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Cite This: Inorg. Chem. 2017, 56, 12938-12951

Elucidation of Binding Site and Chiral Specificity of Oxidovanadium Drugs with Lysozyme through Theoretical Calculations Giuseppe Sciortino,#,§ Daniele Sanna,† Valeria Ugone,§ Giovanni Micera,§ Agustí Lledós,# Jean-Didier Maréchal,*,# and Eugenio Garribba*,§ #

Departament de Química, Universitat Autònoma de Barcelona, Cerdanyola del Vallés, 08193 Barcelona, Spain Dipartimento di Chimica e Farmacia, Università di Sassari, Via Vienna 2, I-07100 Sassari, Italy † Istituto CNR di Chimica Biomolecolare, Trav. La Crucca 3, Baldinca-Li Punti, I-07040 Sassari, Italy Downloaded via UNIV OF TOLEDO on June 29, 2018 at 14:14:58 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

§

S Supporting Information *

ABSTRACT: This study presents an implementation of the protein−ligand docking program GOLD and a generalizable method to predict the binding site and orientation of potential vanadium drugs. Particularly, theoretical methods were applied to the study of the interaction of two VIVO complexes with antidiabetic activity, [V IV O(pic) 2 (H 2 O)] and [V IV O(ma)2(H2O)], where pic is picolinate and ma is maltolate, with lysozyme (Lyz) for which electron paramagnetic resonance spectroscopy suggests the binding of the moieties VO(pic)2 and VO(ma)2 through a carboxylate group of an amino acid residue (Asp or Glu). The work is divided in three parts: (1) the generation of a new series of parameters in GOLD program for vanadium compounds and the validation of the method on five X-ray structures of VIVO and VV species bound to proteins; (2) the prediction of the binding site and enantiomeric preference of [VO(pic)2(H2O)] to lysozyme, for which the X-ray diffraction analysis displays the interaction of a unique isomer (i.e., OC-6−23-Δ) through Asp52 residue, and the subsequent refinement of the results with quantum mechanics/molecular mechanics methods; (3) the application of the same approach to the interaction of [VO(ma)2(H2O)] with lysozyme. The results show that convenient implementation of protein−ligand docking programs allows for satisfactorily reproducing X-ray structures of metal complexes that interact with only one coordination site with proteins and predicting with blind procedures relevant low-energy binding modes. The results also demonstrate that the combination of docking methods with spectroscopic data could represent a new tool to predict (metal complex)−protein interactions and have a general applicability in this field, including for paramagnetic species.

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INTRODUCTION

complementarity between the metal drug and the biological “receptors” at the molecular level. However, the application of these techniques is not always possible, and looking to other methodologies to shed some light on how the metallodrugs bind to biological target(s) is necessary. When thinking about paramagnetic vanadium(IV) compounds, one of the spectroscopic approaches to decode metallodrug−protein interactions is electron paramagnetic resonance (EPR). Over the last years, several studies that combined EPR and computational methods, such as molecular mechanics (MM), quantum mechanics/molecular mechanics (QM/MM), molecular dynamics (MD), and density functional theory (DFT), were published.3−5 For VIV the value of the hyperfine coupling constant, Aiso or Az, can be correlated with the type and number of atoms that coordinate VIVO2+ ion in the equatorial plane by using either the empirical “additivity

In recent years, the development of vanadium-containing drugs became a vivid field of research. Vanadium compounds have shown interesting pharmacological properties and have been proposed as antidiabetic, antiparasitic, spermicidal, antiviral, anti-HIV, antituberculosis, and antitumor potential drugs.1 In particular, bis(maltolato)oxidovanadium(IV) (or BMOV) became the benchmark compound for the new molecules with insulin-mimetic action.2 The discovery and design of novel vanadium derivatives could open major avenues in medicinal inorganic chemistry.1 However, the development of such compounds as drugs is often limited by the lack of knowledge on their interaction with the bioligands of organism, particularly with proteins. In fact, these interactions can influence the transport, uptake, and mechanism of action of the metal compounds with pharmacological action. In this task, X-ray diffraction (XRD) analysis and spectroscopic techniques are of fundamental importance, as they give a precise description of the © 2017 American Chemical Society

Received: July 7, 2017 Published: October 6, 2017 12938

DOI: 10.1021/acs.inorgchem.7b01732 Inorg. Chem. 2017, 56, 12938−12951

Article

Inorganic Chemistry rule”6 or comparative quantum mechanical calculations.7 EPR spectroscopy provided strong experimental evidence of the interaction of VIVO compounds with proteins,8 such as apotransferrin (apo-hTf),9 holo-transferrin (holo-hTf),10 albumin (HSA),11 immunoglobulin G (IgG),12 and hemoglobin (Hb).13 However, EPR gives insights only on the type of amino acids (usually His, Asp, or Glu) that directly interact with the metal but nothing about the exact binding site of the polypeptide chain (in other words, with EPR it is possible to distinguish the binding of a His residue from that of Asp or Glu but not to identify the specific residue involved in the coordination) or about the possible stabilization of the structure through second coordination sphere interactions. This information is fundamental for optimizing V therapeutic agents and studying their biotransformation in the organism. At the beginning of this work, we wondered if EPR data could be combined with computational tools to provide a complete three-dimensional (3D) representation of a protein− (metal complex) interaction. In this regard, molecular modeling has become a major element in drug design pipelines, and protein−ligand14 dockings are probably the techniques that constitute the cornerstone of the field. Docking methods are used to generate fast and accurate enough predictions of small molecules binding orientations into biomolecules and predict both affinities and geometries, even if standardized methods for transition metal complexes have not been set up yet.15 Some of us recently showed the potential of docking algorithms in dealing with metallodrugs when no changes of the first coordination sphere of the metal occurs during binding.16 Nonetheless, the interaction of a metallodrug can also take place with the formation of one or various coordination bonds between the metal and donor atoms of amino acid sidechains.1,8c,17−19 Some commercial docking suits, like GOLD,20 AutoDock,21 Glide,22 HADDOCK,23 or FlexX,24 offer the possibility to simulate systems in which a metal is part of the receptor. Until now, however, standardized methods were not optimized when the metal was part of the ligand, even if this problem could be dealt by using covalent approaches implemented in current software20,21,25 or code modification such as CovalentDock,26 or Docktite.27 In these approaches the formation of the covalent or coordination bond is forced a priori through energetic restraints for a predefined protein− (ligand donor) pair and generally limited to specific bond distances and angles. These schemes are poorly predictive: the limitation is that restraint values are generally empirical, and the scoring function parameters are not optimized for the specific interaction under investigation (in particular, coordination bond parameters are lacking). Our experience showed us that a convenient optimization process of the docking parameters could result in the possibility to take into account direct interactions between a metallodrug and protein binding site(s). Such an approach could be validated only if combined with spectroscopic data such as those collected by EPR analysis. As a consequence, here we present a mixed computational−experimental study, combining EPR data with protein−ligand docking, which allowed us to predict the 3D structure of (VIVO complex)−protein adducts. This work presents a novel series of optimized parameters for vanadium-containing ligands specifically designed for this problem. The new parameters were successfully tested on four VV−protein and one VIVO−protein structures (PDB codes: 2p8o,28 1rpt,29 1b8j,30 3i80,31 and 4c3w32). Our showcase protein receptor is the hen egg white lysozyme

(Lyz), frequently used as a model for the study of (metal complex)−protein interaction, for example, with species formed by Pt33 and Ru.34 In particular, we tried to reproduce the X-ray structure of the adduct formed by Lyz and [VIVO(pic)2(H2O)], where pic indicates picolinate anion, recently reported in the literature (PDB: 4c3w).32 The results were subsequently refined and further validated through QM/ MM methods. After benchmarking our computational tools, we applied the approach to predict the structure and stereospecificity in the lysozyme binding of [VIVO(ma)2(H2O)], where ma is maltolate.2 We believe that the approach here reported, which starts from the spectroscopic data (in this case EPR) and combines them with the docking and QM/MM methods, is easily generalizable to other metal complexes and metallodrugs and could open major avenues in structural drug design and in the study of metal−protein and (metal complex)−protein interactions.

