Activation of Platinum(IV) Prodrugs by Cytochrome c and

Platinum(IV) complexes generally require reduction to reactive Pt(II) species to exert their chemotherapeutic activity. The process of reductive activ...
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Activation of Platinum(IV) Prodrugs by Cytochrome c and Characterization of the Protein Binding Sites Alessia Lasorsa,† Olga Stuchlíková,‡,§ Viktor Brabec,‡ Giovanni Natile,† and Fabio Arnesano*,† †

Department of Chemistry, University of Bari “A. Moro”, via E. Orabona, 4, 70125 Bari, Italy Institute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i, Královopolská 135, CZ-61265 Brno, Czech Republic § Department of Biophysics, Faculty of Science, Palacky University, 17. listopadu 12, CZ-77146 Olomouc, Czech Republic ‡

S Supporting Information *

ABSTRACT: Platinum(IV) complexes generally require reduction to reactive Pt(II) species to exert their chemotherapeutic activity. The process of reductive activation of 15Nlabeled (OC-6−43)-bis(acetato)diamminedichloridoplatinum(IV), in the presence of nicotinamide adenine dinucleotide (NADH) and horse heart cytochrome c (cyt c), was monitored by 1H,15N-HSQC NMR spectroscopy and protein digestion experiments. It has been shown that cyt c plays a catalytic role in the transfer of two reducing equivalents from NADH to Pt(IV) species. Noncovalent interactions between reduced monoaqua cisplatin (cis-[PtCl(15NH3)2(H2O)]+) and the protein, in the proximity of the heme cofactor, and also covalent binding of platinum to the protein region around Met65 and Met80 take place. KEYWORDS: Pt(IV) complexes, anticancer prodrugs, cytochrome c, reductive activation

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INTRODUCTION In order to address issues like the onset of toxic side effects or drug resistance (either de novo or acquired),1,2 anticancer Pt(IV) prodrugs have been developed. The expansion of the coordination sphere with respect to Pt(II) drugs has been exploited to optimize the drug-uptake or to add a second pharmacological moiety so as to increase the efficacy of the platinum-based therapy. The platinum(IV) complexes cis-[Pt(NH3)2Cl4], trans-[Pt(NH3)2Cl4], and [Pt(en)Cl4] were already investigated by Rosenberg along with cisplatin, but they were subsequently abandoned since they were less effective than cisplatin in vivo.3,4 To date, the most successful example of Pt(IV) prodrug is represented by satraplatin, a Pt(IV)-based antineoplastic agent differing from cisplatin for having a cyclohexylamine in place of an ammine ligand and two acetato ligands in axial positions (Figure 1).5 The use of satraplatin as an alternative to cisplatin is particularly attractive because of the possible oral administration, the milder toxicity profile, the lack of crossresistance with cisplatin, the potential use as radiosensitizer, and the activity in cancers nonresponsive to other platinum drugs. Presently, satraplatin is under investigation for the treatment of patients with advanced prostate cancer.6−8 A general assumption is that Pt(IV) complexes require reduction to reactive Pt(II) species to exert their chemotherapeutic activity,9,9,10 which usually implies binding to DNA and induction of cell death by apoptosis.11,12 The reduction of © XXXX American Chemical Society

Figure 1. Structures of satraplatin and of 15N-labeled (OC-6−33)bis(acetato)diamminedichloridoplatinum(IV) used in this investigation.

satraplatin results in the loss of its two axial acetato ligands and formation of a Pt(II) complex, known as JM118, able to react with DNA at the same sites as cisplatin.13 Compared to cisplatin−DNA adducts, the lesions formed by JM118 are more efficient at inhibiting translesion DNA synthesis14 and are less easily recognized by DNA-mismatch repair15 or high mobility group proteins.16 Other results have shown decreased DNA bending, higher DNA−protein cross-linking efficiency, and less Received: May 17, 2016 Revised: July 20, 2016 Accepted: July 29, 2016

