ORIGINAL RESEARCH published: 07 May 2020 doi: 10.3389/fchem.2020.00345 Biospeciation of Potential Vanadium Drugs of Acetylacetonate in the Presence of Proteins Giuseppe Sciortino 1,2 , Valeria Ugone 1 , Daniele Sanna 3 , Giuseppe Lubinu 1 , Simone Ruggiu 1 , Jean-Didier Maréchal 2 and Eugenio Garribba 1* 1 Dipartimento di Chimica e Farmacia, Università di Sassari, Sassari, Italy, 2 Departament de Química, Universitat Autònoma de Barcelona, Cerdanyola del Vallés, Barcelona, Spain, 3 Istituto di Chimica Biomolecolare, Consiglio Nazionale delle Ricerche, Sassari, Italy Among vanadium compounds with potential medicinal applications, [VIV O(acac)2 ] is one of the most promising for its antidiabetic and anticancer activity. In the organism, however, interconversion of the oxidation state to +III and +V and binding to proteins are possible. In this report, the transformation of VIII (acac)3 , VIV O(acac)2 , and VV O2 (acac)− 2 after the interaction with two model proteins, lysozyme (Lyz) and ubiquitin (Ub), was studied with ESI-MS (ElectroSpray Ionization-Mass Spectroscopy), EPR (Electron Paramagnetic Resonance), and computational (docking) techniques. It was shown that, in the metal concentration range close to that found in the organism (15–250 µM), VIII (acac)3 is oxidized to VIV O(acac)+ and VIV O(acac)2 , which—in their turn—interact with proteins Edited by: Arthur David Tinoco, University of Puerto Rico, Puerto Rico to give n[VIV O(acac)]–Protein and n[VIV O(acac)2 ]–Protein adducts. Similarly, the complex Reviewed by: in the +IV oxidation state, VIV O(acac)2 , dissociates to the mono-chelated species Sergio Armando Loza Rosas, VIV O(acac)+ which binds to Lyz and Ub. Finally, VV O2 (acac)− 2 undergoes complete University of Boyaca, Colombia Ritika Gautam, dissociation to give the ’bare’ VV O+ 2 ion that forms adducts n[VV O ]–Protein with n = 2 Indian Institute of Technology 1–3. Docking calculations allowed the prediction of the residues involved in the metal Kanpur, India binding. The results suggest that only the VIV O complex of acetylacetonate survives *Correspondence: Eugenio Garribba in the presence of proteins and that its adducts could be the species responsible of garribba@uniss.it the observed pharmacological activity, suggesting that in these systems VIV O2+ ion should be used in the design of potential vanadium drugs. If VIII or VV O2 potential active Specialty section: complexes had to be designed, the features of the organic ligand must be adequately This article was submitted to Inorganic Chemistry, modulated to obtain species with high redox and thermodynamic stability to prevent a section of the journal oxidation and dissociation. Frontiers in Chemistry Keywords: metal drugs, vanadium, anticancer action, antidiabetic action, proteins, transport in the organism, drug Received: 11 February 2020 design Accepted: 02 April 2020 Published: 07 May 2020 Citation: INTRODUCTION Sciortino G, Ugone V, Sanna D, Lubinu G, Ruggiu S, Maréchal J-D Among the metal-based drugs, vanadium (V) compounds have drawn a growing interest and Garribba E (2020) Biospeciation of Potential Vanadium Drugs of for their pharmacological properties shown both in vivo and in vitro studies. In particular, Acetylacetonate in the Presence of several V complexes have been proposed as antidiabetic and anticancer potential drugs (Sakurai Proteins. Front. Chem. 8:345. et al., 2008; Costa Pessoa et al., 2015a; Kioseoglou et al., 2015; Rehder, 2016) and some of doi: 10.3389/fchem.2020.00345 them reached the clinical trials, for example bis(ethylmaltolato)oxidovanadium(IV) or BEOV Frontiers in Chemistry | www.frontiersin.org 1 May 2020 | Volume 8 | Article 345 Sciortino et al. Biospeciation of Potential Vanadium Drugs (Thompson and Orvig, 2006; Thompson et al., 2009). Over the conditions (Jakusch and Kiss, 2017); the limitation is that it last years, many complexes were studied with vanadium in one of does not give any information on the structure of the revealed the three oxidation states found in biological systems, +III, +IV, vanadium adducts and that the intensity of the peaks cannot be and +V. However, the mechanism of action and active oxidation related to the amount of the species in solution. With regard to state in the organism remain unclear and biospeciation has not EPR spectroscopy, it allows only the recognition of the type of been completely ascertained. This lack of knowledge is one of amino acid donors involved in the metal coordination (His-N, the reasons why V compounds are not considered for clinical Asp/Glu-COO, or carbonyl-CO, etc.), with the limitation that tests by pharmaceutical companies and limits the possibility of the specific site and its three-dimensional structure cannot be a rational design of new potential drugs with more activity and identified (Chasteen, 1981; Smith et al., 2002). lower toxicity than those proposed up to now (Scior et al., 2016). The results of this study on the transformation of VIV O(acac)2 Among these compounds, bis(acetylacetonato)- and—in general—of bis-chelated complexes with stoichiometry oxidovanadium(IV) or VIV O(acac)2 is one of the most studied VIV OL2 , where L is a bidentate monoanionic organic ligand, (Makinen and Brady, 2002; Crans et al., 2018). It favors the could be very useful to ascertain the active species in the organism decrease of blood glucose concentration and is more effective and be used as a guide for a rational design of new potential than the inorganic salt VIV OSO4 in the suppression of hepatic V drugs. glycolysis in streptozotocin-induced diabetic rats, and this effect cannot be related only to an improved intestinal absorption (Reul MATERIALS AND METHODS et al., 1999; Amin et al., 2000). VIV