& Computer Chemistry Structural Biology of Cisplatin Complexes with Cellular Targets: The Adduct with Human Copper Chaperone Atox1 in Aqueous Solution Vania Calandrini, [a, b] Trung Hai Nguyen, [a, b] Fabio Arnesano, [c] Angela Galliani, [c] Emiliano Ippoliti, [a, b] Paolo Carloni,* [a, b] and Giovanni Natile [c] Abstract: Cisplatin is one of the most used anticancer drugs. Its cellular influx and delivery to target DNA may involve the copper chaperone Atox1 protein. Although the mode of binding is established by NMR spectroscopy measurements in solution—the Pt atom binds to Cys12 and Cys15 while re- taining the two ammine groups—the structural determi- nants of the adduct are not known. Here a structural model by hybrid Car–Parrinello density functional theory-based QM/MM simulations is provided. The platinated site minimal- ly modifies the fold of the protein. The calculated NMR and CD spectral properties are fully consistent with the experi- mental data. Our in silico/in vitro approach provides, togeth- er with previous studies, an unprecedented view into the structural biology of cisplatin–protein adducts. Introduction Cisplatin (cis-diamminedichloridoplatinum(II)) is one of the most widely used drugs in anticancer chemotherapy. [1–6] Its an- titumor activity is due to the formation of stable adducts with DNA. [7, 8] Unfortunately, after repeated administrations of the drug, cancer cells develop resistance mechanisms, which strongly limit cisplatin efficacy. [1, 3, 4] The decreased accumula- tion of cisplatin by a decreased uptake and by an increased drug efflux and sequestration is among the proposed mecha- nisms leading to resistance. [3, 4, 9] Cisplatin may enter the cell via human copper transport proteins, [4, 10–14] including the high-af- finity copper transporter Ctr1; [15] while the ATPase copper pumps ATP7A/ATP7B could be responsible for sequestration and efflux of cisplatin; [16, 17] finally, the copper chaperone Atox1 could deliver platinum (Pt) to the ATPases [18, 19] and also be in- volved in the influx of cisplatin by controlling the cisplatin-in- duced down regulation of Ctr1 through ubiquitination. [10, 20] Moreover, Atox1 has been found to translocate to the nucleus in response to copper exposure, [21] which raises the question of whether it could also be involved in the delivery of cisplatin to DNA. Designing new Pt-based compounds able to reduce drug re- sistance problems related to their access to tumor cells re- quires an understanding of the Pt coordination chemistry to the proteins involved in resistance. Recently, using a combina- tion of QM/MM and computational spectroscopy approaches, along with experimental data, we have characterized the bind- ing modes of cisplatin to one of the extracellular methionine- rich motifs of yeast Ctr1 (Met7) in aqueous solution. [22] This study suggested that the platinated peptide [PtX] + -(M*TGM*KGM*S), with X = Cl À or OH À , is the most relevant species occurring in solution. An investigation of cisplatin bind- ing to the first N-terminal copper binding motif of the human ATP7A in aqueous solution (Mnk1) led to the conclusion that, differently from Met7, the Pt substrate keeps the two cis am- mines and coordinates to the sulfur atoms of Cys19 and Cys22 of the highly conserved CxxC sequence. [23, 24] So far, cisplatin binding to Atox1 in water solution has been inferred from electrospray ionization mass spectrometry (ESI MS) and nuclear magnetic resonance (NMR) spectroscopy. [25, 26] These data show that, similarly to what happens in the reaction with the struc- turally similar Mnk1, in solution the [Pt(NH 3 ) 2 ] 2 + moiety binds to Cys12 and Cy15 residues of Atox1 keeping the two am- mines and that the adduct is monomeric [25, 26] (Scheme 1). Note that this binding mode is different from the one evidenced in crystallographic structures, [27] where 1:1 and 1:2 complexes be- tween human Atox1 and cisplatin have been observed with different coordination to that in solution. [27] In two recent re- ports it has been unambiguously shown that Cu I promotes the [a] Dr. V. Calandrini, Dr. T.H. Nguyen, Dr. E. Ippoliti, Prof. Dr. P. Carloni Computational Biophysics German Research School for Simulation Sciences 52425 Jülich (Germany) and Computational Biomedicine Institute for Advanced Simulation IAS-5 Forschungszentrum Jülich, 52425 (Germany) [b] Dr. V. Calandrini, Dr. T.H. Nguyen, Dr. E. Ippoliti, Prof. Dr. P. Carloni Computational Biomedicine, Institute of Neuroscience and Medicine INM-9 Forschungszentrum Jülich, 52425 Jülich (Germany) E-mail : p.carloni@grs-sim.de [c] Dr. F. Arnesano, Dr. A. Galliani, Prof. G. Natile Department of Chemistry, University of Bari “A. Moro” via Edoardo Orabona 4, 70125 Bari (Italy) Supporting information for this article is available on the WWW under http ://dx.doi.org/10.1002/chem.201402834. Chem. Eur. J. 2014, 20, 11719 – 11725 # 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 11719 Full Paper DOI: 10.1002/chem.201402834