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DOI: 10.1021/acs.molpharmaceut.6b00438 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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NADH and incubated overnight at 37 °C in 25 mM phosphate buffer (pH 5.8). Then the reductant was removed under nitrogen atmosphere by using Amicon Ultra centrifugal filters with 3 kDa cutoff (Millipore, US), and the reduced state of the protein was checked by means of UV−vis absorption spectroscopy in the range between 400 and 600 nm. High Performance Liquid Chromatography−Mass Spectrometry (HPLC−MS). The oxidized and reduced forms of cyt c (700 μM) were treated with cisplatin (1:1 molar ratio) and incubated at 37 °C in 25 mM phosphate buffer (pH 5.8). At different reaction times, aliquots (50 μL) were taken out, diluted 2.5-fold, subjected to HPLC separation by using a Zorbax Eclipse Plus-C8 column (4.6 × 250 mm, 100 Å, 5 μm, Agilent), and directly analyzed in positive-ion mode on a coupled Q-TOF mass spectrometer. Mobile phases: A (H2O containing 1% formic acid) and B (CH3CN containing 1% formic acid). The separation of Pt-cyt c adducts was achieved using a 30 min linear gradient from 2% to 98% of B at a rate of 0.5 mL min−1. The eluents were directly infused into the mass spectrometer through the ESI probe. The spray voltage of the mass spectrometer was +3.5 kV, and the pressure of the nebulizer gas was 35 psig. The drying gas was heated to 300 °C at a flow rate of 8 L/min. Full mass spectra were recorded in the mass/charge (m/z) range of 100−7000. In order to perform HPLC−MS analysis of the tryptic digest, a 10-fold excess of cisplatin was allowed to react with cyt c at 37 °C, in 25 mM phosphate buffer (pH 5.8). After 72 h incubation, unbound cisplatin was removed by using Amicon Ultra centrifugal filters with 3 kDa cutoff, and the protein was subjected to trypsin digestion in 40 mM ammonium bicarbonate (pH 8.5). The lyophilized enzyme was dissolved in the same buffer at a concentration of 1 mg/mL immediately prior to use, then a 1:40 (w/w) ratio of enzyme to substrate was used. The incubation time was 12 h keeping the temperature constant at 37 °C. The separation was achieved by using the Zorbax Eclipse Plus-C18 column (4.6 × 250 mm, 100 Å, 5 μm, Agilent) with a 30 min linear gradient from 2% to 70% of B at a rate of 0.5 mL min−1. Nuclear Magnetic Resonance (NMR) Spectroscopy. The protein was treated with 15N-labeled Pt(IV) complex or cisplatin (700 μM concentration, 1:1 ratio), in 25 mM phosphate buffer (pH 5.8), keeping the temperature constant at 37 °C. NMR samples also contained 10% v/v D2O for NMR spectrometer lock. HSQC spectra were acquired using a gradient-enhanced sequence in which coherence selection and water suppression were achieved via gradient pulses. Sixteen transients were acquired over an F2 (1H) spectral width of 14 ppm into 1024 complex data points for each of 256 t1 increments with an F1 spectral width of 40 ppm (centered at 120 ppm for amide 15N), 100 ppm (centered at −50 ppm for 15 NH3), or 70 ppm (centered at 40 ppm for 13C). The sequence was optimized with a delay 1/(4JXH) of 2.78 ms (X = 15 N) or 1.72 ms (X = 13C), and a recycle delay of 1.0 s. All spectra were collected using an Avance 700 MHz UltraShield Plus magnet (Bruker, Germany), equipped with a cryo probe, processed using the standard Bruker software (TOPSPIN), and analyzed with the programs CARA, developed at ETH-Zurich (www.cara.nmr.ch). Resonance assignment of cyt c was carried out by using the available 1H and 13C chemical shifts.32,33

