Novel aureolic acid type antitumor agents

说明书(申请人提供)：金酸类抗癌药物，如米特拉霉素(MTM)、色霉素(CMM)和硬霉素(DHM)，是具有独特作用模式的强效抗癌和抗艾滋病毒药物。它们通过连接富含GC的DNA来抑制癌细胞的生长，从而关闭依赖于特异性蛋白(Sp)的通路，从而通向包括c-myc和c-src在内的各种原癌基因，后者与这些药物独特的降钙活性有关。尤其是MTM非常重要，已成为研究Sp依赖的信号转导途径的常用生化工具，但由于其毒副作用，除了用于治疗其他化疗药物耐药的肿瘤高钙血症外，很少被用作抗癌药物。然而，2011年，NCI确认MTM是50,000种化合物中唯一的铅，这种化合物可以抑制EWS-FLI1转录因子，这种转录因子导致尤文肉瘤的高度恶性表型，这种肿瘤经常影响儿童，在过去40年里无法治疗。此外，另一个NCI小组在2012年6月发现，MTM抑制香烟烟雾诱导的ABCG2外排泵，这些外排泵导致肺癌和食道癌的耐药性，并抑制与此类癌症的肿瘤发生和增殖相关的多个干细胞相关途径。几年前，MTM被确定为治疗神经系统疾病、关节炎和血液病的潜在先导药物。所有这些新的应用只需要非常小的、毒性较低的药物浓度，尽管在这些情况下的作用模式仍然不清楚，特别是影响EWS-FLI1转录因子的模式。MTM的生物合成在本研究项目的前几个资助期已经得到了深入的研究，因此进行了组合生物合成努力，发现了各种生物合成中间体和许多新的MTM类似物，这使得推断重要的结构-活性-关系成为可能。有两个领域特别令人感兴趣：首先，对3-侧链进行修饰，首先在优越的类似物MTM SK和MTM SDK中举例说明，导致更好的抗癌活性，与MTM本身相比，治疗指数大大提高。其次，D-洋地黄糖能交换MTM三糖链E-位的D-麦芽糖，从而获得更好的类似物，毒副作用大大降低。这现在允许新的策略进一步集中在分子的这些区域，以进一步优化药物。在以往的生物合成研究中，生物合成是耐人寻味的关键 发现了一些需要进一步研究的酶，特别是相互依赖的双功能glycosyltransferase/methyltransferase-ketoreductase)，酶MtmGIV/MtmC(C-C键裂解加氧酶/酮还原酶)，它们在3-侧链和三糖链的形成中起着关键作用。此外，PKS的释放还不清楚，推测另一对相互依赖的酶，即加氧酶MtmOII和环化酶MtmX在这一过程中发挥着重要作用。我们的目标是更深入地了解MTM途径中这些关键酶的生物合成作用、机制和相互作用，为这些酶的优化和再工程铺平道路，这些酶是新的、进一步改进的MTM衍生物的关键。计划(A)进一步研究MTM和其他金酸途径的生物合成步骤和机制，(B)进一步开发选择性地产生新的、改进的MTM类似物的策略，包括选择性3侧链和糖交换/糖随机化策略，(C)分析有趣的酶，特别是最近发现的相互依赖的酶对。新的MTM类似物将被提交给NCI DTP(发育疗法计划)进行进一步的机制研究。

DESCRIPTION (provided by applicant): Aureolic acid-type anticancer agents, such as mithramycin (MTM), chromomycin (CMM) and durhamycin (DHM), are potent anticancer and anti-HIV drugs with a unique mode-of-action. They inhibit the growth of cancer cells by cross-linking GC-rich DNA thereby shutting down specificity-protein (Sp)-dependent pathways toward various proto-oncogenes including c-myc and c-src, the latter being associated with the unique hypocalcemic activity found for these drugs. Particularly, MTM is important, and has become a popular biochemical tool to study Sp-dependent signal transduction pathways, but -due to its toxic side effects- is rarely used as anticancer agent, except for the treatment of tumor hypercalcemia refractory to other chemotherapy. However, MTM was identified in 2011 by the NCI as the only lead from a screen of 50,000 compounds that inhibits the EWS-FLI1 transcription factor responsible for the highly malignant phenotype of Ewing sarcomas, which often affect children and were untreatable for the past 40 years. In addition, another NCI group found in June 2012 that MTM represses cigarette smoke induced ABCG2 efflux pumps responsible for the drug resistance of lung and esophageal cancers, and inhibited multiple stem-cell related pathways relevant for tumorigenicity and proliferation of such cancers. A few years ago MTM was identified as a potential lead drug against neurological diseases, arthritis, and for the treatment of hematologic disorders. All these new applications require only very small, less toxic concentrations of the drug, although the mode-of-action in these contexts remains obscure, particularly the mode affecting the EWS-FLI1 transcription factor. MTM's biosynthesis has been studied intensely during the previous funding periods of this research project, and consequently pursued combinatorial biosynthetic efforts revealed various biosynthetic intermediates and many new MTM-analogues, which allowed deducing important structure-activity-relationships. Two regions are of special interest: First, a modification of the 3-side chain, first exemplified in the superior analogues MTM SK and MTM SDK, led to much better anticancer activity profiles with a greatly improved therapeutic index compared to MTM itself. Second, exchange of the D-mycarose sugar in E-position of MTM's trisaccharide chain by D-digitoxose led to superior analogues with greatly reduced toxic side effects. This now allows for new strategies to further concentrate on these regions of the molecule for further drug optimization. During the previous biosynthetic studies biosynthetic intriguing and interesting key enzymes were discovered, which need to be further investigated, particularly the co-dependent enzymes MtmGIV/MtmC (bifunctional glycosyltransferase/methyltransferase-ketoreductase), MtmOIV/MtmW (C-C-bond cleaving oxygenase/ketoreductase), which play key roles for the 3-side chain and the trisaccharide chain formation. Furthermore, the PKS release remains unclear, and it is hypothesized that another co-dependent enzyme pair, namely oxygenase MtmOII and cyclase MtmX play an important role for this process. The goal here is to gain a deeper understanding of the biosynthetic roles, mechanisms and interactions of these key enzymes of the MTM pathway, to pave the way for the optimization and re-engineering of these enzymes that are crucial for novel, further improved MTM derivatives. It is planned to (a) further investigate unclear biosynthetic steps and mechanisms of the MTM and other aureolic acid pathways, (b) to further develop strategies for the selective generation of new, improved MTM analogues including selective 3-side chain and sugar exchange/glycorandomization strategies, (c) to analyze intriguing enzymes, particularly recently discovered co- dependent enzyme pairs. New resulting MTM analogues will be submitted to the NCI DTP (developmental therapeutics program) for further mechanistic investigations.