Biotransformation of BMOV in the presence of blood serum proteins

Abstract

The interaction of the potent anti-diabetic agent bis(maltolato)oxidovanadium(IV) (BMOV) with some proteins of blood serum was studied by EPR spectroscopy, pH-potentiometry and DFT calculations. The formation of cis-VO(ma)₂(hTf), cis-VO(ma)₂(HSA) and cis-VO(ma)₂(IgG), their role in the biotransformation in vivo and the mechanism of transport of BMOV in blood are discussed.

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One of the most important applications of vanadium compounds is their potential use in the therapy of patients suffering from type II diabetes mellitus.¹ It was found that neutral VO²⁺ complexes with bidentate anionic ligands (VOL₂) are more effective, better tolerated, and result in reliable glucose-lowering in all the animal models of diabetes than VO²⁺ and vanadium(V) inorganic salts. Bis(maltolato)oxidovanadium(IV)—[VO(ma)₂], where Hma is maltol—or BMOV became the most widely and extensively tested of a whole series of VOL₂ complexes and now is considered the benchmark for new oral anti-diabetic vanadium compounds.² A derivative, bis(ethylmaltolato)oxidovanadium(IV) or BEOV, arrived at phase IIa of the clinical trials.³ However, their biotransformation in the blood, the mechanism with which vanadium is transported to the target organs and the in vivo form are not fully known and are the subject of much debate. This has been recently reviewed.^4,5 Particularly, the interaction of pharmaceuticals with the serum proteins is an important aspect in the drug metabolism, as it is capable of strongly affecting their distribution, biotransformation, and ultimately mechanism of action.

In this work we examined the interaction of BMOV with some proteins of blood serum (human serum transferrin (hTf), human serum albumin (HSA) and immunoglobulin G (IgG)) through the combined application of electron paramagnetic resonance (EPR) spectroscopy, pH-potentiometry and density functional theory (DFT) methods. The results may allow us to clarify which is the active form under physiological relevant conditions and the biodistribution of VO²⁺ ion among the blood serum components.

VO²⁺, like several metal ions, binds to transferrin in the two Fe³⁺ sites (the N- and C-lobe sites) and needs HCO₃⁻ or other anions, called synergistic anions, for its binding.⁶ The ternary system VO²⁺/hTf/Hma has been previously studied: both Willsky and Crans,⁷ and Orvig and co-workers,⁸ based on EPR measurements, found no interaction between the anti-diabetic complex and the protein at pH 7.4. Subsequently, Kiss and co-workers confirmed these hypotheses with potentiometric and ultra-filtration studies.^4a,9

Anisotropic EPR spectrum, recorded at pH 7.4 in the binary system VO²⁺/Hma, shows the formation of the hexa-coordinated species cis-[VO(ma)₂(H₂O)], with an (equatorial–equatorial) and (equatorial–axial) arrangement of the two ligands and a water molecule bound in the fourth equatorial position (I in Fig. 1). In contrast, in the ternary system containing VO²⁺, hTf and Hma, two different complexes are observed (III and IV in Fig. 1). The predominant species (indicated with IV) can be identified as (VO)hTf and/or (VO)₂hTf, i.e. the complexes in which vanadium is bound to one or both of the iron coordination sites; the other species (indicated with III) is characterized by g_z = 1.944 and A_z = 164.7 × 10⁻⁴ cm⁻¹, see Table S1 of ESI.† The spectrum of this latter species is very similar to that of the mixed complex cis-[VO(ma)₂(1-MeIm)]—where 1-MeIm is 1-methylimidazole, a model for the binding of a histidine residue—in which the equatorially coordinated water molecule of cis-[VO(ma)₂(H₂O)] is replaced by a His-N (Scheme 1a). It is noteworthy that the experimental spectrum recorded in the VO²⁺/hTf/Hma system can be reproduced by adding the spectra obtained in the binary system with transferrin and in the ternary with maltol and 1-methylimidazole (see trace c of Fig. 1).

