Cisplatin handover between copper transporters: the effect of reducing agents

Abstract

Copper (Cu) transporters emerged as key factors at the basis of the biological response to antitumor platinum (Pt) drugs, which are among the most potent and broadly used chemotherapeutics. ATP7A and ATP7B (the Menkes and Wilson disease proteins, respectively) appear to be implicated in promoting tumor cell resistance to cisplatin. Cu-ATPases could bind the drug and, with the alleged involvement of the chaperone ATOX1, contribute to cell detoxification and survival. Here, we report the spectroscopic characterization of cisplatin binding to ATOX1 and MNK1, the first metal-binding domain of ATP7A, in the presence of the physiological reducing agent glutathione, a sulfur-containing molecule responsible for the majority of Pt detoxification in the cytosol. Under conditions mimicking the cellular environment, we show that cisplatin transfer from ATOX1 to MNK1 does not occur at a detectable rate. These results appear to contradict other literature data which, however, were obtained in the presence of exogenous reducing agents such as tris(2-carboxyethyl)phosphine (TCEP) having good coordinating ability for soft metal ions (such as Pt) and strong trans-labilizing effect. A better understanding of Pt drug processing by Cu trafficking proteins under physiological conditions may help to answer key issues, such as drug availability in tumor cells and resistance.

Oxaliplatin Binding to Human Copper Chaperone Atox1 and Protein Dimerization

“…12 Characterization of Atox1 Monomer and Dimer by Solution Nuclear Magnetic Resonance Spectroscopy. An equimolar amount of [Pt(R,R-DACH)(H 2 O)(SO 4 )] was added, under strict anaerobic conditions, to 15 N-labeled Atox1 in 25 mM phosphate buffer (pH 7.0), and after 48 h of incubation, SEC was used to separate and purify the monomeric Atox1 adducts, containing one or two {Pt-(DACH) 2+ } moieties, and the dimeric Atox1 2 ·Pt 2+ 2 adduct.…”

Cellular trafficking, accumulation and DNA platination of a series of cisplatin-based dicarboxylato Pt(IV) prodrugs

“…Mnk1 protein was recombinantly expressed by standard biotechnological techniques, using Escherichia coli BL21DE3-Gold cells (Stratagene, USA) and a pET vector under the control of the IPTG (isopropyl-β-D-1-thiogalactopyranoside) inducible lac (lactose) promoter (Novagen, USA). Purification was achieved by combining immobilized metal affinity and size-exclusion chromatography, as previously described [23]. All steps of purification were carried out in the presence of an excess of the reducing agent dithiothreitol (DTT) to preserve the protein in its active form.…”

Mass spectrometry as a powerful tool to study therapeutic metallodrugs speciation mechanisms: Current frontiers and perspectives

“…[190] Natile et al were able to describe in details the different steps for the coordination of cisplatin to Atox1 in physiological media using in-cell NMR spectroscopy, [191] and also combined NMR and ESI-MS to study cisplatin's interaction to the metal-binding domain of ATP7A (MNK1) and/or Atox1 in the absence or presence of GSH. [192] Cisplatin was found to form the expected first step chelate adducts with both Atox1 and MNK1 ([protein+Pt(NH 3 ) 2 ] 2+ ), with no apoproteins detected after 24 h incubation. When the proteins were exposed to a 10-fold excess of glutathione prior incubation with the metallodrug, Atox1 was found unreactive towards cisplatin (with only cisplatin-GSH adducts detected in the mass spectrum), whereas MNK1 was able to compete with GSH for binding to cisplatin.…”

“…When the proteins were exposed to a 10-fold excess of glutathione prior incubation with the metallodrug, Atox1 was found unreactive towards cisplatin (with only cisplatin-GSH adducts detected in the mass spectrum), whereas MNK1 was able to compete with GSH for binding to cisplatin. [192] Interestingly, when Atox1 was used in its holo-form (pre-incubated with copper), despite the presence of GSH, Cu(I)-Atox1 was able to form adducts corresponding to [Cu(I)-Atox1+Pt(NH 3 ) 2 ] 2+ after 3 h. [192] When glutathione was incubated with the pre-formed platinum adducts, no release of platinum from the protein was observed. The presence of TCEP during incubation of Atox1 with cisplatin induced the formation of the adduct [Atox1+Pt(TCEP) 2 ] 2+ , indicating the coordination of two phosphorus atoms from TCEP on platinum, in addition to two cysteine residues from the protein.…”

“…The presence of TCEP during incubation of Atox1 with cisplatin induced the formation of the adduct [Atox1+Pt(TCEP) 2 ] 2+ , indicating the coordination of two phosphorus atoms from TCEP on platinum, in addition to two cysteine residues from the protein. [192] The influence of copper binding on Atox1 with respect to the reactivity of cisplatin has been further investigated by Liu et al using 2D NMR spectroscopy and ESI-MS. [193] Copper coordination to Atox1 promoted significantly the binding of platinum to the protein and prevented metal release upon incubation with reducing agents such as DTT. [193] The nature of the platination sites on both apo-and holo-Atox1 was investigated using a bottom-up approach.…”