O(acac)2 enhances in cells the IR (insulin receptor) kinase activity more than other VIV O Chemicals chelate species (Makinen and Salehitazangi, 2014), even though it Water was deionized through the purification system Millipore has not been proven if its action is direct through the stimulation MilliQ Academic or purchased from Sigma-Aldrich (LC-MS of the IR kinase activity or indirect through the activation of grade). VIII (acac)3 and VIV O(acac)2 , NH4 VV O3 , acetylacetone other tyrosine kinases or inhibition of tyrosine phosphatases, (Hacac), and 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic for example PTP-1B (Ou et al., 2005; Makinen et al., 2007). acid (HEPES) were Sigma-Aldrich products of the highest grade VIV O(acac)2 exhibits also important anti-proliferative effects: available and used as received. it blocks the G1/S phase of the cell cycle progression via an Chicken egg white lysozyme (Lyz; molecular mass of 14.3 activated ERK (extracellular signal-regulated kinase) signal in kDa, code 62970) and ubiquitin from bovine erythrocytes (Ub; human hepatoma HepG2 cells (Fu et al., 2008), while in human molecular mass of 8.6 kDa, code U6253) were purchased pancreatic cancer cell line AsPC-1 it induces cell cycle arrest from Sigma-Aldrich. at the G2/M phase and causes an increase of ROS (reactive oxygen species) concentration (Wu et al., 2016). These results ESI-MS Measurements indicate that V-based complexes may be potentially applied as The solutions for ESI-MS measurements were prepared as antidiabetic and anticancer agents to treat patients suffering follows. Briefly, the solid complexes VIII (acac)3 and VIV O(acac)2 from both the diseases (Liu et al., 2013). or NH4 VV O3 plus Hacac in molar ratio 1/2 were dissolved in Species related to VIV O(acac)2 in the organism are VIII (acac)3 LC-MS grade water to obtain a V concentration of 1.0–2.0 mM. and VV O2 (acac)− 2 . In fact, on one hand several experimental Subsequently, all the solutions were diluted in LC-MS grade evidences showed that VIV complexes can be rapidly oxidized water with an aliquot of a stock protein solution (500 µM) to to VV in the cell culture medium and, on the other, that +IV reach (V complex)/Protein ratios of 3/1, 5/1, and 10/1 and a state can converge to +III in the gastrointestinal medium, in the protein concentration of 5 µM. In all the solutions pH was in blood and reducing cellular environment; for these reasons, more the range 6–7. Immediately after the preparation of the solutions, than one species could contribute to the global biological action. ESI-MS spectra were acquired. The biotransformation of VIV O(acac)2 is obviously related to the Mass spectra in the positive-ion or negative-ion mode were pharmacological efficacy, since it determines the gastrointestinal recorded on a Q ExactiveTM Plus Hybrid Quadrupole-OrbitrapTM absorption, the transport in blood and—overall—the availability (Thermo Fisher Scientific) mass spectrometer. The solutions of the active species. The interaction with proteins plays a key were infused at a flow rate of 5.00 µL/min into the ESI chamber. role in the biospeciation of a potential metal-based drug in the The spectra were obtained in the m/z range 300–4,500 at a organism due to their high concentration and/or high affinity resolution of 140,000 and accumulated for at least 5 min to toward the metals (Costa Pessoa et al., 2015b). increase the signal-to-noise ratio. The instrumental setting for In this paper, the transformation of VIII (acac)3 , VIV O(acac)2 , the measurement of the spectra in positive-ion mode were: and VV O2 (acac)− 2 in the presence of two model proteins, spray voltage 2,300 V, capillary temperature 250◦ C, sheath gas lysozyme (Lyz) and ubiquitin (Ub), was studied by ESI-MS 10 (arbitrary units), auxiliary gas 3 (arbitrary units), sweep (ElectroSpray Ionization-Mass Spectrometry), EPR (Electron gas 0 (arbitrary units), probe heater temperature 50◦ C. The Paramagnetic Resonance), and computational (docking and QM) setting used for negative-ion mode spectra were: spray voltage techniques. Both ESI-MS and EPR techniques present, for what −1,900 V, capillary temperature 250◦ C, sheath gas 20 (arbitrary concerns this work, pros and cons. The main advantage of ESI- units), auxiliary gas 5 (arbitrary units), sweep gas 0 (arbitrary MS is that the metal concentrations used for experiments are units), probe heater temperature 14◦ C. ESI-MS spectra were in the range 1–100 µM, i.e., that found for V at physiological analyzed by using Thermo Xcalibur 3.0.63 software (Thermo Frontiers in Chemistry | www.frontiersin.org 2 May 2020 | Volume 8 | Article 345 Sciortino et al. Biospeciation of Potential Vanadium Drugs Fisher Scientific) and the average deconvoluted monoisotopic grid point of 2.6–6.4 and 3.4–7.2 Å, respectively (Sciortino et al., masses were obtained through the Xtract tool integrated in 2019b). Both the protonation states at δ and ε nitrogens of His the software. imidazole ring were taken into account during the simulations. Genetic algorithm (GA) parameters have been set to 50 EPR Measurements GA runs and a minimum of 100,000 operations. The other EPR spectra were acquired by dissolving in ultra-pure water parameters of GA were set to default. VIII (acac)3 to obtain a VIII concentration of 1 mM. To the The scoring (Fitness of GoldScore) was calculated with the solutions, HEPES buffer of 0.1 M concentration was added. The modified version of GoldScore scoring function, which was value of pH was brought in the pH range 5–7 and a protein validated in previously published papers (Sciortino et al., 2017, (Lyz or Ub) was added to 1 mL of the solution with the V 2018a, 2019a). The best solutions (binding