efficient removal of JM118 from DNA adducts.17 In addition, the organic cation transporters appear to play a more important role in the mechanism of cytotoxicity of JM118 than in the cytotoxicity of cisplatin.18 However, to date the mechanisms responsible for the conversion of satraplatin to JM118 have not been fully elucidated, and there are many holes in the knowledge of the biological reducing agents involved in the activation of satraplatin in vivo or where, within the human body, it is converted to JM118 or to other active species. Most evidence indicates that Pt(IV) complexes are rapidly reduced under physiological conditions by biologically relevant reducing agents, such as glutathione (GSH) and ascorbic acid, to release the two axial ligands and yield the cytotoxic Pt(II) species.19−22 However, the reduction of satraplatin in extracts from three different cancer cell lines is not performed by low molecular weight (MW) antioxidants, but primarily by cellular components having MW > 3000.23 In a seminal work of Carr et al., the heme proteins hemoglobin and horse heart cytochrome c (hereafter cyt c), in the presence of electron donor nicotinamide adenine dinucleotide (NADH), were shown to be competent to reduce satraplatin, and it was suggested that complexation precedes transfer of electrons from the protein to the Pt(IV) core.8 In addition to functioning as an electron carrier, the protein cyt c promotes the assembly of a caspaseactivating complex to induce cell apoptosis.24 For a better understanding of the redox mechanism, we selected cyt c as test protein25 and an analogous of satraplatin, 15 N-labeled (OC-6−33)-bis(acetato)diamminedichloridoplatinum(IV) (Figure 1), which allowed to use 1H,15N-HSQC NMR spectroscopy for monitoring the reaction course.26 NADH was used as external source of electrons. Additionally, we investigated the interaction between cisplatin and cyt c by performing HPLC−ESI−MS analysis on both the entire protein and the products of trypsin digestion. Presently, the interaction of anticancer metallodrugs with proteins is attracting a considerable interest since it can have relevant pharmacological and toxicological consequences, and a better understanding of such an interaction at the molecular level can inspire the design of new metallodrugs. Cyt c has already been used as a model protein in some platinum− protein interaction studies, suggesting that platinum binding predominantly occurs to methionine 65.27−30 We were also interested to see if this is the only site for platinum binding to cyt c.

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EXPERIMENTAL SECTION Materials. 15N-Labeled cisplatin (cis-diamminedichloridoplatinum(II)) (cis-[PtCl2(15NH3)2]) and 15N-labeled (OC-6− 33)-bis(acetato)diamminedichloridoplatinum(IV) (cis,trans,cis[PtCl2(CH3COO)2(15NH3)2]) were prepared by literature methods.31 Nicotinamide adenine dinucleotide, horse heart cytochrome c, and trypsin from porcine pancreas (proteomics grade) were purchased from the Sigma Chemical Company (St Louis, MO, USA). Preparation of Incubation Solutions. 15N-Labeled cisplatin and 15N-labeled Pt(IV) complex were dissolved immediately prior to use in pure deoxygenated water at 2 mM final concentration. Both Pt-complex solutions were extensively vortexed and sonicated, and the exact metal concentration was determined by using a Varian 880Z atomic absorption spectrometer. Reduction of Oxidized Cyt c. The oxidized protein (2 mM final concentration) was treated with a 10-fold excess of B

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Figure 2. Percentages of Pt(IV) complex (a) and cisplatin ([PtCl2(NH3)2]) (b) as a function of incubation time, obtained from integration of a series of 1H,15N-HSQC NMR spectra, in the absence of cyt c and in the presence of different equivalents of cyt c with respect to starting Pt(IV) complex (700 μM).

