Anticancer drug impact on DNA – a study by neutron spectroscopy coupled with synchrotron-based FTIR and EXAFS

Complementary structural and dynamical information on drug-DNA interplay has been achieved at a molecular level, for Pt/Pd-drugs, allowing a better understanding of their pharmacodynamic profile which is crucial for the development of improved chemotherapeutic agents. The interaction of two cisplatin-like dinuclear Pt(ii) and Pd(ii) complexes with DNA was studied through a multidisciplinary experimental approach, using quasi-elastic neutron scattering (QENS) techniques coupled with synchrotron-based extended X-ray absorption fine structure (SR-EXAFS) and Fourier-Transform Infrared Spectroscopy-Attenuated Total Reflectance (SR-FTIR-ATR). DNA extracted from drug-exposed human triple negative breast cancer cells (MDA-MB-231) was used, with a view to evaluate the effect of the unconventional antineoplastic agents on this low prognosis type of cancer. The drug impact on DNA's dynamical profile, via its hydration layer, was provided by QENS, a drug-triggered enhanced mobility having been revealed. Additionally, an onset of anharmonicity was detected for dehydrated DNA, at room temperature. Far- and mid-infrared measurements allowed the first simultaneous detection of the drugs and their primary pharmacological target, as well as the drug-prompted changes in DNA's conformation that mediate cytotoxicity. The local environment of the absorbing Pd(ii) and Pt(ii) centers in the drugs' adducts with adenine, guanine and glutathione was attained by EXAFS.


Introduction
Cancer is a leading cause of death worldwi de, with a growi n g incidence: 12.7 million people per year are diagnosed wi th cancer, from which more than 50% die from the disease. Earl y diagnosis and chemothera peuti c treatment can lower this mortality rate by 30 to 40 %, and may lead to the cure of ca. 30 % of the patients. Anticancer platinum drugs have been introduced in the clinics since the serendipitous discovery of cisplati n (cis-dichlorodi a mi ne platinum(II), cis-(NH3)2PtCl2) in the 1970s , which was the first inorganic compound displaying a hi gh antineoplastic activity towards human tumours. 1,2 The three currently approved platinum agents -cisplatin, carboplatin (ci sdiamine(1,1-cyclobutanedica rboxyla to) platinum(II)) and oxaliplatin ([(1R,2R)-cyclohexa ne-1,2-dia mi ne] (etha nedi oa to-O, O' ) platinum(II)) -are applied worldwi de in chemothera pe uti c regimes. However, their clinical application is still restricted by dose-limiting deleterious side effects and acquired resista nce upon prolonged administrati on, 3 as well as by lack of specifici ty against several cancer types (e.g. metastatic). Hence, considerable effort has been put into the development of novel metal-based drugs, including cisplatin-like agents, aiming at an improved antitumor efficiency. [4][5][6] Among these, polynucl ea r multifuncti onal Pt(II) and Pd(II) chelates with flexible biogeni c polyamines as bridging ligands have been synthetized by the authors and evaluated as to their effect on several types of human neoplasias, namely the low prognosis , highly metasta ti c triple negative breast cancer against which a Pd(II) agent (Pd2Spm, Spm=spermine, H2N(CH2)3N H(CH2)4N H(CH2)3NH2) has yielded particularly promising results. 7 A number of studies have been conducted in the last few years on these Pt-and Pd-based complexes, reporting conf ormational profiles [8][9][10][11] and pharmacoki netic/pharmacodyna mi c behaviour, 12,13 and the effect on different human cancers 7, [14][15][16][17][18][19][20][21][22] including their impact on cellular biochemical profile. [23][24][25] Unconventi onal pathways of cytotoxici ty were disclosed, whi ch are recognized to lead to an improved therapeuti c efficiency coupled to a lower toxicity. In addition, the data gathered so far suggests distinct mechanisms of action for the Pt(II) versus the Pd(II) agents, as well as for the dinuclear polyamine chelates relative to the mononucl ear lead drug cisplatin. The activity of this kind of agents was found to be mediated by selecti ve covalent binding of the metal centres to DNA bases (mainly the purines at their most nucleophilic nitrogen atom, N7), yieldi ng long-range intra-and interstrand adducts responsible for cell growth arrest and apoptotic death. Adducts with DNA are formed upon drug activation, according to the following steps : transport into the cell, hydrolysis (in the cytoplasm) by sequential loss of the chloride ligands and aquation, uptake into the nucleus and reaction of the unstable diaquo species wi th DNA (the drug´s main pharmacol ogi cal target). 26 Howeve r, apart from this interplay with DNA, during the pharmacoki neti c stage drug interaction may also occur with amine and sulfur groups from proteins and other cellular constituents. In particular, endogenous thiols such as glutathione (L-γ-gluta m ylcysteinyl-gl yci ne (Glu-Cys-Gl y), GSH, a key cellular antioxida nt ) are known to intercept this type of agents owing to the hi gh affinity of the soft Pt(II) and Pd(II) cations towards S-donor ligands, 27,28 which may significantly lower the drug´s bioavailability at the main target and lead to acqui red resistance. Since the mechanisms underlying drug resistance as well as the molecular basis of cytotoxici ty are still not full y understood for these DNA-da ma gi ng agents, the present study aims at contributi ng to their elucidation by monitori ng the interaction of spermine Pt(II) and Pd(II) complexes (Pt2Spm and Pd2Spm) with DNA.