poses) were evaluated species, to reach a concentration of 0.5 mM and a molar ratio (V through the mean (F mean ) and the highest value (F max ) of the complex)/Protein of 2/1. Subsequently, an aliquot of the solution Fitness associated with each pose, the population of the cluster with V complex was added to bring the ratio (V complex)/Protein containing the best pose and the position in the Fitness ranking to 3/1. of the computed cluster. EPR spectra were recorded at 120 K with an X-band Bruker EMX spectrometer equipped with a HP 53150A microwave frequency counter using a microwave frequency of 9.40–9.41 RESULTS AND DISCUSSION GHz, microwave power of 20 mW, time constant of 81.92 ms, modulation frequency of 100 kHz, modulation amplitude of Interaction of VIII (acac)3 and VIV O(acac)2 0.4 mT, and resolution of 4,096 points. The spectra were With Proteins immediately recorded after the preparation of the samples. The species distribution diagram of the VIII /acac system with To increase the signal to noise ratio, signal averaging was molar ratio 1/3 and V concentration of 150 µM, that does not employed (Sanna et al., 2009). account for the oxidation process to VIV , can be built using the data of the thermodynamic stability constants reported in the DFT and Docking Calculations literature (Brito et al., 2009). The diagram is shown in Figure S1 All the DFT calculations were performed with Gaussian 09 of the Supplementary Material and suggests that the main (revision D.01) (Frisch et al., 2010). The geometries and species in the pH range 6.0–7.0 is VIII (acac)3 . ESI-MS spectrum harmonic frequencies of VIV O and VV O2 complexes were was recorded on VIII (acac)3 at pH 6.2 in a degassed aqueous computed at the level of theory B3P86/6-311++g(d,p) with the solution to evaluate its stability in the absence of a protein SMD model (Marenich et al., 2009) for water. (Figure S2). The results indicate that the oxidation to VIV O is Docking calculations were carried out with GOLD 5.8 only partial: even though the intensity of the MS signal cannot software (Jones et al., 1997) on the X-ray structure available in be directly related to the concentration in solution, the peaks the Protein Data Bank (PDB) of lysozyme [PDB code: 2LYZ of the adducts [VIII (acac)3 +H+ ] and [VIII (acac)3 +Na+ ] (m/z (Diamond, 1974)] and ubiquitin [3H1U (Qureshi et al., 2009)]. = 349.09 and 371.07, respectively) are much more intense than The PDB structure was cleaned removing all the small molecules those assigned to [VIV O(acac)2 +H+ ] and [VIV O(acac)2 +Na+ ] and crystallographic waters, and hydrogen atoms were added (m/z = 266.04 and 288.02). The simulation of the two with the UCSF Chimera (Pettersen et al., 2004). peaks of VIII (acac)3 is shown in Figures S3, S4 and confirms The docking simulations were carried out constructing in the attribution. the region of interest an evaluation sphere of 12 Å. The When the spectra were measured on the system coordination docking calculations (with an active binding VIII (acac)3 /Lyz the transformation to VIV O is complete and no V–donor) were performed on the DFT optimized moieties peaks assignable to the active or inert binding of VIII (acac)3 to the VIV O(acac)+ and cis-VV O+ 2 , activating one, two or three protein could be revealed. With a molar ratio of 3/1 and a protein equatorial coordination positions, respectively, with dummy concentration of 5 µM, the peaks of the free protein at 14304 Da hydrogen atoms (Sciortino et al., 2017, 2018a,b, 2019a). All the plus the signals of the adducts n[VIV O(acac)]–Lyz with n = 1–4 possible moieties VV O2 (H2 O)n + with n = 0–2 were taken into (with mass of 14469, 14635, 14800 and 14965 Da, respectively) account during dockings and the proposed solutions in the text were observed (Figure S5). For these species the equatorial are those with higher scoring. For coordination dockings the interaction with one or two protein residues is possible. Similar regions with the potential coordinating residues were examined results were obtained at (V complex)/Protein ratio of 5. When with the GOLD rotamers libraries (Lovell et al., 2000). For higher protein concentration was used (50 µM), the spectra the non-coordination dockings (inert binding V···donor) with show a series of peaks belonging to n[VIV O(acac)]–Lyz, with n VIV O(acac)2 , the whole rigid protein was considered. The = 1–4, while the signals at 14570 and 14836 Da can be attributed solutions were analyzed by means of GaudiView (Rodríguez- to [VIV O(acac)2 ]–Lyz and 2[VIV O(acac)2 ]–Lyz (Figure 1A). Guerra Pedregal et al., 2017). Moreover, the adduct [VIV O(acac)+VIV O(acac)2 ]–Lyz at 14735 To identify potential binding sites, the protein space was first Da is detected with the contemporaneous coordination of probed with multisite.py for zones where at least two potential mono- and bis-chelated moieties (Figure 1A). Therefore, the coordinating residues (Asp, Glu, Asn, Gln, and His) featured an increase of the V concentration favors the interaction of the α- and a β-carbon within a range of distances from each queried bis-chelated complex VIV O(acac)2 with lysozyme, even though Frontiers in Chemistry | www.frontiersin.org 3 May 2020 | Volume 8 | Article 345 Sciortino et al. Biospeciation of Potential Vanadium Drugs FIGURE 1 | Deconvoluted ESI-MS spectrum recorded on the systems: (A) VIII (acac)3 and lysozyme, pH 6.2, and (B) VIII (acac)3 and ubiquitin, pH 6.3. The molar ratio VIII /Protein was 3/1 and Protein concentration 50 µM. Under these experimental conditions, VIII species fully oxidizes to VIV O. the binding of the fragment VO(acac)+ appears to be favored compared to VIV O(acac)2 . Concerning the interaction between [VO(acac)2 ] with lysozyme, no