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RESULTS AND DISCUSSION Pt(IV)-based prodrugs require activation by reduction to Pt(II) compounds. In the literature there are only few studies addressing the role of redox proteins, while most data concern the reduction by GSH or ascorbic acid. In the present study we have investigated the reduction of a Pt(IV) complex, a cisplatin prodrug, mediated by cyt c, an electron transfer protein, under physiologically relevant conditions. Our measurements are aimed at both the chemical and molecular characterization of the metallodrug and the protein for a deeper understanding of the complex biological reduction process. Catalytic Reduction of Pt(IV) Complex by Cyt c in the Presence of NADH. As a first check, it was verified that NADH alone is unable to promote reduction of Pt(IV). Thus, the 15N-labeled Pt(IV) complex (700 μM) was incubated at 37 °C with a 10-fold excess of NADH (7 mM) in the absence of cyt c, and the reaction monitored by 1H,15N-HSQC NMR spectroscopy (0 equiv, Figure 2). The NMR spectra, acquired from 2 to 16 h of incubation, displayed a major peak corresponding to the Pt(IV) complex and a tiny peak corresponding to cisplatin, both having ammines trans to chlorido ligands, as confirmed by 15NH3 chemical shifts (δ 15N ≈ −37 and −64 ppm, respectively). Therefore, NADH alone seems to be unable to promote Pt(IV) reduction in reasonable time and yield (the cross-peak corresponding to the Pt(IV) complex was by far the major one up to the end of the incubation). In a second experiment, an equimolar amount of cyt c (700 μM) was added to the Pt(IV) complex (1 equiv, Figure 2) in the presence of a 10-fold excess of NADH. The same two peaks were detected as in the previous experiment; however, after 16 h of incubation, the intensity of the cross-peak corresponding to the Pt(IV) complex was less than 10% of the initial one, while the peak of cisplatin remained rather small. In a third experiment, only a catalytic amount of cyt c was used (70 μM), corresponding to 10% of the starting Pt(IV) complex concentration (0.1 equiv, Figure 2), always operating in the presence of 10-fold excess of NADH. The cross-peak relative to the Pt(IV) complex completely disappeared after 16 h of incubation, while there was an intense cross-peak corresponding to cisplatin. In order to better highlight the role of cyt c as catalyst, a fourth experiment was carried out in which the concentration of cyt c (7 μM) was only 1% of that of the Pt(IV) complex (0.01 equiv, Figure 2). Also in this case it was possible to

observe the almost complete disappearance of the cross-peak corresponding to the Pt(IV) complex and a strong cross-peak relative to cisplatin. All the NMR spectra obtained after 2 and 16 h of incubation are reported in Figure SI1 of the Supporting Information. Finally, it was checked whether a stoichiometric amount of reduced cyt c (1.4 mM) can be effective in reducing Pt(IV) (700 μM) to Pt(II). In this case, no decrease of the cross-peak relative to the Pt(IV) complex was detected even after 24 h of incubation, indicating that the presence of an external source of electrons, like NADH, is crucial for the cyt c-catalyzed reduction of Pt(IV) (data not shown). Noncovalent Interaction between Platinum Substrate and Cyt c. By comparing the data of the curves at 1 and 0.1 equiv of cyt c, we noticed that, although the decrease in intensity of the peak of the Pt(IV) complex is identical after 2 h incubation, the intensity of the peak of cisplatin is far greater in the case of 0.1 equiv of cyt c than in the case of equimolar amount of cyt c and platinum substrates. It is reasonable to admit that the greater concentration of cyt c in the latter case is responsible for sequestering cisplatin from the reaction mixture. Moreover, the extent of reduction of Pt(IV) to Pt(II) appears to be complete after 5 h incubation in the case of lower cyt c concentration, while in the case of higher cyt c concentration some Pt(IV) (ca. 20%) is still present after 5 h incubation. The latter result can be accounted for by assuming that, like cisplatin, also the Pt(IV) substrate can be adsorbed on the protein surface becoming more resistant to reduction. The possible interaction between cisplatin and cyt c was confirmed by looking at the cross-peaks of ammine ligands in Pt(II) species. The peaks typical of dichloro and monoaqua-monochloro cisplatin are reported in blue in Figure 3. In the case of reaction between the Pt(IV) complex and equimolar cyt c (700 μM each) in excess NADH (7 mM), after 4 h incubation there was a cross-peak (δ 15N = −82.5 ppm and δ 1H = 3.97 ppm, 15NH3 trans to H2O) that can be associated with monoaqua cisplatin (cis-[PtCl(15NH3)2(H2O)]+) but resonating at slightly different values of chemical shift (red cross-peaks in Figure 3). The observed variations in chemical shift can be taken as evidence for the occurrence of a noncovalent interaction between cyt c and aquated cisplatin. Such a shift of the cross-peak of the Pt(II) solvato species is not observed in the case of a catalytic amount of cyt c. C

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Figure 3. Overlay of 1H,15N-HSQC spectra of solutions (4 h incubation at 37 °C in 25 mM phosphate buffer, pH 5.8) containing 15 N-labeled cisplatin 700 μM (blue contours) or 15N-labeled Pt(IV) complex (700 μM) and NADH (7 mM) in the presence of cyt c 700 μM (red contours).

