence of lysine supplemented media H. pylori was unable to grow. Therefore, DapE9s are essential for cell growth and proliferation. A major limitation in developing a novel class of antibiotics that target DapE enzymes is the lack of X-ray crystallographic information. Recently, the PI in collaboration with Dr. Boguslaw Nocek at Argonne National Laboratory, solved the 2.0 and 2.3 E resolution structures of the mono and dinuclear Zn(II) DapE enzymes from the pathogenic bacterium Haemophilus influenzae. These structures provide the foundation needed to examine the binding of several new, potent inhibitors of DapE that were recently discovered by the PI, some of which have been shown to possess antimicrobial activity towards Escherichia coli. The specific aims of this proposal are: i) Discover novel DapE inhibitors by high throughput screening, ii) Analyze the determinants of substrate/inhibitor binding to DapE, and iii) Examine active site residues that are involved in substrate/inhibitor binding. Results obtained from these studies will identify new medicinal chemistry leads for optimization as well as mechanistic insight into the determinants of substrate recognition and binding. These data will provide insight into which functional groups are most important to increase inhibitor binding enthalpy to DapE. The most promising compounds discovered will be tested for antimicrobial properties in vivo against several pathogenic bacterial strains providing an avenue to translate newly discovered antimicrobial leads into the clinic. It is anticipated that the successful completion of the studies described in this proposal will provide benefits to healthcare and the general welfare of society given that multidrug-resistant organisms pose a serious and increasing treat to human health. PUBLIC HEALTH RELEVANCE: The importance of this project is underscored by the emergence of several pathogenic bacterial strains that are resistant to all currently available antibiotics. To address this problem, we propose to identify new medicinal chemistry leads for optimization and analyze the determinants of substrate/inhibitor binding to the dapE-encoded N- succinyl-L,L-diaminopimelic acid desuccinylase (DapE) from Haemophilus influenzae, a novel antimicrobial target that is apart of the lysine biosynthetic pathway in bacteria. The most promising compounds will be tested for antimicrobial properties in vivo against several pathogenic bacterial strains providing an avenue to translate newly discovered antimicrobial leads into the clinic.