equatorial sites are accessible and so only two possibilities exist: an inert binding with the protein surface or a weak axial active binding through an amino acid residue. In the analysis of the results on the system VIV O(acac)2 /Lyz, it must be considered that in the examined pH range VIV O(acac)2 is the major species in aqueous solution and that it coexists with 1:1 complex VIV O(acac)+ . The diagram with the distribution of the species is represented in Figure 2. The ESI- MS spectra are in agreement with these data and show similar results with formation of the adducts n[VIV O(acac)]–Lyz and n[VIV O(acac)2 ]–Lyz (Figure S6). EPR spectroscopy confirms the oxidation of VIII and the resonances due to the VIV O ion (3d1 electronic configuration) are FIGURE 2 | Distribution diagram of the species as a function of pH in a revealed (Figure 3A). Beside the absorption due to VIV O(acac)2 solution containing VIV O2+ ion and acac (VIV O/acac = 1/2, VIV O = 1.50 × not covalently bound to lysozyme, the signals were attributed 10−4 M). The stability constants of the VIV O–acac complexes were taken from Crans et al. (2001) and of the hydrolytic species from Buglyo et al. (2000). to [VIV O(acac)]–Lyz adducts with the equatorial binding of two protein residues (g z ∼ 1.943 and Az ∼ 171–172 × 10−4 cm−1 ). This is illustrated in Figures S7A,B. The deconvoluted ESI-MS spectrum of the system to those revealed with VIII and recently published, that indicate VIII (acac)3 /Ub 3/1 using an Ub concentration of 5 or the formation of the adducts n[VIV O(acac)]–Ub, with n = 1–3 50 µM (Figure 1B) showed the presence of [VIV O(acac)]– (Ugone et al., 2019). For the system containing Ub as well, Ub, 2[VIV O(acac)]–Ub and 3[VIV O(acac)]–Ub. This means EPR measurements indicated the complete transformation of that three moieties VIV O(acac)+ bind to ubiquitin, which VIII (acac)3 to VIV O(acac)+ moiety, which in solution interacts replaces two weak water ligands in the two adjacent equatorial with ubiquitin (Figure 3B). positions of VIV O(acac)(H2 O)+ 2 . When the interaction between Overall, the behavior of the VIII and VIV O systems with VIV O(acac)2 and ubiquitin is considered, the results are similar the two model proteins is very similar. The results indicate Frontiers in Chemistry | www.frontiersin.org 4 May 2020 | Volume 8 | Article 345 Sciortino et al. Biospeciation of Potential Vanadium Drugs that, in the metal concentration range 15–250 µM, i.e., that are as follows: found in the organism (Jakusch and Kiss, 2017), VIII (acac)3 is quantitatively oxidized to VIV O(acac)+ and VIV O(acac)2 , VIII (acac)3 + H2 O + Protein → which—in their turn—interact with proteins. Compared to [VIV O(acac)+ ]–Protein + 2Hacac + e− (1) the binary systems without proteins, the oxidation is favored VIII (acac)3 + H2 O + Protein → probably due to the stabilization of the VIV O adducts upon the binding with the amino acid residues. So, the results obtained [VIV O(acac)2 ]–Protein + Hacac + H+ + e− (2) with VIII can be interpreted considering the species distribution The number of VIV O(acac)+ and VIV O(acac)2 moieties bound diagram shown in Figure 2 for VIV O–acac system, postulating depends on the vanadium concentration and type of protein; in that the oxidation process in the presence of the proteins is particular, the binding of VIV O(acac)2 is favored with increasing quantitative. The reactions which account for the formation of V concentration and going from Ub to Lyz. the adducts [VIV O(acac)+ ]–Protein and [VIV O(acac)2 ]–Protein Docking calculations were performed to confirm ESI-MS and EPR findings and get information on the residues involved in the VIV binding. For lysozyme, the results indicate that four- five sites are available for VIV O(acac)+ interaction, in agreement with ESI-MS measurements (Table 1 and Figure 4). In the region 44–52 one or two VIV O(acac)+ can bind to protein with the simultaneous coordination of two residues. The affinity order is as follows: (Asn46, Asp48) > (Asn46, Asp52) ∼ (Asn44, Asp52), the F mean values being in the range 35.9–40.8 with a population between 41/50 and 50/50. In all the three sites, named A, the donor set is (NCO, COO− ) and this accounts for the Az value measured in the EPR spectra (Az ∼ 171–172 × 10−4 cm−1 , see above). It must be observed that the contemporaneous coordination to both (Asn46, Asp52) and (Asn44, Asp52) donor set is not compatible and the binding to the first site excludes the second one. The second site (site B) is located in the N- terminal region, where His15 plus a water ligand are coordinated to the VIV O2+ ion, and a hydrogen bond between V=O group and Thr89 stabilizes the binding. Interestingly, the His15 equatorial binding is not expected for bis-chelated cis moieties FIGURE 3 | EPR spectra recorded on frozen solutions (120 K) of the systems: such as cis-VIV O(picolinato)2 or cis-VIV O(maltolato)2 due to the (A) VIII (acac)3 and lysozyme and (B) VIII (acac)3 and ubiquitin. VIII /Protein molar steric hindrance of the second ligand with an (equatorial-axial) ratio was 3/1 and V concentration 1 mM. pH was 5.70 and 5.15, respectively. arrangement (Sciortino et al., 2017). The third and fourth sites The region at high field was amplified 10 times. At these conditions, VIII species fully oxidizes to VIV O. are weaker than these ones and are based on the coordination of (Asp18, Asn19) with F mean = 30.9 (site C) and (Asp119, TABLE 1 | Docking solutions for the active binding of the VIV O(acac)+ moiety with Lyz and Ub. Protein Site Residues V–donora 2nd interaction F bmax F cmean Pop.d Rank.e Lyz A Asn46, Asp48 2.082, 2.255 Oacac ···Ser50 41.8 40.8 50/50 I A Asn44, Asp52 2.315, 2.117 – 37.1 35.9 45/50 I A Asn46, Asp52 