Probing the Interaction of Cisplatin with the Human Copper Chaperone Atox1 by Solution and In-Cell NMR Spectroscopy

作者：Arnesano Fabio; Banci Lucia; Bertini Ivano; Felli Isabella C; Losacco Maurizio; Natile Giovanni^*

来源：Journal of the American Chemical Society, 2011, 133(45): 18361-18369.

DOI：10.1021/ja207346p

Among anticancer therapeutics, platinum-based drugs have a prominent role. They carry out their antitumor activity by forming stable adducts with DNA, thus interfering with replication and transcription processes. Cellular uptake of these drugs is tightly connected to copper transport. The major Cu(I) influx transporter Ctrl has been found to mediate transport of cisplatin and its analogues. Evidence also suggests that ATP7A and ATP7B mediate cisplatin sequestration and efflux from cells, thus influencing drug resistance. The copper-chaperone Atox1, which normally binds Cu(I) via two cysteines and delivers the metal to ATP7A/B, has also been reported to interact with cisplatin in in vitro experiments. In the present investigation we apply a combined approach, using solution and in-cell NMR spectroscopy methods, to probe intracellular drug delivery and interaction of cisplatin with Atox1. The intracellular environment provides itself the suitable conditions for the preservation of the protein in its active form. Initially a {Pt(NH3)(2)}-Atox1 adduct is formed. At longer reaction time we observed protein dimerization and loss of the ammines. Such a process is reminiscent of the copper-promoted formation of Atoxl dimers which have been proposed to be able to cross the nuclear membrane and act as a transcription factor. We also show that overexpression of Atoxl in E. coli reduces the amount of DNA platination and, consequently, the degree of cell filamentation.

• 出版日期2011-11-16