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COMPUTATIONAL AND EXPERIMENTAL METHODS

Computational Section. Computational Approach. The computational part of this work stands on (1) optimization of isolated metal complexes (ligands in the docking terminology14) using DFT; (2) preparation of the metal binding site for docking experiments to explore the direct interaction of the V drug to the protein; (3) blind docking without any constraint of the V compounds to Lyz using a customized version of GOLD 5.2,20,35 that accounts for the formation of coordination bonds during the docking process. Metal-Containing Ligand Preparation: DFT Optimization. The structures of cis-[VO(pic)2(H2O)] and cis-[VO(ma)2(H2O)] were optimized with Gaussian 09 software36 in the gas phase through DFT methods at the level of theory B3P86/6-311g.37 The H2O ligand (which occupies the fourth equatorial site in the complexes) was removed, and a fictitious (dummy) hydrogen atom was added to mimic the ability of vanadium to interact with a Lewis base (that, in our case, is a side chain protein donor) by hydrogen bond-like interactions and to reproduce the directionality of the V−(Lewis base) coordination bond. Protein−(Metal-Containing Ligand) Docking. Once we obtained a series of optimized parameters for VIVO complexes (discussed in detail in the first section of Results and Discussion), all the calculations reported in this manuscript were performed on the lysozyme structure (PDB: 4c3w32). The set up of the receptor, including the addition of hydrogen atoms on the titratable residues, was performed with the UCSF Chimera program. The dockings were performed with an evaluation space defined by a 16 Å radius sphere centered on the Oγ oxygen of the Asp52 residue, which includes almost entirely the protein space. Genetic algorithm (GA) parameters were set with a number of GA runs equal to 100 and a minimum of 100 000 operations. The rest of the parameters, including pressure, number of islands, niche size, crossover, mutation, and migration were set to default. The docking solutions were analyzed by means of GaudiView, an in-house graphical interface developed as an extension for UCSF Chimera.39 QM/MM Refinement. All QM/MM calculations were performed with ONIOM method implemented in Gaussian 09. The structures were optimized using the B97D functional and the 6-311g(d,p) basis set. The Amber force-field was used for the MM part, including additional parameters for the metal complexes computed by Seminario method. 40 Gasteiger charges were computed through UCSF Chimera38 for the protein and restrained electrostatic potential (RESP) charges through antechamber for the complex and the amino acid involved in the bond. The side chains of the amino acids close (∼5 Å) to the metal complex were set as flexible (see Supporting Information). Experimental Section. Chemicals. Water was deionized prior to use through the purification system Millipore Milli-Q Academic. Hen 12939


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Inorganic Chemistry Table 1. Docking Results for VIV and VV Structures, Used for the Validation of the Method with GOLD 5.2 with the Implementation Described in the Text donor

V−Xcalcd a

V−Xexptl a

RMSDb

Fmaxc

Smax(hbond_ext)d

Smax(vdW_ext)e

Popf

O (Ser195) N(His12) O−(Ser102) S−(Cys215) γ-COO−(Asp52)

2.278 2.367 1.865 2.401 2.066

2.030 1.966 1.721 2.514 1.885

0.163 0.465 0.555 0.709 1.035

55.38 28.37 41.62 36.20 49.58

20.45 18.58 36.32 31.39 13.89

24.03 6.92 3.86 4.21 27.46

100/100 100/100 100/100 100/100 58/100

structure 28

2p8o 1rpt29 1b8j30 3i8031 4c3w32

−

a Distance in angstroms. bThe RMSD value (in Å) was obtained from the simple docking calculation in which the metal complex was redocked to the protein using its relative coordinates extracted from the PDB structure. cFitness value of the GoldScore scoring function (see eq 1); for each structure the scoring of the most stable pose is reported. dScoring term related to the intermolecular hydrogen bonds. eScoring term related to the intermolecular van der Waals interactions. fNumber of solutions in the identified cluster.

egg white lysozyme (Lyz) was purchased from Sigma (molecular mass of 14.3 kDa). Other chemicals, that is, VOSO4·3H2O, picolinic acid, maltol, 1-methylimidazole (MeIm), and 4-(2-hydroxyethyl)piperazine1-ethanesulfonic acid (HEPES), were Aldrich products of the highest grade available and used as received. EPR Measurements and Prediction of 51V Hyperfine Coupling Constants. EPR spectra were recorded on frozen solutions at 120 K with an X-band (9.4 GHz) Bruker EMX spectrometer equipped with an HP 53150A microwave frequency counter. The microwave frequency used for EPR measurements was in the range of 9.40− 9.41 GHz; microwave power was 20 mW, time constant 81.92 ms, modulation frequency 100 kHz, modulation amplitude 0.4 mT, resolution 4096 points. To extract spin Hamiltonian parameters, the spectra were simulated with WinEPR SimFonia software;41 in the simulations rhombic symmetry was assumed in agreement with DFT optimization and X-ray crystallographic analysis of the structures. The solutions were prepared dissolving in ultrapure water VOSO4·3H2O to obtain a VIVO2+ concentration in the range of (0.3−1.0) × 10−3 M. Argon was bubbled through the solutions to avoid the oxidation of VIVO2+ ion. To the solution containing the metal ion, the ligand L (pic or ma) and HEPES (1.0 × 10−1 M) were added; the molar ratio L/ VIVO2+ was 2. Subsequently, to 1 mL of this solution Lyz was added, pH was adjusted to ca. 4.5 or 7.4, and the EPR spectra were immediately recorded. The model systems VIVO2+/L and VIVO2+/L/ MeIm were previously examined in the literature.9 The 51V hyperfine coupling constants (A) of the isomers of [VOL2(H2O)], [VOL2(PrO)]−, and [VOL2(MeIm)], where L = pic or ma, PrO− = propionate anion, and MeIm = 1-methylimidazole, were calculated with Gaussian 09 using the functional BHandHLYP and the basis set 6-311g(d,p) according to previously published procedures.7

considered as a hydrogen bond donor that interacts with the protein hydrogen bond acceptor side chains. The computational procedure consists of favoring the protein binding to the metal complex through the addition, in the coordination vacancy left by the water ligand, of a fictitious hydrogen, a fundamental artifact in the GOLD architecture and, more in general, in the docking methods.26,43 It is important to highlight that this dummy hydrogen serves only to allow the metal ion to form a standard hydrogen bond with the Lewis bases of protein and to ensure the directionality of the binding or, in other words, to “activate” the metal−protein interaction. Transforming this concept into GOLD software implied two steps: (1) the introduction in the GOLD parameter libraries of a series of novel atom types for vanadium and the atoms that can interact with it, like the keto oxygen of maltol (listed in Supporting Information) and (2) the implementation of the corresponding “coordination” energetic terms in the GoldScore scoring function. Principally, the hydrogen bond (hbond) donor parameters were optimized, including the distance between the metal and the hydrogen that acts as a dummy atom. As a result, the metal interaction with the protein can be evaluated through the intermolecular hydrogen bond term of the scoring function. The capability in prediction of the method was evaluated through five different criteria: (a) the scoring (Fitness F of GoldScore) associated with each pose, as reported in eq 1; (b) the energy of the hbond formed, as shown in eqs S1−S3 of Supporting Information;20,35 (c) the root-mean-square deviation (RMSD) value of each docked pose calculated on the heavy atom, as reported in eq S4 of Supporting Information; (d) the population of the cluster containing the better docked structure, and (e) the ranking of the clusters.

