SYNTHESIS AND BIOLOGICAL - ACTIVITY ASSESSMENT OF FUSAROCHROMANONE AMIDE ANALOGS

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 Fusarochromanone（FC-101 a）是由真菌木贼镰刀菌（Fusarium equiseti）产生的一种黄酮类化合物。 FC-101 a诱导幼龄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，由此在鸡饲料上生长的霉菌产生FC-101 a，其抑制肉鸡骨骼发育中血管的生长。 FC-101 a对人微血管内皮细胞的IC 50为50 nM。 因此，它也可能是一种有效的抗血管生成剂。 后来发现FC-101 a也直接作用于癌细胞。 该分子的抗癌效力最好通过在体外诱导细胞凋亡时对人黑色素瘤和小细胞肺癌的IC 50值小于10 nM来证明。 因此，我们认为FC-101 a是发现新治疗药物的一个很好的主要候选者。 这种先导化合物作为药物的进一步开发需要我们进行其全合成和一组新的FC-101 a类似物，这些类似物具有相似的体外活性，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许多新的FC 101 a类似物，并进行了一系列初步试验，以确定这些类似物对细胞活力的影响。 我们已经分析了人皮肤鳞状细胞癌（SCC）细胞（SRB 12-p9细胞系）和一个克隆的SRB 12-p9细胞，已稳定转染显性负作用形式的Stat 3（p9 dn 5），人角质形成细胞衍生的细胞（HaCaT细胞系），和人乳腺癌细胞（MDA-MB-231细胞系）。 令人惊讶的是，与未处理的对照相比，大多数FC 101 a类似物引起所有细胞系的细胞活力的增加，如通过MTT细胞活力测定所确定的。 用10 μ M FC 101 a类似物处理HaCaT细胞的时间过程进一步证实了对细胞活力的积极作用。 对于测试的七种类似物，我们观察到细胞数量的时间依赖性增加长达4天，范围从29.8%至66.7%。 实验正在进行中，以确定增强细胞活力效应的机制。 正在进行荧光激活细胞分选（FACS）分析和凋亡测定，以确定增加的增殖和/或减少的细胞死亡对观察到的经处理细胞的增加的细胞数量的相对贡献。 此外，我们正在探索类似物的潜在抗血管生成作用。 HaCaT细胞可被诱导分泌血管内皮生长因子（VEGF），一种促血管生成生长因子，以响应紫外光，并且这种作用可通过用全反式视黄酸处理来抑制。 我们将测试FC 101 a类似物在该模型中类似地抑制UV诱导的VEGF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.