High throughput screening of the Prp8 intein splicing inhibitors for pathogenic fungi

病原真菌 Prp8 内含肽剪接抑制剂的高通量筛选

基本信息

批准号：
10382470
负责人：
HONGMIN LI
金额：
$ 56.93万
依托单位：
UNIVERSITY OF ARIZONA
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-12-01 至 2024-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10382470
关键词：
Affect Affinity Animal Model Antifungal Agents Antineoplastic Agents Binding Biochemical Biological Assay Biology Biophysics C-terminal Cause of Death Cells Cessation of life Chemical Structure Cisplatin Combined Modality Therapy Complex Cryptococcal Meningitis Cryptococcosis Cryptococcus Cryptococcus gattii Cryptococcus neoformans Crystallization Crystallography Development Disease Disease Outbreaks Drug Kinetics Drug Synergism Drug Targeting Drug resistance Elements Essential Genes Exteins FDA approved Genes Goals Growth HIV Human Immune Immune system Immunocompetent Immunocompromised Host In Vitro Individual Infection Lead Ligation Luciferases Lung Microbe Mutagenesis N-terminal Organ Transplantation Organism Pathogenicity Pathway interactions Peptides Persons Pharmaceutical Preparations Pharmacology Pharmacotherapy Protein Splicing Proteins Public Health RNA Splicing Resistance Severe Combined Immunodeficiency Site Speed Spliceosomes Structure Structure-Activity Relationship Testing Toxic effect Trypsin Validation Variant Work analog base chemical synthesis combat cytotoxicity drug testing fungus high throughput screening improved in silico in vivo inhibitor innovation intein invention microbial mortality new therapeutic target novel novel therapeutics pathogen pathogenic fungus resistant strain screening small molecule

项目摘要

Abstract: The fungi Cryptococcus neoformans and C. gattii cause cryptococcal meningitis (CM) and pulmonary cryptococcosis, which mainly occurs in immunocompromised people, such as those with HIV, severe combined immunodeficiency, or post organ-transplant status. Recent outbreaks of infections of C. gattii in immune competent people raise significant concerns about the overall threat of cryptococcal species to public health. There are over 1 million annual cases of CM worldwide, with estimated deaths of 700,000 per year. Treatment of CM has become increasingly difficult with a mortality rate over 50%, due to the emergence of drug-resistant strains, making the development of new treatments for cryptococcal infections imperative. Cryptococcal fungi contain protein self-splicing elements, called inteins, which are internal protein elements that self-excise from their host proteins and catalyze ligation of the flanking sequences (exteins) with a natural peptide bond. Because inteins do not exist in multi-cellular organisms, such as humans, and often disrupt the functions of critical microbial genes, they are attractive drug targets. Cryptococcal species have inteins in the essential gene, Prp8, a component of the spliceosome. Intein inhibitors have many advantages over traditional anti-fungal drugs. Because the Prp8 inteins of cryptococcal species share high sequence similarity and have similar splicing mechanisms, it is highly possible that a single intein splicing inhibitor can inhibit the splicing activities of all Prp8 inteins. Thus, Prp8 intein splicing inhibitors will be “broad-spectrum” against all Prp8 intein-containing fungal pathogens. On the other hand, because neither human nor microbes normally associated with humans have inteins, intein inhibitors would be also function as “narrow-spectrum” antifungals specific only for intein-containing pathogens such as cryptococcal species. In addition, inteins as novel drug targets have an inhibition mechanism different from those of all known drugs. Thus, intein inhibitors will provide a potentially fresh approach and may synergize with existing drugs in treating the infected. In our preliminary results, cisplatin was found not only to inhibit the intein splicing in vitro, but also to reduce the fungal burden in vivo. The major goal of this proposal is to develop and perform high throughput screening assays to identify and characterize compounds that inhibit the Prp8 intein splicing by a combination of biophysical, biochemical, computational, pharmacological, medicinal, cellular and in vivo approaches. Hit compounds with activity better than the existing drugs will be tested for reduction of growth of cryptococcal species using various assays and resistant variants. This study will generate information on inhibitor-intein interaction at structurally significant sites and potentially lead to novel therapies for cryptococcal infections.

摘要：真菌加密球菌和C. gattii引起的加密型脑膜炎（CM）和肺部炎隐球菌病主要发生在免疫功能低下的人，例如患有艾滋病毒的人，严重合并免疫缺陷或在器官移植后状态。近期在免疫中爆发C. gattii感染有能力的人对隐球菌对公共卫生的整体威胁提出了重大关注。全球CM年度有超过100万例，估计每年死亡700,000例。治疗由于耐药性的出现，CM的死亡率超过50％变得越来越困难菌株，使得必须开发用于隐球菌感染的新疗法。隐球菌真菌包含蛋白质自剪式元素，称为内膜，它们是内部蛋白质元素，可以自我验证他们的宿主蛋白质并用天然肽键催化侧翼序列（EXTEINS）的连接。因为多细胞生物（例如人类）不存在直肠素，并且经常破坏关键的功能微生物基因，它们是有吸引力的药物靶标。加密秒物种在基本基因PRP8中具有整数剪接体的组成部分。与传统抗真菌药物相比，内部抑制剂具有许多优势。因为隐球菌物种的PRP8整数具有较高的序列相似性，并且具有相似的剪接机制，很有可能单个内部剪接抑制剂可以抑制所有PRP8的剪接活性 inteins。那就是PRP8内素剪接抑制剂对所有含Prp8 intein的真菌的“广谱” 病原体。另一方面，因为人类和通常与人类相关的微生物都没有内膜，内因抑制剂也将作为仅针对含内丁蛋白的抗真菌剂的“狭窄光谱”抗真菌剂病原体，例如隐球菌物种。另外，作为新型药物靶标的intein具有抑制作用机制与所有已知药物的不同。那是内素抑制剂将提供一种潜在的新方法，并且可能与现有药物协同治疗感染。在我们的初步结果中，还发现顺铂不仅是在体外抑制内部剪接，也可以减轻体内的真菌负担。该提议的主要目标是开发和执行高吞吐量筛选分析，以识别和表征抑制的化合物生物物理，生化，计算，药物，医学，细胞和体内方法。与现有药物相比，具有更好活性的命中化合物将被测试使用各种测定和抗性变体降低隐球菌物种的生长。这项研究将产生有关抑制剂 - inintein相互作用在结构上有重要位点的信息，并有可能导致新的疗法隐球菌感染。