Targeting cofactor biosynthesis in biodefense pathogens

描述（由申请人提供）：需要探索新的抗菌药物靶点，以建立针对具有天然或工程抗生素耐药性的病原体构成的威胁的新防御。在我们之前的工作中，我们使用比较基因组学和功能基因组学来确定、优先排序和验证生物合成中必不可少的含腺苷酸辅助因子的关键酶中的候选药物靶点。烟酸单核苷酸腺苷转移酶（Nicotinate Mononucleotide adenylylytransferase， NMNAT）是NAD生物合成的关键酶，在广泛的细菌病原体中高度保守，被认为是最有希望的靶点。该项目的主要目标是开发针对细菌病原体的有效抑制剂，作为新抗生素的原型，潜在的生物战剂。为了实现这一目标，我们将使用基于比较结构、功能和计算分析技术的综合方法。我们的初步结果支持了NMNAT家族的选择，包括细菌同源物和类似的人类酶（反靶标）的动力学和结构分析，以及通过化合物文库的计算机筛选预测的第一个经过验证的抑制剂。最近，其中一种抑制剂在培养中被证明能强烈抑制革兰氏阳性菌的生长。我们的研究工作将围绕以下具体目标进行：(1)代表性靶酶的功能分析。NIAID优先病原菌A、b类中10个代表性NMNAT靶酶的克隆、过表达、纯化及动力学分析（II）选定靶酶的三维结构分析及基于结构的抑制剂设计。将使用从代表性细菌靶点和人类反靶点的比较分析中获得的结构模板进行300万种已知化合物的计算机筛选。(2)酶抑制剂相互作用的实验测试、动力学和结构分析。计算机辅助建模、虚拟筛选、抑制分析、共结晶和3D结构分析相结合，将用于高排名NMNAT抑制剂的迭代优化。抑制剂测试将包括革兰氏阳性和革兰氏阴性细菌模型的离体实验。除了为新抗生素的开发奠定基础之外，这项研究还将影响我们对细菌病原体维生素/辅助因子代谢中一类重要的关键生物合成酶的理解。

DESCRIPTION (provided by applicant): Exploration of novel antimicrobial drug targets is required for building a new defense against threats posed by pathogens with natural or engineered antibiotic-resistance. In our previous work, we used comparative and functional genomics to identify, prioritize, and validate candidate drug targets among the key enzymes in the biosynthesis of indispensable adenylate-containing cofactors. Nicotinate Mononucleotide Adenylyltransferase (NMNAT), a key enzyme in NAD biosynthesis, highly conserved in a broad range of bacterial pathogens, was chosen as the most promising target. The major goal of the proposed project is to develop efficient inhibitors targeting this enzyme in bacterial pathogens, potential agents of biological warfare, as prototypes for new antibiotics. To address this goal, we will use an integrated approach based on comparative structural, functional, and computational analysis techniques. A choice of NMNAT family is supported by our preliminary results, which include kinetic and structural analysis of bacterial orthologs and analogous human enzymes (counter-targets), and the first verified inhibitors predicted by in silico screening of compound libraries. Recently, one of these inhibitors was shown to strongly suppress the growth of gram-positive bacteria in culture. Our research work will be structured around the following Specific Aims: (I) Functional analysis of representative target enzymes. Cloning, overexpression, purification and kinetic analysis of 10 representative NMNAT target enzymes tentatively identified by genomic searches in NIAID priority bacterial pathogens of categories A and B. (II) 3D structural analysis of selected target enzymes and structure-based inhibitor design. An in silico screening of 3,000,000 known chemical compounds will be performed using structural templates derived from the comparative analysis of representative bacterial targets and human counter-targets. (Ill) Experimental testing, kinetic and structural analysis of enzyme-inhibitor interactions. A combination of computer-assisted modeling, virtual screening, inhibitory analysis, co-crystallization, and 3D structural analysis will be used for iterative optimization of highly ranked NMNAT inhibitors. Inhibitor testing will include ex vivo experiments in gram-positive and gram-negative bacterial models. In addition to setting the stage for the development of new antibiotics, this study will impact our understanding of an important class of key biosynthetic enzymes in the vitamin/cofactor metabolism of bacterial pathogens.