Signal Transduction Enzyme Inhibitors from Extremophilic Microbes as Anticancer A

描述（由申请人提供）：该项目的总体目标是从酸性矿山废水湖中分离的极端微生物中发现具有抗癌活性的新天然产物。化合物将基于其抑制与几种不同类型癌症的发作和转移有关的特异性信号转导酶的能力来分离。基质金属蛋白酶-3（MMP-3）和半胱天冬酶-1（casp-1）已显示在某些癌症中上调。在过去的五年里，我们已经开发了一种生物测定指导的分离方案，导致了几种新的化合物，抑制这些酶之一或两者的分离和纯化。通过光谱技术鉴定了这些化合物的结构。为了建立酶抑制和抗癌活性之间的相关性，将这些纯酶抑制剂提交给国家癌症研究所发育治疗计划（NCI/DTP），用于对60种人类癌细胞系进行测试。这种方法已经导致了几种先导化合物的分离和鉴定，这些化合物具有纳摩尔或低微摩尔活性，并且对卵巢癌、非小细胞肺癌和白血病具有高选择性。从酸性矿山废物中分离出的微生物已被证明是以前未报道的生物活性代谢物的丰富来源。由美国国立卫生研究院-IDEA生物医学研究卓越网络（INBRE）资助的初步研究从富含金属硫酸盐的沃茨和深层基底沉积物中产生了70多种不同的微生物。在已经在小型中试发酵研究中检查的30种微生物中，超过50%的提取物抑制MMP-3和/或Casp-1。对这些生物体中的五种的深入探索已经产生了几种新的化合物，包括对OVCAR-3表现出选择性纳摩尔活性的伯克利酸和对非小细胞肺癌菌株NCI-H460表现出选择性低微摩尔活性的伯克利二酮。有了这些早期的成功，我们想继续研究这些微生物，具体目标如下：1。利用信号传导酶抑制作为分离指导，从目标极端微生物中分离化合物。2.使用光谱或X射线方法阐明这些化合物的结构。3.在NIH/DTP细胞系筛选中测试活性化合物以确定体外活性。4.将对特定人类癌细胞系具有酶抑制活性和纳摩尔-低微摩尔活性的化合物送至Riskai研究所进行进一步评估。5.让本科生参与微生物药物发现的各个方面这项研究已经显示出希望，为RO 1申请的资金将用于继续这项探索。它解决了一个关键的需求领域，并结合了独特的分析工具和独特的极端环境，用于药物发现。公共卫生相关性：预计今年将有超过565，000名美国人死于癌症，预计将诊断出超过2，437，000例新的癌症病例。尽管我们有很多抗癌药物，但癌症现在是西方世界的第二大死亡原因。不幸的是，目前的治疗方式使用细胞毒性剂，这可能导致有害的副作用。本申请描述了一种药物发现的新方法，使用信号转导酶抑制作为从极端微生物中分离选择性、有效抗癌剂的指导。酶抑制剂将由NCI/DTP针对60种人类癌细胞系进行检测，并由Riskai Research Institute进行检测，以确定抗癌活性。新型化合物将有助于满足对具有高选择性和效力以及比当前治疗选择更低的细胞毒性的新治疗方式的日益增长的需求。

DESCRIPTION (provided by applicant): The overall goal of this project is the discovery of new natural products with anticancer activity from extremophilic microbes isolated from an acid mine waste lake. Compounds will be isolated based on their ability to inhibit specific signal transduction enzymes that have been implicated in the onset and metastasis of several different types of cancer. Matrix metalloproteinase-3 (MMP-3) and caspase-1 (casp-1) have been shown to be up-regulated in certain cancers. For the past five years we have developed a bioassay-guided isolation scheme that has resulted in the isolation and purification of several novel compounds that inhibit one or both of these enzymes. The structures of these compounds were elucidated through spectral techniques. To establish a correlation between enzyme inhibition and anticancer activity, these pure enzyme inhibitors were submitted to the National Cancer Institute Developmental Therapy Program (NCI/DTP) for testing against 60 human cancer cell lines. This methodology has resulted in the isolation and identification of several lead compounds with nanomolar or low micromolar activity and high selectivity towards ovarian cancer, non-small cell lung cancer, and leukemia. Microbes isolated from acid mine waste have proven to be a rich source of previously unreported, bioactive metabolites. Preliminary studies, which have been funded through the National Institutes of Health-IDeA Networks of Biomedical Research Excellence (INBRE), yielded over 70 different microorganisms from the metal sulfate rich waters and deep basal sediment. Of the 30 microorganisms already examined in small pilot fermentation studies, over 50% of the extracts inhibited MMP-3 and/or Casp-1. In- depth exploration of five of these organisms has yielded several novel compounds including berkelic acid, which exhibited selective nanomolar activity against OVCAR-3 and berkeleydione which exhibited selective low micromolar activity against nonsmall cell lung cancer strain NCI-H460. With these early successes we would like to continue studying these microbes with the following specific aims: 1. Isolate compounds from targeted extremophilic microorganisms using signal transduction enzyme inhibition as an isolation guide. 2. Elucidate the structures of these compounds using spectroscopic or x-ray methodology. 3. Test active compounds in the NIH/DTP cell line screen to determine in vitro activity. 4. Send compounds with enzyme inhibitory activity and nanomolar - low micromolar activity against specific human cancer cell lines to Eisai Research Institute for further evaluation. 5. Involve undergraduate students in all aspects of microbial drug discovery This research has shown promise and the funds requested for this RO1 would be used to continue this exploration. It addresses a critical need area and combines both unique assay tools and a unique extreme environment for drug discovery. PUBLIC HEALTH RELEVANCE: Over 565,000 Americans are expected to die of cancer this year, and more than 2,437,000 new cancer cases are expected to be diagnosed. Despite our arsenal of anticancer agents, cancer is now the second leading cause of death in the western world. Unfortunately, current treatment modalities use cytotoxic agents that can cause deleterious side-effects. This application describes a novel approach to drug discovery, using signal transduction enzyme inhibition as a guide to the isolation of selective, potent anticancer agents from extremophilic microbes. Enzyme inhibitors will be tested by NCI/DTP against 60 human cancer cell lines and by Eisai Research Institute to determine anticancer activity. Novel compounds will help meet the increasing need for new treatment modalities with high selectivity and potency, and lower cytotoxicity than current treatment options.