Recifin A, Initial Example of the Tyr-Lock Peptide Structural Family, Is a Selective Allosteric Inhibitor of Tyrosyl-DNA Phosphodiesterase 1

摘要

Tyrosyl-DNA phosphodiesterase 1 (TDP1) is a molecular target for the sensitization of cancer cells to the FDA-approved topoisomerase inhibitors topotecan and irinotecan. High-throughput screening of natural product extract and fraction libraries for inhibitors of TDPl activity resulted in the discovery of a new class of knotted cyclic peptides from 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 an unprecedented 42-residue cysteine-rich peptide named recifin A. The native NMR structure revealed a novel fold comprising a four strand antiparallel β-sheet and two helical turns stabilized by a complex disulfide bond network that creates an embedded ring around one of the strands. The resulting structure, which we have termed the Tyr-lock peptide family, is stabilized by a tyrosine residue locked into three-dimensional space. Recifin A inhibited the cleavage of phosphodiester bonds by TDPl in a FRET assay with an IC_(50) of 190 nM. Enzyme kinetics studies revealed that recifin A can specifically modulate the enzymatic activity of full-length TDPl while not affecting the activity of a truncated catalytic domain of TDPl lacking the N-terminal regulatory domain (Δ 1-147), suggesting an allosteric binding site for recifin A on the regulatory domain of TDPl. Recifin A represents both the first of a unique structural class of knotted disulnde-rich peptides and defines a previously unseen mechanism of TDPl inhibition that could be productively exploited for potential anticancer applications.