Targeting DHFR to Design Antimicrobial Agents

描述(申请人提供)：真菌感染已成为严重疾病和死亡的一个重要且不断增加的原因。光滑念珠菌正在成为一种致命的真菌病原体，极大地增加了流行病原体--白色念珠菌已经造成的死亡。不幸的是，光滑假单胞菌对包括两性霉素B和唑类化合物在内的几种可用的抗真菌药物具有固有的抗药性。治疗光滑毛囊线虫感染是非常必要的。此外，由于系统性真菌感染通常会迅速走向死亡，而正式诊断需要关键时间，因此理想的治疗方法也将是一种广谱抗真菌药物，对主要的真菌病原体白色念珠菌也有效。在以前针对病原体二氢叶酸还原酶(DHFR)的成功的基础上，我们合成了一类多功能的DHFR抑制剂。我们最初的几个抑制剂系列显示出对真菌DHFR酶的强大效力，对哺乳动物酶的良好选择性，在生物培养中的良好抗真菌活性，以及几乎不明显的哺乳动物细胞毒性。我们还结晶了我们的一种与光滑毛囊线虫DHFR结合的抑制剂，并确定了其结构，分辨率为1.6E。在本申请的第一个目标中，我们建议使用结构指导的方法来设计和合成新的类似物，这些类似物能够有效和选择性地针对光肩星天牛DHFR。在第二个平行目标中，我们将确定这些化合物在细胞和动物中的功效，阐明耐药机制，并确定耐药酶的结构。在第三个目标中，我们将评估这些抑制剂对白色念珠菌的作用，并确定有效和选择性抑制剂与白色念珠菌DHFR的晶体结构。在第四个目标中，我们建议产生一种有效的和选择性的广谱抑制剂，对两种念珠菌都表现出良好的抗真菌活性，并保持对哺乳动物细胞的毒性。公共卫生相关性：系统性真菌感染已成为严重疾病和死亡的重要和破坏性原因。由光滑假丝酵母菌引起的感染已经变得越来越普遍，并对许多标准的抗真菌治疗药物产生抗药性。在这项建议中，我们的目标是使用结构指导的方法来合成对光滑线虫有效的二氢叶酸还原酶(DHFR)抑制剂，并在细胞和动物中对这些抑制剂进行评估。我们还旨在创造一种有效的、选择性的光滑念珠菌和白色念珠菌DHFR的广谱抑制剂，可以在正式诊断念珠菌之前使用。感染。

DESCRIPTION (provided by applicant): Fungal infections have become a significant and increasing cause of severe illness and death. Candida glabrata is emerging as a lethal fungal pathogen, contributing significantly to the mortality already caused by the prevalent pathogen, Candida albicans. Unfortunately, C. glabrata is inherently resistant to several of the available antifungal therapeutics, including amphotericin B and the azole compounds. Therapeutics to treat C. glabrata infections are critically necessary. Furthermore, since systemic fungal infections often proceed rapidly toward death and formal diagnosis requires critical time, an ideal therapeutic would also be a broad spectrum antifungal agent that is also effective against the primary fungal pathogen, C. albicans. Building on previous success to target dihydrofolate reductase (DHFR) in pathogenic organisms, we have synthesized a class of versatile DHFR inhibitors. Several of an initial series of our inhibitors exhibit strong potency against the fungal DHFR enzymes, good selectivity against the mammalian enzyme, good antifungal activity in cultures of the organisms and little appreciable mammalian cell toxicity. We have also crystallized one of our inhibitors bound to C. glabrata DHFR and determined the structure to 1.6 E resolution. In the first aim of this application, we propose to use a structure-guided approach to design and synthesize new analogs that potently and selectively target C. glabrata DHFR. In a second parallel aim, we will determine efficacy of the compounds in cells and animals, elucidate resistance mechanisms and determine structures of resistant enzymes. In a third aim, we will evaluate these inhibitors against C. albicans and determine crystal structures of potent and selective inhibitors with C. albicans DHFR. In a fourth aim, we propose to generate a potent and selective broad spectrum inhibitor that exhibits excellent antifungal activity against both Candida species and maintains a lack of toxicity against mammalian cells. PUBLIC HEALTH RELEVANCE: Systemic fungal infections have become a significant and devastating cause of severe illness and mortality. Infections caused by Candida glabrata have become increasingly prevalent and are resistant to many of the standard antifungal therapeutics. In this proposal we aim to use a structure-guided approach to synthesize inhibitors of dihydrofolate reductase (DHFR) effective against C. glabrata and to evaluate those inhibitors in cells and animals. We also aim to create a potent and selective broad spectrum inhibitor of C. glabrata and C. albicans DHFR that can be used prior to formal diagnosis of the Candida spp. infection.