SYNTHESIS AND BIOLOGICAL - ACTIVITY ASSESSMENT OF FUSAROCHROMANONE AMIDE ANALOGS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 赤霉素酮(Fusarochromanone，FC-101a)是由木质镰刀菌Fusum equiseti产生的一种黄酮类化合物。FC-101a可诱导雏鸡胫骨软骨发育不良。这种活性是强大的抗血管生成活性的结果，在鸡饲料上生长的霉菌产生FC-101a，它抑制肉鸡发育中骨骼中血管的生长。FC-101a对人微血管内皮细胞的IC50为50 nM。因此，它也可能是一种有效的人类抗血管生成剂。后来发现，FC-101a也直接作用于癌细胞。该分子在体外对人黑色素瘤和小细胞肺癌诱导细胞凋亡的IC50值均小于10 NM，最能证明该分子的抗癌效力。因此，我们认为FC-101a是发现新治疗剂的极佳候选药物。要进一步开发这种先导化合物作为药物，我们需要进行它的全合成和一组新的FC-101a类似物，这些类似物在体外具有类似的活性，但在体内也表现出更大的活性。 我们的研究小组已经合成了一些新的FC101a类似物，并进行了一系列初步分析，以确定这些类似物对细胞活力的影响。我们分析了人皮肤鳞状细胞癌(SCC)细胞(SRB12-p9细胞系)和稳定转染显性-负性作用形式的STAT3(P9DN5)的SRB12-p9细胞克隆、人角质形成细胞来源的细胞(HaCaT细胞系)和人乳腺癌细胞(MDA-MB-231细胞系)。令人惊讶的是，与未经处理的对照组相比，大多数FC101a类似物引起了所有细胞系的细胞存活率的增加，这是通过四甲基偶氮唑盐细胞存活率测定确定的。用10M的FC101a类似物处理HaCaT细胞的一段时间进一步证实了对细胞活力的积极作用。对于测试的七个类似物，我们观察到长达4天的细胞数量随时间的增加而增加，从29.8%到66.7%不等。 实验正在进行中，以确定增强细胞存活率效应的机制。荧光激活细胞分选(FACS)分析和细胞凋亡分析正在进行中，以确定促进增殖和/或减少细胞死亡对观察到的处理细胞数量增加的相对贡献。此外，我们正在探索类似物潜在的抗血管生成作用。紫外线可诱导HaCaT细胞分泌促血管生成生长因子--血管内皮生长因子，全反式维甲酸可抑制这种作用。我们将在这个模型中测试FC101a类似物抑制紫外线诱导的血管内皮生长因子表达的能力。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Fusarochromanone (FC-101a) is a flavinoid produced by the mold, Fusarium equiseti. FC-101a induces tibial dyschondroplasia in young chickens. This activity is the result of powerful anti-angiogenic activity, whereby mold growing on chicken feed produces FC-101a, which inhibits the growth of blood vessels in the developing bones of broiler chicks. FC-101a has an IC50 of 50 nM against human microvascular endothelial cells. Thus it could be a potent anti-angiogenic agent in man as well. It was later discovered that FC-101a also acts directly on cancer cells. The molecule's anti-cancer potency is best demonstrated by IC50 values of less than 10 nM against both human melanoma and small cell lung cancer in vitro for the induction of apoptosis. Thus, we viewed FC-101a to be an excellent lead candidate for the discovery of new therapeutic agents. Further development of this lead compound as a drug requires that we undertake its total synthesis and a set of new FC-101a analogs that possess similar in vitro activity, but also manifest greater activity in vivo. Our research group has synthesized a number of novel analogs of FC101a and conducted a series of preliminary assays to determine the effect of these analogs on cell viability. We have analyzed human skin squamous cell carcinoma (SCC) cells (SRB12-p9 cell line) and a clone of SRB12-p9 cells that has been stably transfected with a dominant-negative acting form of Stat3 (p9 dn5), human keratinocyte-derived cells (HaCaT cell line), and human breast carcinoma cells (MDA-MB-231 cell line). Surprisingly, most of the FC101a analogs caused an increase in cell viability for all cell lines as determined by the MTT cell viability assay, compared to untreated controls. A time course of treatment of HaCaT cells with 10¿M of FC101a analogs further confirmed the positive effect on cell viability. For the seven analogs tested we observed a time dependent increase in cell number for up to 4 days, ranging from 29.8% to 66.7%. Experiments are in progress to determine the mechanism of the enhanced cell viability effect. Fluorescence activated cell sorting (FACS) analysis and apoptosis assays are underway to determine the relative contribution of increased proliferation and/or decreased cell death to the enhanced cell numbers observed for treated cells. In addition, we are exploring the potential anti-angiogenic effect of the analogs. HaCaT cells can be induced to secrete vascular endothelial growth factor (VEGF), a pro-angiogenic growth factor, in response to ultraviolet light, and this effect can be suppressed by treatment with all-trans retinoic acid. We will test the ability of the FC101a analogs to similarly suppress UV-induced VEGF expression in this model.