2.279, 2.306 – 37.4 36.3 41/50 II B His15 2.351 VO···Thr89 40.5 38.7 8/50 I C Asp18, Asn19 2.134, 2.179 – 32.1 30.9 49/50 I D Asp119, Gln121 2.162, 1.946 – 33.7 30.7 38/50 I Ub 1 Glu16, Glu18 2.300, 2.342 – 46.2 44.3 7/50 I 1’ Glu18, Asp21 2.382, 2.236 – 42.8 39.3 10/50 II 2 Glu24, Asp52 2.328, 2.353 – 38.2 37.2 44/50 I 2’ Glu51, Asp52 2.373, 2.373 – 36.3 34.9 6/50 II a Distance between V and protein donors in Å. b GoldScore Fitness value obtained for the more stable pose of each cluster (Fmax ). c Mean value of GoldScore Fitness for each cluster (F mean ). d Population of the cluster. e Ranking of the identified cluster. Frontiers in Chemistry | www.frontiersin.org 5 May 2020 | Volume 8 | Article 345 Sciortino et al. Biospeciation of Potential Vanadium Drugs FIGURE 4 | Most stable structures obtained by docking methods for the active binding of VIV O(acac)+ to Lyz: (A) site A with (Asn44, Asp52) and (Asn46, Asp52); (B) site B with His15; (C) site C with (Asp18, Asn19); and (D) site D with (Asp119, Gln121). The hydrogen bonds are indicated with the full blue lines. FIGURE 5 | Most stable site obtained by docking methods for the inert binding of VIV O(acac)2 to Lyz: (A) V=O···HN–Trp63 is 2.029 Å and V···OCN–Ala107 3.370 Å and (B) V=O···HN–Trp63 is 1.644 Å and V···OCN–Ala107 3.875 Å. The weak bonds with Trp63 and Ala107 are indicated with the full blue lines. Gln121) with F mean = 30.7 (site D). The four identified sites When the interaction of VIV O species and ubiquitin is are represented in Figure 4. For the interaction of VIV O(acac)2 , examined, the data indicated that two main sites exist. The only inert binding must be considered: the best site is based first is based on the coordination of (Glu 16, Glu18; site 1) on a hydrogen bond stabilization between the VIV =O and NH or (Glu18, Asp21; site 1’) (Ugone et al., 2019); the two sites group of Trp63 residue and a weak axial interaction of NCO are mutually exclusive and have F mean in the range 39.3– of Ala107 with vanadium. The two solutions are displayed in 44.3 with population between 14 and 20%. The second site Figures 5A,B, showing that with increasing the strength of the is due to the equatorial binding of the couple (Glu24, Asp52; axial interaction with Ala107 decreases the strength of the contact site 2) or (Glu51, Asp52; site 2’) (Ugone et al., 2019); in of V=O with Trp63 and viceversa; in Figure 5A V=O···HN– this case too, these sites are not independent with F mean = Trp63 is 2.029 Å and V···OCN–Ala107 is 3.370 Å, while in 34.9–37.2 and population = 12–88%. The two sites are shown Figure 5B V=O···HN–Trp63 is 1.644 Å and V···OCN–Ala107 in Figure 6 and the values of F max , F mean and population is 3.875 Å. in Table 1. Frontiers in Chemistry | www.frontiersin.org 6 May 2020 | Volume 8 | Article 345 Sciortino et al. Biospeciation of Potential Vanadium Drugs Interaction of VV O2 (acac)− 2 With Proteins et al., 1996); the amount of VV O2 (maltolato)− 2 increases Positive-ion mode and negative-ion mode ESI-MS spectra significantly with V concentration and overcomes 70% when were recorded on the system H2 VV O− 4 /Hacac 1/2 with V it is ten times larger, i.e., 1.5 mM. This demonstrates that the concentration of 150 µM at pH 7.0 (Figure S8). In the literature, hydrolytic processes cannot be neglected with decreasing V the thermodynamic stability constants on the system VV /acac concentration. A similar behavior is expected for VV -acac system: are lacking. The data for maltol, a (O,O) ligand with similar specifically, the complex VV O2 (acac)−2 is formed only at high V basicity [pK a is 8.44 for maltol (Buglyo et al., 2002) and 8.76 concentration (around some mM), but it is not stable when the for acetylacetone (Crans et al., 2001)], indicate that at these metal concentration is in the order of µM. experimental conditions, the percentage of V in solution as ESI-MS results indicated the presence of [Hacac+H+ ] and VV O2 (maltolato)−2 in the pH range 6–7 is <30% (Elvingson [Hacac+Na+ ] in the positive-ion mode (Figure S8A), and of FIGURE 6 | Most stable structures obtained by docking methods for the active binding of VIV O(acac)+ to Ub: (A) site 1 with (Glu16, Glu18) and (B) site 2 with (Glu24, Asp52). The hydrogen bonds are indicated with the full blue lines. FIGURE 7 | Deconvoluted ESI-MS spectrum recorded on the systems containing: (A) VV O2 (acac)− V 2 and lysozyme, pH 6.4, and (B) V O2 (acac)2 and ubiquitin, pH 6.4. − The molar ratio VIII /Protein was 3/1 and Protein concentration 50 µM. With the asterisks the adducts with the binding of one and two water molecules are indicated. Frontiers in Chemistry | www.frontiersin.org 7 May 2020 | Volume 8 | Article 345 Sciortino et al. Biospeciation of Potential Vanadium Drugs [H2 VV O− 4 ] in the negative-ion mode (Figure S8B), derived ligand is not coordinated to vanadium(V), confirming that from the hydrolysis of VV O2 (acac)− 2 . In the deconvoluted the hydrolysis causes the removal of acac− from the first spectrum recorded in the system containing VV O2 (acac)− 2 coordination sphere of the metal; interestingly, the free and lysozyme (Figure 7A), the signal corresponding to the VV O+2 ion interacts with lysozyme which behaves as a mass of Lyz at 14304 Da and the series of signals of the polydentate ligand. Moreover, the adducts with one or two adducts between the protein and one, two or three VV O+ 2 water ligands were revealed (peaks indicated by the asterisks ions are revealed. In all these adducts the acetylacetonato in Figure 7). FIGURE 8 | Most stable structures obtained by docking methods for the active binding of VV O+ 2 to Lyz (A–C) and Ub (D–F). The hydrogen bonds are highlighted with the full blue lines. TABLE 2 | Docking solutions for