Structural Biology of Cisplatin Complexes with Cellular Targets: The Adduct with Human Copper Chaperone Atox1 in Aqueous Solution (original) (raw)
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Journal of Chemical Theory and Computation, 2012
The cellular uptake of cisplatin and of other platinum-based drugs is mediated by the high-affinity copper transporter Ctr1. The eight-residue long peptide called Mets7 (MTGMKGMS) mimics one of extracellular methionine (Met)rich motifs of Ctr1. It is an excellent model for investigating the interaction of platinum drugs with Ctr1 under in vitro and in vivo conditions. Some of us have shown that (i) Cisplatin loses all of its ligands upon reaction with Mets7 and the metal ion binds to the three Met residues and completes its coordination shell with a fourth ligand that can be a chloride or a water/hydroxyl oxygen. (ii) Transplatin loses only the chlorido ligands, which are replaced by Met residues. Here, we provide information on the structural determinants of cisplatin/Mets7 and transplatin/Mets7 adducts by computational methods. The predictions are validated against EXAFS, NMR, and CD spectra. While EXAFS gives information restricted to the metal coordination shell, NMR provides information extended to residue atoms around the coordination shell, and finally, CD provides information about the overall conformation of the peptide. This allows us to elucidate the different reaction modes of cisplatin and transplatin toward the peptide, as well as to propose the platinated peptides [PtX] + −(M*TGM*KGM*S) (X = Cl − , OH − ) and trans[Pt(NH 3 ) 2 ] 2+ −(M*TGM*KGMS) as the most relevant species occurring in water solution.
Journal of the American Chemical Society, 2011
is an essential element in living organisms and is required as a cofactor of a variety of enzymes that regulate key biological processes, such as cellular respiration and oxidative stress defense. 1,2 At the same time, altered states of its intracellular concentrations are extremely dangerous. Accumulating evidence also suggests its fundamental role in the regulation of cell growth, in important processes such as angiogenesis, and in several pathologies including cancer and neurodegenerative diseases. 3À8 Thus, a fine balance between Cu demand and supply is crucial for survival and is tightly controlled by several transport proteins. 9,10 The soluble cytosolic Cu chaperone Atox1 (antioxidant-1, 68 amino acids), previously known as Hah1 (human atx1 homologue 1), mediates Cu(I) delivery to the Cu-transporting P-type ATPases ATP7A and ATP7B (the Menkes and Wilson disease proteins, respectively), which are responsible for Cu release to the secretory pathway of the trans-Golgi network. 11À13 Recently, a novel role for Atox1 as a nuclear Cu-dependent transcription factor has been revealed, demonstrating its implication both in activation of cell proliferation (positive expression of cyclin D1) and moderation of oxidative stress in the cardiovascular system (positive expression of extracellular superoxide dismutase SOD3). 14,15 Atox1, which is ubiquitously expressed, has a β 1 α 1 β 2 β 3 α 2 β 4 ferredoxin-like structure. The metal-binding site, CxxC, is located in the loop between ' REFERENCES
J Mol Biol, 2001
A 5 ns unrestrained molecular dynamics (MD) simulation of the DNA duplex d(GCCG*G*ATCGC)-d(GCGATCCGGC), bearing a cis-Pt(NH 3) 2 2 unit crosslinking the two G* guanine bases, is reported. The MD trajectory was a posteriori correlated with NMR data determined for the same adduct, and it is shown that interproton distances and the characteristic chemical shifts are accounted for by the simulation. The simulation and its confrontation with the NMR data have con®rmed the ®nding derived early from static models that the cytosine complementary to the 5 H G*, C17, is mobile with respect to its adjacent bases. However, in contrast to our previous description of this mobility, which included rupture of the Watson-Crick hydrogen bonds and formation of non-Watson-Crick hydrogen bonds, the MD simulation indicated that the G*4-C17 pair moves continuously along a trajectory roughly perpendicular to the local helix axis, with retention of all three Watson-Crick hydrogen bonds. The simulation indicated the reversible formation of a hydrogen bond between the 5 H oriented NH 3 ligand of platinum and the C3pG*4 phosphate group, in accord with our former prediction. Furthermore, the simulation has disclosed previously undetected BI BII transitions at the G*5pA6 and A6pT7 steps, connected to formation/rupture of a hydrogen bond between the 3 H oriented NH 3 ligand of platinum and the N7 atom of A6. All these conformational equilibria affect the form of the minor groove and increase the conformational¯exibility at the platination site, and are thus likely to facilitate recognition by cellular proteins.