Figure 4. Overlay of 1H,15N-HSQC spectra of solutions (4 h incubation at 37 °C in 25 mM phosphate buffer, pH 5.8) containing 15 N-labeled cisplatin 700 μM in the absence of cyt c (blue contours) or in the presence of equimolar amounts of oxidized (red contours) or reduced (green contours) cyt c.

Hyperfine Coupling between Oxidized Cyt c and Cisplatin. An equimolar amount of 15N-labeled cisplatin was added to the protein, either in the oxidized or in the reduced state, and the solution monitored by 1H,15N-HSQC NMR spectroscopy. The reduced form of cyt c was obtained by treating the oxidized protein with a 10-fold excess of NADH. The reaction was monitored by UV−vis absorption spectroscopy in the region ranging from 400 to 600 nm. After overnight incubation, the absorption bands were those typical of the reduced form (data not shown). In particular, it was possible to appreciate a red shift of the Soret band at 420 nm and the appearance of two maxima at 520 and 550 nm, corresponding to the β and α bands, respectively. The oxidation state of the protein was also checked by 1H NMR, focusing on the region of the hyperfineshifted signals ranging from 10 to 40 ppm (Figure SI2, Supporting Information). In reduced cyt c residual paramagnetic signals were not detectable, indicating that no significant oxidation took place while removing NADH under anaerobic conditions, and no oxidation signals were observed after addition of cisplatin. Initially we investigated the interaction between equimolar amounts of oxidized cyt c and cisplatin. The 1H,15N-HSQC NMR spectrum acquired after 4 h incubation at 37 °C showed the presence of two cross-peaks, in addition to those of cisplatin, corresponding one to an ammine ligand trans to a chloride (δ 15N = −63.0 and δ 1H = 4.19 ppm) and the other to an ammine ligand trans to oxygen (δ 15N = −82.5 and δ 1H = 3.96 ppm) (Figure 4, red cross-peaks). This species resonates at different chemical shifts (δ 15N ≈ −83.3 ppm and δ 1H ≈ 4.04 ppm) with respect to pure monoaqua/monochlorido species generated by cisplatin hydrolysis under strictly analogous conditions, but in the absence of the protein, and corresponds to the species observed by reduction of Pt(IV) in the presence of 1:1 molar ratio of cyt c with excess NADH (Figure 3, red cross-peaks). An analogous experiment was carried out by treating reduced cyt c with cisplatin in 1:1 molar ratio. In this case, together with cisplatin cross-peak, the appearance of a cross-peak at −83.1 and 4.03 ppm (for 15N and 1H, respectively) was observed (Figure 4, green cross-peak). This peak, relative to an ammine

trans to oxygen, appears only slightly upfield shifted along the proton dimension with respect to the peak of pure monoaqua cisplatin (δ 1H ≈ 4.04 ppm) (Figure 4, blue cross-peak). Thus, it is possible to conclude that the chemical shift of the ammine trans to oxygen is strongly affected by oxidized cyt c, due to the presence of paramagnetic Fe(III) (hyperfine shift), whereas it is little affected by diamagnetic Fe(II) in the reduced form of the protein. The small shift observed in the case of reduced cyt c could also be ascribed to traces of the oxidized form still present in the sample. Assuming no contact contribution to the hyperfine shift of ammine ligands, the observed negative difference in 1H chemical shift of ammine trans to oxygen in the case of oxidized vs reduced cyt c (Δδ 1H = δpara − δdia = 3.96 − 4.03 = −0.07 ppm) would indicate a proximity of the Pt(II) complex to the xy plane of the magnetic susceptibility tensor, which roughly corresponds to the porphyrin plane of the heme cofactor.34−36 Moreover, the intensity of monoaqua cisplatin cross-peaks is greater in the presence of cyt c. This could imply that cisplatin hydrolysis is fostered by electrostatic interactions between the metal complex and the basic surface areas of the protein (e.g., the ring of positively charged residues surrounding the heme cleft). An increased reactivity of the metal complex also emerged from a work of Casini et al.,25 concerning the interaction between Pt(II) iminoether compounds and cyt c, even if no direct evidence of the electrostatic interaction between the metal complex and the protein was reported. Covalent Interaction between Platinum Substrate and Cyt c. So far, evidence has been gained that the monoaqua/monochlorido species derived from the first aquation step of cisplatin is involved in a noncovalent interaction with cyt c so to feel the effect of the paramagnetic Fe(III). However, at this stage of the investigation, it is not possible to exclude that cyt c is also able to bind cisplatin covalently. The absence of specific cross-peaks in the NMR spectra indicative of covalent interaction could be the consequence of too small concentration of such covalent adducts. Therefore, in order to verify if cyt c is able to bind covalently to platinum in our experimental conditions, we D