DNA extracted from human triple negative breast cancer cells (MDA-MB-231 cell line) and commercial DNA were used as target models, while cisplatin was taken as a drug reference. Drug concentra tions and incubation times were chos en according to previous results, corresponding to an opti ma l cytotoxic effect towards this particular neoplastic cell line 7 : 4 and 8 μM (up to ca. 2xIC50 level), for a 48 h exposure time. A multidisciplinary experimental approach was followed, through the application of: (i) Quasi-elastic neutron scattering (QENS ), suitable for directly accessing different spatially resol ved dynamical processes (at subnanometer lengthscale and subnanosecond timescale), under distinct conditions, mainly fo r hydrogen-rich systems; 25,[29][30][31] (ii) synchrotron-ra dia ti on Fouri ertransform Infrared Spectroscopy-Attenua ted Total Reflecta nce (SR-FTIR-ATR), recognized as a cutting-edge non-destructi v e tool for obtaining spectral signatures of molecular compon en ts in biological samples, so as to relate structural to functiona l data; 32,33 (iii) synchrotron-based Extended X-ray Absorpti o n Fine Structure (EXAFS) and X-ray Absorption Near-Edge Structure (XANES), that are methods of choice for obtai ni ng detailed information on the local structure of bioinorga ni c noncrystalline materials. 34,35 Water supports vital biochemi cal processes in livi ng organisms, and is responsible for the maintenance of the functional three-dimensi onal architecture of biopoly me rs through a tight interplay within their hydration shells. 36 Actually, mobility changes in these hydration layers may affect the biopolymer´s conforma tional and dynamical profile, whi ch rule biofunctionality. It is a well-recognized fact that water within hydration sheets is retarded with respect to the bul k, although its dynamical properties are not yet completel y understood. Hence, elucidation of water dynamics in biologi ca l systems and its impact on activity and function is of the utmos t relevance in drug development, for an improved understan di n g of a drugs´ mode of action via interaction with all its possibl e pharmacol ogi cal targets that may include the water molecul es wrapping their conventi onal receptors. Neutron techni ques such as high resolution quasielastic scattering are particul a rl y suited for selectively probing water dynamical behavi ou r, namely within biomolecular hydration shells, at a nano-to picosecond timescale (ca. 10 -9 -10 -13 s) and a 1 to 30 Å lengthscale (correspondi ng to inter-and intramolec ul a r distances, e.g. H-bonding) 37 , yielding results not achievable by any other methods. QENS measurements at different temperatures allow to characterize the translational and rotational modes of water hydrogens within a biological matri x (e.g. biomolecul es, cells or even tissues), and determine how this dynamical behaviour may be disturbed by the presence of an external entity such as a drug. A few QENS experiments on water dynamics in living cells have been reported, 29 but drug effects were first investigated by the authors in cisplati nexposed human cancer cells using QENS and inelastic neutron scattering spectroscopy (INS). 25 FTIR spectroscopy is an extremely powerful, sensitive and non-invasive analytical tool for interrogati ng the chemi ca l compositi on of biological systems, delivering unique spectra l signatures for a particular biomolecul e at specific conditi ons . The synchrotron beam available at the MIRIAM (Multi m od e InfraRed Imaging And Microspectroscopy) beamline from the Diamond Light Source (DLS, United Kingdom), currently used, provides stable, broadband and extremely bright IR radiati on and delivers FTIR data with an unmatched signal-to-nois e ratio. In addition, MIRIAM spans the largest infrared spectral rangeextending from the near-up to the far-IR (or THz) region -and is one to two orders of magnitude brighter in the mid-/fa r-IR than any other conventi onal thermal IR source. Furtherm or e, the FTIR attenuated total reflection (ATR) mode used in the present measurements allows to directly probe DNA without any particular sample preparation, with the added advanta ges of high spatial resolution and absence of resonant Mi e scattering effects on the collected spectral data. 38 In particul a r, probing the far-infrared region (< 300 cm -1 ) that comprises the internal vibrational modes of the molecule, enabled to detect H-bonds and weak non-bondi ng interactions within the nuclei c acid, with very high specificity, thus unveiling specific druginduced conformati onal changes that underlie cytotoxicity.
EXAFS, in turn, allows the direct observation of meta l coordinati on and elucidation of the local environment of the absorbing metal centre in inorganic compounds , particul a rl y when good quality crystals are unavailable, and has been successfully applied to Pt(II) compounds specifically for assessing their degradati on in the presence of S-contai ni n g molecules. 34 , 35 In the present study, synchrotron-ba s e d EXAFS/XANES measurements yielded detailed information on the first coordinati on shell of Pd(II) and Pt(II) within the drugs´ adducts with adenine (A), guanine (G) and glutathione (GSH).
Complementa ry structural and dynamical information wa s therefore achieved for the drug-DNA systems presently studi ed. These results provide a more comprehensi ve understanding of the pharmacodyna mi c profile of the dinuclear Pt-and Pd-anticancer agents, at a molecular level, particularly regarding thei r effect on vital biomolecules through an impact on thei r hydration water, apart from a direct perturbation of their nati ve conformati on. This knowledge is paramount for a rationa l

Experimental
The list of chemicals, the experimental details regarding the synthesis and characterization of the Pt2Spm and Pd2Spm complexes, the preparation of drug solutions, and the cell culture protocol are extensively described in the Electroni c Supplementary Information, as well as the pre-processi ng and analysis procedures of the QENS, FTIR-ATR and EXAFS/XA N E S data. Synthesis and Characterization of the Drug-Purine and Drug-Glutathione Adducts. Drug-Purine Adducts: The (1:4) drugadenine and drug-gua ni ne adducts (MA4 and MG4, M=Pt(II) or Pd(II)) were synthesized following a synthetic route previous l y optimized by the authors 39,40 (see details in the Supporti n g Information, Figs. S1 and S2). Drug-Glutathione Adducts: In order to prepare (1:4) drug-G S H adducts, 4 molar equivalents of glutathione were added to a solution of each of the drugs -Pt2Spm-0.45 mM or Pd2Spm-0 .2 5 mM -and stirring was maintained in the dark, at room temperature, for 24 h. The initial colourless reaction mixture became orange over time (a characteristic colour of compl exes containing Pt-S covalent bonds). The resulting drug-GS 4 solutions (MGS4, M=Pt(II) or Pd(II)) were then concentra ted by rotary evaporation under vacuum and lyophilized to obtain an orange powder. Titration of Drug-Purine Adducts with Glutathione , Adenine or Guanine: The Mbase4 (M=Pt(II) or Pd(II), base=A