Development of Natural Product Leads as Anticancer Therapeutics

根据叶下珠树皮有机提取物在NCI-60细胞筛选中的活性，分离得到恩格列菌素A。这种提取物是在对68,000种提取物的测试数据进行的回溯性生物信息学分析中确定的，该分析试图确定对肾癌细胞株具有最高选择性的样品。生物测定导向分级分离得到了新的倍半萜二酯--恩格列林A。恩格列林A产量高(1-4g/kg干重)。由坦桑尼亚树干树皮和根皮制成，叶下珠(大戟科)。分离需要三个纯化步骤。最初收集的其他植物部分不含明显数量的工程师素，也没有抗癌活性。从坦桑尼亚伊林加省原址采集的三种树皮都产生了类似数量的英格列尔素A，表明自然采集是临床前开发的一个可行来源。NCI目前拥有6克从坦桑尼亚树皮中分离出来的可用于开发活动的纯英格灵A。我最近报告了一系列来自英格列林A的氯化类似物，其中一种的活性仅比天然产物弱2.5倍。结构-活性研究已经确定了几个重要的点：a)细胞生长抑制不仅仅是由于释放乙醇酸，乙醇酸是一种众所周知但低效的肾脏毒素，因为一种不能产生乙醇酸的反转酯类似物是有效的。B)肉桂酸部分耐受较大的变化而不丧失活性。肉桂酸双键起着刚性作用，但其电子贡献并不重要。C)异丙基在活性中起重要作用，因为它被简化为乙基和甲基迅速降低效力。D)肉桂酸苯环不要求是芳香的。我们目前的假设是，englerin A的净作用是同时使细胞缺乏葡萄糖，同时造成对葡萄糖的上瘾。我们认为这种选择性取决于同时表达PKC-theta和HSF1的细胞和/或高度依赖葡萄糖的细胞。对Englerin A的敏感性也与对2-脱氧葡萄糖的敏感性直接相关，进一步突出了Englerin A敏感性、葡萄糖依赖和PKC-theta激活之间的联系。Engerin A在两种不同的异种移植模型中被证明是活性的。药物配方和其他临床前开发正在进行中。与三个合成化学小组的合作产生了一些活性类似物，这些类似物正在与天然产品一起进行评估。此外，我们还发现，在一大批尤文肉瘤细胞系中，Englerin A具有良好的体外活性。我从非洲植物Macaranga schweinFurthii Pax中分离出SchweinFursins A和B。在NCI-60细胞实验中，这些化合物对中枢神经系统、肾脏和乳腺癌细胞显示出很强的选择性活性，对四种敏感的中枢神经系统肿瘤细胞的GI50值在10-25 nm范围内。抗癌活性的光谱与目前使用的任何药物的光谱都不匹配，表明这些化合物可能作用于以前未识别的目标，或通过新的机制发挥作用。到目前为止，总共有11种雪威因富士从大自然中分离出来。Wiemer实验室(爱荷华大学)已经开发出合成策略，为进一步的生物测试提供可靠的天然舒维辛素和合成类似物来源。以SchweinFurtin F为例，通过全合成(R，R，R)和(S，S，S)对映体，并与报道的天然产物的光谱数据、旋光度和生物测定数据进行比较，确定天然化合物为(R，R，R)异构体。这些合成努力一直在继续，现在大多数自然生成的施韦因富辛都是通过全合成获得的。对SchweinFursins作用机制的研究还没有确定一个接近的分子靶点；然而，在胶质母细胞瘤细胞系中，似乎有缺陷的神经纤维瘤病1型(NF1)途径赋予了敏感性。在儿童肿瘤基金会的资助下，我们正在进行大规模的siRNA筛查，以寻找CRO的潜在靶点。我与洛基特实验室(FNLCR)的合作重点是天然雪魏因呋喃对敏感细胞系细胞肌动蛋白细胞骨架的明显影响。细胞图像的模式识别算法的发展使得能够量化药物治疗过程中F-肌动蛋白分布的变化。一种合成的神经营养因子A的类似物在由NF1缺陷驱动的外周神经鞘肿瘤的同种异体移植模型中显示出活性。药物开发正在进行中，计划在转基因迷你猪身上进行研究，以检查三种有希望的化合物的有效性和毒性。

ENGLERINS: Englerin A was isolated based on the activity of the organic extract of the bark of Phyllanthus engleri Pax in the NCI 60 cell screen. The extract was identified in a retrospective bioinformatic analysis of testing data for 68,000 extracts, which sought to identify samples with the most selectivity against renal cancer cell lines. Bioassay guided fractionation of the extract led to isolation of the novel sesquiterpene diester, englerin A. Englerin A was isolated in high yield (1-4 g/kg dry wt.) from stem bark and root bark of the Tanzanian tree, Phyllanthus engleri Pax (Euphorbiaceae). Isolation required three purification steps. Other plant parts from the initial collection did not contain appreciable amounts of englerins and were devoid of anticancer activity. Three collections of bark collected from the original location in Iringa Province, Tanzania have all yielded similar amounts of englerin A, showing that natural collections are a viable source for preclinical development. The NCI currently possesses 6 g of pure englerin A which is available for development activities, isolated from Tanzanian bark. I recently reported a series of chlorinated analogues derived from englerin A, one of which has activity only 2.5-fold weaker than the natural product. Structure-activity studies have established several important points: a) Cell growth inhibition is not simply due to release of glycolate, a well-known but low-potency renal toxin, since a reverse ester analogue which cannot generate glycolate is active. b) The cinnamate moiety tolerates substantial variation without loss of activity. The cinnamate double bond plays a rigidifying role but its electronic