Novel Inhibitors to DHPS to Probe Catalytic Mechanism & Therapeutic Potential - r

描述（由申请人提供）：叶酸生物合成是高等动物缺乏的重要细菌途径，70多年来一直是抗菌剂的靶点。磺胺类药物通过作为对氨基苯甲酸（pABA）的非生产性底物类似物来抑制该途径中的二氢蝶呤合成酶（DHPS）。然而，柔性pABA结合位点在结构上易受耐药突变的影响，磺胺类药物正在迅速变得无效。相比之下，第二种DHPS底物pterin-焦磷酸的结合位点被埋在一个保守的口袋中，不太可能耐受突变。我们建议产生新的有效的DHPS抑制剂，专门参与这个口袋。这些抑制剂可以潜在地开发成新的治疗药物，仍然针对叶酸合成，但避免了耐药性的问题。为了产生有效的酶抑制剂，了解其活性位点的结构和机制至关重要。这些信息在很大程度上是DHPS缺失的，了解DHPS如何在分子水平上进行催化将是该提案的中心目标。另一个目标将是了解磺胺耐药性的分子和功能基础，以避免在我们未来的药物设计中出现这一问题。这种综合方法将需要结构生物学、药物化学、生物化学、计算生物学和微生物学方面两位主要研究人员的联合专业知识。该项目还将结合相对较新的基于片段的药物发现技术，以确定新的小型抑制化学支架，这些支架可以很容易地通过化学合成来阐述。应用程序中描述的令人兴奋的新数据为该项目能够实现其最终目标提供了强有力的支持，该目标是快速开发新的抗微生物药物，最大限度地减少耐药性的出现。虽然我们的目标是开发广谱抗感染药物，但我们的微生物筛选将重点放在近年来在美国出现的特别有问题的传染病病原体金黄色葡萄球菌和耶氏疟原虫上。

DESCRIPTION (provided by applicant): Folate biosynthesis is an essential bacterial pathway that is absent in higher animals, and it has been a target of antibacterial agents for over 70 years. Sulfa drugs inhibit the enzyme dihydropteroate synthase (DHPS) in the pathway by acting as non-productive substrate analogs of p-aminobenzoic acid (pABA). However, the flexible pABA binding site is structurally susceptible to resistance mutations, and the sulfa drugs are rapidly becoming therapeutically ineffective. In contrast, the binding site of the second DHPS substrate, pterin-pyrophosphate, is buried in a conserved pocket that is less likely to tolerate mutations. We propose to generate new classes of potent DHPS inhibitors that specifically engage this pocket. These inhibitors can potentially be developed into novel therapeutic agents that still target folate synthesis but which avoid the problems of resistance. To generate effective inhibitors of any enzyme, it is crucial to understand the structure and mechanism of its active site. This information is largely absent for DHPS, and understanding how DHPS performs catalysis at the molecular level will be a central goal of the proposal. Another goal will be to understand the molecular and functional basis of sulfa drug resistance to avoid this problem in our future drug designs. This comprehensive approach will require the joint expertise of the two principal investigators in structural biology, medicinal chemistry, biochemistry, computational biology and microbiology. The project will also incorporate the relatively new technique of fragment based drug discovery to identify novel small inhibitory chemical scaffolds that can readily be elaborated by chemical synthesis. Exciting new data described in the application provide strong support that the project can realize its ultimate goal which is to rapidly develop new antimicrobials that minimize the emergence of drug resistance. Although we aim to develop broad-spectrum anti-infective agents, our microbiological screening will focus on S. aureus and P. jirovecii that have emerged as particularly problematical infectious disease agents in the U.S. in recent years. PUBLIC HEALTH RELEVANCE: This proposal is aimed at structurally understanding and exploiting the enzyme target of the sulfonamide antibacterial agents, dihydropteroate synthase; to study enzyme mechanism, drug resistance and inhibitor design. It is expected that these studies will lead to new antimicrobial agents and a new understanding of target based sulfonamide resistance.