or G) adducts were titrated with GSH. A glutathione solution was added, in a (1:4) molar ratio, to an aqueous solution of 10 mg of each Mbase4 adduct. The reaction mixtures were kept in the da rk with stirring, at 30 °C for 24 hours, after which they were concentrated under vacuum, at 30 °C until precipitation occurred and then vacuum filtered through a 0.22 μm filter to remove precipitated adenine or guanine. Finally, they were concentrated under vacuum (at 30 °C) and lyophilized to yield the soli d products (hereafter denomina ted MA4+GSH and MG4+GSH). Preparation of Cell Pellets for QENS measurements. Cell pellets (100 mg/1 cm 3 , ca. 5×10 8 cells per sample), were prepared by cell harvesting (through trypsinisation) followed by repea ted (2×) PBS washing and centrifugati on (at 195× g, for 15 min). PBS was used as an isotonic medium in order to avoid water exchange from the inside to the outside of the cell (leading to cell shrinkage). The drugs (cisplatin, Pt2Spm and Pd2Spm at 4 and 8 μM) were added to the cells during their logarithmic phase of growth, and left to incubate for 48 h. In order to completel y remove the extracellular water component (less than 5%), the cell pellets were washed with deuterated PBS by resuspension (1×) followed by centrifugati on (at 195× g) for 5 min, which was then repeated for 15 min (after removal of the first supernatant). Fibrous commerci al human DNA (from calf-thymus) was also analysed -samples without and with drug were prepared, the latter by solubilizing 100 mg of DNA fibres in 50 mL of either cisplatin, Pd2Spm or Pt2Spm at 8 μM, with stirring (at 4 °C) for ca. 24 h. Aqueous drug solutions were used (instead of saline solutions) in order to ensure a prompt hydrolysis of the chlorides, that is essential for drug activation prior to DNA bindi ng. 5 mL of 3 M-sodium acetate and 150 mL of ethanol (≥99.8% ) were then added, followed by a 2 h incubation (at -20 °C). The solutions were centrifuged at 4075×g for 20 min (4 °C), the pellets were washed twice with ethanol (70%) and centrif uge d again (under the same conditions ). After discarding the supernatant, the obtained DNA samples were air dri ed completel y in a desiccator -hereafter denomina ted DNAdehyd . Apart from this dehydrated DNA, H2O-and D2O-hydrated DNA were prepared at a r.h. >80%, in order to ensure the stability of the native B conforma tion -yielding the samples hencef ort h denoted as DNAhyd (either H2O-DNAhyd or D2O-DNAhyd). This wa s achieved by placing DNAdehyd in a desiccator (clos ed environment) with a saturated KCl solution (in either H2O or D2O) until attaining a stable weight (correspondi ng to a r.h. of 84.34-83.62%, at 25 °C). 41 QENS Measurements. QENS data were acquired at the ISIS Pulsed Neutron and Muon Source of the Rutherford Appl eton Laboratory (United Kingdom), 42 on the OSIRIS spectromete r 43 (see details in the Supporti ng Information). H2O-DNAhyd and D2O-DNAhyd samples were measured, as well as dehydra te d DNA (DNAdehyd), both untreated and drug-expos ed -to cisplati n or Pd2Spm at 8 μM (for a 48 h incubation time). The sampl es were mounted in indium-sealed 0.1 mm-thick (3×5 cm) flat Al cans (the beam size at the sample being 2.2×4.4 cm), and were oriented at -30° with respect to the incident beam. A vana di um sample (purely incoherent elastic scatterer) was also measured, to define the instrument resolution and correct for detector efficiency. Experiments were carried out at 150 and 298 K. Synchrotron-based FTIR-ATR Measurements. FTIR-ATR spectra were recorded at the MIRIAM beamline B22 of DLS, 44

Results and Discussion
The present study aimed at obtaining detailed information on the pharmacoki netic and pharmacodyna mic profiles of two polynuclear cisplatin-like Pt(II) and Pd(II) anticancer agents: thei r impact on DNA´s dynamical and conforma tional preferences was evaluated by QENS techniques and synchrotron-ra dia ti o n FTIR-ATR, while their interaction with DNA purine bases, as wel l as competiti on from glutathione, were tackled by synchrotr o nbased EXAFS/XANES measurements (Scheme 1). QENS. It is a well-recogniz ed fact that biomolecules are subject to a strict relationship between structure/conf orma tion and activity, as well as between dynamical behaviour and functi on. Hydration is critical for bioactivity, the first hydration shell bei ng an essential part of a biomolecul e´s structure, strictly regula ti n g its conforma ti onal and dynamical preferences and cons equently its physiologi cal role. 47,48 Regarding DNA, interacti ons with water are crucial to ensure the native B-conformati on of the double helix, thus modulating functionality. 49 The dyna mi cs of water within the hydration layer differ from that of bul k water, and are dependent on the biomolecule´s conformati o na l fluctuations as well as on the chemical and topological heterogeneity of its surface (e.g. phosphate groups or DNA minor vs major grooves ). Conversel y, even minor variations in hydra ti on water dynamics may prompt significant rearrangements in DNA that can severely distress the normal cellular function and lea d to growth inhibition and cell death. An external entity such as a drug may affect the H-bonding network of water in these first hydration shells, both amid water molecules and between water and the biomolecule. Hydration water (as well as intracellular water, in a cellular or tissue matrix) may thus constitute a potential secondary pharmacol ogical target, namely for anticancer chemothera peuti c compounds . 25 This concept wa s currently applied for the elucidation of the drug-target intera ction of metal-based agents, which is an innovative approach to monitor pharmacodyna mics and better interpret a drug´s mechanis m of action, with a view to improve cytotoxicity.
DNA hydration water is mostly adsorbed, in a coopera ti v e way, at the outer double helix surface, with a higher dens i ty around the phosphate groups and more ordered near the bases (ca. 18-30 water molecules per nucleotide 50 ). In this first hydration shell, H-bond interactions are stronger than in bul k water and water mobility is significantly reduced, wi th residence times about 10 times larger. 51 Although relaxati on within DNA and its hydration layer is slower than for RNA and proteins, a dynamical transition was detected for this nuclei c acid at 200-230 K, similarly to other hydrated biopoly me rs (globular proteins, GFP, RNA). 47,52,53 Below this temperat ur e, flexibility is significantly reduced and functionality is lost. This transition is thought to be triggered by strong coupling with the hydration water molecules, that undergo mobility changes at the same temperature (via H-bonding and translati ona l processes). Consequentl y, it is strongly affected by the properties of the surroundi ng medium, which may vary wi th temperature, pressure, pathological conditions or the presence of non-endogenous compounds such as a drug. 52,54 Despite the still poorly understood nature of this dynamical transition, it is generally acknowledged as essential for biological activity.