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RESULTS AND DISCUSSION 1. Establishing the Docking Strategy for the Binding of VIVO Derivatives to Proteins. As protein−ligand docking programs, including GOLD, do not provide parameters that explicitly consider the interaction of metal-containing ligands with protein amino acids throughout a metal−protein coordination bond, an effort in atom typing and parametrization was necessary at the beginning of our study. To the best of our knowledge, it represents the first extensive attempt to generate coordination scoring parameters into docking programs. As the rest of the present manuscript would not stand without it, we present here briefly the ground of our approach and its validation. The reader could refer to the Supporting Information for further details. We adapted GOLD software to the particular needs of predicting the coordination of a metal complex to a protein by expanding some of our previous intents,42 which consist fundamentally in mimicking the ability of metals to interact with Lewis bases amino acid side chains (i.e., Glu, Asp, His) by mean of a hydrogen bond-like interaction, one of the cornerstones in docking scoring functions. The metal is

ext ext int int F = α ·S hbond + β ·SvdW + γ ·S hbond + δ·(SvdW − Stors)

(1) ext where Sext hbond and SvdW are the scoring terms related to the hbond and van der Waals (vdW) intermolecular interactions, int Sint hbond and SvdW represent the intramolecular interactions, Stors evaluates the change in stability due to the molecular torsions, and α, β, γ, and δ are empirical parameters optimized to weigh the different interactions. 2. Validation of the Method. To test the new parameters and validate them, we tried to reproduce five structures where VV and VIV species form only one coordination bond with a protein donor: VVO3(benzohydroxamate) bound to chymotrypsin A with the coordination of a Ser-O−(PDB: 2p8o28); VVO4 moiety bound to acid phosphatase, alkaline phosphatase, and tyrosine phosphatase, which give a trigonal bipyramidal arrangement with the axial binding of a His-N, Ser-O−, and

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Inorganic Chemistry

Figure 1. Superimposition of the calculated (yellow) and X-ray (green) structures for VV-protein and VIV-protein adducts: (a) VVO4 moiety bound to acid phosphatase (PDB: 1rpt); (b) VVO4 moiety bound to alkaline phosphatase (PDB: 1bj8); (c) VVO4 moiety bound to tyrosin phosphatase (PDB: 3i80); (d) VVO3(benzohydroxamate) bound to chymotrypsin A (PDB: 2p8o); and (e) VO(pic)2 moiety bound to lysozyme (PDB: 4c3w). In all the cases the protein and the metal species used for the blind redocking were considered as two separated rigid structures with the bond lengths and angles being the same as in the X-ray structure.

Cys-S− donors (PDB: 1rpt,29 1b8j,30 and 3i80,31 respectively); the unique structure for a VIVO species, in which the cisVO(pic)2 moiety is bound to lysozyme through an Asp-γCOO− donor to yield a cis-octahedral geometry (PDB: 4c3w32). The validation was performed through simple docking calculations: the XRD relative coordinates of the metal complex were extracted, randomized, and used as input to perform a blind redocking with the protein whose coordinates were not changed. In other words, the protein and the metal-containing ligand (i.e., the metal complex) were placed at “infinite” distance and were blind docked without imposing any restraints: in this manner, it is possible to assess the viability of this method to reproduce an experimental X-ray crystallographic structure. Our GOLD calculations with the GoldScore scoring function and our set of optimized parameters led to low-energy docking solutions with reasonably predicted binding affinities (Fmax in the range 28.4−55.4, Table 1). The calculated binding modes are in good agreement with the experimental binding orientation, and the RMSD between the best-predicted pose and the experimental coordinates is, for all the structures, lower than 1.035 Å, a very satisfactory value considering that in the literature a prediction is considered successful if a deviation below 3.0 Å is obtained.44−48 In addition to the agreement between the experimental and calculated geometries and the good value predicted for the binding affinity, the solutions close to the X-ray structures represent more than ca. 60% of those identified in the docking assay. This shows that, more than an accident, the docking methods identify a clear minimum associated with the structure experimentally observed. Comparisons between the experimental X-ray and calculated structures using the protocol described above are shown in Figure 1. The fact that the docking calculations allowed us to predict the structure of the five V-protein species, characterized by the monodentate coordination of four different side-chain donors, such as Ser-O−, His-N, Cys-S−, and Asp-COO−, means that the

method works independently of the protein donor involved in the metal binding and oxidation state of the metal, and that the parameters are well-balanced and implicitly take into account the difference in basicity of such donors. The results reached with the five V compounds are further supported by the successful prediction by blind docking simulations of the X-ray structures of other (metal complex)−proteins with the monodentate binding of an amino acid residue to several metal species (Mg, Cr, Mn, Cu, Fe, Co, Ni, Cu, Zn, Ru, Rh, Re, Os, Pt, and Au);49 for example, the X-ray structure of CuII(Sal-Leu)(apo-Mb), where Sal-Leu is Nsalicylidene-L-leucinato and apo-Mb is apo-myoglobin (PDB: 2eb950), was reproduced with an RMSD value of 0.431 Å.49 This demonstrates that the method is generalizable and applicable, in principle, to any (metal complex)−protein system in which the protein binds the metal with only one donor. In the next section we will discuss how the developed docking method with the new parameters could be applied to systems for which only spectroscopic information is available. 3. Predicting Isomeric and Chiral Discrimination of [VO(pic)2(H2O)] Binding to Lysozyme in the Absence of X-ray Diffraction Analysis. In most of cases the X-ray determination of a (metal complex)−protein system is not possible, and only spectroscopic data are available. Someone could wonder which results would be obtained if the XRD analysis was lacking and if one would like to predict the interaction of a metal species with a specific protein with known structure. In other words, someone could wonder if the low-energy mode of binding could be obtained only with theoretical calculations. We could start with a hypothetical exercise and imagine that the X-ray structure of VO(pic)2−Lyz adduct was not known. Therefore, the approach used could be as follows: (i) collect some instrumental data (EPR in this case) to evaluate if an interaction between the metal complex and the protein exists and if the spectroscopy can suggest the specific donor involved in the metal binding; (ii) examine all the possible isomers and enantiomers of metal species taking into 12941


