Structural Biological Development of Fungal-Specific Calcineurin Inhibitors

描述(申请人提供)：侵袭性真菌感染是免疫功能低下患者死亡的主要原因。目前的抗真菌药物临床疗效有限，杀菌力差，在某些情况下是有毒的，并且由于新出现的耐药性而越来越无效。我们已经证实，侵袭性真菌疾病广泛需要保守的磷酸酶钙调神经磷酸酶。FDA批准的钙调神经磷酸酶抑制剂FK506在体外对主要侵袭性真菌病原体具有活性，但也抑制宿主免疫。我们的方法寻求克服真菌对人类的特异性障碍，以显著推进抗真菌治疗。本研究的目的是利用一种基于结构生物学的策略 确定真菌-哺乳动物钙调神经磷酸酶的结构差异，验证真菌特异性靶点，并产生和优化用于治疗侵袭性真菌疾病的新型FK506类似物。我们的中心假设是，通过使用结晶学和核磁共振光谱的结构生物学方法，我们将在钙调神经磷酸酶复合体中定义对真菌发病至关重要的新的可靶向的真菌特异性区域。为了最大限度地扩大临床广度，我们将重点介绍两种主要的临床病原体：白色念珠菌和霉菌烟曲霉菌。我们的初步研究证明了具有强大抗真菌活性的非免疫抑制FK506类似物的原理。我们假设，钙调神经磷酸酶A和B复合体的结构与钙调蛋白和免疫亲和素复合体(FKBP12-FK506)相结合，将揭示新的真菌特异性抑制靶点。我们最近已经解决了白色念珠菌的结构，现在将单独解决钙调神经磷酸酶异二聚体的结构，并与来自烟曲霉菌的FKBP12-FK506/类似物复合。多个分子视图将允许识别可用于靶向抑制剂开发的人和真菌钙调神经磷酸酶复合体之间的不同位点。动态或抵抗结晶的蛋白质区域将通过核磁共振进行结构表征。推测的抑制结构域将通过遗传和生化分析来验证，利用关键接触和表面残基的定点突变来检查结构稳定性、真菌表型和药物作用/耐药性。Amplyx制药公司将根据我们第一次迭代的SAR结果生成非免疫抑制真菌特异性FK506类似物。这将指导第二代类似物的生产，通过利用宿主和真菌酶之间独特的结构差异来保持抗真菌活性和消除免疫抑制。将根据筛选结果进行药物化学和抑制剂对接实验，以改变类似物。先导化合物将在体外和侵袭性念珠菌病和侵袭性曲霉菌病的小鼠模型中使用迭代方法进行测试。我们将利用结构生物学作为一种新的靶向钙调神经磷酸酶的方法，通过定义真菌特有的特征而不是哺乳动物的相应特征来产生新的抗真菌治疗药物。

DESCRIPTION (provided by applicant): Invasive fungal infections are a leading cause of death in immunocompromised patients. Current antifungals have limited clinical efficacy, are poorly fungicidal, are in some cases toxic, and are increasingly ineffective due to emerging resistance. We have established that the conserved phosphatase calcineurin is broadly required for invasive fungal disease. The FDA-approved calcineurin inhibitor FK506 is active in vitro against major invasive fungal pathogens, but also suppresses host immunity. Our approach seeks to overcome the fungal versus human specificity barrier to significantly advance antifungal treatment. The objective of this study is to utilize a structural biology-based strategy to define fungal-mammalian calcineurin structural differences, validate fungal-specific targets, and generate and optimize novel FK506 analogs to treat invasive fungal diseases. Our central hypothesis is that by employing a structural biological approach using both crystallography and NMR spectroscopy that we will define novel targetable fungal-specific areas in the calcineurin complex critical for fungal pathogenesis. For maximum clinical breadth, we will focus on the two major clinical pathogens: the yeast Candida albicans and the mold Aspergillus fumigatus. Our preliminary studies document proof of principle non-immunosuppressive FK506 analogs with robust antifungal activity. We hypothesize that structures of the calcineurin A and B complex, coupled with calmodulin and the immunophilin complex (FKBP12-FK506), will reveal novel fungal-specific targets for inhibition. We have recently solved the structure for C. albicans, and will now solve structures for the calcineurin heterodimer alone and complexed with FKBP12-FK506/analogs from A. fumigatus. The multiple molecular views will allow identification of sites that are distinct between human and fungal calcineurin complexes that can be exploited for targeted inhibitor development. Protein regions that are dynamic or resist crystallization will be structurally characterized by NMR. Putative inhibitory domains will be validated via genetic and biochemical assays, utilizing site-directed mutagenesis of key contact and surface residues to examine structural stability, fungal phenotype, and drug action/resistance. Non-immunosuppressive fungal-specific FK506 analogs will be generated by Amplyx Pharmaceuticals based on the SAR results of our first iteration. This will guide the production of second generation analogs optimized for retention of antifungal activity and abrogation of immunosuppression by capitalizing on the unique structural differences between the host and fungal enzymes. Medicinal chemistry and inhibitor docking experiments will be conducted to alter analogs based on screening results. Lead compounds will be tested using an iterative approach both in vitro and in murine models of invasive candidiasis and invasive aspergillosis. We will capitalize on structural biology as a new approach to targeting calcineurin by defining fungal-specific features with no mammalian counterpart to generate novel antifungal therapeutics.