the active binding of the VV O+ 2 ion with Lyz and Ub. Protein Site Residues V–donora 2nd interaction F bmax F cmean Pop.d Rank.e Lyz A Asn46, Asp52, Asn59 2.161, 2.157, 2.073 VO···Ser50 48.5 47.3 50/50 I A Asn44, Asp52, Gln57 2.271, 2.415, 2.304 VO···Gln57 31.5 30.6 48/50 I B His15, Asp87 2.095, 2.257 VO···Thr89 41.0 40.2 31/50 I C Asp18, Asn19 2.196, 1.904 VO···Asn19 22.4 19.6 17/50 I Ub 1 Glu16, Glu18 2.303, 2.309 VO···Lys29 45.2 44.5 49/50 I 1’ Glu18, Asp21 1.958, 2.448 VO···Lys29 43.3 43.3 1/50 II 2 Glu24, Asp52 2.915, 1.810 VO···Thr22 25.6 25.6 1/50 I 2’ Glu51, Asp52 2.528, 2.213 VO···Lys27 25.2 24.9 19/50 II 2’ Glu51, Asp52 2.368, 2.481 VO···Arg72 24.2 24.1 25/50 III His68 2.977 VO···Lys6 22.3 22.1 50/50 I a Distance between V and protein donors in Å. b GoldScore Fitness value obtained for the more stable pose of each cluster (Fmax ). c Average value of GoldScore Fitness for each cluster (F mean ). d Population of the cluster. e Ranking of the identified cluster. Frontiers in Chemistry | www.frontiersin.org 8 May 2020 | Volume 8 | Article 345 Sciortino et al. Biospeciation of Potential Vanadium Drugs The behavior of the system with ubiquitin is similar and the residues involved in the metal binding and three-dimensional adducts n[VV O2 ]–Ub are detected with n = 1–3 (Figure 7B). structure of the formed adducts. Three important sites were This means that three sites of Ub are available for the predicted both for lysozyme and ubiquitin, A (with Asn44, metal binding. Asn46, Asp48, Asp52, and Asn59), B (His15), and C (Asp18, Asn The docking results confirmed the suggestions of ESI-MS data 19) for Lyz, and 1/1’ (Glu16, Glu18, Asp21), 2/2’ (Glu24, Glu51, and indicate that three sites are available both for Lyz and Ub. Asp52), and His68 for Ub. One, two or three donors are coordinated to V, which shows a The results suggest that, at the studied conditions, only VIV O slightly distorted trigonal bipyramidal environment (Figure 8). species of acetylacetonate survive in the presence of proteins With lysozyme the binding occurs at site A (Asn46, Asp52, and these could be the species responsible of the observed Asn59), B (His15, Asp87), C (Asp18, Asn 19) with different pharmacological activity. Starting from VIII or VV may be affinity and F max of 48.5, 41.0, and 22.4 (Table 2). With ubiquitin useless, since VIII gives quantitatively VIV O, while VV undergoes the coordination to sites 1 (Glu16, Glu18) and 2′ (Glu24, Asp52) hydrolysis to the inorganic ions and the active species should plus to His68 with F max in the range 22.3–45.2 is predicted be the same as when inorganic vanadium(V) was administered. (Table 2). The monodentate coordination of Ub at His68 is This finding allow us to suggest that in this system and in those stabilized by a V=O· · ·H3 N–Lys6 hydrogen bond and reminds containing a bidentate organic ligand, VIV O ion should be used that of His12 in acid phosphatase (Lindqvist et al., 1994). in the design of potential vanadium drugs, and that, if VIII or VV O2 potential active complexes had to be designed, species with CONCLUSIONS high redox and thermodynamic stability should be synthesized to prevent oxidation and dissociation processes. To reach this aim, Even though over the recent years many studies in bioinorganic it is necessary to modulate adequately the features of the organic and medicinal inorganic chemistry were dedicated to the ligand and ligands with weak or intermediate strength such as development of vanadium-based potential drugs, the active acetylacetonate or maltolate should not be associated to VIII and oxidation state in the organism and mechanism of action are VV but only with VIV . still elusive and their biospeciation has not completely explained. This lack of knowledge is one of the reasons why the number DATA AVAILABILITY STATEMENT of V complexes under clinical tests is significantly lower than those of other metal ions. For example, while the oxidation state All relevant data is contained within the article. All datasets of Pt remains stable under physiological conditions (+II), for analyzed in this study are included in the article and the V three oxidation states are possible (+III, +IV, and +V) with Supplementary Material. an interconversion among them in the body. Therefore, in the design of vanadium drugs, such biotransformation and active AUTHOR CONTRIBUTIONS state in the organism must be taken into account. In this study, the V complexes with acetylacetonate, which EG conceived this study and designed the experiments and displayed very promising antidiabetic and anticancer activity, calculations. GS, SR, and J-DM performed the computational were examined. It may be considered just as an example for the calculations. DS and GL performed the EPR measurements behavior of other V compounds. It was shown that, in the metal and VU the ESI-MS experiments. All authors contributed concentration range close to that found in the organism (15– to the manuscript revision, read, and approved the 250 µM), the species with +III state, VIII (acac)3 , is completely submitted version. oxidized to VIV O(acac)+ and VIV O(acac)2 and this process is favored—with respect to the binary system without protein—by ACKNOWLEDGMENTS the binding of one or more amino acid side-chains. VIV O(acac)+ and VIV O(acac)2 , in their turn, interact with proteins: up to EG, GS, VU, DS, GL, and SR thank Regione Autonoma della four VIV O(acac)+ can bind to proteins to yield n[VIV O(acac)]– Sardegna (grant RASSR79857) and Università di Sassari (fondo Ub/Lyz adducts, the binding of VIV O(acac)+ being favored with di Ateneo per la ricerca 2019) for financial support. GS and decreasing the vanadium concentration. At high concentration, J-DM thank also