Abstract
Copper transporter 1 (CTR1) represents an important determinant of cisplatin resistance. A series of 35 semi-substituted steroids were recently investigated on cisplatin-resistant CTR1-expressing A2780cis ovarian carcinoma cells as well as their parental sensitive counterparts regarding their cytotoxic and resistance-reversing features. In the present investigation, three compounds (4, 5, 25) were selected for molecular docking analysis on the homology-modelled human CTR1 transmembrane domain. Steroids 4, 5 and 25 interacted with CTR1 at a similar docking pose and with even higher binding affinities than the known CTR1 inhibitor, cimetidine. Applying the defined docking mode, the binding energies were found to be −7.15±<0.001 kcal/mol (compound 4), −8.71±0.06 kcal/mol (compound 5), −7.63±0.01 kcal/mol (compound 25), and −5.05±0.02 kcal/mol (for cimetidine). These steroids have the potential for further development as CTR1 inhibitors overcoming cisplatin resistance.
Abbreviations: ABC, ATP-binding cassette; CTR 1/2, copper transporter 1/2; MDR, multidrug resistance.
Cancer mortality accounts for approximately one eighth of all deaths worldwide. The annual cancer incidence doubled during the past 30 years (1). Around 1.5 million new cases and 0.6 million deaths are projected in the United States for 2015 (2). Carcinogenesis underlies complex mechanisms and single-target approaches are inadequate to fight cancer. Drug resistance caused by ATP-binding cassette (ABC) or copper transporters (3-5) hamper successful cancer chemotherapy (6). ABC-transporters cause multi-drug resistance (MDR) (7-9). Cisplatin resistance is another type mediated by copper transporters (CTRs or SLC31) and P-type transporters, that are localized in the plasma membrane (e.g. CTR1) or intracellular membranes of late endosomes and lysosomes (CTR2 or P-type copper transporters α and β (ATP7A/B)) (10, 11).
Copper is essential for growth and development. However, its cellular accumulation beyond homeostatic limits is toxic (12). CTR1 is the main copper uptake transporter. It consists of 190 amino acids with three transmembrane domains, whose gene is localized on chromosome 9. CTR1 forms oligomeric (mostly homo-trimer) complexes facilitating pore formation for copper uptake (13, 14). The methionine- and histidine-rich amino terminus is important for copper binding (15). CTR1 represents an ubiquitously expressed high-affinity importer of reduced copper (Cu+), which regulates the sensitivity to platinum-containing drugs. The associations between CTR1, immune function, and disorders such as Alzheimer's disease or cancer make this protein an attractive target for therapeutic interventions (16). CTR1 confers cancer drug resistance (11, 17, 18). Decreased platin accumulation is related to up-regulated CTR1 in ovarian, testicle, lung, head and neck carcinoma (11).
The CTR2 gene is localized on chromosome 9 and encodes a 143-amino-acid protein. CTR2 functions as homo-multimer similar to CTR1. Low CTR2 levels increased the cellular uptake and cytotoxicity of cisplatin and carboplatin (19, 20).
Several studies have demonstrated that down-regulation of CTR1 reduced uptake and sensitivity, whereas down-regulation of CTR2 enhanced both (21-24). Cisplatin degraded CTR1 in ovarian cancer cells, whereas it increased CTR2 expression. Copper depletion caused CTR2 disappearance, whereas excessive copper increased it. CTR1 and CTR2 revealed opposite effects (25). CTR1 inhibitors open new strategies in reversing platinum resistance (15).
Structures of steroids 4, 5 and 25.
Herein, we generated a homology-based three-dimensional CTR1 model and performed in silico molecular docking studies to predict docking poses (binding position) and binding energies of the selected steroid compounds that we previously reported to inhibit CTR1 and overcome CTR1-mediated cisplatin resistance (26).
Materials and Methods
Compounds. Semi-substituted steroids and their derivatives in complexes with platinum and copper (I). Previously, we synthesized and investigated 35 modified steroid derivatives (1-35) regarding their CTR1 inhibition and modulation of cisplatin resistance (26-31). The best three compounds (4, 5, 25) were selected for the present investigation (Figure 1).