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Inorganic Chemistry

the “additivity rule”,6 an Asp-γCOO− or a Glu-δCOO− should cause an increase of Az of ∼3 × 10−4 cm−1 in comparison with a His-N, in agreement with the experimentally observed change. All the spectra were simulated with WinEPR SimFonia software to extract spin Hamiltonian parameters, which are listed in Table S1 of Supporting Information; the simulations of the spectra were reported in Figures S1−S4. Therefore, the EPR spectra indicate that cis-VO(pic)2 moiety interacts with Lyz through a direct coordination bond between the metal and a negatively charged oxygen donor of a carboxylate group. These results must be considered as the starting point on which is built the subsequent computational study. As for any other bis-chelated VIVO species, for [VO(pic)2(H2O)] three structural variations, depending on the presence or absence of a coordinated solvent molecule (water in this case) and its relative position in the first coordination sphere of the metal, are virtually possible: cis-octahedral, transoctahedral, and square pyramidal arrangement.54,55 Computational and structural analyses of a large data set of compounds formed by picolinate derivatives have shown a clear preference for the cis-octahedral isomers,11b,54,56−58 and, among the four possible geometrical isomers of cis-[VO(pic)2(H2O)], that is, OC-6−23, OC-6−24, OC-6−42, and OC-6−44 (Scheme 1),59 those with two equatorial nitrogen atoms are strongly favored (OC-6−23 and OC-6−24), each of them with their respective Δ and Λ enantiomers. However, experimental and computational data indicate that the difference in the Gibbs energy of OC-6−23 and OC-6−24 is small, and they are likely in equilibrium in aqueous solution.11b,54 In the next step, all the most stable isomers of cis[VO(pic)2(H2O)] (OC-6−24-Δ, OC-6−24-Λ, OC-6−23-Δ, and OC-6−23-Λ, Scheme 1) were optimized through DFT calculations with the functional B3P86, the equatorial water molecule was removed, and each energy minimum was subsequently blind docked to the lysozyme structure (PDB: 4c3w32), using our optimized parameters for vanadium (see Supporting Information). In other words, the moiety cisVO(pic)2without the weak water ligand that leaves an empty equatorial siteis left free to explore the proteic space and interact with lysozyme. It is important to highlight once again that no restraints were used in the calculation and that the moiety cis-VO(pic)2 could interact, in principle, with any side chain donor of the protein. The best result, obtained with the OC-6−23-Δ isomer, is shown in the animated video in the Supporting Information. In general, all the lowest-energy solutions of the four isomers have good GoldScore values (from 33.5 to 48.3 units, Table 2). Most of the 100 solutions generated for each isomer show a direct interaction between the metal and one amino acid side chain of the protein. However, only two residueswhich act as Lewis basesare predicted to bind the metal throughout: one of them is Asp52 (γ-COO− group), and the other is Glu35 (δCOO− group). Observe that our docking is consistent with the EPR results, which indicate that with lysozyme the binding of the metallodrug does not occur with the only one residue of histidine (His15). In contrast, up to now, spectroscopic evidence has shown that cis-[VIVOL2(H2O)] complexes interact with proteins such as apo-hTf, holo-hTf, HSA, Hb, and IgG forming adducts cis-VIVOL2(Protein) with the direct coordination to the metal of nitrogen atom of an accessible His residue.9−13 In all the cases, the docking binding modes present the VIVO complex in the same binding pocket as the one observed in the

account all the information available in the literature; (iii) optimize by DFT methods the structures of all the enantiomers that could interact with the protein; (iv) perform blind docking calculations with the new series of parameters developed for GOLD program. At the end of the study it is obviously desirable that the structure obtained is close to that existing in the real biological system (not known a priori in this hypothetical exercise). [VIVO(pic)2(H2O)] is one of the most promising V drugs with potential antidiabetic application,51 and the study of its interaction with the proteins of blood or cells is fundamental to understand the biotransformation in the organism, the uptake by the target cells, and the mechanism of action.52,53 As pointed out in the literature,33,34 hen egg white lysozyme can be taken as a model protein to examine such an interaction. EPR spectrum recorded in the system VIVO2+/pic/Lyz is shown in trace b of Figure 2, where it is compared with those of

Figure 2. High-field region of anisotropic X-band EPR spectra recorded at 120 K on aqueous solution containing (a) VIVO2+/pic 1/2 at pH 4.5 (VIVO2+ 1.0 × 10−3 M); (b) VIVO2+/pic/Lyz 1/2/8 at pH 4.5 (VIVO2+ 4.4 × 10−4 M); (c) VIVO2+/pic/MeIm 1/2/4 at pH 7.4 (VIVO2+ 1.0 × 10−3 M), and (d) VIVO2+/pic/IgG 1/2/1 at pH 7.4 (VIVO2+ 3.0 × 10−4 M). The MI = 7/2 resonances of the VIVO species are indicated as follows: with I cis-[VO(pic)2(H2O)] with equatorial water-O coordination, with II cis-VO(pic)2(Lyz), with III cis[VO(pic)2(MeIm)] with equatorial imidazole-N coordination, and with IV cis-VO(pic)2(IgG) with equatorial His-N coordination. The position of the MI = 7/2 resonance of cis-VO(pic)2(Lyz) is shown with an arrow. The weak shoulder indicated with IIa in the trace d could suggest the coordination of a COO− group belonging to an Asp or Glu residue of IgG to VO(pic)2 moiety in cis-VO(pic)2(IgG).

the species cis-[VO(pic)2(H2O)] with the coordination (N, COO−); (N, COO−ax); H2O (I in Figure 2, trace a), [VO(pic)2(MeIm)] and cis-VO(pic)2(IgG) (III and IV in Figure 2, traces c and d), which have the same coordination mode, i.e. (N, COO−); (N, COO−ax); imidazole-N/His-N.12 The variation of Az of cis-VO(pic)2(Lyz) (163.3 × 10−4 cm−1) with respect to cis-[VO(pic)2(H2O)] (165.0 × 10−4 cm−1 54) and to the mixed species formed by MeIm and IgG (158.8 × 10−4 cm−1 and 160.0 × 10−4 cm−1 12) indicates without any doubt that, in contrast to other proteins,9−13 lysozyme does not bind the VIVO2+ ion with a His residue (see the position of MI = 7/2 resonances in Figure 2) but with a γ/δ-carboxylate group, stemming from an Asp or Glu residue. Indeed, on the basis of 12942


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Inorganic Chemistry Scheme 1. Possible Isomers/Enantiomers of cis-[VO(pic)2(H2O)]a

a

The more stable OC-6−23 and OC-6−24 isomers (with the corresponding enantiomers) with the two nitrogen atoms in the equatorial position are highlighted with the blue square.

