Research for dinoflagellates producing cytotoxic macrolide using DNA probes and development of leads for anticancer drugs

在从海洋甲藻中寻找生物活性代谢物的过程中，我们从海洋共生甲藻中分离出了一系列具有细胞毒性的大环内酯类化合物，命名为两性内酯。两性内酯类化合物具有很强的细胞毒性和抗肿瘤活性。然而，具有大环内酯类或长链多酮类等生物活性物质产生能力的两性藻，很难通过光学显微镜观察进行尖锐和常规的区分。发展传统的方法来鉴定和筛选产生这些次级代谢物的两性虫物种是有用的。本研究以核编码部分小亚基(SSU)rDNA为基础，对产生细胞毒性大环内酯类等生物活性次生代谢物的冲绳双鞭毛藻进行了分子系统发育研究。此外，从产生细胞毒性大环内酯类化合物的Amphiddium物种的18S rDNA序列中开发了特异的DNA探针，这是我们首次尝试将分子探针应用于甲藻类产生生物活性物质的鉴别。在我们从海洋甲藻中寻找生物活性物质的过程中，从一株海洋甲藻中分离到了新的具有细胞毒性的25元大环内酯化合物，即两性内酯C2。另一方面，从海洋扁虫Amphiscolops sp.(菌株Y-100)中分离得到两个新的具有细胞毒性的大环内酯类化合物B4和B5。根据核磁共振数据研究了抗肿瘤大环内酯类化合物H在氯仿和二甲基亚砜中的溶解构象。

In our searches for bioactive metabolites from marine dinoflagellates, we have isolated a series of cyototoxic macrolides, named amphidinolides, from marine symbiotic dinoflagellates Amphidinium species separated from acoel flatworms Amphiscolops species. Amphidinolides exhibit potent cytotoxicity and antitumor activity. Nevertheless, Amphidinium species with producing ability of bioactive substances such as macrolides or long-chain polyketides are difficult to discriminate acutely and conventionally by light microscopic observation. Development of the conventional method for identification and screening of Amphidinium species to produce these secondary metabolites was useful. In this study, the molecular phylogenetic investigations of Okinawan Amphidinium dinoflagellates producing bioactive secondary metabolites such as cytotoxic macrolides were examined on the basis of nuclear-encoded partial small subunit (SSU) rDNA. Furthermore, specific DNA probes were developed from 18S rDNA sequences of the Amphidinium species producing cytotoxic macrolides, and we represent the first attempt to apply molecular probes for the discrimination of dinoflagellates producing bioactive substancesDuring our continuing search for bioactive substances from marine dinoflagellates, new cytotoxic 25-membered macrolides, amphidinolide C2 have been isolated from a marine dinoflagellate Amphidinium sp.(strain Y-72). On the other hand, two new potent cytotoxic macrolides, amphidinolides B4 and B5, have been isolated from a marine dinoflagellate Amphidinium sp.(strain Y-100) separated from marine flatworm Amphiscolops sp.Solution conformation of antitumor macrolide amphidinolide H in chloroform and dimthyl sulfoxide wer investigated on the basis of NMR data.