Journal of Molecular Biology, 2001
A 5 ns unrestrained molecular dynamics (MD) simulation of the DNA duplex d(GCCG*G*ATCGC)-d(GCGATCCGGC), bearing a cis-Pt(NH 3) 2 2 unit crosslinking the two G* guanine bases, is reported. The MD trajectory was a posteriori correlated with NMR data determined for the same adduct, and it is shown that interproton distances and the characteristic chemical shifts are accounted for by the simulation. The simulation and its confrontation with the NMR data have con®rmed the ®nding derived early from static models that the cytosine complementary to the 5 H G*, C17, is mobile with respect to its adjacent bases. However, in contrast to our previous description of this mobility, which included rupture of the Watson-Crick hydrogen bonds and formation of non-Watson-Crick hydrogen bonds, the MD simulation indicated that the G*4-C17 pair moves continuously along a trajectory roughly perpendicular to the local helix axis, with retention of all three Watson-Crick hydrogen bonds. The simulation indicated the reversible formation of a hydrogen bond between the 5 H oriented NH 3 ligand of platinum and the C3pG*4 phosphate group, in accord with our former prediction. Furthermore, the simulation has disclosed previously undetected BI BII transitions at the G*5pA6 and A6pT7 steps, connected to formation/rupture of a hydrogen bond between the 3 H oriented NH 3 ligand of platinum and the N7 atom of A6. All these conformational equilibria affect the form of the minor groove and increase the conformational¯exibility at the platination site, and are thus likely to facilitate recognition by cellular proteins.
Experimental and Computational Investigations of Carboplatin Supramolecular Complexes
ACS omega, 2020
Supramolecular systems (macromolecules), such as calix[n]arenes (SCn), cyclodextrins (CDs), and cucurbiturils (CBs), are promising vehicles for anticancer drugs. In this work, guest−host complexes of carboplatin, a second-generation platinum-based anticancer drug, and p-4-sulfocalix[n]arenes (n = 4 and 6; PS4 and PS6, respectively) were prepared and studied using 1 H NMR, UV, Job's plot analysis, HPLC, and densityfunctional theory calculations. The experimental and the computational studies suggest the formation of 1:1 complexes between carboplatin and each of PS4 and PS6. The stability constants of the formed complexes were estimated to be 5.3 × 10 4 M −1 and 9.8 × 10 4 M −1 , which correspond to free energy of complexation of −6.40 and −6.81 kcal mol −1 , in the case of PS4 and PS6, respectively. The interaction free energy depends on the different inclusion modes of carboplatin in the host cavities. UV−vis findings and atoms in molecules analysis showed that hydrogen bond interactions stabilize the host−guest complexes without the full inclusion in the host cavity. The in vitro anticancer study revealed that both complexes exhibited stronger anticancer activities against breast adenocarcinoma cells (MCF-7) and lung cancer cells (A-549) compared to free carboplatin, preluding to their potential use in cancer therapy.
