Structural Biological Development of Fungal-Specific Calcineurin Inhibitors

描述（由申请人提供）：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，杀真菌效果差，在某些情况下有毒，并且由于出现耐药性而越来越无效。我们已经确定，保守的磷酸酶钙调神经磷酸酶是广泛需要的侵袭性真菌疾病。FDA批准的钙调磷酸酶抑制剂FK 506在体外对主要的侵袭性真菌病原体有活性，但也抑制宿主免疫力。我们的方法旨在克服真菌与人类特异性的障碍，以显着推进抗真菌治疗。本研究的目的是利用基于结构生物学的策略 确定真菌-哺乳动物钙调磷酸酶结构差异，验证真菌特异性靶点，并产生和优化新型FK 506类似物以治疗侵袭性真菌疾病。我们的中心假设是，通过采用结构生物学方法，使用晶体学和NMR光谱，我们将定义新的靶向真菌特异性领域的钙调磷酸酶复合物的真菌发病机制的关键。为了最大限度地扩大临床范围，我们将重点关注两种主要的临床病原体：酵母菌白色念珠菌和霉菌烟曲霉。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性FK 506类似物的原理。 我们假设钙调磷酸酶A和B复合物与钙调蛋白和亲免疫素复合物（FKBP 12-FK 506）的结构将揭示新的真菌特异性抑制靶点。我们最近解决了C的结构。albicans，并且现在将解析单独的钙调磷酸酶异二聚体和与来自A.烟熏。多分子的观点将允许识别的网站是不同的人类和真菌钙调磷酸酶复合物，可以利用有针对性的抑制剂的发展。动态或抗结晶的蛋白质区域将通过NMR进行结构表征。将通过遗传和生物化学试验验证推定的抑制结构域，利用关键接触和表面残基的定点诱变来检查结构稳定性、真菌表型和药物作用/耐药性。非免疫抑制性真菌特异性FK 506类似物将由Ambenix Pharmaceuticals基于我们第一次迭代的SAR结果生成。这将指导第二代类似物的生产，该第二代类似物通过利用宿主和真菌酶之间的独特结构差异来优化以保留抗真菌活性和消除免疫抑制。将进行药物化学和抑制剂对接实验，以根据筛选结果改变类似物。将使用迭代方法在体外和侵袭性念珠菌病和侵袭性曲霉病的小鼠模型中检测先导化合物。我们将利用结构生物学作为一种新的方法来靶向钙调神经磷酸酶，通过定义真菌特异性特征，而没有哺乳动物对应物来产生新的抗真菌疗法。

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.