Selective inhibition of fungal BET protein Bdf1

 描述（由申请方提供）：寄生性真菌感染是免疫功能低下个体发病和死亡的主要原因。念珠菌属是主要的医院病原体，这些生物体的血流感染与30- 40%的死亡率相关。白色念珠菌和光滑念珠菌在分离频率上分别排名第一和第二，占所有播散性念珠菌病病例的70%。许多菌株已经获得对现有药物的抗性，这些药物也具有毒性、成本和窄活性谱的限制。因此，迫切需要新的抗真菌药物， 战略布局在这个R21项目中，其特点是一个跨学科的联盟，结合了药物化学，高通量筛选（HTS）技术，结构生物学，真菌遗传学和念珠菌感染的研究专长，我们提出了一种新的表观遗传方法，抗真菌剂的基础上选择性抑制念珠菌Bdf 1溴结构域（BD）。布罗莫结构域和末端外（BET）蛋白是染色质相关因子，调节基因转录和染色质重塑。BET蛋白通过其两个BD（BD 1和BD 2）识别染色质，BD是特异性结合组蛋白肽上的乙酰化赖氨酸的小螺旋结构域。溴结构域因子1（Bdf 1）是一种真菌BET蛋白，调节500多个基因的转录，对真菌的正常发育和生存至关重要。高分辨率晶体结构的证据S。cerevisiae和C.白色念珠菌Bdf 1-BD 1和BD 2的研究表明，配体结合口袋与它们的人BD对应物显著不同，为选择性抑制真菌BD提供了基础。HTS研究的原理证明已经确定了一个具有共同结构基序的小分子家族，其抑制C。白色念珠菌Bdf 1-BD 1（IC 50 < 6 µM），但不与人直系同源物Brd 4的相应结构域结合。在拟议的研究中，将筛选一个更广泛的库（约1，000，000种化合物），以寻找对C.白色念珠菌和C. glabrata Bdf 1-BD相对于人BD，使用均相时间分辨荧光（HTRF）测定。将在二次荧光偏振、AlphaScreen和下拉试验中确认命中并确定IC 50值。最有效的抑制剂与真菌Bdf 1-BD的共结晶将建立它们的结合模式，允许通过计算机建模和修饰的配体SAR研究进行优化。基于细胞的测定将建立人类细胞毒性和抑制真菌生长的功效。这项研究将提供有关真菌Bdf 1-BD的重要新信息，因为它们在相对未开发的真菌表观遗传学领域中作为药物设计的新策略的翻译潜力，并将创建一套新的和有效的抑制剂，用于在动物模型中进一步评估。

 DESCRIPTION (provided by applicant): Opportunistic fungal infections are a major cause of morbidity and mortality in immunocompromised individuals. Candida species are major nosocomial pathogens, and bloodstream infections with these organisms are associated with a mortality rate of 30-40%. Candida albicans and Candida glabrata rank first and second in isolation frequency, respectively, and are responsible for 70% of all cases of disseminated candidiasis. Many strains have acquired resistance to existing drugs, which also have limitations of toxicity, cost, and narrow activity spectra. Thus, there is an urgent need for novel anti-fungal strategies. In this R21 project, which features an interdisciplinary consortium combining research expertise in medicinal chemistry, high-throughput screening (HTS) technology, structural biology, fungal genetics and Candida infection, we propose a new epigenetic approach to anti-fungal agents based on selective inhibition of Candida Bdf1 bromodomains (BDs). Bromodomain and Extra-Terminal (BET) proteins are chromatin-associated factors that regulate gene transcription and chromatin remodeling. BET proteins recognize chromatin through their two BDs (BD1 and BD2), which are small helical domains that specifically bind acetylated lysines on histone peptides. Bromodomain factor 1 (Bdf1) is a fungal BET protein that regulates the transcription of over 500 genes and is essential for correct fungal development and survival. Evidence from high-resolution crystal structures of S. cerevisiae and C. albicans Bdf1-BD1 and BD2 demonstrates that the ligand binding pockets differ significantly from their human BD counterparts, providing the basis for selective inhibition of the fungal BDs. A proof of principle HTS study has identified a family of small molecules sharing a common structural motif, that inhibit C. albicans Bdf1-BD1 (IC50 < 6 µM), but do not bind to the corresponding domain from the human ortholog Brd4. In the proposed research, a more extensive library (~1,000,000 compounds) will be screened for strongly bound ligands that are highly selective for both C. albicans and C. glabrata Bdf1-BDs relative to human BDs, using a homogeneous time-resolved fluorescence (HTRF) assay. Hits will be confirmed and IC50 values determined in secondary fluorescence polarization, AlphaScreen, and pull-down assays. Co- crystallization of the most potent inhibitors with the fungal Bdf1-BDs will establish their binding modes, permitting optimization by computer modeling and modified ligand SAR studies. Cell-based assays will establish human cytotoxicity and efficacy in inhibiting fungal growth. This research will provide important new information about fungal Bdf1-BDs in terms of their potential for translation as a novel strategy for drug design within the relatively unexplored field of funga epigenetics, and will create a suite of novel and potent inhibitors for further evaluation in an animal model.