Lipid flippase in echinocandin drug resistance in Cryptococcus neoformans

脂质翻转酶在新型隐球菌棘白菌素耐药性中的作用

基本信息

批准号：
10170266
负责人：
Chaoyang Xue
金额：
$ 19.6万
依托单位：
RBHS-NEW JERSEY MEDICAL SCHOOL
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-05-22 至 2023-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10170266
关键词：
3-Dimensional AIDS/HIV problem ATP phosphohydrolase Acquired Immunodeficiency Syndrome Amphotericin B Antibodies Antifungal Agents Antifungal Therapy Bacteria Binding Biochemical Genetics Calcineurin Pathway Calcium Caspofungin Cell membrane Cell surface Cellular Assay Cellular Membrane Cessation of life Chemicals Combined Modality Therapy Cryptococcosis Cryptococcus Cryptococcus neoformans Data Development Disease Drug Targeting Drug resistance Drug resistance pathway Enzymes Epitopes Fab Immunoglobulins Fluconazole Fungal Drug Resistance Future Generations Goals Homeostasis Human Immunoglobulin Fragments Impairment Industrial fungicide Infection Lead Lipid Bilayers Lipids Liposomes Mediating Membrane Membrane Proteins Mission Modeling Molecular Molecular Weight Monoclonal Antibodies Mus Mycoses Pharmaceutical Preparations Phosphatidylserines Phospholipids Polyenes Reporting Research Resistance Role Sequence Homology Surface System Testing Therapeutic Toxic effect Treatment Protocols Triazoles Ubiquitin United States National Institutes of Health Vesicle Virulence base disorder control disorder prevention drug development drug sensitivity echinocandin resistance extracellular fungus genetic approach glucan synthase human pathogen in vivo inhibitor/antagonist macrophage mouse model mutant new therapeutic target novel pathogenic fungus polyclonal antibody pre-clinical research and development resistance mechanism success tool trafficking yeast two hybrid system

项目摘要

Abstract Cryptococcosis is a deadly fungal disease that accounts for over 15% HIV/AIDS related deaths. Treatment options for cryptococcosis are limited. Currently available antifungal drugs are either highly toxic (polyenes) or exert a fungistatic effect (triazoles), necessitating long treatment regimens and leaving open the avenue for emergence of drug resistance. The third major antifungal drug class, the echinocandins, show low toxicity and are fungicidal against several other prevalent fungal pathogens. However, Cryptococci are resistant to echinocandins and the mechanisms of this resistance remain unknown. We recently reported that loss of lipid flippase, the enzyme responsible for maintaining the asymmetry of membrane lipid bilayers and normal intracellular vesicle trafficking, sensitizes C. neoformans to caspofungin, a drug of the echinocandin class, as well as to several triazoles. We also found that lipid flippase was essential for virulence in a murine model of cryptococcosis and sensitized C. neoformans to killing by macrophages, suggesting that it may be a novel antifungal drug target. In this project, we propose to decipher the mechanism of lipid flippase in cryptococcal echinocandin resistance and to conduct proof-of-principle studies inhibiting flippase function in C. neoformans. In the first Aim, we will test two related, non-mutually exclusive hypotheses regarding the role of lipid flippase in drug resistance: (1) that loss of lipid flippase changes membrane structure, e.g. PS distribution on membrane, to promote the interaction of caspofungin with its target β-1,3-D-glucan synthase (Fks1), and (2) that in the absence of lipid flippase certain drug resistance pathways, such as calcineurin pathway are compromised, disrupting cellular calcium homeostasis and inducing killing by drugs. We will test these hypotheses by employing a host of cellular, molecular, biochemical, and genetic approaches. In the second Aim, we propose to develop an antibody Fab fragment against the extracellular loop of lipid flippase, which is essential for flippase function, and to test its ability to sensitize C. neoformans to antifungal drugs and to killing by macrophages. The region of lipid flippase targeted by this antibody-based approach has low sequence homology to its human counterpart, and our preliminary studies showed that an antibody raised against this region is fungal-specific. The success of this study will lead to a better understanding of lipid flippase mediated drug resistance in C. neoformans, which could help expand the use of echinocandin drugs against Cryptococci and other resistant fungal pathogens. Furthermore, generation of flippase inhibitory antibodies will provide a valuable research tool and may lead to future development of novel combination therapy approaches. Finally, successful development of antibody- based inhibitors could open a new avenue of research and drug development against other membrane proteins in fungi and bacteria.

抽象的隐孢子虫是一种致命的真菌疾病，占艾滋病毒/艾滋病相关的死亡人数超过15％。治疗隐球菌病的选择有限。目前可用的抗真菌药物要么是剧毒（polyeners）或发挥真实效应（三轮唑），必要的长期治疗方案，并为耐药性的出现。第三个主要抗真菌药物类别Echinocandins显示出低毒性和对其他几种流行的真菌病原体是真菌。但是，加密环球具有抗性 eChinocandins和这种抗性的机制仍然未知。我们最近报道了脂质的损失 Flippase，负责维持膜脂质双层不对称和正常的酶细胞内囊泡运输，感应新生虫的c。以及几个三轮唑。我们还发现，在鼠模型的鼠模型中，脂质小裂对于病毒至关重要。隐球菌病和对巨噬细胞杀死的敏感梭菌新生虫，这表明它可能是一种新颖的抗真菌药物靶标。在这个项目中，我们建议在隐球菌中破译脂质氟脂肪的机理 eChinocandin耐药性并进行原本研究抑制新梭菌中Flippase功能的原则研究。在第一个目标中，我们将测试有关脂质Flippase在耐药性：（1）脂质flippase的丧失会改变膜结构，例如PS分布在膜上，促进Caspofungin与靶β-1,3-D-葡聚糖合酶（FKS1）的相互作用，（2）缺乏脂质Flippase某些耐药性途径，例如钙调神经蛋白途径，受到损害，破坏细胞钙稳态并被药物杀死。我们将通过采用许多细胞，分子，生化和遗传方法。在第二个目标中，我们建议开发针对脂质氟脂酶的细胞外环的抗体Fab片段，这对于Flippase功能至关重要，并测试其将新生甲梭菌吸引到抗真菌药物并被巨噬细胞杀死的能力。区域这种基于抗体的方法靶向的脂质Flippase与其人类对应物具有较低的序列同源性，我们的初步研究表明，针对该区域提出的抗体是真菌特异性的。成功的成功这项研究将导致对脂质Flippase介导的耐药性抗药性的了解，而新甲状腺梭菌的耐药性。可以帮助扩大针对加密球动和其他抗性真菌病原体的echinocandin药物的使用。此外，Flippase抑制性抗体的产生将提供有价值的研究工具，并可能导致新型组合疗法方法的未来发展。最后，成功开发抗体 - 基于其他膜蛋白的抑制剂可以开放研究和药物开发的新途径在真菌和细菌中。