Signal Transduction Enzyme Inhibitors from Extremophilic Microbes as Anticancer A

描述(申请人提供)：该项目的总体目标是从从酸性矿山废湖中分离出来的极端微生物中发现具有抗癌活性的新天然产物。化合物将根据它们抑制特定信号转导酶的能力进行分离，这些信号转导酶与几种不同类型癌症的发生和转移有关。基质金属蛋白酶-3(MMP-3)和半胱氨酸天冬氨酸氨基转移酶-1(Casp-1)在某些癌症中表达上调。在过去的五年里，我们开发了一种生物测定指导的分离方案，该方案已经分离和纯化了几种抑制这两种酶中的一种或两种的新化合物。通过波谱技术对化合物的结构进行了鉴定。为了建立酶抑制和抗癌活性之间的相关性，这些纯酶抑制剂被提交给国家癌症研究所开发治疗计划(NCI/DTP)，以针对60种人类癌细胞进行测试。这一方法已经导致分离和鉴定了几种具有纳摩尔或低微摩尔活性以及对卵巢癌、非小细胞肺癌和白血病具有高选择性的先导化合物。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。通过国家卫生研究院-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水域和深层基底质沉积物中产生了70多种不同的微生物。在小型中试发酵研究中已经检查的30种微生物中，超过50%的提取物对基质金属蛋白酶-3和/或Casp-1有抑制作用。对其中五种生物的深入研究已经产生了几个新的化合物，包括对OVCAR-3显示出选择性纳米分子活性的伯克酸，以及对非小细胞肺癌NCI-H460显示出选择性低微摩尔活性的伯克利酮。有了这些早期的成功，我们将继续研究这些微生物，具体目标如下：1.以信号转导酶抑制为分离指南，从目标极端微生物中分离出化合物。2.用光谱学或X-射线衍射法确定化合物的结构。3.在NIH/DTP细胞株筛选中检测活性物质，以确定其体外活性。4.将具有酶抑制活性和纳摩尔-低微摩尔活性的化合物送卫材研究所进一步评价。5.让本科生参与微生物药物发现的各个方面这项研究显示出了希望，为这项RO1申请的资金将用于继续这一探索。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的药物发现。与公共健康相关：预计今年将有超过56.5万美国人死于癌症，预计将有超过243.7万例新癌症病例被诊断出来。尽管我们有大量的抗癌药物，但癌症现在是西方世界的第二大死因。不幸的是，目前的治疗方式使用的细胞毒剂可能会引起有害的副作用。本申请描述了一种新的药物发现方法，使用信号转导酶抑制作为从极端微生物中分离选择性、有效抗癌药物的指导。酶抑制剂将由NCI/DTP和卫材研究所对60种人类癌细胞株进行测试，以确定抗癌活性。新的化合物将有助于满足对新的治疗方式的日益增长的需求，这些新的治疗方式具有高选择性和有效性，并且比目前的治疗方案具有更低的细胞毒性。

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.