Building on the success of the first QENS experi men t performed by the authors on a drug´s impact in human brea s t cancer cells, that unveiled a noticeable dose-depen d en t cisplatin effect on intracellular water dynamics (both for the cytoplasm and hydration layers), 25 the present study appli ed quasi-elastic neutron scattering spectroscopy (with isotope labelling) to probe hydrated DNA, upon chemothera pe uti c exposure. This allowed us to detect drug-elicited dynami ca l changes, that were assessed through variations in the mobil i ty of the labile protons from the macromol ecul e and its hydra ti on layer -observable within the time-and lengthscales of the OSIRIS spectrometer, ca. 4-200 ps and 4-20 Å. DNA sampl es (prepared from fibrous calf thymus DNA) were measured before and after incubation with either cisplatin or Pd2Spm. Both H2Oand D2O-hydrated samples were probed (DNAhyd), with a vi ew to differentiate between the drug effects on: (i) DNA´s exchangea ble hydrogens and hydration water; (ii) DNA´s backbone and non-labile hydrogens . The hydration degree was kept constant (r.h. >80%), ensuring that the measured variations in the QENS profiles were solely due to the effect of the drug and not to transitions induced by differences in the macromol e -cule´s hydration. The H2O-DNAhyd samples comprised all dynamical contributi ons (hydration water, exchangea bl e and non-excha ngeabl e H´s and DNA´s skeleton), while the slower motions from the non-labile hydrogens and DNA phosphori b os e backbone were retrieved from the D2O-DNA samples. Dehydrated DNA (DNAdehyd) was also analysed, with and without the tested compounds. Any effect of the experimental protocol on the dynamical behaviour of the drug-treated DNAdehyd sampl es was ruled out by comparing the QENS profiles of DNAdehyd as received (from Sigma) and a corresponding sample prepared in the same way as those exposed to either cisplatin or Pd 2 Spm (see Experimental section). For these dehydrated samples, the main foreseen dynamical processes are the global modes encompassi ng the macromol ecule´s heavy atoms (sl ow backbone motions) not expected to be detected within the OSIRIS time window. The experiments were performed at 150 and 298 K, that span below and above the dynamical transiti on temperature reported for B-DNA (222 K 52 ). In contrast to room temperature, no significant differences were observed between the dynamical profiles of H2O-and D2O-DNAhyd at 150 K, i.e. below the dynamical crossover temperature the hydration layer is more rigid and the H2O-hydra ted nucleic acid gradua l l y reaches a dynamical profile similar to that of the D2O-hydra te d molecule (the internal DNA dynamics being undetecta ble in OSIRIS).  Please do not adjust margins Please do not adjust margins Figure 1 depicts the QENS profiles (Figure 1(A)) and elasti c scan plots (elastic intensity vs temperature for the whol e temperature range probe, Figure 1(B) to (D)) for H2O-and D2O-DNAhyd as well as for DNAdehyd, clearly showing a dynami ca l transition taking place at ca. 225 K for H2O-DNAhyd and at ca. 260 K for D2O-DNAhyd. A gentle linear-like tempera tur e dependence was detected below the cross-over temperat ur e, while above it different behaviours were observed for each sample: (i) for D2O-DNAhyd a dynamic component was observed, driven by its D2O-hydrati on shell, and an anticipated mobil i ty reduction relative to H2O-DNAhyd was verified (Figure 1(B)); (ii ) regarding dehydrated DNA (lyophilized DNA, lacking an hydration layer), some dynamical contributi on was still detected within the OSIRIS timescale (Figure 1(C) and (D)). This was also evidenced through the QENS profiles for H2O-DNAh y d and D2O-DNAhyd versus DNAdehyd samples (Figure 1(A)). This dynamic behaviour currently unveiled for dehydrated DNA is surprising, and not in complete accordance with previ ous studies that reported an harmonic (temperature independ en t ) profile for dry DNA (commerciall y available lyophil i z ed sample) 55,56 and identified hydration water motions as the sol e responsible for the dynamical transition in this system. However, anharmoni city has been formerly reported for dry lysozyme and assigned to methyl group relaxation-l i ke motions. 57,58 . Accordingl y, we tentatively ascribe the onset of anharmonicity presently detected for dehydrated DNA to intrinsic relaxation processes, namely rotational motions of CH 3 as well as of H-bond free NH2 groups within the nucleic aci d molecule (at the nitrogen bases). In fact, methyls have been recognized as plasticizers of proteins, with a high impact on their dynamics and activity. 56 Although DNA contains a much lower amount of CH3 moieties as compared to proteins (one CH3 (in thymine) per each four nitrogen bases), its effect should be similar even if to a much lower extent. Since methyl groups are symmetric tops (C3v symmetry), they have a very low rotationa l energy barrier and therefore facilitate the dynamics of biomolecules, namely DNA, even for low hydration levels.
The drug influence through an effect on DNA´s hydra ti o n layer was only detected at 298 K, which appears to indicate tha t hydration water and DNA motions, in the ps timescale, are not directly coupled at low temperatures. Furthermore, at 150 K (well below the dynamical transition temperature) the hydration shell is considerabl y less fluid and its dynamics are too slow to be probed with the OSIRIS instrument (Figure 2(A)). Also, no drug effect was found for dehydrated DNA (Figure 2(B)).
For the hydrated nucleic acid, drug exposure was found to prompt a faster DNA dynamics, justified by the disruption of the ordered hydration shell and of the native conformati on of the nucleic acid upon drug binding, known to affect the native basepair and base packing arrangements : for drug-incubated DNA, a broader QENS profile was measured (Figure 3(A)), as well as a slight deviation to faster kinetics above the dynamical transiti on temperature. The effect of cisplatin was found to be somewha t stronger than that of Pd2Spm (Figure 3(A) vs (B)), which is in accordance with previous studies by vibrational microspect r oscopy on these drugs´ influence on the cellular metabol i c profile. 23 Interestingl y enough, the drug impact on DNA´s hydration layer was not found to affect the biomolecul e´s dynamical transition temperature, neither for cisplatin nor for the dinuclear Pd-agent. It should be emphasised that drug exposure, either to cisplatin or to Pd2Spm, was found to have a negligible effect on DNA`s hydration level (Fig.S4, Supporti n g Information), thus ascribing the measured mobility increa s e exclusively to a drug-mediated effect on the nucleic acid.
No drug effect was found for either D2O-hydra ted DNA pellet or DNAdehyd (Figure 2(B)), suggesting that the slow motions of the nucleic acid skeleton (backbone dynamics ), although expected to be affected by the DNA-bindi ng compou n ds presently studied, are not detected within the OSIRIS timesca l e. In addition, deuteration of the hydration shell in D2O-DNAh y d macromolecul e, evidencing the key role of mobile H´s and hydration water molecules as key mediators in external appea r to hinder a significant drug impact on the perturbations to the biomolecule. Nevertheless, these are only samples and the absence of an hydration layer in DNAdehyd tentative conclus i ons that require confirmation on a higher resolution QENS spectrometer, allowing the detection of the slower dynami ca l  The neutron diffraction plots obtained for H2O-DNAh y d display two noticeable Bragg peaks (at ca. 0.35 and 0.55 Å -1 , detected at both 298 and 150 K, Figure 4) that reflect the ordered structure of the nucleic acid molecule. In the presence of the drug (either cisplatin or Pd2Spm), the most intense pea k is clearly shifted from 0.35 to 0.3 Å -1 , which corresponds to a variation in distance of ca. 3 Å (from 17.9 to 20.9 Å), that ma y be related to both the gap between stacked bases (ca. 3.3 Å) and the distance between H-bonded base pairs (ca. 2.8 to 2.95 Å) in dsDNA helices. This effect (found to be independent of temperature) supports a drug-elicited structure disruption of the B-DNA molecule, associated to a perturbati on of the nuclei c acid´s base packing and pairing arrangements.
