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

摘要：新型隐球菌和隐球菌C. gattii引起隐球菌性脑膜炎（CM）和肺 隐球菌病，这主要发生在免疫功能低下的人，如那些与艾滋病毒，严重结合 免疫缺陷或器官移植后状态。近年来爆发的C.免疫学 有能力的人对隐球菌物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100万例CM病例，估计每年有70万人死亡。治疗 CM的治疗变得越来越困难，死亡率超过50%，这是由于耐药菌株的出现。 菌株，使隐球菌感染的新疗法的发展势在必行。隐球菌真菌 含有蛋白质自我剪接元件，称为内含肽，这是内部蛋白质元件， 它们的宿主蛋白质并催化侧翼序列（外显蛋白）与天然肽键的连接。因为 内含肽不存在于多细胞生物体（如人类）中，并且经常破坏关键的细胞的功能。 微生物基因，它们是有吸引力的药物靶标。隐球菌物种在必需基因Prp 8中具有内含肽， 剪接体的组成部分。内含肽抑制剂与传统的抗真菌药物相比具有许多优势。 由于隐球菌属物种的Prp 8内含肽具有高度的序列相似性并且具有相似的剪接， 由于Prp 8的剪接机制不同，因此很有可能单一内含肽剪接抑制剂可以抑制所有Prp 8的剪接活性。 内含肽。因此，Prp 8内含肽剪接抑制剂将是“广谱的”针对所有含有Prp 8内含肽的真菌 病原体另一方面，因为无论是人类还是通常与人类相关的微生物， 内含肽、内含肽抑制剂也可以作为仅对含内含肽 病原体如隐球菌属。此外，内含肽作为新的药物靶点具有抑制机制 与所有已知药物不同。因此，内含肽抑制剂将提供一种潜在的新方法， 与现有药物协同治疗感染者。在我们的初步结果中，发现顺铂不仅 在体外抑制内含肽剪接，还能减轻体内真菌负担。这项建议的主要目的是 开发和执行高通量筛选试验，以鉴定和表征抑制 Prp 8内含肽通过生物物理学、生物化学、计算、药理学、医学 细胞和体内方法。比现有药物活性更好的命中化合物将被测试 使用各种测定和抗性变体减少隐球菌物种的生长。这项研究将产生 在结构上重要的位点上的内含子-内含肽相互作用的信息，并可能导致新的治疗方法， 隐球菌感染

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.