Fig. 1 High-field region of anisotropic X-band EPR spectra recorded at pH 7.4 in aqueous solution containing: (a) VO²⁺/Hma 1/2 (VO²⁺ 1.0 × 10⁻³ M); (b) VO²⁺/Hma/1-MeIm 1/2/4 (VO²⁺ 1.0 × 10⁻³ M); (c) sum of the spectra obtained in the systems VO²⁺/Hma/1-MeIm (trace b) and VO²⁺/hTf (trace e); (d) VO²⁺/hTf/Hma 2/1/4 (VO²⁺ 5.0 × 10⁻⁴ M) and (e) VO²⁺/hTf 2/1 (VO²⁺ 5.0 × 10⁻⁴ M). The M_I = 7/2 resonances of cis-[VO(ma)₂(1-MeIm)] and cis-VO(ma)₂(hTf) (broken line), of (VO)hTf/(VO)₂hTf (dotted line), and of cis-[VO(ma)₂(H₂O)] (full line) are indicated; I, II, III and IV denote the species cis-[VO(ma)₂(H₂O)], cis-[VO(ma)₂(1-MeIm)], cis-VO(ma)₂(hTf), and (VO)hTf/(VO)₂hTf, respectively.

Scheme 1 Structure of the species cis-[VO(ma)₂(1-MeIm)] (a) and cis-VO(ma)₂(protein) (b), where protein indicates hTf, HSA or IgG.

Therefore, we suppose that hTf binds a VO²⁺ ion with an imidazole nitrogen of a histidine residue exposed on the protein surface, originating a complex with composition cis-VO(ma)₂(hTf) (Scheme 1b). The possible interaction with the other potential donors present in the amino acid side chains (for example, Tyr-O⁻, Ser-O⁻, Asp-COO⁻ and Cys-S⁻) can be ruled out on the basis of the EPR results obtained in the ternary systems formed by VO²⁺, maltol and a model ligand, such as 4-hydroxybenzoic acid, methanol, acetic acid and ethanethiol, that should mimic the coordination of tyrosine, serine, aspartic acid and cysteine residues, respectively.

These results are in contrast with those recently published by Jakusch et al.,⁵ who propose that Hma binds VO²⁺ at the iron binding sites, even if maltol does not have the features of a synergistic anion (it does not possess a carboxylate nor an electron withdrawing group).¹⁰

The log β value of cis-VO(ma)₂(hTf) (19.2 ± 0.6, Table 1) can be estimated by measuring the ratio between the intensities of the M_I = 5/2, 7/2 EPR resonances of cis-VO(ma)₂(hTf) and binary species formed by transferrin, (VO)hTf and (VO)₂hTf (Fig. S5 of ESI†).

Table 1 Stability constants of the species formed for reaction of BMOV with histidine models and blood serum proteins

Species	log β	Method
cis-[VO(ma)2(1-MeIm)]	19.12 ± 0.01	pH-potentiometry
cis-[VO(ma)2(Ac-his)]	18.96 ± 0.04	pH-potentiometry
cis-VO(ma)2(hTf)	19.2 ± 0.6	EPR spectroscopy
cis-VO(ma)2(HSA)	19.5 ± 1.0	EPR spectroscopy
cis-VO(ma)2(IgG)	19.6 ± 1.0	EPR spectroscopy

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pH-potentiometry confirms unambiguously that in the ternary system VO²⁺/Hma/1-MeIm a main species with composition VO(ma)₂(1-MeIm) is formed. Potentiometric titrations indicate that another model for monodentate histidine coordination, acetyl-histamine (Ac-his), interacts with BMOV in the same way forming VO(ma)₂(Ac-his). The obtained values of log β are summarized in Table 1, together with the stability constants for cis-VO(ma)₂(protein) species, with protein = hTf, HSA, IgG, measured from EPR spectra.

The fact that A_z (both experimental and calculated by DFT methods, see below) and the log β values of cis-VO(ma)₂(hTf) (and of cis-VO(ma)₂(HSA) and cis-VO(ma)₂(IgG)) are comparable with those of the models cis-[VO(ma)₂(1-MeIm)] and cis-[VO(ma)₂(Ac-his)] (coordination mode [(O_ket, O⁻_phen); (O_ket, O⁻_phen^ax); N_imid]) confirms that in all the species the coordination mode is [(O_ket, O⁻_phen); (O_ket, O⁻_phen^ax); N_His]. In other words, a histidine residue on the protein surface behaves like a simple monodentate imidazole nitrogen, as that present on the model ligands (1-methylimidazole and acetyl-histamine, for example).