The experimental data was best fit using one δ-functi on (elastic component) convoluted with two Lorentz i a ns (quasielastic contributions ) (eq. (5), Figure 5). The very slow global motions of the macromol ecule are defined by the Delta function (slower than the longest observable time defined by the instrument resolution), while the narrow (Γglobal) and broader (Γlocal) Lorentzians represent, respectivel y: (i) slow motions (Q-dependent) from the biopolymer´s hydration water and H-bond restricted moieties located at the molecul e´s surface (side-chain motions); (ii) fast localized moti ons (Q-independent) ascribed to the rotation of CH3 groups and also of amine moieties (not involved in H-bonds).
The Q-dependent dynamical processes were consistent wi th a jump-diffusion reorientation model (via large-amplitude cooperative jumps, see Supporti ng Information), 59, 60 as reflected in the plots depicting the correspondi ng FWHM (Γglobal) as a function of Q 2 for both treated and untreated H2O-hydra te d DNA, that show an asymptotic approach to a plateau at high Q values ( Figure 6(A)). This behaviour agrees with the water distribution in the first hydration layer of nucleic acids, predominantly binding to their outer surface (unlike for proteins were it may be enclosed between polypeptide layers or in hydrop h obic pockets) and therefore allowing the formation and brea ki ng of H-bonds between the biopolymer and its neighbou ri n g waters. In turn, the fast motions of the nucleic acid´s amine and hydroxyl groups (not restricted by H-bondi ng) were found to be independent of the scattering vector ( Figure 6(B)), evidencing a localized dynamical process with relaxation times given by τ=(Γlocal) -1 . Table 1 comprises the values presently obtained for the translational jump-diffusion coefficients (DT) and residence times between jumps (τT), as well as for the correlation times of the fast localized processes, for each system studied. These results reflect the effect of the tested Pt-and Pd-agents on DNA´s dynamical behaviour. For the dynamical processes associated to    The τT value (10 ps) currently obtained for the dynami ca l processes taking place within DNA´s first hydration layer, in the absence of drug, agrees with previousl y reported data 36,49 and is one order of magnitude higher than the value for bulk water (1.1 ps 61 ). This clearly evidences the restricted dynamics of water molecules in the close vicinity of DNA. This retarda ti on relative to bulk water (up to a 6-fold slowdown at room temperature) is due to interactions of the water molecules with hydrophobic moieties and phosphate groups of the biopolymer, 49 , 62 a small number of particularly slow water molecules havi ng been found in DNA´s minor groove (bridging the nitrogen and oxygen atoms of complementa ry bases), with reorienta ti o n times between 60 and 85 ps -the so-called "spine of hydration". 63 In addition, the lower residence times presently measured for drug-incubated DNA may be partially due to a cha rge screening effect of the metal complexes under study (compri sing partial positive charges on the Pt(II) and Pd(II) ions), tha t may weaken the electrostatic interactions between hydra ti on water and negatively charged phosphates at the DNA surface, thus allowing an increased flexibility of the system. In fact, apart from the major role of hydration dynamics, electrostatic factors have also been suggested to influence the dynamical behavi ou r of biopolymers. 58 In sum, drug exposure was found to disrupt the nati ve ordered B-conforma tion of DNA, prompti ng a higher flexibili ty of the nucleic acid. In the light of the results currently gathered, this is suggested to be associated to a drug impact on the biomolecule´s first hydration layer, which is prompted into a faster dynamics. This corroborates and complements the effect observed for cisplatin 25 and currently measured for Pd2Spm on the intracellular milieu, in human breast MDA-MB-231 cancer cells: cytoplasmi c water was rendered more rigid by the presence of the metal complexes, while hydration water was dri ven into a more mobile state, as presently verified for H2O-DNAh y d (Figure 7). Additionally, the more moderate effect on DNA dynamics revealed for Pd2Spm relative to cisplatin is in accordance with the influence on intracellular water that was also shown to be more significant for the mononucl ea r Pt-agent. Synchrotron-based FTIR-ATR. SR-FTIR-ATR measurem en ts allowed access to the spectral signatures of DNA in the absence and presence of the tested antitumor agents, thus revea l i ng their effect on the nucleic acid´s conforma tion which is the basis for their cytotoxicity. The impact of the dinuclear Pt2Spm and Pd2Spm compounds was assessed, in both the far-and midinfrared domains, the conventi onal mononuclea r drug cisplati n having been measured for comparison purposes. Apart from the free Pt-and Pd-agents, drug-treated and untreated DNA samples were analysed (at room temperature): for DNA extracted from MDA-MB-231 cells (DNApellet) and for commer ci a l DNA (calf thymus DNA fibres, DNAdehyd) taken as a reference.
This was intended as a target-oriented study on the pharmacodynami cs of Pt2Spm and Pd2Spm, the vibrational fingerpri n t of drug-expos ed DNA having been probed with a view to: (i ) evaluate the drug effect on DNA -relating the induced changes in its infrared fingerprint to conformati onal rearrangem en ts (e.g. B-DNA to A-or Z-DNA); (ii) detect each tested drug and determine its structural variations due to interaction with the nucleic acid -via the measured deviations in its characteri s ti c vibrational profile, particularly in the low frequency region. To the best of the authors´ understandi ng, this is the first time the simultaneous detection of metal-based anticancer compou n ds and their pharmacol ogical target is achieved, which constitutes

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Please do not adjust margins an innovative way of directly monitoring a drug´s pharmaco d ynamics at a molecular level. Actually, although the full vibrational profile of the antitumor agents currently tackled (cisplatin, Pt2Spm and Pd2Spm) has been previously assigned by the team, [8][9][10][11] these compounds had never been detected within a biological matrix. This was currently made possible through the use of a high flux far-IR coherent synchrotron radiation, ensuring a high signal quality even in the low frequency region (<600 cm -1 ). Indeed, this spectral interval was particularly relevant to the study, as it allowed to simultaneousl y observe characteri s ti c low energy vibrational bands from both the drug and the nuclei c acid. Interpretati on of the measured data was assisted by the extensive spectroscopi c results formerly obtained by the authors for cisplatin, Pt2Spm and Pd2Spm, 8 -11 as well as by reported low frequency vibrational data on DNA encompas s i n g H-bond vibrations, base-twisting (breathing) and librationa l modes, known to be strongly conformati on-dependent 64 , 65 and thus prone to be significantly affected by drug binding. The metal complexes under study yield low frequency IRactive signals, assigned to vibrational modes involving the meta l centre  (Figure 8). This constitutes solid evidence of the drug´s cellular uptake and conformati onal rearrangement upon binding to the nucleic acid, the main observed changes bei ng (Figures 8 and 9): (i) absence of the strong ν(Cl-Pt-Cl ) signal (ca. 320 cm -1 ), as expected in view of the drug activation process by intracellular chloride hydrolysis prior to interaction with the target 13 ; (ii) blue shift of the (Pt-NH) and ν(CN/CC) signals from Pt2Spm and Pd2Spm (at ca. 1050 and 1070-1085 cm -1 , respectively); (iii) shifts of the CH2 and NH2 deformation bands from the spermine ligand (at 1150-1460 and 1585 cm -1 ), more noticeable for the Pd(II) system and reflecting a conformati o na l reorganizati on of the alkylamine chain upon formation of the drug-DNA adduct. Similarly, for cisplatin the NH3 torsi on, rocking and symmetric deformation modes (at ca. 210, 800 and 1300-1325 cm -1 ) were found to be severely affected, which ca n be justified by the significantly restricted conformati o na l freedom of the drug´s NH3 moieties within the DNA adducts.