Spanish MINECO (grant CTQ2017-87889-P) also n[VIV O(acac)2 ]–Protein adduct may be revealed with an and Generalitat de Catalunya (2017SGR1323). axial active or a surface inert interaction with the protein. The results with the complex in the +IV oxidation state are the same, SUPPLEMENTARY MATERIAL with VIV O(acac)2 undergoing dissociation to the mono-chelated species VIV O(acac)+ moiety which binds to the proteins. Finally, The Supplementary Material for this article can be found VV O2 (acac)− 2 undergoes complete dissociation to give the “bare” online at: https://www.frontiersin.org/articles/10.3389/fchem. VV O+ 2 ion. Docking calculations allowed us to predict the 2020.00345/full#supplementary-material REFERENCES Bis(acetylacetonate)oxovanadium(IV) and derivatives. Inorg. Chem. 39, 406–416. doi: 10.1021/ic9905897 Amin, S. S., Cryer, K., Zhang, B., Dutta, S. K., Eaton, S. S., Anderson, Brito, F., Araujo, M. L., Martínez, J. D., Hernández, Y., Moh, A., and O. P., et al. (2000). Chemistry and insulin-mimetic properties of Lubes, V. (2009). Speciation of the vanadium(III)–acetylacetone system Frontiers in Chemistry | www.frontiersin.org 9 May 2020 | Volume 8 | Article 345 Sciortino et al. Biospeciation of Potential Vanadium Drugs in 3.0 M KCl ionic medium at 25◦ C. J. Coord. Chem. 62, 52–62. Makinen, M. W., and Salehitazangi, M. (2014). The structural basis of doi: 10.1080/00958970802474763 action of vanadyl (VO2+ ) chelates in cells. Coord. Chem. Rev. 279, 1–22. Buglyo, P., Kiss, E., Fabian, I., Kiss, T., Sanna, D., Garribba, E., et al. (2000). doi: 10.1016/j.ccr.2014.07.003 Speciation and NMR relaxation studies of VO(IV) complexes with several O- Marenich, A. V., Cramer, C. J., and Truhlar, D. G. (2009). Universal donor containing ligands: oxalate, malonate, maltolate and kojate. Inorg. Chim. solvation model based on solute electron density and on a continuum Acta 306, 174–183. doi: 10.1016/S0020-1693(00)00168-7 model of the solvent defined by the bulk dielectric constant and atomic Buglyo, P., Kiss, T., Kiss, E., Sanna, D., Garribba, E., and Micera, G. (2002). surface tensions. J. Phys. Chem. B 113, 6378–6396. doi: 10.1021/jp81 Interaction between the low molecular mass components of blood serum and 0292n the VO(IV)-DHP system (DHP=1,2-dimethyl-3-hydroxy-4(1H)-pyridinone). Ou, H., Yan, L., Mustafi, D., Makinen, M. W., and Brady, M. J. (2005). J. Chem. Soc. Dalton Trans. 11, 2275–2282. doi: 10.1039/b200688j The vanadyl (VO2+ ) chelate bis(acetylacetonato)oxovanadium(IV) potentiates Chasteen, D. N. (1981). “Vanadyl(IV) EPR spin probe. Inorganic and biochemical tyrosine phosphorylation of the insulin receptor. J. Biol. Inorg. Chem. 10, aspects,” in Biological Magnetic Resonance, eds L. J. J. Berliner and J. Reuben 874–886. doi: 10.1007/s00775-005-0037-x (New York, NY: Plenum Press), 53–119. doi: 10.1007/978-1-4613-3201-5_2 Pettersen, E. F., Goddard, T. D., Huang, C. C., Couch, G. S., Greenblatt, D. M., Costa Pessoa, J., Etcheverry, S., and Gambino, D. (2015a). Vanadium compounds Meng, E. C., et al. (2004). UCSF Chimera-A visualization system for exploratory in medicine. Coord. Chem. Rev. 30, 24–48. doi: 10.1016/j.ccr.2014.12.002 research and analysis. J. Comput. Chem. 25, 1605–1612. doi: 10.1002/jcc. Costa Pessoa, J., Garribba, E., Santos, M. F. A., and Santos-Silva, T. (2015b). 20084 Vanadium and proteins: uptake, transport, structure, activity and function. Qureshi, I. A., Ferron, F., Seh, C. C., Cheung, P., and Lescar, J. (2009). Coord. Chem. Rev. 30, 49–86. doi: 10.1016/j.ccr.2015.03.016 Crystallographic structure of ubiquitin in complex with cadmium ions. BMC Crans, D. C., Khan, A. R., Mahroof-Tahir, M., Mondal, S., Miller, S. M., la Cour, Res. Notes 2:251. doi: 10.1186/1756-0500-2-251 A., et al. (2001). Bis(acetylamido)oxovanadium(IV) complexes: solid state and Rehder, D. (2016). Perspectives for vanadium in health issues. Future Med. Chem. solution studies. J. Chem. Soc. Dalton Trans. 3337–3345. doi: 10.1039/b101718g 8, 325–338. doi: 10.4155/fmc.15.187 Crans, D. C., Yang, L., Haase, A., and Yang, X. (2018). “Health benefits of vanadium Reul, B. A., Amin, S. S., Buchet, J.-P., Ongemba, L. N., Crans, D. C., and Brichard, and its potential as an anticancer agent,” in Metallo-Drugs: Development and S. M. (1999). Effects of vanadium complexes with organic ligands on glucose Action of Anticancer Agents, eds A. Sigel, H. Sigel, E. Freisinger, and R. K. O. metabolism: a comparison study in diabetic rats. Br. J. Pharmacol. 126, 467–477. Sigel (Berlin: De Gruyter GmbH), 251–280. doi: 10.1515/9783110470734-009 doi: 10.1038/sj.bjp.0702311 Diamond, R. (1974). Real-space refinement of the structure of hen egg-white Rodríguez-Guerra Pedregal, J., Sciortino, G., Guasp, J., Municoy, M., and lysozyme. J. Mol. Biol. 82, 371–391. doi: 10.1016/0022-2836(74)90598-1 Maréchal, J.-D. (2017). GaudiMM: a modular multi-objective platform for Elvingson, K., González Baró, A., and Pettersson, L. (1996). Speciation in molecular modeling. J. Comput. Chem. 38, 2118–2126. doi: 10.1002/jcc. vanadium bioinorganic systems. 2. An NMR, ESR, and potentiometric study 24847 of the aqueous H+ -vanadate–maltol system. Inorg. Chem. 35, 3388–3393. Sakurai, H., Yoshikawa, Y., and Yasui, H. (2008). Current state for the development doi: 10.1021/ic951195s of metallopharmaceutics and anti-diabetic metal complexes. Chem. Soc. Rev. Frisch, M. J., Trucks, G. W., Schlegel, H. B., Scuseria, G. E., Robb, M. A., 37, 2383–2392. doi: 10.1039/b710347f Cheeseman, J. R., et al. (2010). Gaussian 09, Revision D.01. Wallingford, CT: Sanna, D., Garribba, E., and Micera, G. (2009). Interaction of VO2+ ion with Gaussian, Inc. human serum transferrin and albumin. J. Inorg. Biochem. 103, 648–655. Fu, Y., Wang, Q., Yang, X.