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Figure 5. HPLC−ESI−MS spectrum of oxidized cyt c (retention time, tR, of 17.7 min) from m/z 750 to 1750 (a) and close-up from m/z 920 to 1080 (b). HPLC−ESI−MS spectrum of oxidized cyt c-Pt(NH3)2Cl+ adduct (tR of 17.2 min) from m/z 750 to 1750 (c) and close-up from m/z 920 to 1080 (d). The spectra were obtained after 4 h of incubation at 37 °C in 25 mM phosphate buffer, pH 5.8.

in the experiment performed with 1 equiv of oxidized cyt c (more than 30% in intensity with respect to the dichloro species), further confirms that the latter peak does not belong to Pt(II) covalently bound to cyt c, but most likely to monoaqua cisplatin adsorbed on the protein (Figure 4). Furthermore, the absence in the 1H,15N-HSQC spectrum of cross-peaks assignable to ammine ligands trans to sulfur atoms in the above experiment also rules out the formation of significant amount of platinum/cyt c-methionine adducts, corroborating the idea of prevalent formation of noncovalent Pt-cyt c interactions. An analogous experiment was performed starting from reduced cyt c. Therefore, after overnight incubation at 37 °C of oxidized cyt c with a 10-fold excess of NADH in 25 mM phosphate buffer (pH 5.8), the NADH was removed, and an equimolar amount of cisplatin (with respect to the protein) was added. The progress of the reaction was monitored by HPLC− ESI−MS analysis. The ESI−MS spectra of the fractions eluted at tR of 17.7 and 17.3 min indicated the presence of free cyt c and cyt c-Pt(NH3)2Cl+ adduct, respectively, analogously to what observed in the reaction performed with the oxidized form of cyt c (data not shown).

performed further experiments using the more sensitive HPLC−ESI−MS technique. The chromatogram (UV detector, 280 nm) of oxidized cyt c exhibits one peak (retention time, tR, of 17.7 min). The corresponding ESI−MS spectrum showed a series of multicharged states, from +21 to +5, corresponding to a molecular weight of 12360 Da. Following addition of an equimolar amount of cisplatin to oxidized cyt c (300 μM), aliquots of the reaction solution were taken off from time to time, diluted 2.5 times, and analyzed by HPLC−ESI−MS. After 2 h of incubation, it was possible to observe two main peaks (tR of 17.7 and 17.2 min), corresponding to free cyt c and cyt cPt(NH3)2Cl+ adduct (molecular weights of 12360 and 12624 Da, respectively). The peak relative to the cyt c-Pt(NH3)2Cl+ adduct increased in intensity in the first 4 h incubation (Figure 5). For longer reaction times, the loss of the remaining ligands together with the replacement of some ligands by water molecules occurred; however, even after 24 h of incubation, the amount of Pt-cyt c adduct (ca. 10%) was still much lower than that of the free protein (data not shown). The small intensity of the HPLC peak corresponding to the cyt c-Pt(NH3)2Cl+ adduct with respect to the strong 1H/15N cross-peak, characteristic of ammine trans to oxygen observed E

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Figure 6. 1H/13C peak intensity ratios between cisplatin-bound and free cyt c (oxidized form) as a function of protein residue (a). Ribbon diagram of horse heart cyt c generated with PyMOL (1HRC pdb). Most affected residues are colored in red (b).

Figure 7. HPLC−ESI−MS spectra at different retention times (tR) of Pt-bound peptides derived from tryptic digestion of cyt c reacted with a 10-fold excess of cisplatin (a,c,e) and enlargements of multicharged peaks (labeled with the appropriate m/z ratio on the left plots) corresponding to platinated peptides that are indicated on the top of each graph (b,d,f).

a 1:1 mixture of oxidized cyt c and cisplatin incubated for 24 h at 37 °C. From the intensity variations of 1H/13C cross-peaks, it was possible to identify the most affected regions of the protein.