Regarding the main drug target, DNA, the typical phospha t e stretching signals from the B-conformati on -at 1092 (νs(PO2 ) ) and 1237 cm -1 (νas(PO2)) -were found to undergo drug-ind u ce d deviations and intensity changes, which were much more significant upon Pt2Spm-treatment (Figure 9). This is indicati ve of a distinct type of DNA interplay for this Pt(II) agent as compared to its Pd(II) homologue, which is in agreement wi th previously reported evidence obtained by both FTIR and Ra ma n microspectroscopy in drug-expos ed human cancer cells. 23 By comparison of the PO2 vibrational features for the three drugs under analysis, it is noteworthy that DNA´s phospha te groups are affected by these agents in the following order: Pt2Spm > Pd2Spm > cisplatin. In addition, the DNA samples exposed to Pt2Spm and Pd2Spm evidenced a noticeable variati on of the vibrational features at 1380 cm -1 and 1550-1620 cm -1 , assigned to the ν(CC) and δ(NH2) modes of the double helix puri ne and pyrimidine bases, these bands being considerably less disturbed in the presence of cisplatin (Figure 9) which appea rs to reflect a distinct interaction of this compound with the bases . Actually, while cisplatin can only establish two covalent bonds with DNA´s bases (usually within the same helix), the dinucl ea r Pt-and Pd-spermi ne agents are capable of binding to four different sites simultaneously, and may yield interstrand adducts (apart from intrastrand) which cause a more severe damage.   Please do not adjust margins Please do not adjust margins Spectral changes were also detected in the low frequenc y bands ascribed to collective vibrations within double stranded DNA (phonon modes), such as helical motions, base twisti ng and DNA breathing (Figure 8). The latter, in particular, are associated to intrinsic fluctuational processes involving intermol ecular hydrogen bonds (e.g. breaking of base-pair interacti ons creating transient DNA "bubbles"), that are highl y conformati on-dependent. 6 7, 68 Clear variations were meas ured in the spectral range 400 to 600 cm -1 (Figure 8), also compri s i n g ring and carbonyl deformation modes (both in-plane and outof-plane) from DNA bases. These are prone to be strongl y affected by DNA metalation upon drug exposure, which occurs at the most nucleophilic nitrogens (N7) of the purine and pyrimidine bases. Actually, distinct spectral patterns are observed in this region for cisplatin as compared to the dinuclear agents, with a well-defined and intense band bei ng detected at 594 cm -1 for cisplatin-DNA. Although this feature was also seen for the other drug-DNA pellets, it displayed a quite lower intensity suggesting a different interplay of the polynuclea r agents with the double helix as compared to mononuclear cisplatin. In fact, for the spermine chelates a preaggregati on process is known to precede the metal-ta rget covalent binding due to a hydrophobi c attraction between the drug´s polyamine ligand and DNA´s apolar backbone. This type of aggregation mechanism is absent for cisplatin. As to the spectral profile around 200 cm -1 , it is markedly different for the cisplatin-vs the Pt2/Pd2Spm-contai ni ng samples. The DNA ri ng puckering modes assigned to this low frequency interval are overruled by characteristic water-breathi ng vibrations, mainl y in the samples exposed to the spermine complexes ( Figure 8). The region below ca. 100 cm -1 , in turn, encompass es skeleta l modes from the nucleic acid´s solid lattice.
The spectroscopic data currently gathered is a clear evidence of the strong interaction between the Pt(II) and Pd(II) agents presently probed and DNA -via metal coordination to its nitrogen bases upon a hydrophobic-driven pre-aggregati on between the drug and DNA in the case of the polyamine chelates. This interplay is reflected in the noticeable narrowing of the vibrational bands measured for the drug-treated DNA vs the control (Fig. 9), which reflects a significantly lower flexibility of the former. Furthermore, since binding of these metal-based agents to DNA triggers a severe damage of the nucleic acid´s native helica l B-conformati on, mainly through disruption of the double heli x H-bonded base-pairs (DNA unzipping, at temperatures bel ow the DNA melting value), the low energy intrahelical vibrati ona l modes ascribed to DNA breathing (closed-open base-pair fluctuations) were visibly affected. These low frequency DNA modes (<300 cm -1 ), that are critical to biological functi on, display a marked cooperative nature and are particul a rl y sensitive to structural reorientations, thus allowing to identi f y different conformati onal states of the biomolecul e (e.g. B-vs Aor Z-DNA). Hence, the data currently reported offers uni que information on the drug´s impact on DNA and helps to establi s h a connection between the observed low frequency vibrati ona l fingerprint of the nucleic acid in the presence of each compound and DNA´s conforma tional changes that underl i e cytotoxicity. In addition, it should be emphasized that the FTIR pattern obtained for the commercial DNAdehyd sample differs from that of the DNA pellets, as expected attending to the change in the hydration degree of the biomolecule.
These results, including complementary data regarding the lower frequency spectral range not previously probed, are in good accordance with the information formerly obtained (by EXAFS, FTIR, Raman and INS) for DNA´s purine bases upon cisplatin binding. 13,23 In addition, valuable information wa s gathered on a different interplay with the nucleic acid regardi n g the dinuclear agents Pt2Spm and Pd2Spm as compared to the mononuclear (clinically used) drug cisplatin. EXAFS. EXAFS/XANES analysis provided a molecular picture of drug-DNA interplay for Pt2Spm and Pd2Spm, specifical l y regarding their interaction with DNA purine bases recognized to be the main binding sites for cisplatin-like agents. The major goal of the proposed study was attained: to unequivoca l l y determine the local environment of the absorbing Pt(II) and Pd(II) centres in the drugs´ adducts with adenine and guani ne (1:4), with a view to determine the precise first coordina ti o n sphere content in these entities. Additionally, interaction wi th glutathione was assessed, since this ubiquitous sulfurcontaining tripeptide can compete for the drug relative to its major pharmacological target (DNA), thus being implicated in acquired resistance to metal-based chemothera peuti c drugs . 28 The current experiments were performed for both the soli d samples and the aqueous solutions, the results thus obtai ned showing no significant differences in accordance with previ ous EXAFS measurements for the homologous cisplatin-adducts 13 . Regarding the effect of temperature , it was verified that the data at 90 K agreed well with that measured at room temperature, corroborati ng the stability of these spermi ne complexes under physiological conditions, a major requirem en t for their in vivo application as anticancer agents.