-G., Yang, X.-D., and Wang, K. (2008). Vanadyl doi: 10.1016/j.jinorgbio.2009.01.002 bisacetylacetonate induced G1/S cell cycle arrest via high-intensity ERK Scior, T., Guevara-Garcia, J. A., Do, Q.-T., Bernard, P., and Laufer, S. (2016). phosphorylation in HepG2 cells. J. Biol. Inorg. Chem. 13, 1001–1009. Why antidiabetic vanadium complexes are not in the pipeline of “big doi: 10.1007/s00775-008-0387-2 pharma” drug research? A critical review. Curr. Med. Chem. 23, 2874–2891. Jakusch, T., and Kiss, T. (2017). In vitro study of the antidiabetic doi: 10.2174/0929867323666160321121138 behavior of vanadium compounds. Coord. Chem. Rev. 351, 118–126. Sciortino, G., Garribba, E., and Maréchal, J.-D. (2019a). Validation and doi: 10.1016/j.ccr.2017.04.007 applications of protein–ligand docking approaches improved for Jones, G., Willett, P., Glen, R. C., Leach, A. R., and Taylor, R. (1997). Development metalloligands with multiple vacant sites. Inorg. Chem. 58, 294–306. and validation of a genetic algorithm for flexible docking1. J. Mol. Biol. 267, doi: 10.1021/acs.inorgchem.8b02374 727–748. doi: 10.1006/jmbi.1996.0897 Sciortino, G., Garribba, E., Rodríguez-Guerra Pedregal, J., and Marechal, J.- Kioseoglou, E., Petanidis, S., Gabriel, C., and Salifoglou, A. (2015). The chemistry D. (2019b). Simple coordination geometry descriptors allow to accurately and biology of vanadium compounds in cancer therapeutics. Coord. Chem. Rev. predict metal-binding sites in proteins. ACS Omega 4, 3726–3731. 87–105. doi: 10.1016/j.ccr.2015.03.010 doi: 10.1021/acsomega.8b03457 Lindqvist, Y., Schneider, G., and Vihko, P. (1994). Crystal structures Sciortino, G., Rodríguez-Guerra Pedregal, J., Lledós, A., Garribba, E., and of rat acid phosphatase complexed with the transition-state Maréchal, J.-D. (2018a). Prediction of the interaction of metallic moieties with analogs vanadate and molybdate. Eur. J. Biochem. 221, 139–142. proteins: an update for protein-ligand docking techniques. J. Comput. Chem. doi: 10.1111/j.1432-1033.1994.tb18722.x 39, 42–51. doi: 10.1002/jcc.25080 Liu, J.-C., Yu, Y., Wang, G., Wang, K., and Yang, X.-G. (2013). Sciortino, G., Sanna, D., Ugone, V., Lledós, A., Maréchal, J.-D., and Garribba, E. Bis(acetylacetonato)-oxovanadium(IV), bis(maltolato)-oxovanadium(IV) (2018b). Decoding surface interaction of VIV O metallodrug candidates with and sodium metavanadate induce antilipolytic effects by regulating hormone- lysozyme. Inorg. Chem. 57, 4456–4469. doi: 10.1021/acs.inorgchem.8b00134 sensitive lipase and perilipin via activation of Akt. Metallomics 5, 813–820. Sciortino, G., Sanna, D., Ugone, V., Micera, G., Lledós, A., Maréchal, J.-D., et al. doi: 10.1039/c3mt00001j (2017). Elucidation of binding site and chiral specificity of oxidovanadium Lovell, S. C., Word, J. M., Richardson, J. S., and Richardson, D. C. drugs with lysozyme through theoretical calculations. Inorg. Chem. 56, (2000). The penultimate rotamer library. Proteins 40, 389–408. 12938–12951. doi: 10.1021/acs.inorgchem.7b01732 doi: 10.1002/1097-0134(20000815)40:3<389::AID-PROT50>3.0.CO;2-2 Smith, T. S. II, LoBrutto, R., and Pecoraro, V. L. (2002). Paramagnetic spectroscopy Makinen, M. W., and Brady, M. J. (2002). Structural origins of the insulin- of vanadyl complexes and its applications to biological systems. Coord. Chem. mimetic activity of Bis(acetylacetonato)oxovanadium(IV). J. Biol. Chem. 277, Rev. 228, 1–18. doi: 10.1016/S0010-8545(01)00437-4 12215–12220. doi: 10.1074/jbc.M110798200 Thompson, K. H., Lichter, J., LeBel, C., Scaife, M. C., McNeill, J. H., and Orvig, C. Makinen, M. W., Rivera, S. E., Zhou, K. I., and Brady, M. J. (2007). “Enhancement (2009). Vanadium treatment of type 2 diabetes: a view to the future. J. Inorg. of insulin action by bis(Acetylacetonato)oxovanadium(IV) occurs through Biochem. 103, 554–558. doi: 10.1016/j.jinorgbio.2008.12.003 uncompetitive inhibition of protein Tyrosine phosphatase-1B,” in Vanadium: Thompson, K. H., and Orvig, C. (2006). Vanadium in diabetes: 100 The Versatile Metal, eds K. Kustin, D. C. Crans and J. Costa Pessoa (American years from Phase 0 to Phase I. J. Inorg. Biochem. 100, 1925–1935. Chemical Society), 82–92. doi: 10.1016/j.jinorgbio.2006.08.016 Frontiers in Chemistry | www.frontiersin.org 10 May 2020 | Volume 8 | Article 345 Sciortino et al. Biospeciation of Potential Vanadium Drugs Ugone, V., Sanna, D., Sciortino, G., Marechal, J. D., and Conflict of Interest: The authors declare that the research was conducted in the Garribba, E. (2019). Interaction of vanadium(IV) species with absence of any commercial or financial relationships that could be construed as a Ubiquitin: a combined instrumental and computational approach. potential conflict of interest. Inorg. Chem. 58, 8064–8078. doi: 10.1021/acs.inorgchem.9b 00807 Copyright © 2020 Sciortino, Ugone, Sanna, Lubinu, Ruggiu, Maréchal and Garribba. Wu, J.-X., Hong, Y.-H., and Yang, X.-G. (2016). Bis(acetylacetonato)- This is an open-access article distributed under the terms of the Creative Commons oxidovanadium(IV) and sodium metavanadate inhibit cell proliferation Attribution License (CC BY). The use, distribution or reproduction in other forums via ROS-induced sustained MAPK/ERK activation but with is permitted, provided the original author(s) and the copyright owner(s) are credited elevated AKT activity in human pancreatic cancer AsPC-1 cells. and that the original publication in this journal is cited, in accordance with accepted J. Biol. Inorg. Chem. 21, 919–929. doi: 10.1007/s00775-016- academic practice. No use, distribution or reproduction is permitted which does not 1389-0 comply with these terms. Frontiers in Chemistry | www.frontiersin.org 11 May 2020 | Volume 8 | Article 345