Targeting Bacillus DHFR: Structural Studies and Synthesis of Inhibitors

描述（申请人提供）：炭疽杆菌是炭疽的病原体，已成为严重的生物恐怖威胁。由于这种微生物的高毒性，预防性抗生素治疗是至关重要的，因此有效的药物需要便宜且在大量人群中耐受。二氢叶酸还原酶（DHFR）抑制剂已用于治疗细菌感染数十年，然而，临床上使用的DHFR抑制剂甲氧苄啶由于与芽孢杆菌酶相互作用差而对炭疽芽孢杆菌无效。然而，对芽孢杆菌酶表现出高效力的其他DHFR抑制剂应该是治疗炭疽感染的良好疗法。我们设计了许多新的DHFR抑制剂，发现有几种抑制炭疽芽孢杆菌DHFR，促进炭疽芽孢杆菌细胞死亡，同时保持低哺乳动物细胞毒性。合成这些化合物的新方法已经产生了一种亚微摩尔IC50值和MIC99值为205g /mL的抑制剂，没有哺乳动物细胞毒性。最近的研究已经提供了MIC99值低至3.5 g/mL的更有效的化合物。本研究的目标是开发一种先导化合物和一种后备化合物，这两种化合物是有效的、选择性的炭疽芽孢杆菌生长抑制剂。为此，我们有四个具体目标：第一，合成一个集中的抑制剂文库，旨在提高效力并确定耐药性的产生；二是在有效抑制剂的基础上合成选择性抑制剂库；三、测定炭疽芽孢杆菌DHFR与先导化合物结合的晶体结构，以评价蛋白与配体的相互作用；第四，根据蛋白质结合和P450酶活性分析的结果以及抗性蛋白的额外晶体结构，开发和合成先进的先导物。公共卫生相关性：迫切需要对炭疽感染有效的新型抗生素。一种关键代谢酶的小分子抑制剂已经被合成并证明对哺乳动物细胞有效而无毒。本课题的目标是利用该酶的结构进一步开发出能在低浓度下有效抑制炭疽芽孢杆菌生长的化合物。

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.