Interpretati on of the data was based on spectra meas ured for solid PtA4, PdA4, PtGS4 and PdGS4, taken as references for the metal centre with distinct first coordinati on spheres , respectively (4N) and (4S). EXAFS fit of these standards allowed us to optimise the bond length values and obtain the ampli tude (S0 2 ), energy shift (E0) and Debye-Wall er (σ 2 ) factors that were then used for fitting the metal adducts (displaying coordina ti o n environments with either equal or different ligands).
The parameters of the nearest coordination shell around Pd and Pt were obtained, namely the number and type of neighbour atoms, and their distance from the selected meta l ( Table 2). It was clearly observed that stoichiometric amounts of either adenine or guanine led to a complete coordinati on to the metal centres in both the Pt and Pd adducts -MA4 and MG4, displaying a (4N) environment per metal as opposed to (2N+2Cl ) in the free complexes (Figure 10 (A) and (B)). When comparing both purine bases as binding sites for the Pt2Spm and Pd2Spm agents, the symmetry was found to be significa ntl y higher for the Pt(II) adducts (reflected by the stronger intens i ty of the EXAFS signals). When comparing the Pt(II) and Pd(II) adducts with DNA´s purine bases, the latter displayed slightl y shorter Pd-N values in both the adenine and guanine systems (respectivel y 2.027±0.007 vs 2.039±0.005 and 2.035±0.006 vs 2.047±0.005 Å, Table 2). In addition, it should be noted that the  For each sample, the fit was performed on the first shell of the EXAFS signal with all parameters free (first row) and with coordination numbers fixed to expected values (second row). S0 2 : passive electron reduction factor; E0: energy shift; CNX: number of X atoms bond to the metal (either Pt or Pd); RX: M-X distances (M=Pt or Pd); σX 2 : X atom Debye-Waller factor.

ARTICLE Chemical Sciences
Pd-N bond lengths within PdA4 and PdG4 were also shortened relative to the Pt(NH3)4 reference sample previously measured 13 (2.039±0.004 Å). For the drug-gluta thi one (1:4) adducts, a (4S) first coordination shell was detected for each of the meta l centres (Figure 10  The slight shift of the edge (to higher values) in the XANES profiles observed for the distinct systems under study evidenced a variation in the degree of electron donation from the ligand atoms to the metal centre (Table 3). While this cha rge transfer process was similar for both Pd(II)-puri ne adducts differing only for PdGS4, for the Pt(II) systems this ligand-to-me ta l electron delocalisation was found to increase from the free complex ((2N+2Cl ) environment) to the PtA4/PtG4 ((4N)) and PtGS4 ((4S)) adducts, as expected attending to the increasi ngl y softer nature of the surrounding ligands. This fact has releva nt implications regarding the glutathione-i nduced resista nce associated to this type of metal-agents . Furthermore, the marked intensity increase detected for PtA4 and PtGS4 (Fi gure 10(C)) reflects a significantly lower symmetry of these speci es as compared to PtG4 (and to the free drug), since this pre-edge intensity is strongly dependent on the size and geometry of the molecular cage surrounding the metal absorber. For the Pd(II) systems, in turn, this symmetry loss was hardly noticea bl e, which may suggest a more labile coordinati on of this cati on relative to Pt(II), the latter displaying a clear preference for guanine -yielding PtG4 stable adducts with a defined and qui te rigid geometry.    Apart from the purine and glutathione adducts of Pt 2 Spm and Pd2Spm, the drug´s preference for glutathione versus DNA binding sites (purine´s N7 atoms) was assessed. For the PdA4 and PdG4 systems, the presence of glutathione (1:4) was found to lead to a N by S substitution, a (4N) metal coordina ti o n sphere giving rise to ca. (3N+1S) and (4S) patterns for the meta l centres within the adenine and guanine adducts, respecti vel y ( Figure 11(A), Table 2). This clearly evidences a lower stability of the Pd2Spm-gua nine adducts, for which there was a tota l substitution of the nitrogen ligand atoms by sulfur, leading even to a disruption of the Pd2Spm complex (Pd(II) being deta ched from the spermine NH2 ligand moieties, at both metal centres ). Interestingl y enough, this is contrary to what had been previously found for the mononucl ear cisplatin-A4 and cisplati n-G4 systems, the latter having shown to be stabilised 13 (as expected, through an intramolecular NHdrug … O=Cg uani ne hydrogen clos e contact). A possible justification for the behaviour presentl y found for the dinuclear spermine agents may be the less favourable H-bond between their amine groups and the guani ne´s carbonyl as compared to cisplatin, coupled to a higher steri c hindrance between the Pd2Spm-bas e adducts and glutathi on e on account of the bulkier polyamine ligand and the presence of two metal centres. The Pt2Spm-base adducts, in turn, revea l ed a quite distinct behaviour relative to their Pd homologues in the presence of glutathione, both the adenine and guanine systems displaying a similar degree of S-substituti on yielding an approxima te (1N+3S) shell (Figure 11(B)). Overall, PdA4 wa s shown to be the most stable adduct relative to glutathi on e competition, only one nitrogen ligand having been substituted by sulfur. These results were confirmed by the XANES linea r combinati on fits, comprised in Table 3. Upon GSH coordina ti o n to the drug adducts, the M-S bond lengths were found to be identical for all systems under study (2.31 to 2.33 Å, Table 2). Moreover, S-binding did not affect the metal-nitro ge n coordinati on within the adducts, the M-N values being equa l for PtA4+GSH, PtG4+GSH and PdA4+GSH (2.03 Å, Table 2).
Glutathione competition for the drugs, well justified by the higher affinity of Pt(II) and Pd(II) for GSH´s sulfur over adeni ne´s and guanine´s nitrogens, has been previously found for cisplatin 13 and constitutes experimental proof of glutathi on emediated drug inactivation (underlyi ng acquired resista nce) also for the dinuclear agents presently investigated.

Conclusions
The dinuclear complexes Pt2Spm and Pd2Spm have been studi ed by the authors in the last few years and have been shown to act as promising anticancer agents towards human metasta ti c breast cancer 7,14,20,22,23 and osteosarcoma 24 . A multidisci pl inary study was presently carried out to attain complementa r y structural and dynamical information on their interplay wi t h DNA (their main pharmacol ogi cal target), using QENS measurements, SR-FTIR and SR-EXAFS/XANES.
