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.

抽象的隐球菌病是一种致命的真菌病，占艾滋病毒/艾滋病相关死亡的 15% 以上。目前可用的抗真菌药物要么是剧毒的（多烯），要么是治疗隐球菌病的选择有限。发挥抑制真菌作用（三唑类），需要长期治疗方案并为第三种主要抗真菌药物棘白菌素表现出低毒性和耐药性。对其他几种常见的真菌病原体具有杀菌作用，但隐球菌具有抗药性。我们最近报道了棘白菌素及其耐药机制。翻转酶，负责维持膜脂双层不对称性和正常的酶细胞内囊泡运输，使新型隐球菌对卡泊芬净（一种棘白菌素类药物）敏感，我们还发现脂质翻转酶对于小鼠模型中的毒力至关重要。隐球菌病并使新型隐球菌对巨噬细胞的杀伤敏感，这表明它可能是一种新的在这个项目中，我们建议破译隐球菌中脂质翻转酶的机制。棘白菌素耐药性并进行抑制新型隐球菌翻转酶功能的原理验证研究。在第一个目标中，我们将测试关于脂质翻转酶在耐药性：（1）脂质翻转酶的丢失改变了膜结构，例如膜上的 PS 分布，促进卡泊芬净与其靶标 β-1,3-D-葡聚糖合酶 (Fks1) 的相互作用，以及 (2) 缺乏脂质翻转酶某些耐药途径，例如钙调磷酸酶途径受到损害，我们将通过使用药物来破坏细胞钙稳态并诱导杀伤。在第二个目标中，我们建议开发一系列细胞、分子、生物化学和遗传方法。针对脂质翻转酶细胞外环的抗体 Fab 片段，这对于翻转酶功能至关重要，并测试其使新型隐球菌对抗真菌药物敏感并被巨噬细胞杀死的能力。这种基于抗体的方法靶向的脂质翻转酶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，针对该区域产生的抗体具有真菌特异性。这项研究将有助于更好地了解脂质翻转酶介导的新型隐球菌耐药性，可以帮助扩大棘白菌素药物针对隐球菌和其他耐药真菌病原体的使用。此外，翻转酶抑制抗体的产生将提供有价值的研究工具，并可能导致新型联合治疗方法的未来发展最后，抗体的成功开发。基于抑制剂可以开辟针对其他膜蛋白的研究和药物开发的新途径在真菌和细菌中。