Table 2. Docking Results of the Interaction of VO(pic)2 Isomers with Lysozyme Using GOLD 5.2 isomera

donor

dist V−Ob

Fmaxc

Smax(hbond_ext)d

Smax(vdW_ext)e

Popf

OC-6−23-Δ

γ-COO−(Asp52) δ-COO−(Glu35) δ-COO−(Glu35) γ-COO−(Asp52) δ-COO−(Glu35) γ-COO−(Asp52)g,h γ-COO−(Asp52)g,h γ-COO−(Asp52)h δ-COO−(Glu35) γ-COO−(Asp52)

1.978 2.273 2.185 1.935 2.012 2.170 2.023 2.017 1.965 2.066

48.33 44.19 42.97 37.44 37.89 34.68 34.39 33.46 43.40 37.31

14.11 15.15 15.02 14.46 15.05 14.74 12.88 7.61 15.03 14.29

26.25 21.16 20.34 22.07 16.64 16.15 16.78 19.41 28.83 16.74

98/100 2/100 98/100 2/100 10/100 7/100 15/100 10/100 71/100 15/100

OC-6−23-Λ OC-6−24-Δ

OC-6−24-Λ a

Isomer structure simulated with Gaussian 09. bDistance in angstroms (experimental V−O distance is 1.885 Å). cFitness value of the GoldScore scoring function (see eq 1); for each isomer the scoring of the most stable pose is reported. dScoring term related to the intermolecular hydrogen bonds. eScoring term related to the intermolecular van der Waals interactions. fNumber of solutions in the identified cluster. gIn the solution there are interatomic contacts between the metal complex and the protein. hThe three clusters reported for the isomer OC-6−24-Δ present a coordination bond V−γCOO−(Asp52), but they are rather different for the spatial orientation of the metal complex.

crystal structure 4c3w32 (shown in Figure S5 of Supporting Information). From an examination of the data, it can be also argued that OC-6−23-Δ presents the best affinity for lysozyme among the four possible isomers, both as scoring value (Fmax = 48.33) and population of the most stable cluster (98/100); furthermore, the preferred site is the Oγ of Asp52. Therefore, even in the absence of an X-ray structure, we should state that lysozyme interacts with cis-VO(pic)2 moiety, binding V through the carboxylate of Asp52 residue. The data also show that the isomers OC-6−23-Λ, OC-6−24Δ, and OC-6−24-Λ interact less favorably with Asp52 or Glu35 than OC-6−23-Δ (lower Fmax values, in the range of 33.5− 43.4). The capability of our docking approach to discriminate among the binding of the four isomers was further validated through QM/MM and non-covalent interactions (NCI) calculations (see the last paragraphs of this section). Now, our hypothetical exercise can be considered concluded, and the results obtained can be compared with the X-ray structure of the complex (Figure 3). In this structure cisVO(pic)2 is in a small and relatively exposed pocket with an equatorial coordination bond between V and Oγ of Asp52 that replaces the water ligand of the complex to give a distorted

Figure 3. X-ray structure of the adduct formed by lysozyme with cisVO(pic)2 moiety taken from the PDB structure 4c3w.32

octahedral geometry, the adduct being stabilized through electrostatic interactions with Glu35, Asn46, and Val109 residues. 12943


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To confirm the agreement between the docking calculations and EPR spectra, the 51V hyperfine coupling constants (A) were calculated by DFT methods. The calculations were performed on cis-[VO(pic)2(H2O)], cis-[VO(pic)2(PrO)]−, and cis-[VO(pic)2(MeIm)], where PrO− is propionate anion and MeIm is 1-methylimidazole, the two latter complexes representing a model for the equatorial coordination of a γ/δcarboxylate group from an Asp or Glu residue and of an imidazole nitrogen from a His residue. The results are reported in Table S2 of Supporting Information and show that, for OC6−23 isomer (which binds to lysozyme), the order of Az predicted[VO(pic)2(H2O)] < [VO(pic)2(PrO)]− < [VO(pic)2(MeIm)]is exactly that experimentally observed; that is, Az for VO(pic)2(Lyz) with an equatorial carboxylate bound to V is expected to be intermediate between [VO(pic)2(H2O)] and [VO(pic)2(MeIm)]. It is interesting to observe that our docking calculations indicate that cis-VO(pic)2 moiety preferentially binds to the protein in the form of OC-6−23-Δ isomer (O donor trans to water, Scheme 1) and not as one of the enantiomers OC-6−24 (N donor trans to water, Scheme 1), even if the two isomers should have a comparable stability in aqueous solution as demonstrated by DFT calculations.11b,54 This observation has two chemical implications: (1) the equilibrium in solution between OC-6−23 and OC-6−24 and their corresponding enantiomers (OC-6−23 ⇄ OC-6−24) can be shifted toward OC-6−23-Δ upon the interaction with the lysozyme and (2) the specific conformation of lysozyme allows the isomer OC6−23-Δ to interact preferentially with the residue of Asp52. To further validate the reliability of the docking approach and test the discriminative capabilities of our parameters, the most stable solutions of the docking runs (Table 2) were refined by QM/MM method. The ΔG for the reaction [VO(pic)2(H2O)] + Lyz ⇄ VO(pic)2(Lyz) + H2O was computed for all the solutions with the four isomers covalently bound with Asp52 or Glu35. The Gibbs energy in the gas phase (ΔGtotgas) was calculated as

If the structure of the (metal complex)−protein adduct, calculated using the DFT optimization of VO(pic)2, is compared with the X-ray diffraction analysis, it can be noticed that there is a good agreement (Figure 4). The final RMSD

Figure 4. Adduct formed by cis-VO(pic)2 moiety (coordinates optimized by DFT methods) with lysozyme (relative coordinates taken from X-ray diffraction analysis). The experimental X-ray structure (green) and the pose with the highest fitness value (Fmax) predicted by GOLD 5.2 for the DFT optimized isomer OC-6−23-Δ (light blue) are shown.

between the best pose and the experimental coordinates is satisfactory with a value of 0.788 Å (taking into account the differences between the crystallographic and the optimized coordinates of the free complex; for further details see Supporting Information). The predicted bond length V− O(Asp52) of 1.978 Å is very close to the experimental value (1.885 Å) with an absolute error of 0.093 Å. Concerning the other isomers with lower fitness values (Fmax) than OC-6−23Δ, OC-6−24-Λ shows the best affinity to δ-COO−(Glu35), while OC-6−23-Λ and OC-6−24-Δ have similar affinities for γCOO−(Asp52) and δ-COO−(Glu35), suggesting that these sites are equally valid from a coordination point of view. It is interesting to observe that these energetic trends, which suggest the binding of OC-6−23-Δ-VO(pic)2 to lysozyme, are also coherent with the cluster population of the entire set of 100 solutions generated in the docking (Table 2).

tot ΔGgas = ΔE ele + ΔGtherm

(2)

The obtained results are in good agreement with those based on pure scoring and population of GoldScore terms. The best

Figure 5. Gradient isosurfaces (s = 0.3 au) analysis of the better solution of (a) OC-6−23-Δ and (b) OC-6−23-Λ. NCIPlot surfaces show only the intermolecular interactions. The surfaces are reported in a blue-green-red scale according to values of sign(λ2) × ρ. Blue surfaces indicate strong attractive interactions (such as dipole−dipole or hydrogen bond), red indicates repulsion, while green means van der Waals interaction. For further details on NCIPlot the readers can refer to the Supporting Information. 12944


