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

• 批准号: 10319642
• 负责人: HONGMIN LI
• 金额: $ 58.27万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-12-01 至 2022-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10319642
• 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; 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/antagonist; innovation; intein; invention; microbial; mortality; new therapeutic target; novel; novel therapeutics; pathogen; pathogenic fungus; resistant strain; screening; small molecule

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：新型隐球菌属和加蒂隐球菌属可引起隐球菌性脑膜炎和肺炎性疾病。 隐球菌病，主要发生在免疫功能低下的人，如艾滋病毒携带者，严重合并 免疫缺陷，或器官移植后的状态。新近暴发的加蒂亚氏弧菌免疫感染 有能力的人对隐球菌种对公共卫生的总体威胁表示严重关切。 全世界每年有超过100万的CM病例，估计每年有70万人死亡。治疗 由于出现耐药性，CM的死亡率超过50%，变得越来越困难 菌株，使得开发新的治疗隐球菌感染的方法势在必行。隐球菌类 包含蛋白质自剪接元件，称为内含子，它是自切除的内部蛋白质元件 它们的宿主蛋白和催化连接的侧翼序列(外显子)与一个天然的多肽键。因为 内含子不存在于多细胞生物体中，例如人类，并且经常破坏关键蛋白的功能 微生物基因，它们是有吸引力的药物靶点。隐球菌种在必需基因Prp8中有内含子， 剪接体的一部分。与传统的抗真菌药物相比，Intein抑制剂具有许多优势。 因为隐球菌种的Prp8内含子具有很高的序列相似性和相似的剪接 机制，单个内含子剪接抑制物很有可能抑制所有Prp8的剪接活性 INTINT。因此，Prp8内含子剪接抑制剂对所有含有Prp8内含子的真菌都是“广谱”的 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 内含素、内含素抑制剂也将作为仅针对含有内含素抗真菌药物发挥“窄谱”作用 病原体，如隐球菌种。此外，作为新的药物靶点的内含素具有抑制机制。 与所有已知的药物不同。因此，内含素抑制剂将提供一种潜在的新方法，并可能 与现有药物协同治疗感染者。在我们的初步结果中，顺铂不仅被发现 在体外抑制内含子的剪接，也能降低体内的真菌负荷。这项提议的主要目标是 开发和执行高通量筛选试验，以鉴定和表征抑制 Prp8内含子通过生物物理、生化、计算、药理、药物、 细胞和体内的方法。活性好于现有药物的Hit化合物将接受测试 使用各种检测方法和耐药变种减少隐球菌种的生长。这项研究将产生 关于抑制物-整合素在结构上有意义的位点相互作用的信息，并可能导致新的治疗方法 隐球菌感染。