Discovery and characterisation of recifin

Recifin 的发现和表征

• 批准号: 10487083
• 负责人: Ingrid Schroeder
• 金额: $ 21.32万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10487083
• 关键词: Axinella; Binding Sites; Biochemical; Biological Assay; Camptothecin; Complex; Cysteine; Enzyme Kinetics; Fractionation; Length; Libraries; Mass Spectrum Analysis; Molecular Target; Names; Natural Products; Pattern; Peptide Sequence Determination; Peptides; Porifera; Source; Structure; Structure-Activity Relationship; Topoisomerase Inhibitors; aqueous; beta pleated sheet; cancer cell; chemical synthesis; disulfide bond; high throughput screening; improved; inhibitor/antagonist; novel; pharmacophore; tyrosyl-DNA phosphodiesterase

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.

酪氨酸-DNA磷酸二酯酶1（TDP 1）是肿瘤细胞对拓扑异构酶抑制剂喜树碱及其衍生物敏感的分子靶点。高通量筛选天然产物提取物库的TDP 1活性抑制剂，导致发现了一种生物活性的水提取物的海绵，Axinella sp.生物测定引导分馏的源提取物导致分离的活性成分，这被确定为一种新的，42个残基的富含半胱氨酸的肽，我们命名为recifin。recifin的一级序列和二硫键模式，确定使用Edman降解和串联质谱从头蛋白质测序的组合。NMR结构揭示了一种新的折叠，包括一个四链反平行β-折叠和两个由复杂的二硫键网络稳定的螺旋转角，该网络在其中一条链周围产生嵌入的环。Recifin抑制全长TDP 1，但不N-末端截短的TDP 1在生化测定。酶动力学研究表明，recifin可以特异性地调节全长TDP 1的酶活性，而不影响TDP 1的截短形式，这表明recifin在TDP 1上的变构结合位点。