Development of Natural Product Leads as Anticancer Therapeutics

ENGLERINS：利用Phyllanthus engleri Pax树皮有机提取物在NCI 60细胞筛选中的活性分离得到Englerin A。该提取物是在对68,000种提取物的测试数据进行回顾性生物信息学分析后确定的，该分析旨在确定对肾癌细胞系具有最高选择性的样品。在生物测定法的指导下，从坦桑尼亚树Phyllanthus engleri Pax （Euphorbiaceae）的茎皮和根皮中分离出了一种新型倍半萜二酯englerin A。分离需要三个纯化步骤。最初收集的其他植物部位不含相当数量的englerins，也没有抗癌活性。从坦桑尼亚伊林加省原产地采集的三组树皮都产生了相似数量的englerin A，表明天然树皮是临床前开发的可行来源。NCI目前拥有6克可用于开发活动的纯englerin A，从坦桑尼亚树皮中分离出来。我最近报道了一系列由englerin a衍生的氯化类似物，其中一种活性仅比天然产物弱2.5倍。结构-活性研究已经确立了几个重要的观点：a)细胞生长抑制不仅仅是由于乙醇酸（一种众所周知的低效肾毒素）的释放，因为一种不能产生乙醇酸的反向酯类似物是有活性的。b)肉桂酸部分耐受大量变异而不丧失活性。肉桂酸双键起硬化作用，但其电子贡献不重要。c)异丙基在活性中起重要作用，因为其简化为乙基和甲基会迅速降低效力。d)肉桂酸苯环不要求是芳香的。我们目前的假设是，englerin A的净效应是在使细胞缺乏葡萄糖的同时产生对葡萄糖的依赖。我们认为，这种选择性取决于同时表达PKC-theta和HSF1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，进一步强调了englerin A敏感性、葡萄糖依赖性和PKC-theta活化之间的联系。Englerin A在两种不同的异种移植物模型中显示出活性。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已经产生了一些活性类似物，这些类似物正在与天然产物一起进行评估。此外，我们发现englerin A在大量的尤文氏肉瘤细胞系中具有出色的体外活性。SCHWEINFURTHINS：我从非洲植物Macaranga schweinfurthii Pax中分离出SCHWEINFURTHINS A和B。在NCI 60细胞实验中，化合物对中枢神经系统、肾脏和乳腺癌细胞系显示出有效的选择性活性，在10-25 nM范围内对四种敏感的中枢神经系统肿瘤细胞系具有GI50值。抗癌活性谱与目前使用的任何药物都不匹配，这表明这些化合物可能作用于以前未被识别的靶点或通过一种新的机制。到目前为止，已经从自然界中分离出了11只施魏因芬。Wiemer实验室（爱荷华大学）开发了合成策略，为进一步的生物测试提供了天然schweinfurins和合成类似物的可靠来源。在schweinfurthin F的情况下，（R，R，R）和（S，S，S，S）对映体的全合成，以及与天然产物的光谱数据、旋光度和生物测定数据的比较，导致天然化合物被指定为（R，R，R）异构体。这些合成努力一直在继续，大多数天然存在的施魏因芬素现在都是通过全合成得到的。对schweinfurthins的作用机制的研究尚未确定一个近似的分子靶点；然而，在胶质母细胞瘤细胞系中，似乎有缺陷的1型神经纤维瘤病（NF1）通路赋予其敏感性。在儿童肿瘤基金会的资助下，我们正在进行大规模的siRNA筛选，寻找CRO的潜在靶点。我与Lockett实验室（FNLCR）的合作主要集中在天然schweinfurins对敏感细胞系的细胞肌动蛋白细胞骨架的明确影响上。细胞图像的模式识别算法的发展使得药物治疗中f -肌动蛋白分布变化的定量成为可能。一种合成的schweinfurthin A类似物在NF1缺陷驱动的周围神经鞘肿瘤同种异体移植模型中显示出活性。药物开发正在进行中，计划对转基因迷你猪进行研究，以检查三种有希望的化合物的功效和毒性。

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.