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ΔGtot gas value of the set is found for the isomer OC-6−23-Δ bound with γ-COO−(Asp52) (experimental solution). The other isomers have positive ΔG with γ-COO−(Asp52) and good values of ΔG with δ-COO−(Glu35) but less stable than that of OC-6−23-Δ bound to γ-COO−(Asp52). The affinity order, reported in Table S3 of Supporting Information, results: OC-6−23-Δ-VO(pic)2(Lyz-Asp52) (−63.8 kcal/mol) > OC6−24-Δ-VO(pic)2(Lyz-Glu35) (−57.6 kcal/mol) > OC-6−23Λ-VO(pic)2(Lyz-Glu35) (−55.6 kcal/mol) > OC-6−24-ΛVO(pic)2(Lyz-Glu35) (−41.3 kcal/mol) > OC-6−23-Δ-VO(pic)2(Lyz-Glu35) (−40.9 kcal/mol). Furthermore, the analysis of the energetic breakdown shows that the main contribution in the stabilization of the OC-6−23Δ complex interacting with γ-COO−(Asp52) is mainly due to van der Waals contacts (Table 2), a parameter that was not optimized in the generation of our metal atom parameters. In fact, the examination of the second coordination sphere interactions, performed with Non-Covalent Interactions Plot (NCIPlot),60 finds an elegant illustration for the different behavior of OC-6−23-Δ and OC-6−23-Λ enantiomers (Figure 5): only the Δ structure can coordinate the Oγ of Asp52 preserving the stabilization in the binding pocket through secondary interactions given by the lysozyme conformation (Figure 5a). The Λ isomer, due to the steric hindrance generated by the orientation of the picolinate ligands, rotates ∼90° with respect to the Oγ−V axis with the consequent loss of various favorable interactions (Figure 5b). Summarizing this part of the study, we tested to which extent our expanded GOLD parameters could confirm the spectroscopic (EPR) data, rationalize the location of VIVO complex binding site, identify the specific amino acid interacting with the metal and discriminate between different isomers/enantiomers. The possibility to reproduce the experimental structure, optimizing first by DFT methods the several isomers of cis[VO(pic)2(H2O)] and then blind docking the moiety cisVO(pic)2 with Lyz (lysozyme must be considered only a representative example of a protein) using no restraints is a promising step forward in the drug design and medicinal inorganic chemistry landscape. 4. Predicting Interaction of [VO(ma)2(H2O)] with Lysozyme. X-ray structures of metallodrugs bound to proteins are extremely rare and, therefore, we decided to put into

practice our combined EPR-docking analysis, validated in the previous sections, on a vanadium compound whose interaction with lysozyme has not been analyzed yet. In particular, we focused on the most important V compound developed in the fight against diabetes, bis(maltolato)oxidovanadium(IV) (BMOV).1,2 The bis-chelated VIVO formed by maltol exists in aqueous solution as a cis-octahedral structure,9d,55,61 and the four possible geometric isomers (OC-6−23, OC-6−24, OC-6−32, and OC-6−3459) and the corresponding enantiomers are reported in Scheme 2. DFT calculations showed that in aqueous solution the stability of the isomers with cis-octahedral structure (coordination modes (Oket, Ophen); (Oket, Ophenax); H2O or (Oket, Ophen); (Oketax, Ophen); H2O) is comparable with an energy difference less than 1 kcal/mol.55 For this reason, when the interaction of [VO(ma)2(H2O)] with a protein occurs, one or more of the eight isomers could be stabilized, as discussed above in case of picolinate. EPR spectra recorded in the systems VIVO2+/ma/Lyz, IV 2+ V O /ma, VIVO2+/ma/MeIm, and VIVO2+/ma/IgG are reported in Figure 6. It can be noticed that the MI = 7/2 resonance of cis-[VO(ma)2(H2O)] (I in Figure 6) falls at highest value of magnetic field (Az 170.8 × 10−4 cm−1) due to the equatorial coordination of the weakest donor (water-O). In contrast, the MI = 7/2 resonances in the spectra of cis[VO(ma)2(MeIm)] and cis-VO(ma)2(IgG) (III and IV in Figure 6) fall at lowest magnetic field (Az 164.8 × 10−4 cm−1 and 164.6 × 10−4 cm−1, respectively), because the fourth equatorial position is occupied by the strongest donor (His-N). An accurate analysis of the spectra shows that (1) the intensity of the signal attributable to the mixed species increases by increasing the ratio between Lyz and VIVO2+ ion (cf. traces b and c in Figure 6); (2) the MI = 7/2 resonance (II in Figure 6) is intermediate between those of the species formed by water-O and imidazole-N coordination (Az 167.6 × 10−4 cm−1). This experimental evidence suggests that the cis-VO(ma)2 moiety is covalently bound to lysozyme and that its fourth equatorial position is occupied by a donor with an intermediate strength with respect to H2O and an aromatic N: the logical conclusion is that this is a carboxylate-O, whose contribution to Az (42.1 × 10−4 cm−1 6b,c) is situated halfway between water-O (45.6 × 10−4 cm−1 6) and His-N (∼40 × 10−4 cm−1 6b,c). Therefore, the 12945


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Furthermore, the 51V hyperfine coupling constants were calculated by DFT methods for all the isomers OC-6−23, OC6−24, OC-6−32, and OC-6−34 and compared with the experimental data (Table S2 of Supporting Information). In particular, |Az|calcd for cis-[VO(ma)2(H2O)] is in the range (169.1−171.4) × 10−4 cm−1 (|Az|exptl = 170.8 × 10−4 cm−1), while |A z | calcd for cis-[VO(ma) 2 (PrO)] − and cis-[VO(ma)2(MeIm)] are in the ranges (164.8−165.3) × 10−4 cm−1 and (159.5−160.5) × 10−4 cm−1, respectively, to be compared with the experimental value of 167.6 × 10−4 cm−1 for the adduct VO(pic)2(Lyz). These results confirm the probable coordination to cis-VO(ma)2 moiety of a γ/δ-COO− group stemming from an Asp or Glu residue of lysozyme. The entire set of eight isomers of cis-[VO(ma)2(H2O)] (Scheme 2) was optimized by DFT methods, the water molecule in the equatorial plane was removed, and the moiety cis-VO(ma)2 was blind docked on the lysozyme structure (PDB: 4c3w32), using the same procedure as described above. The results are listed in Table 3. Similarly to VO(pic)2, the two side chain residues involved in the metal coordination, on the basis of the low-energy solutions, are δ-COO−(Glu35) and γ-COO−(Asp52). The backbone carbonyl oxygen of Ala107 also generates docking solutions of low energy, but such solutions are not consistent with the EPR results shown in Figure 6, because the contribution of a carbonyl-O to Az is comparable to that of water-O,6 and thus the species with an equatorial CO should not be distinguishable from cis-[VO(ma)2(H2O)]; therefore, these solutions can be discarded. By comparing the GOLD results obtained with the different enantiomers, it can be observed that all the best scoring solutions are obtained for Δ series; Λ enantiomers display always lower scoring values of ∼5 units. In most cases their structures exhibit clashes as reported in Table 3. Therefore, the analysis of the EPR pattern, binding energy (Fmax), population of the clusters and clashes, leads to the conclusion that the preferred coordination between the

Figure 6. High-field region of anisotropic X-band EPR spectra recorded at 120 K and pH 7.4 on aqueous solution containing (a) VIVO2+/ma 1/2 (VIVO2+ 1.0 × 10−3 M); (b) VIVO2+/ma/Lyz 1/2/3 (VIVO2+ 1.0 × 10−3 M); (c) VIVO2+/ma/Lyz 1/2/8 (VIVO2+ 4.4 × 10−4 M); (d) VIVO2+/ma/MeIm 1/2/4 (VIVO2+ 1.0 × 10−3 M); and (e) VIVO2+/ma/IgG 1/2/1 (VIVO2+ 3.0 × 10−4 M). The MI = 7/2 resonances of the VIVO species are indicated as follows: with I cis[VO(ma)2(H2O)] with equatorial water-O coordination, with II cisVO(ma)2(Lyz), with III cis-[VO(ma)2(MeIm)] with equatorial imidazole-N coordination, and with IV cis-VO(ma)2(IgG) with equatorial His-N coordination. The position of the MI = 7/2 resonance of cis-VO(ma)2(Lyz) is shown with an arrow.

system VIVO2+/ma/Lyz (Figure 6) behaves similarly to VIVO2+/ pic/Lyz (Figure 2), and the spectra are compatible with the replacement of the equatorial H2O of cis-[VO(ma)(H2O)] with a γ/δ-carboxylate oxygen donor belonging to an Asp or Glu residue. The spin Hamiltonian parameters, obtained simulating the spectra with WinEPR SimFonia software, are reported in Table S1 of Supporting Information, while the comparison of the experimental and simulated spectra is shown in Figures S6− S9.

