Targeting Bacillus DHFR: Structural Studies and Synthesis of Inhibitors

描述(申请人提供)：炭疽芽孢杆菌，炭疽病的病原体，已成为严重的生物恐怖主义威胁。由于这种微生物的高度毒力性质，预防性开始抗生素治疗是至关重要的，因此有效的药物需要廉价并在大量人群中耐受。二氢叶酸还原酶(DHFR)抑制剂几十年来一直被用于治疗细菌感染，然而，临床上使用的DHFR抑制剂甲氧普林由于与芽孢杆菌酶的相互作用较差而对炭疽杆菌无效。然而，替代的DHFR抑制剂显示出对芽孢杆菌酶的高效力，应该是治疗炭疽感染的优秀疗法。我们设计了一些新的DHFR抑制剂，发现有几种可以抑制炭疽杆菌的DHFR，并在保持低哺乳动物细胞毒性的同时促进炭疽杆菌细胞死亡。这些化合物的新合成方法已经得到了一种IC50值为亚微摩尔，MIC99值为20.5g/mL的抑制剂，对哺乳动物细胞没有毒性。最近的努力提供了更有效的化合物，其MIC99值低至3.5 5g/mL。本申请工作的目标是开发一种前导化合物和后备化合物，它们是炭疽杆菌生长的有效和选择性抑制物。为此，我们有四个具体目标：第一，合成一个旨在提高效力和确定耐药性产生的重点抑制剂文库；第二，在有效抑制剂的基础上合成一个选择性抑制剂文库；第三，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酶活性分析的结果以及耐药蛋白质的额外晶体结构，开发和合成高级先导化合物。与公共卫生相关：迫切需要有效对抗炭疽感染的新抗生素。一种关键代谢酶的小分子抑制剂已经被合成，并被证明是有效的，对哺乳动物细胞没有毒性。这项提议的目的是利用酶的结构进一步开发这些化合物，在低浓度下有效地抑制炭疽芽孢杆菌的生长。

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.