Targeting Bacillus DHFR: Structural Studies and Synthesis of Inhibitors

靶向芽孢杆菌 DHFR：抑制剂的结构研究和合成

• 批准号: 7842528
• 负责人: Amy C. Anderson
• 金额: $ 33.09万
• 依托单位: UNIVERSITY OF CONNECTICUT STORRS
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-06-01 至 2013-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7842528
• 关键词: Anthrax disease; Anti-Bacterial Agents; Antibiotic Therapy; Antibiotics; Bacillus (bacterium); Bacillus anthracis; Bacterial Infections; Binding; Biological Assay; Bioterrorism; Cell Death; Cells; Communicable Diseases; Cytochrome P450; DHFR gene; Dihydrofolate Reductase; Dihydrofolate Reductase Inhibitor; Docking; Enzyme Inhibitor Drugs; Enzyme Inhibitors; Enzymes; Exhibits; Generations; Goals; Growth; Human; In Vitro; Infection; Inhibitory Concentration 50; Lead; Libraries; Ligands; Malignant Neoplasms; Mammalian Cell; Metabolic; Methodology; Methods; Methotrexate; Nature; Pathway interactions; Pharmaceutical Preparations; Population; Property; Protein Binding; Proteins; Pyrimethamine; Resistance; Structure; Therapeutic; Therapeutic Agents; Toxic effect; Trimethoprim; Virulent; Work; base; biodefense; cell growth; clinical application; combat; design; diaminopyrimidine; enzyme activity; enzyme structure; in vitro testing; inhibitor/antagonist; lead series; microorganism; novel; pathogen; public health relevance; resistance mutation; scaffold; small molecule; success

DESCRIPTION (provided by applicant): Bacillus anthracis, the causative agent of anthrax, has become a serious bioterrorism threat. Due to the highly virulent nature of this microorganism, it is crucial that antibiotic therapy begins prophylactically, therefore effective drugs need to be inexpensive and tolerated in a large population. Inhibitors of dihydrofolate reductase (DHFR) have been used for decades to treat bacterial infections, however, trimethoprim, a clinically used DHFR inhibitor, is ineffective against B. anthracis due to poor interactions with the Bacillus enzyme. However, alternative DHFR inhibitors that exhibit high potency against the Bacillus enzyme should be excellent therapeutics to treat anthrax infections. We have designed a number of new DHFR inhibitors and found that several inhibit DHFR from B. anthracis and promote B. anthracis cell death while maintaining low mammalian cell toxicity. Novel methods for the syntheses of these compounds have resulted in an inhibitor with a submicromolar IC50 value and a MIC99 value of 20 5g/mL with no mammalian cell toxicity. Recent efforts have delivered more potent compounds with MIC99 values as low as 3.5 5g/mL. The goal of the work in this application is to develop a lead and a backup compound that are potent and selective inhibitors of B. anthracis growth. To do this, we have four specific aims: one, synthesize a focused library of inhibitors intended to increase potency and determine resistance generation; two, build on the potent inhibitors to synthesize a library of selective inhibitors; three, determine the crystal structure of Bacillus anthracis DHFR bound to a lead compound in order to evaluate protein:ligand interactions; and four, develop and synthesize advanced leads based on results from protein binding and P450 enzyme activity assays as well as additional crystal structures of resistant proteins. PUBLIC HEALTH RELEVANCE: There is a critical need for new antibiotics effective against anthrax infections. Small molecule inhibitors of a key metabolic enzyme have been synthesized and proven to be potent without being toxic to mammalian cells. The goal of this proposal is to use the structure of the enzyme to further develop those compounds to effectively inhibit the growth of Bacillus anthracis Sterne at low concentrations.

描述（由申请人提供）：炭疽杆菌是炭疽病的病原体，已成为严重的生物恐怖主义威胁。由于这种微生物具有高毒力，预防性开始抗生素治疗至关重要，因此有效的药物需要价格便宜且能够在大量人群中耐受。二氢叶酸还原酶（DHFR）抑制剂几十年来一直用于治疗细菌感染，然而，临床上使用的DHFR抑制剂甲氧苄啶由于与芽孢杆菌酶的相互作用较差，对炭疽芽孢杆菌无效。然而，对芽孢杆菌酶表现出高效力的替代 DHFR 抑制剂应该是治疗炭疽感染的优秀疗法。我们设计了许多新的 DHFR 抑制剂，并发现其中几种能够抑制炭疽芽孢杆菌的 DHFR 并促进炭疽芽孢杆菌细胞死亡，同时保持较低的哺乳动物细胞毒性。合成这些化合物的新方法已产生一种具有亚微摩尔IC50值和20 5g/mL MIC99值且无哺乳动物细胞毒性的抑制剂。最近的努力已经提供了更有效的化合物，其 MIC99 值低至 3.5 5g/mL。本申请的工作目标是开发一种先导化合物和备用化合物，它们是炭疽芽孢杆菌生长的有效和选择性抑制剂。为此，我们有四个具体目标：一是合成一个重点抑制剂库，旨在提高效力并确定耐药性的产生；二、以强效抑制剂为基础，合成选择性抑制剂库；三、确定与先导化合物结合的炭疽芽孢杆菌DHFR的晶体结构，以评估蛋白质：配体相互作用；第四，根据蛋白质结合和 P450 酶活性测定的结果以及抗性蛋白质的其他晶体结构开发和合成先进的先导化合物。公共卫生相关性：迫切需要有效对抗炭疽感染的新型抗生素。一种关键代谢酶的小分子抑制剂已被合成，并被证明是有效的，且对哺乳动物细胞无毒。该提案的目标是利用该酶的结构进一步开发这些化合物，以在低浓度下有效抑制炭疽杆菌的生长。