Table 3. Docking Results of the Interaction of VO(ma)2 Isomers with Lysozyme Using GOLD 5.2 isomera OC-6−23-Δ OC-6−23-Λ OC-6−24-Δ OC-6−24-Λ

OC-6−32-Δ OC-6−32-Λ OC-6−34-Δ OC-6−34-Λ

donor −

γ-COO (Asp52) δ-COO−(Glu35)g γ-COO−(Asp52)g δ-COO−(Glu35)g γ-(COO−)Asp52 δ-(COO−)Glu35 γ-COO−(Asp52)g,h γ-COO−(Asp52)g,h γ-COO−(Glu35)g CO(Ala107) γ-COO−(Asp52) CO(Ala107)g γ-COO−(Asp52)g CO(Ala107) γ-COO−(Asp52) CO(Ala107) γ-COO−(Asp52)

dist V−Ob

Fmaxc

Smax(hbond_ext)d

Smax(vdW_ext)e

Popf

2.019 2.276 2.313 1.880 2.072 2.471 2.358 2.127 1.994 2.05 1.945 2.121 2.636 2.063 2.055 2.091 2.547

40.83 36.03 35.48 31.44 42.07 34.53 36.01 35.15 35.02 42.54 35.53 41.03 33.30 44.38 41.06 41.54 34.97

10.53 12.68 15.42 12.72 12.80 10.77 13.57 14.40 13.14 10.05 10.05 10.00 10.44 10.03 18.31 9.88 10.96

23.21 19.62 17.14 13.62 22.97 17.56 19.77 15.11 15.95 25.69 19.89 23.89 17.81 25.88 20.78 25.43 19.23

89/100 11/100 38/100 1/100 96/100 2/100 12/100 2/100 3/100 95/100 4/100 85/50 9/100 16/100 82/100 87/100 4/100

a

Isomer structure simulated with Gaussian 09. bDistance in angstroms. cFitness value of the GoldScore scoring function (see eq 1); for each isomer the scoring of the most stable pose is reported. dScoring term related to the intermolecular hydrogen bonds. eScoring term related to the intermolecular van der Waals interactions. fNumber of solutions in the identified cluster. gIn the solution there are interatomic contacts between the metal complex and the protein. hThe two clusters reported for the isomer OC-6−24-Λ present a coordination bond V−γCOO−(Asp52), but they are rather different for the spatial orientation of the metal complex. 12946


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Figure 7. Adducts formed by cis-VO(ma)2 moiety with lysozyme. The better docking solutions for the isomers OC-6−24-Δ (a), OC-6−23-Δ (b), and OC-6−34-Δ (c) are reported.

In the absence of an X-ray structure, other experimental resources can give valuable insight, although often partial, on (metal species)−protein interactions such as NMR, EPR, ESEEM, ENDOR, UV−vis, and CD spectroscopy.63 EPR has been applied to systems in which the proteins interact with VIVO2+ ion with only one donor,8a,b,d,e,9−13,17 and if on one hand it allows obtaining strong evidence on the type of amino acids involved in the interaction (mainly, His and Asp/Glu residues), on the other it does not give any information on the specific residue bound to V or on the possible stabilization of the structure through second coordination sphere contacts. Computation can provide interesting information on the interaction of metallodrugs with their receptors or binding sites at the molecular level. Nonetheless, up to now, there are no examples in which the docking methods have been applied to the simulation of coordination bonds with a metal species and, less again, with an approach that escapes the usual covalent methods, which force the system toward the exact location of the hypothesized interaction. In this study, we used a generalizable docking strategy to identify trustable low-energy binding orientation of systems in which a metal complex interacts with various amino acids of a protein through a direct coordination bond. The experimental EPR spectroscopic data serve to identify the type of amino acids involved in the metal binding, and the docking to identify the exact location of the metal species and chemically reliable 3D solutions. After a new set of parameters was developed, the computational approach was tested on five X-ray structures of vanadium (VIV and VV) species, and the results are in good agreement with the experimental data. Then, this approach was used to predict the structure and enantiospecificity of (metal complex)−protein interactions for two VIVO complexes with

protein and the metal occurs through a bond with the Oγ atom of Asp52. The isomer that presents the best score with Asp52 γ-COO− side chain coordination is OC-6−24-Δ (Fmax = 42.1). Nonetheless, the energy difference below 2 GoldScore units between the three Δ isomers OC-6−23-Δ, OC-6−24-Δ, and OC-6−34-Δ (Figure 7a−c), makes it difficult to ascertain if any of those is clearly favored, or if, instead, a mixture of these species exists in aqueous solution. The EPR technique is not able to discriminate between them; in fact, in the three cases, the four equatorial donors give comparable contributions to hyperfine coupling constant Az (see the structures in Scheme 2). Nevertheless it can be noticed that, similarly to what was done for the bis-chelated species of picolinate, in aqueous solution the eight isomers are in equilibrium, and, upon the interaction with the protein, the equilibrium is shifted toward the Δ series for a more favorable interaction with lysozyme, which, in its turn, is related to the conformation of the protein and to the relative arrangement of the two maltolate anions around the V center. The structural details of the highestaffinity solutions of the three Δ enantiomers are reported in Table S4 of Supporting Information.

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CONCLUSIONS AND OUTLOOK The study of the interaction of metal complexes with the proteins is of fundamental importance in biology, pharmacy, medicine, and even in enzyme design.62 In many cases, the metal ion binds to the protein through direct coordination bonds with donor atoms of the side chains of specific amino acids, which usually replace weak ligands in the metal coordination sphere (water molecules, for instance). 12947


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high antidiabetic activity, cis-[VO(pic)2(H2O)] and cis-[VO(ma)2(H2O)]. To the best of our knowledge, this study presents the first extensive effort to generate coordination scoring parameters into docking programs, and the combination of this new computational framework with the EPR capabilities opens major avenues in metallodrug design and (metal complex)−protein interaction. It also escapes the standard mechanism for the modeling of covalent binding in protein−ligand dockings by offering calculations without imposition of structural and energetic restraints and maintaining first coordination sphere geometries. Obviously, there is still much to do to account for all the possible electronic and structural variables involved in the binding mechanism of a metal compound with a proteic host. For example, the calculations can only be performed when the vacant site of the metal complex that could interact with the protein is clearly known. Another aspect is the full relaxation of the first coordination sphere of the metal during the binding. Even if major changes of this environment are rarely observed in the structures of metallodrugs bound to their targets, this aspect could be studied, as demonstrated in the literature,42,64 through QM/MM refinement.