The QENS experiments currently performed provided accurate and unique data on the impact of metal-based antica ncer drugs on DNA via the biopolymer´s first hydration layer, whi ch is known to be closely coupled to DNA function. Indeed, any drug-triggered perturbati on on the nucleic acid´s hydra ti on shell is expected to influence the biomolecule´s dynami ca l profile and consequentl y its biological function. Measurem en ts for drug-treated and untreated H2O-hydra ted DNA, at room temperature, revealed a clear effect of both cisplatin and Pd2Spm on the nucleic acid´s hydration shell dynami cs , reflected in an increased flexibility for the drug-incuba te d samples. Two dynamical processes were discriminated (in the ps timescale), ascribed to: (i) the biopolymer´s hydration water; (ii) fast localized rotations of specific groups within DNA -CH3, as well as NH2 not restricted by hydrogen-bonds . The intrins i c stiffness of native DNA appears to be disrupted by the drug, underlining the influence of the water molecules from the first hydration sheath. It should be emphasized that this effect wa s only observed (in the ps timescale) for H2O-hydra ted DNA and not for the D2O-hydra ted nucleic acid, which also evidenced distinct dynamical transition temperatures . Apart from the hydration-i nduced flexibility increase, an electrosta ti cmediated dynamic enhancement was unveiled for drugexposed H2O-hydra ted DNA. These conclusions are in good agreement with the previously reported drug impact on the intracellular milieu -cytoplasmic water and biomolecul es´ hydration layers: 25 while the former showed a restrai ned dynamics upon drug exposure, the hydration shells were prompted into a more mobile state, as presently corrobora te d for DNA. These combined results support the pronounced effect of metal-based chemotherapeuti c agents on water dynami cs , and should help to gain a more thorough understandi ng of the drug-induced cytotoxicity via the water molecules both in the intracellular medium and in the close vicinity of biopolymers. In addition, an interesting onset of anharmoni city was detected for dehydrated DNA (in the ps timescale) in contrast wi th previous studies on dehydrated biopolymers (e.g. proteins, RNA and DNA), and it was tentatively assigned to rotational moti ons of methyl groups (1 per 4 bases within the double helix) and, to a smaller extent, of H-bond free NH2 moieties.
Infrared spectroscopy with synchrotron radiation wa s confirmed as a powerful non-invasive molecular probe of biosamples, allowing to interroga te and obtain an accura te insight on the biochemical impact of chemothera py drugs at the cellular level. Following previous studies on the effect of the presently tested compounds in cancer cells, 23 their impact on DNA was currently probed, for DNA samples extracted from Please do not adjust margins Please do not adjust margins treated and untreated human MDA-MB-231 cells. The twof ol d purpose of this study was accomplished, since the simultaneo us detection of the drug and its pharmacol ogi cal target wa s achieved, specific spectral biomarkers having been identif i ed reflecting changes in both the metal agent and DNA, prompte d by drug-target interaction. Furthermore, the vibrational modes comprised in the far-infrared region, which are a spectral signature of the nucleic acid conformati onal state, allowed to unvei l drug-elicited perturbati ons in this biomolecule recognized to be the basis of cytotoxici ty: an altered pattern was detected for the bands ascribed to DNA breathing, as a consequence of a drugtriggered disruption of the double helix H-bonded base-pai rs .
To the best of the authors´ knowledge, this is the first experimental observation of the DNA breathing process in the presence of a chemotherapeutic agent. Additionally, analysis of the variations occurring in the characteristic bands of the complexes provided evidence of structural variations upon accumulation at the biological target. Moreover, sampling DNA extracted from drug-expos ed human cancer cells enabled to qualitatively probe the drug´s cellular uptake and its activati on process (through intracellular chloride hydrolysis) prior to DNA interaction, apart from its impact on the nucleic acid upon binding to the purine bases. These results could only be successful l y achieved through FTIR spectroscopy with a coherent synchrotron radiation that guarantees both broadband spectra l coverage and a high signal quality. The SR-FTIR results presentl y gathered complement previous SR-based infrared data obtained at the MIRIAM beamline for the same compounds and cell line (at equal concentrati ons and incubation time): 23 comparing these results on whole cells with those measured for DNA isolated from cells pre-incubated with the chemothe rapeutic agents is of key biological significance for discrimina ti n g drug effects regarding other potential cellular targets (e.g. DNA vs proteins, membrane lipids or intracellular water). The successful EXAFS/XANES results gathered for the adenine, guanine and glutathione adducts with Pt2Spm and Pd2Spm allowed an accurate assessment of their pharmaco d ynamics (interaction with DNA) and pharmacoki netics : the loca l environment of the metal centres was obtained, for each system, and GSH-metal binding (solely via glutathione´s sulfur atom) was clearly evidenced thus justifying the glutathi on emediated drug resistance mechanisms occurring in vivo. A slightly weaker glutathione coordinati on was unveiled for Pd2Spm as compared to Pt2Spm, which may be relevant with a view to overcome in vivo GSH-media ted drug resistance. Additionally, EXAFS measurements for the adducts in the presence of glutathione revealed a lower stability of the Pd2Spm-gua ni ne adducts relative to their adenine homolog u es (contrary to what had been previously found for cisplatin 13 ), while their Pt counterpa rts showed to have an identical stabili ty to the mononucl ear clinical drug. Coupled to the data previous l y reported for cisplatin, 13 these results provided accura te information on the chemical composition, structure and relati ve stability of the adducts formed between the Pd2Spm and Pt2Spm complexes and DNA purine bases, leading to a better understanding of the mode of action of this type of polynucl ea r metal agents at the molecular level.
The present multidisciplinary study on the impact of cisplati n and cisplatin-like dinuclear agents on DNA´s dynamical and structural profiles constitutes an innovative way of tackling a drug´s mode of action. A comprehensi ve and reliable set of data was gathered on the molecular basis of their cytotoxicity towards the very low prognosis human metastatic breast cancer, allowing to better clarify the unconventi onal interaction of Pt2Spm and Pd2Spm with their main target. Combined with biological assays for evaluation of antitumor activity, the current results are expected to provide valuable clues for the rationa l design of metal-based compounds with improved therapeuti c properties, acting through a multistep process that leads to function loss of vital biomolecules and ultimately to cell death: (i) direct binding to DNA, causing disruption of its native conf ormation and prompti ng biofunctional disability; (ii) continui n g effect on the nucleic acid´s hydration layer, which is prompte d into a faster dynamics, triggering changes in the biopol y me r with consequences at the functional level; (iii) impact on intracellular water (cytosol), with an expected global effect on essential cellular components thus hindering normal cellular functi on.

Conflicts of interest
There are no conflicts to declare.