In an attempt to localize the platinum-binding sites on the protein, natural abundance 1H,13C-HSQC spectra were recorded on a sample of oxidized cyt c and a sample containing F

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The residues that decreased in intensity upon cisplatin addition were those numbered 10, 26, 36, 38, 39, 55, 59, 65, 66, 67, 68, 73, 78, 79, 80, 81, and 97, which define two main stretches: 65−68 and 78−81, containing Met65 and Met80, respectively. Thus, these two methionines represent the potential binding sites for platinum (Figure 6). Confirming the Platinum-Binding Sites by Protein Digestion Experiments. In order to better identify the platinum-binding sites on the protein, a 10-fold excess of cisplatin was added to cyt c. After 72 h incubation at 37 °C, unbound cisplatin was removed and the protein was subjected to trypsin digestion. The HPLC chromatograms of untreated and Pt-treated cyt c appeared very different (Figure SI3, Supporting Information); this was not the case for the chromatogram derived from cyt c reacted with an equimolar amount of cisplatin, which did not show significant differences with respect to that of untreated cyt c. From a careful comparison of the data, it was possible to identify two peptides as main platinum-binding sites. The first one, 61EETLMEYLENPK, gave two elution peaks at tR of 19.2 and 22.0 min corresponding to peptides bearing [Pt(NH3)2]2+ and [Pt(NH3)]2+ moieties, respectively (ESI−MS spectra). Most likely in this case, platinum binding occurs to Met65. The second peptide identified in the tryptic digest of Pt-treated cyt c was 80 MIFAGIK eluting at tR of 21.4 min and corresponding (ESI− MS spectrum) to the peptide carrying a [Pt(NH3)]2+ moiety (Figure 7). In the latter case, it is most likely that platinum binding occurs to Met80, an axial ligand of iron ion. Notably, the 80MIFAGIK peptide was not observed in the tryptic digest of the free protein, maybe because it is poorly ionizable in the absence of a doubly charged platinum ion. In summary, both NADH (as a source of reducing equivalents) and cyt c appear to be essential for promoting Pt(IV) reduction to Pt(II). The experiments performed with substoichiometric amounts of cyt c indicate that the protein plays a catalytic role in the electron-transfer reaction. Direct evidence of noncovalent interaction between monoaqua cisplatin and cyt c is provided by NMR spectroscopy, which suggests proximity of the Pt(II) complex to the heme cofactor plane. Moreover, HPLC−MS analysis and natural abundance 1 13 H, C-HSQC spectra performed on the entire protein reveal formation of minor amounts of covalent Pt-cyt c adducts and allow to identify regions around Met65 and Met80 as most probable binding sites. This is confirmed by HPLC-MS analysis of peptides resulting from trypsin-digestion of free and Pttreated protein.

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the University of Bari, the Consorzio Interuniversitario di Ricerca in Chimica dei Metalli nei Sistemi Biologici (C.I.R.C.M.S.B.), and E.U. Cost Action CM1105. V.B. and O.S. gratefully acknowledge the support by the Czech Science Foundation (project 14-21053S) and the Student Project No. IGA_PrF_2016_013 of Palacký University in Olomouc.

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ABBREVIATIONS Cyt c, horse heart cytochrome c; ESI-MS, electrospray mass spectrometry; GSH, glutathione; HPLC, high-performance liquid chromatography; HSQC, heteronuclear single quantum coherence; MW, molecular weight; NADH, nicotinamide adenine dinucleotide; NMR, nuclear magnetic resonance; QTOF, quadrupole-time-of-flight

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.molpharmaceut.6b00438. 1 15 H, N-HSQC spectra of 15N-labeled Pt(IV) complex treated with excess NADH at different concentrations of cyt c; 1H NMR spectra of oxidized and reduced cyt c; HPLC of tryptic digest of free cyt c and cyt c reacted with cisplatin (PDF)

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DOI: 10.1021/acs.molpharmaceut.6b00438 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.molpharmaceut.6b00438 Mol. Pharmaceutics XXXX, XXX, XXX−XXX