DFT calculations of the ⁵¹V hyperfine coupling tensor A further confirm our suppositions. It has been recently demonstrated that it is possible to calculate A_z for VO²⁺ species with Gaussian 03 and ORCA softwares with a percent deviation from the experimental value below 3 and 4%, respectively.¹¹ The simulations were performed on the model cis-[VO(ma)₂(1-MeIm)] and on the species where the fourth equatorial site is occupied by the coordinating side chain of some amino acids, like His-N, Tyr-O⁻, Ser-O⁻, Asp-COO⁻ and Cys-S⁻. The results, listed in Table 2, demonstrate that only the binding of an aromatic imidazole nitrogen (belonging to a model ligand, such as 1-MeIm, or to a His residue of a protein chain, mimicked by Ac-His-NH₂) gives a value of A_z comparable to that experimentally observed. This implies that, when the anti-diabetic compound adopts in aqueous solution under physiological conditions the cis-octahedral geometry, the binding of the blood proteins in the fourth equatorial position through a surface histidine residue is strongly favoured.

Table 2 ⁵¹V A_z values calculated by DFT methods for the species formed for reaction of BMOV with the coordinating side chains of some amino acids^a

Species^b	A ^calcd z ^c	A ^calcd z ^d	A ^exptl z
a Values reported in 10^−4 cm^−1. b Ac indicates acetyl group. c Calculated by Gaussian software. d Calculated by ORCA software. e Values to be compared with the experimental Az obtained for cis-VO(ma)2(hTf), cis-VO(ma)2(HSA) and cis-VO(ma)2(IgG), that are 164.7, 165.5 and 164.6 × 10^−4 cm^−1, respectively.
cis-[VO(ma)2(1-MeIm)]	162.6	161.2	164.8
cis-[VO(ma)2(Ac-His-NH2)]	163.4	162.4	^e
cis-[VO(ma)2(Ac-Tyr-NH2)]	159.7	158.1	^e
cis-[VO(ma)2(Ac-Ser-NH2)]	157.7	156.6	^e
cis-[VO(ma)2(Ac-Asp-NH2)]	170.5	169.9	^e
cis-[VO(ma)2(Ac-Cys-NH2)]	155.2	153.2	^e

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Unlike transferrin, that has specific sites for metal ion coordination, albumin interacts with vanadium with His-N and Asp/Glu-COO⁻ side chains; it has been demonstrated that it forms a dinuclear species (VO)₂^dHSA‡ in equimolar solution and (VO)_x^mHSA‡ when VO²⁺/HSA > 1.¹² Orvig and co-workers showed that maltol can form ternary complexes with HSA with compositions VO(ma)(HSA) and VO(ma)₂(HSA).⁸

In this work we re-examined the ternary system VO²⁺/HSA/Hma. The anisotropic spectrum, recorded at pH 7.4 in aqueous solution containing VO²⁺/HSA/Hma with molar ratio 4/1/8, shows the presence of only one species characterized by g_z = 1.945 and A_z = 165.5 × 10⁻⁴ cm⁻¹ and rather wide resonances (Fig. S6 of ESI†). However, if the spectra obtained in the two systems VO²⁺/Hma/1-MeIm (model for VO²⁺/HSA/Hma) and VO²⁺/HSA are compared, it can be noticed that cis-[VO(ma)₂(1-MeIm)] (model for cis-VO(ma)₂(HSA)) and (VO)_x^mHSA have similar hyperfine coupling constants, 164.8 and 164.6 × 10⁻⁴ cm⁻¹, respectively. Therefore, it is likely that in the ternary system VO²⁺/HSA/Hma cis-VO(ma)₂(HSA) and (VO)_x^mHSA coexist. A similar species, cis-VO(dhp)₂(HSA), was found in the system with 1,2-dimethyl-3-hydroxy-4(1H)-pyridinone (Hdhp).^13,14 To obtain log β of cis-VO(ma)₂(HSA), EPR spectra were recorded with VO²⁺/HSA/Hma 1/1/2: at this molar ratio, albumin forms only (VO)₂^dHSA,¹² and the relative amounts of the latter species and cis-VO(ma)₂(HSA) can be measured. Using the value of log β for (VO)₂^dHSA,¹⁴ it was found that log β(VO(ma)₂(HSA)) = 19.5 ± 1.0. In this case too, log β is comparable with that of cis-[VO(ma)₂(1-MeIm)] and cis-[VO(ma)₂(Ac-his)] (Table 1). Thus, our results confirm those previously obtained by Orvig and co-workers,⁸ that albumin can replace the equatorial water molecule of cis-[VO(ma)₂(H₂O)] through a superficial histidine residue, forming cis-VO(ma)₂(HSA) (Scheme 1b).