Cisplatin binds human copper chaperone Atox1 and promotes unfolding in vitro
Proceedings of the National Academy of Sciences, 2011
Cisplatin (cisPt), PtðNH 3 Þ 2 Cl 2 , is a cancer drug believed to kill cells via DNA binding and damage. Recent work has implied that the cellular copper (Cu) transport machinery may be involved in cisPt cell export and drug resistance. Normally, the Cu chaperone Atox1 binds Cu(I) via two cysteines and delivers the metal to metal-binding domains of ATP7B; the ATP7B domains then transfer the metal to the Golgi lumen for loading on cuproenzymes. Here, we use spectroscopic methods to test if cisPt interacts with purified Atox1 in solution in vitro. We find that cisPt binds to Atox1's metal-binding site regardless of the presence of Cu or not: When Cu is bound to Atox1, the near-UV circular dichroism signals indicate Cu-Pt interactions. From NMR data, it is evident that cisPt binds to the folded protein. CisPt-bound Atox1 is however not stable over time and the protein begins to unfold and aggregate. The reaction rates are limited by slow cisPt dechlorination. CisPt-induced unfolding of Atox1 is specific because this effect was not observed for two unrelated proteins that also bind cisPt. Our study demonstrates that Atox1 is a candidate for cisPt drug resistance: By binding to Atox1 in the cytoplasm, cisPt transport to DNA may be blocked. In agreement with this model, cell line studies demonstrate a correlation between Atox1 expression levels, and cisplatin resistance.
Journal of Physical Chemistry B, 2006
Dinuclear Pt-containing compounds might be used to overcome the intrinsic and acquired cell resistance of widely used anticancer drugs such as cisplatin. Recently, the complexes [{cis-Pt(NH 3 ) 2 } 2 (µ-OH)(µ-pz)](NO 3 ) 2 (with pz ) pyrazolate) (1), [{cis-Pt(NH 3 ) 2 } 2 (µ-OH)(µ-1,2,3-ta-N(1),N(2))](NO 3 ) 2 (with ta ) 1,2,3-triazolate) (2), and the binding of 1 to d(CpTpCpTpG*pG*pTpCpTpCp) have been characterized. Here we provide the structural and electronic properties of the free drugs, of the intermediates of binding to guanine bases, and of the products, by performing DFT calculations. Our results show that in 2 an isomerization of the Pt-coordination sphere from N(2) to N(3) of the triazolate unit determines a thermodynamic stabilization of ∼20 kcal/mol as a consequence of the formation of an allylic structure. In addition, hybrid quantum-classical molecular dynamics simulations of 1 and 2 DNA adducts have shed light on the structural distortions that the drugs induce to the DNA duplex. Our calculations show that the rise and the tilt of the two adjacent guanines are identical in the presence of 1 and 2, but they markedly increase when 2 binds in the N(1),N(3) fashion. In addition, the drugs do not provoke any kink upon binding to the double-stranded DNA, suggesting that they may act with a mechanism different than that of cisplatin. The accuracy of our calculations is established by a comparison with the NMR data for the corresponding complex with 1. † Part of the special issue "Michael L. Klein Festschrift".
Journal of Inorganic Biochemistry, 2015
Cisplatin, cis-diammineplatinum(II) dichloride, is a metal complex used in clinical practice for the treatment of cancer. Despite its great efficacy, it causes adverse reactions and most patients develop a resistance to cisplatin. To overcome these issues, a multi-drug therapy was introduced as a modern approach to exploit the drug synergy. A synergistic effect had been previously found when testing binary combinations of cisplatin and three copper complexes in vitro, namely, Cu(phen)(OH 2 ) 2 (OClO 3 ) 2 , [Cu(phen) 2 (OH 2 )](ClO 4 ) 2 and [Cu(phen) 2 (H 2 dit)](ClO 4 ) 2 ,(phen = 1,10phenanthroline, H 2 dit = imidazolidine-2-thione), against the human acute T-lymphoblastic leukaemia cell line (CCRF-CEM). In this work [Cu(phen) 2 (OH 2 )](ClO 4 ) 2 was also tested in combination with cisplatin against cisplatin-resistant sublines of CCRF-CEM (CCRF-CEM-res) and ovarian (A2780-res) cancer cell lines. The tested combinations shown a synergistic effect against both the types of resistant cells. The possibility that this effect was caused by the formation of new adducts was considered and mass spectra of solutions containing cisplatin and one of the three copper complexes at a time were measured using electrospray ionisation at atmospheric-pressure mass spectroscopy (ESI-MS). A mixed complex was detected and its stoichiometry was assessed on the basis of the isotopic pattern and the results of tandem mass spectrometry experiments. The formed complex was found to be [Cu(phen)(OH)µ-(Cl) 2 Pt(NH 3 )(H 2 O)] + .