Structural Biological Development of Fungal-Specific Calcineurin Inhibitors

真菌特异性钙调神经磷酸酶抑制剂的结构生物学发展

基本信息

批准号：
8745170
负责人：
JOSEPH HEITMAN
金额：
$ 63.75万
依托单位：
DUKE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-08-04 至 2018-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8745170
关键词：
Affect Antifungal Agents Area Aspergillosis Aspergillus fumigatus Binding Binding Sites Biochemical Biological Biological Assay Calcineurin Calcineurin inhibitor Calmodulin Candida albicans Candidiasis Cause of Death Cell division Chemicals Chemistry Clinical Collaborations Collection Complex Coupled Crystallization Crystallography DNA Development Disease Docking Drug effect disorder Enzyme Inhibitor Drugs Enzyme Inhibitors Enzymes FDA approved FK506 Generations Genetic Genetic Transcription Growth HIV-1 Health Human Immune system Immunity Immunocompromised Host Immunophilins Immunosuppression Immunosuppressive Agents In Vitro Industrial fungicide Knowledge Lead Maps Medical Modeling Modification Molds Molecular Molecular Target Mus Mutagenesis Mycoses NMR Spectroscopy Pathogenesis Patients Pharmaceutical Chemistry Pharmaceutical Preparations Pharmacologic Substance Phenotype Physiology Production Protein Region Proteins Research Resistance Resistance development Screening Result Signal Transduction Site Site-Directed Mutagenesis Solutions Specificity Structural Biologist Structure Surface Tacrolimus Binding Protein 1A Techniques Testing The science of Mycology Therapeutic Titrations Translating Treatment Efficacy Work Yeasts analog base calcineurin phosphatase clinical efficacy cross reactivity drug development env Gene Products fungus immunogenic improved in vitro testing inhibitor/antagonist novel novel strategies pathogen public health relevance research study segregation structural biology

项目摘要

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.

描述（由申请人提供）：侵入性真菌感染是免疫功能低下的患者死亡的主要原因。当前的抗真菌性临床功效有限，杀真菌性较差，在某些情况下是有毒的，并且由于新兴的耐药性而越来越无效。我们已经确定，保守的磷酸酶钙调蛋白是侵入性真菌疾病所必需的。由FDA批准的钙调神经酶抑制剂FK506在体外对主要侵入性真菌病原体具有活性，但也抑制了宿主的免疫力。我们的方法旨在克服真菌与人体特异性障碍，以显着提高抗真菌治疗。这项研究的目的是利用基于结构生物学的策略定义真菌哺乳动物钙调蛋白的结构差异，验证真菌特异性靶标，并生成和优化新颖的FK506类似物来治疗侵入性真菌疾病。我们的中心假设是，通过使用晶体学和NMR光谱使用结构生物学方法，我们将在钙调神经素复合物中定义新型的可靶向真菌特异性区域，这对于真菌发病机理至关重要。为了获得最大的临床广度，我们将重点关注两种主要的临床病原体：酵母念珠菌和霉菌烟曲霉。我们的初步研究记录了具有强大抗真菌活性的原理非免疫抑制FK506类似物的证明。我们假设钙调神经素A和B复合物的结构，再加上钙调蛋白和免疫光蛋白复合蛋白（FKBP12-FK506），将揭示新的新型真菌特异性抑制靶标。我们最近解决了白色念珠菌的结构，现在将单独求解钙调神经蛋白异二聚体的结构，并与烟曲霉的FKBP12-FK506/类似物复合。多个分子视图将允许鉴定可用于靶向抑制剂发育的人类和真菌钙调神经酶复合物之间与众不同的位点。动态或抵抗结晶的蛋白质区域将在结构上以NMR为特征。推定的抑制域将通过遗传和生化测定法进行验证，利用关键接触和表面残基的位置定向诱变，以检查结构稳定性，真菌表型以及药物作用/耐药性。不免疫抑制真菌特异性的FK506类似物将由我们的第一次迭代的SAR结果产生。这将通过利用宿主和真菌酶之间独特的结构差异来指导优化的第二代类似物的生产，以保留抗真菌活性和废除免疫抑制。将根据筛查结果进行药物化学和抑制剂对接实验以改变类似物。铅化合物将在体外和浸润性念珠菌病和浸润性曲霉病的鼠模型中使用迭代方法进行测试。我们将通过定义没有哺乳动物对应物来生成新型抗真菌治疗剂的真菌特异性特征来利用结构生物学作为一种靶向钙调神经酶的新方法。