Crans and Willsky found no interaction of BMOV with IgG by EPR.⁷ However, it has been recently demonstrated that IgG is able to bind VO²⁺ with at least three unspecific sites of comparable strength (called sites 1, 2 and 3, and indicated with IVa, IVb and IVc in Fig. 2), forming a species with composition (VO)_xIgG with x = 3–4.¹⁵ The anisotropic EPR spectrum, recorded in the aqueous solution at pH 7.4, of the ternary system VO²⁺/IgG/Hma with molar ratio 1/1/2, shows the presence of a predominant species (III in Fig. 2)—not observed in the binary systems VO²⁺/IgG and VO²⁺/Hma—characterized by a spectrum similar to that of cis-[VO(ma)₂(1-MeIm)] (II in Fig. 2). The value of A_z calculated by DFT methods for cis-[VO(ma)₂(Ac-His-NH₂)] deviates with respect to the experimental one for the ternary species formed by IgG of 0.7 (Gaussian) and 1.3% (ORCA), suggesting a probable equatorial coordination of a His-N. This indicates that, in this case too, the water molecule has been replaced by an imidazole nitrogen belonging to a surface histidine, and that the mixed species cis-VO(ma)₂(IgG) is formed (Scheme 1b).

Fig. 2 High-field region of anisotropic X-band EPR spectra recorded at pH 7.4 in aqueous solution containing: (a) VO²⁺/Hma 1/2 (VO²⁺ 1.0 × 10⁻³ M); (b) VO²⁺/Hma/1-MeIm 1/2/4 (VO²⁺ 1.0 × 10⁻³ M); (c) VO²⁺/IgG/Hma 1/1/2 (VO²⁺ 3.0 × 10⁻⁴ M) and (d) VO²⁺/IgG 1/1 (VO²⁺ 3.0 × 10⁻⁴ M). The M_I = 7/2 resonances of cis-[VO(ma)₂(1-MeIm)] and cis-VO(ma)₂(IgG) (broken line), of (VO)_xIgG (dotted line), and of cis-[VO(ma)₂(H₂O)] (full line) are indicated; I, II, III, IVa, IVb and IVc denote the species cis-[VO(ma)₂(H₂O)], cis-[VO(ma)₂(1-MeIm)], cis-VO(ma)₂(IgG) and the sites 1, 2 and 3 of (VO)_xIgG, respectively.

Therefore, the behaviour of the system VO²⁺/IgG/Hma resembles that of VO²⁺/hTf/Hma and VO²⁺/HSA/Hma. The stability constant of such a mixed complex can be determined analogously as previously described in the case of transferrin and albumin (Table 1). The obtained value is log β(VO(ma)₂(IgG)) = 19.6 ± 1.0. It is interesting to observe that also actin binds to VO²⁺ in a monodentate manner with a metal–protein ratio of 1 : 1.¹⁶

The values calculated for log β of cis-VO(ma)₂(protein), those present in the literature for the binary VO²⁺ complexes formed by Hma,¹⁷hTf,¹⁴ HSA,¹⁴ IgG,¹⁵ and for the binary and ternary species formed by the two most important low molecular mass bioligands of blood serum, citrate and lactate,¹⁸ allow us to predict the biodistribution of BMOV, administrated orally, between the components of the blood. In Table 3 three VO²⁺ concentrations were examined, 1, 10 and 50 μM; the results indicate that, where the concentration is in the range 1–10 μM, the predominant species in solution should be (VO)hTf, favoured by the ratio VO²⁺/hTf < 1. This is in agreement with the conclusions of Kiss and co-workers, who foresee that transferrin is the preferred bioligand for vanadium at very low concentrations.^4a,5,9 However, an amount of VO²⁺ (∼4–5%) should be present as (VO)hTf(lact) in which lactate, behaving as a synergistic anion, replaces the bicarbonate anion in the iron binding sites. When the concentration of VO²⁺ in the blood serum increases (50 μM), the percentage of vanadium bound in binary species with transferrin decreases and, contemporaneously, increases the importance of the cis-octahedral mixed species formed by maltolate and proteins, cis-VO(ma)₂(hTf), cis-VO(ma)₂(HSA) and cis-VO(ma)₂(IgG). Their total amount reaches 10.7%. Among these three compounds, that with albumin seems to be the most important, not because of the high thermodynamic stability, but due to the high concentration of HSA in blood serum (630 vs. 84 of IgG and 37 μM of hTf).