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REFERENCES

(1) (a) Costa Pessoa, J.; Tomaz, I. Transport of Therapeutic Vanadium and Ruthenium Complexes by Blood Plasma Components. Curr. Med. Chem. 2010, 17, 3701−3738. (b) Rehder, D. The potentiality of vanadium in medicinal applications. Future Med. Chem. 2012, 4, 1823−1837. (c) Costa Pessoa, J.; Etcheverry, S.; Gambino, D. Vanadium compounds in medicine. Coord. Chem. Rev. 2015, 301−302, 24−48. (d) Rehder, D. Perspectives for vanadium in health issues. Future Med. Chem. 2016, 8, 325−338. (2) (a) Thompson, K. H.; Orvig, C. Vanadium Compounds in the Treatment of Diabetes. In Metal Ions in Biological Systems; Sigel, H., Sigel, A., Eds.; Marcel Dekker: New York, 2004; Vol. 41, pp 221−252. (b) Thompson, K. H.; Orvig, C. Vanadium in diabetes: 100 years from Phase 0 to Phase I. J. Inorg. Biochem. 2006, 100, 1925−1935. (3) (a) Comba, P.; Lampeka, Y. D.; Prikhod’ko, A. I.; Rajaraman, G. Determination of the Solution Structures of Melamine-Based Bis- and Tris-Macrocyclic Ligand Copper(II) Complexes. Inorg. Chem. 2006, 45, 3632−3638. (b) Comba, P.; Daumann, L.; Lefebvre, J.; Linti, G.; Martin, B.; Straub, J.; Zessin, T. Mono- and Dinuclear Copper(II) and Iron(III) Complexes of a Tetradentate Bispidine-diacetate Ligand. Aust. J. Chem. 2009, 62, 1238−1245. (4) (a) Watly, J.; Simonovsky, E.; Wieczorek, R.; Barbosa, N.; Miller, Y.; Kozlowski, H. Insight into the Coordination and the Binding Sites of Cu2+ by the Histidyl-6-Tag using Experimental and Computational Tools. Inorg. Chem. 2014, 53, 6675−6683. (b) Watly, J.; Simonovsky, E.; Barbosa, N.; Spodzieja, M.; Wieczorek, R.; Rodziewicz-Motowidlo, S.; Miller, Y.; Kozlowski, H. African Viper Poly-His Tag Peptide Fragment Efficiently Binds Metal Ions and Is Folded into an α-Helical Structure. Inorg. Chem. 2015, 54, 7692−7702. (5) (a) Riplinger, C.; Bill, E.; Daiber, A.; Ullrich, V.; Shoun, H.; Neese, F. New Insights into the Nature of Observable Reaction Intermediates in Cytochrome P450 NO Reductase by Using a Combination of Spectroscopy and Quantum Mechanics/Molecular Mechanics Calculations. Chem. - Eur. J. 2014, 20, 1602−1614. (b) Kochem, A.; Weyhermüller, T.; Neese, F.; van Gastel, M. EPR and Quantum Chemical Investigation of a Bioinspired Hydrogenase Model with a Redox-Active Ligand in the First Coordination Sphere. Organometallics 2015, 34, 995−1000. (6) (a) Chasteen, D. N. Vanadyl(IV) EPR spin probe. Inorganic and Biochemical Aspects. In Biological Magnetic Resonance; Berliner, L. J. J., Reuben, J., Eds.; Plenum Press: New York, 1981; Vol. 3, pp 53−119. (b) Smith, T. S., II; LoBrutto, R.; Pecoraro, V. L. Paramagnetic spectroscopy of vanadyl complexes and its applications to biological systems. Coord. Chem. Rev. 2002, 228, 1−18. (c) Garribba, E.; LodygaChruscinska, E.; Micera, G.; Panzanelli, A.; Sanna, D. Binding of oxovanadium(IV) to dipeptides containing histidine and cysteine residues. Eur. J. Inorg. Chem. 2005, 2005, 1369−1382. (7) (a) Micera, G.; Garribba, E. Is the spin-orbit coupling important in the prediction of the 51V hyperfine coupling constants of VIVO2+ species? ORCA versus Gaussian performance and biological applications. J. Comput. Chem. 2011, 32, 2822−2835. (b) Sanna, D.; Pecoraro, V.; Micera, G.; Garribba, E. Application of DFT methods to the study of the coordination environment of the VO2+ ion in V proteins. JBIC, J. Biol. Inorg. Chem. 2012, 17, 773−790. (c) Sanna, D.; Sciortino, G.; Ugone, V.; Micera, G.; Garribba, E. Nonoxido VIV Complexes: Prediction of the EPR Spectrum and Electronic Structure of Simple Coordination Compounds and Amavadin. Inorg. Chem. 2016, 55, 7373−7387. (8) (a) Liboiron, B. D.; Thompson, K. H.; Hanson, G. R.; Lam, E.; Aebischer, N.; Orvig, C. New Insights into the Interactions of Serum Proteins with Bis(maltolato)oxovanadium(IV): Transport and Biotransformation of Insulin-Enhancing Vanadium Pharmaceuticals. J. Am. Chem. Soc. 2005, 127, 5104−5115. (b) Jakusch, T.; Hollender, D.; Enyedy, E. A.; Gonzalez, C. S.; Montes-Bayon, M.; Sanz-Medel, A.; Costa Pessoa, J.; Tomaz, I.; Kiss, T. Biospeciation of various antidiabetic VIVO compounds in serum. Dalton Trans. 2009, 2428− 2437. (c) Vincent, J. B.; Love, S. The binding and transport of alternative metals by transferrin. Biochim. Biophys. Acta, Gen. Subj. 2012, 1820, 362−378. (d) Correia, I.; Jakusch, T.; Cobbinna, E.;

ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.inorgchem.7b01732. Implemented parameters used for docking calculations, supplementary equations, spin Hamiltonian parameters for VIVO species, values of Ax, Ay, and Az calculated by DFT methods, Gibbs energy for the formation of VO(pic)2(Lyz) adduct calculated by QM/MM methods, comparison of experimental and simulated EPR spectra recorded in the systems with picolinate and maltolate, binding pocket of lysozyme, details on QM/MM calculations, determination of RMSD value and NCIPlot method, and examples of input and output GOLD 5.2 files (PDF) Animated video (AVI)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. (J-D.M.) *E-mail: [email protected]. (E.G.) ORCID

Daniele Sanna: 0000-0001-9299-0141 Agustí Lledós: 0000-0001-7909-422X Eugenio Garribba: 0000-0002-7229-5966 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS J-D.M., A.L., and G.S. are thankful for the support given by the Spanish MINECO (Grant No. CTQ2014-54071-P) and the Generalitat de Catalunya (Grant No. 2014SGR989). Support of COST Action CM1306 is kindly acknowledged. G.S. thanks the Universitat Autònoma de Barcelona for its support to his Ph.D. grant. D.S., V.U., G.M., and E.G. thank Fondazione di Sardegna (Project FdS15Garribba) for the financial support. 12948


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