Targeting DHFR to Design Antimicrobial Agents

描述（由申请人提供）：真菌感染已成为严重疾病和死亡的重要且日益增加的原因。光秃念珠菌正在成为一种致命的真菌病原体，对已经由流行的白色念珠菌引起的死亡率起着重要的作用。不幸的是，C. glabrata对几种可用的抗真菌治疗药物具有固有的耐药性，包括两性霉素B和唑类化合物。治疗裸露锥体感染是非常必要的。此外，由于全身性真菌感染通常会迅速导致死亡，而正式诊断需要关键时间，因此理想的治疗方法是使用广谱抗真菌药物，同时对主要真菌病原体白色念珠菌也有效。在先前成功靶向致病性生物中的二氢叶酸还原酶（DHFR）的基础上，我们合成了一类多功能DHFR抑制剂。我们最初的几个系列抑制剂对真菌DHFR酶表现出很强的效力，对哺乳动物酶有很好的选择性，在生物培养中有很好的抗真菌活性，对哺乳动物细胞几乎没有明显的毒性。我们还结晶了一种与C. glabrata DHFR结合的抑制剂，并确定了1.6 E分辨率的结构。在本应用的第一个目的中，我们建议使用结构导向的方法来设计和合成新的类似物，这些类似物可以有效地和选择性地靶向光棘草DHFR。在第二个平行目标中，我们将确定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，并确定有效的和选择性的白色念珠菌DHFR抑制剂的晶体结构。在第四个目标中，我们建议产生一种有效的选择性广谱抑制剂，该抑制剂对两种念珠菌都具有出色的抗真菌活性，并且对哺乳动物细胞缺乏毒性。公共卫生相关性：全身性真菌感染已成为严重疾病和死亡的重要和破坏性原因。由念珠菌引起的感染已经变得越来越普遍，并且对许多标准的抗真菌治疗具有耐药性。在本研究中，我们的目标是利用结构导向的方法合成二氢叶酸还原酶（DHFR）抑制剂，并在细胞和动物中对这些抑制剂进行评价。我们还旨在创造一种有效的、选择性的广谱的光念珠菌和白色念珠菌DHFR抑制剂，可以在念珠菌感染的正式诊断之前使用。

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.