Table 3 Predicted percent distribution of the VO²⁺ species formed from the biotransformation of BMOV in the blood serum at concentrations of 1, 10 and 50 μM and pH 7.4^a,b

VO^2+ concentration/μM	1	10	50
a Concentration of the blood components taken from ref. 19. b For transferrin, since under physiological conditions 30% of the binding sites is occupied by Fe^3+, the concentration of 25.9 μM was used.
(VO)hTf	91.9	74.3	16.4
(VO)2hTf	2.7	20.4	63.5
(VO)hTf(lact)	4.4	3.5	0.8
(VO)2hTf(lact)2	0.0	0.1	0.1
(VO)2^dHSA	0.0	0.0	0.8
(VO)x^mHSA	0.3	0.3	1.0
(VO)xIgG	0.6	0.7	2.0
VO(ma)2(hTf)	0.0	0.0	0.5
VO(ma)2(HSA)	0.0	0.2	7.6
VO(ma)2(IgG)	0.0	0.1	2.6
[VO(ma)2]	0.0	0.0	1.2
[VO(lactH−1)2]^2^−	0.1	0.1	0.3
[(VO)2(citrH−1)2]^4^−	0.0	0.0	0.3
[VO(ma)(citr)]^2^−	0.0	0.2	2.2
[VO(ma)(citrH−1)]^3^−	0.0	0.1	0.7

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On the basis of the results discussed in this work, the following conclusions can be reached: (i) differently from what has been previously reported,^4,5,7–9 the potent anti-diabetic agent BMOV can interact with both hTf and IgG forming mixed complexes; (ii) maltol, not possessing the features of a synergistic anion,¹⁰ cannot coordinate vanadium at the specific iron sites of transferrin,⁵ but hTf behaves as a simple monodentate ligand and binds VO²⁺ with a His-N exposed on the protein surface forming cis-VO(ma)₂(hTf); (iii) analogously to hTf, also HSA and IgG interact with the metal ion with an exposed His-N, yielding cis-VO(ma)₂(HSA) and cis-VO(ma)₂(IgG); (iv) every protein provided with surface histidines may replace the equatorial water molecule of cis-[VO(ma)₂(H₂O)] to give cis-VO(ma)₂(protein) and an analogous behaviour is expected for the parent anti-diabetic compounds formed by ethylmaltol, kojic acid, etc.; (v) the importance of such species increases for metal concentration larger than 10 μM; (vi) the formation of the ternary species cis-VO(carrier)₂(protein), where carrier is a bidentate monoanionic organic ligand, can contribute significantly to the transport processes of the anti-diabetic VO²⁺ drugs toward the target organs and may explain the significant difference in their pharmacological activity.

Therefore, the interaction of whatever vanadium containing drug (anti-parasitic, anti-viral, anti-HIV, anti-tuberculosis, anti-cancer and spermicidal agents ^4b) with the surface histidines of the serum proteins must be considered when its transport in the blood is examined.

Notes and references

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9. (a) T. Kiss, T. Jakusch, D. Hollender and A. Dörnyei, in Vanadium: the Versatile Metal, ed. K. Kustin, J. Costa Pessoa and D. C. Crans, American Chemical Society, Washington, 2007, pp. 323–339; (b) T. Jakusch, D. Hollender, E. A. Enyedy, C. Sánchez Gonzáles, M. Montes-Bayón, A. Sanz-Medel, J. Costa Pessoa, I. Tomaz and T. Kiss, Dalton Trans., 2009, 2428–2437.
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Footnotes

† Electronic supplementary information (ESI) available: Experimental and computational section, tables showing the experimental and calculated EPR parameters, figures showing the EPR spectra of the ternary systems and distribution of VO²⁺ ion among the components of blood serum at pH 7.4. See DOI: 10.1039/c1mt00161b.

‡ (VO)₂^dHSA indicates the species in which the two VO²⁺ ions are interacting and the EPR spectrum is characteristic of a dinuclear complex with a spin state S = 1, whilst (VO)_x^mHSA (where x = 5–6 and the superscript m denotes a multinuclear complex) the species in which the VO²⁺ ions do not interact.

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