contributions are not important. c) The isopropyl group plays an important role in activity, since its simplification to ethyl and methyl groups rapidly decreases potency. d) The cinnamate benzene ring is not required to be aromatic. Our current hypothesis is that the net effect of englerin A is to simultaneously starve the cells of glucose while creating an addiction to glucose. We believe that the selectivity depends on cells expressing both PKC-theta and HSF1 and/or being highly glucose dependent. Sensitivity to englerin A also correlates directly with sensitivity to 2-deoxyglucose, further highlighting the link between englerin A sensitivity, glucose dependence and PKC-theta activation. Englerin A was shown to be active in two different xenograft models. Pharmaceutical formulation and other preclinical development is ongoing. Collaborations with three synthetic chemistry groups have resulted in a number of active analogues which are being evaluated in tandem with the natural product. In addition, we have discovered that englerin A has excellent in vitro activity in a large panel of Ewing's sarcoma cell lines. SCHWEINFURTHINS: I isolated schweinfurthins A and B from the African plant Macaranga schweinfurthii Pax. The compounds displayed potent and selective activity against central nervous system, renal, and breast cancer cell lines in the NCI 60 cell assay, with GI50 values for four sensitive CNS tumor cell lines in the 10-25 nM range. The spectrum of anticancer activity did not match that of any currently used agent, indicating that these compounds might be acting at a previously unrecognized target or through a novel mechanism. Thus far, a total of 11 schweinfurthins have been isolated from nature. Synthetic strategies have been developed by the Wiemer lab (University of Iowa) to provide a reliable source of natural schweinfurthins and synthetic analogues for further biological testing. In the case of schweinfurthin F, total synthesis of the (R,R,R) and (S,S,S) enantiomers and comparisons of spectral data, optical rotations, and bioassay data with those reported for the natural product have resulted in assignment of the natural compounds as the (R,R,R) isomers. These synthetic efforts have continued, and most of the naturally occurring schweinfurthins have now been obtained by total synthesis. Investigations into the mechanism of action of schweinfurthins have yet to identify a proximate molecular target; however, in glioblastoma cell lines, it appears that a defective neurofibromatosis type 1 (NF1) pathway confers sensitivity. With a grant from the Children's Tumor Foundation, we are conducting a massive siRNA screen for potential targets at a CRO. My collaboration with the Lockett lab (FNLCR) focused on the clear effect of natural schweinfurthins on the cellular actin cytoskeleton in sensitive cell lines. Development of pattern recognition algorithms for cellular images has enabled quantitation of changes in F-actin distribution with drug treatment. A synthetic analogue of schweinfurthin A has shown activity in an allograft model of peripheral nerve sheath tumor driven by an NF1 defect. Pharmaceutical development is ongoing, with planned studies in genetically altered mini-pigs to examine three promising compounds for efficacy and toxicity.