Development of Natural Product Leads as Anticancer Therapeutics

恩格勒蛋白：恩格勒蛋白A是基于在NCI 60细胞筛选中的叶下珠树皮的有机提取物的活性而分离的。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中确定的，该分析旨在确定对肾癌细胞系具有最大选择性的样本。生物测定引导的提取物分级分离导致新的倍半萜二酯，englerin A的分离。以高产率（l-4g/kg干重）分离恩格勒蛋白A。来自坦桑尼亚树，叶下珠（Phyllanthus engleri Pax）（大戟科）的茎皮和根皮。 分离需要三个纯化步骤。来自最初收集的其他植物部分不含有可感知量的englerins，并且缺乏抗癌活性。 从坦桑尼亚伊林加省的原始地点收集的三个树皮收集物都产生了相似量的englerin A，表明天然收集物是临床前开发的可行来源。NCI目前拥有6克纯englerin A，可用于开发活动，从坦桑尼亚树皮中分离出来。我最近报道了一系列来自englerin A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盐（一种众所周知但效力低的肾毒素）的释放，因为不能产生乙醇酸盐的反向酯类似物是有活性的。B）肉桂酸酯部分耐受实质性变化而不丧失活性。肉桂酸酯双键起刚性化作用，但其电子贡献并不重要。c）异丙基在活性中起重要作用，因为其简化为乙基和甲基会迅速降低效力。d）肉桂酸苯环不需要是芳香的。我们目前的假设是englerin A的净效应是在使细胞对葡萄糖成瘾的同时使细胞缺乏葡萄糖。我们认为选择性取决于表达PKC-θ和HSF 1和/或高度葡萄糖依赖性的细胞。 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、葡萄糖依赖性和PKC-θ激活之间的联系。在两种不同的异种移植物模型中显示恩格勒蛋白A具有活性。Schweinfurthins：我从非洲植物Macaranga schweinfurthii Pax中分离出schweinfurthins A和B。这些化合物在NCI 60细胞测定中显示出对中枢神经系统、肾癌和乳腺癌细胞系的有效和选择性活性，其中四种敏感CNS肿瘤细胞系的GI 50值在10-25 nM范围内。抗癌活性谱与目前使用的任何药物都不匹配，表明这些化合物可能作用于以前未识别的靶点或通过新的机制发挥作用。到目前为止，共有11个schweinfurthins从自然界中分离出来。Wiemer实验室（爱荷华州大学）开发了合成策略，为进一步的生物学测试提供了天然schweinfurthins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）对映异构体的全合成和光谱数据，旋光度和生物测定数据的比较与那些报道的天然产物的天然化合物的分配（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schweinfurthins现在已经通过全合成获得。对schweinfurthins作用机制的研究尚未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（NF 1）通路赋予敏感性。我与Lockett实验室（FNLCR）的合作主要集中在天然schweinfurthins对敏感细胞系中细胞肌动蛋白细胞骨架的明显影响上。细胞图像模式识别算法的发展使得药物治疗后F-actin分布的变化能够定量。schweinfurthin A的合成类似物在由NF 1缺陷驱动的外周神经鞘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。

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.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. My collaboration with the Lockett lab (FNLCR) has 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.