Discovery and characterisation of recifin

点击翻译按钮获取中文摘要

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.