Molecular docking. Transmembrane domains of human CTR1 were compiled as single-structure models by homology modelling (32). PubChem was referred for the 3-D structure of the control drug, cimetidine (https://pubchem.ncbi.nlm.nih.gov/). Blind and defined molecular docking calculations were performed with AutoDock4, as previously described (33, 34).
Initial blind dockings were performed with cimetidine and compounds 4, 5 and 25. Blind docking calculations were performed by covering the whole protein. The number of energy evaluations was set to 25,000,000 and number of runs 250. Defined docking calculations were performed by covering the residues involved in hydrogen bonds or hydrophobic interactions with each ligand by blind docking. Docking parameters of defined dockings were set to 250 runs and 2,500,000 energy evaluations. Lamarckian Genetic Algorithm was chosen for docking calculations. For visualization, AutodockTools-1.5.7rc1 was used. The surface representation image showing the binding pocket of transmembrane domains of human CTR1 was created with Visual Molecular Dynamics (VMD) software developed by the Theoretical and Computational Biophysics group at the Beckman Institute, University of Illinois at Urbana-Champaign (http://www.ks.uiuc.edu/Research/vmd/).
Results and Discussion
In order to investigate the binding of steroids 4, 5 and 25 to transmembrane domains of human CTR1, we performed in silico molecular docking. We compiled CTR1 transmembrane domains to a single structure by homology modeling. Since cimetidine reduced cisplatin uptake, we chose it as a control drug (35, 36). All three steroids bound to the same pharmacophore as cimetidine with high binding affinities (i.e. low kcal/mol values) in both blind and defined docking approaches (Figure 2 and Tables I and II). Binding affinity was considered high at binding energies around −8 kcal/mol (37).
Molecular docking studies on the homology-modelled CTR1 transmembrane domain (surface and ribbon representation). Docking poses of compound 4, 5, 25 and control drug cimetidine are depicted.
Cimetidine revealed low binding affinities (blind docking: −4.65±0.03 kcal/mol, defined docking: −5.03±0.02 kcal/mol). However, compound 4 (blind docking: −7.10±0.00 kcal/mol, defined docking: −7.15±<0.001 kcal/mol), compound 5 (blind docking: −8.78±0.06 kcal/mol, defined docking: −8.71±0.06 kcal/mol) and compound 25 (blind docking: −7.63±0.01 kcal/mol, defined docking: −7.62±0.01 kcal/mol) bound to CTR1 with even higher affinities than cimetidine (Tables I and II). Cimetidine did not form hydrogen bonds with CTR1, whereas compound 4 did (blind docking: Phe74 and Trp177, defined docking: Trp177). This was also observed for compound 5 (defined docking: Trp177) and compound 25 (blind docking: Val75, defined docking: Val75) (Tables I and II).
In silico blind molecular docking to transmembrane domains of human copper transporter 1 (CTR1). Dockings were performed three times with 250 numbers of runs by covering the whole protein.
In silico defined molecular docking to transmembrane domains of human CTR1. Dockings were performed three times with 250 numbers of runs by covering the residues found by blind docking.
In the present study, three selected semi-substituted steroids (4, 5, 25) previously described by our group (26) were subjected to in silico molecular docking studies. They interacted with CTR1 transmembrane domain with stronger affinity than the known inhibitor, cimetidine (35). The potential of these compounds is highlighted by the fact that the predicted binding energies were even higher than those for cimetidine. Further pre-clinical and clinical studies are warranted to clarify the therapeutic potential of compounds 4, 5 and 25.
On the other hand, the toxicity of platinum compounds has to be also taken into account. Ototoxicity, nephrotoxicity, and hepatotoxicity are serious side-effects of cisplatin therapy, which may be managed by natural compounds (36, 38, 39). Therefore, CTR1 inhibitors such as natural or synthetic ones, as the ones introduced by our group, have to be carefully investigated to ensure their safety.
Footnotes
Conflicts of Interest
The Authors declare that there exist no conflicts of interest.
- Received August 5, 2015.
- Revision received October 9, 2015.
- Accepted October 16, 2015.
- Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
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