Discovery and characterisation of recifin

酪氨酰-DNA磷酸二酯酶1（TDP 1）是致敏的分子靶点， 癌细胞对拓扑异构酶抑制剂喜树碱及其衍生物的作用。高通量 筛选天然产物提取物文库中的TDP1活性抑制剂， 海洋海绵Axinella sp.的生物活性水提取物的发现 源提取物的分级分离导致活性组分的分离， 被确定为一种新的，42个残基的富含半胱氨酸的肽，我们命名为recifin。主 序列和recifin的二硫键模式确定使用Edman的组合， 降解和串联质谱从头蛋白质测序。NMR结构 揭示了一种新的折叠，包括四股反平行β-折叠和两个螺旋转角 通过复杂的二硫键网络稳定，该网络在其中一个 股。Recifin抑制全长TDP1，但不抑制N-末端截短的TDP1在生化 测定。酶动力学研究表明，recifin可以特异性地调节 全长TDP1的活性，而不影响TDP1的截短形式，这表明 TDP1上recifin的变构结合位点。

Tyrosyl-DNA phosphodiesterase 1 (TDP1) is a molecular target for the sensitization of cancer cells to the topoisomerase inhibitor camptothecin and its derivatives. High-throughput screening of natural product extract libraries for inhibitors of TDP1 activity resulted in the discovery of a bioactive aqueous extract of the marine sponge, Axinella sp. Bioassay-guided fractionation of the source extract resulted in the isolation of the active component which was determined to be a novel, 42-residue cysteine-rich peptide we named recifin. The primary sequence and disulfide bonding pattern of recifin was determined using a combination of Edman degradation and tandem mass spectroscopy de novo protein sequencing. The NMR structure revealed a novel fold comprising a four strand anti-parallel beta-sheet and two helical turns stabilized by a complex disulfide bond network that creates an embedded ring around one of the strands. Recifin inhibited full-length TDP1 but not N-terminally truncated TDP1 in biochemical assays. Enzyme kinetics studies revealed that recifin can specifically modulate the enzymatic activity of full-length TDP1 while not affecting a truncated form of TDP1, suggesting an allosteric binding site for recifin on TDP1.