The Role of Vanadium in Metallodrugs Design and Its Interactive Profile with Protein Targets

Abstract

Metallodrugs represent a critical area of medicinal chemistry with the potential to address a wide range of diseases. Their design requires a multidisciplinary approach, combining principles of inorganic chemistry, pharmacology, and molecular biology to create effective and safe therapeutic agents. Vanadium, the element of the fifth group of the first transition series (3d metals), has been already detected as a crucial species in the biological action of some enzymes, e.g., nitrogenases and chloroperoxidase; furthermore, vanadium-based compounds have recently been described as physiologically stable with therapeutic behavior, e.g., having anticancer, antidiabetic (insulin-mimicking), antiprotozoal, antibacterial, antiviral, and inhibition of neurodegenerative disease properties. Since the binding of metallodrugs to serum albumin influences the distribution, stability, toxicity (intended and off-target interactions), and overall pharmacological properties, the biophysical characterization between serum albumin and vanadium-based compounds is one of the hot topics in pharmacology. Overall, since vanadium complexes offer new possibilities for the design of novel metallodrugs, this review summarized some up-to-date biological and medicinal aspects, highlighting proteins as the main targets for the inorganic complexes based on this transition metal.

1. Introduction: The Vanadium Element

Vanadium is an element from the fifth group of the first transition series (3d metals) with electronic configuration [Ar]3d³4s², which results in several oxidation states, although 3+, 4+ and 5+ are the most biologically relevant [1,2]. In this sense, the 4+ and 5+ oxidation states are the most explored in drug discovery due to their prodrug character in diverse biological systems [3]. These higher oxidation states are from the strong oxophilicities and Lewis’s acidity, e.g., in the species oxidovanadium (IV/V) ([V^IVO]²⁺ and [V^VO]³⁺, respectively) and cis-dioxidovanadium(V) ([cis-V^VO₂]⁻) [3,4,5].

The vanadium element receives its name in allusion to Vanadis, a Scandinavian goddess of beauty and fertility, also referring to Freyja [6,7]. Vanadium found in the Earth’s crust, water reserves, and atmosphere has a total concentration of 135 ppm, being the twenty-first most abundant element in the external regions of planet Earth [8,9]. Despite its low concentration in the crust when compared to other elements, vanadium, in different oxidation states, is found in a very wide range of minerals presenting different oxidation states, many with economic interests (

Table 1. Some minerals containing vanadium in composition and their respective oxidation states.

Complex	Mineral Name	Vanadium Oxidation State
Pb5[(VO4)3]Cl	Vanadinite [10]	V
K2(UO2)2[VO4]2·3H2O	Carnotite [11]	V
VS4	Patronite [12]	IV
K(V,Al)2(AlSi3O10)(OH)2	Roscoelite [13]	III

).

In marine environments, vanadium is the second most abundant transition metal, presenting concentrations of 30 to 35 nM in the form of Mⁿ⁺H₂VO₄, where M is the metallic counterion, such as sodium. This concentration is lower than those reported with molybdenum, at an average concentration of 100 nM in the form of molybdates (MoO₄²⁻) [8,9]. Interestingly, some marine invertebrates, such as tunicates, which belong to the subphylum Tunicata (or Urochordata), also called Ascidiacea (sea squirts or marine tunicates), have an unusual capacity about accumulating vanadium cations in high concentrations. There is evidence that some of these tunicates can concentrate vanadium at up to 350 mM, levels dozens-fold higher than those found in seawater [14,15].

In ascidians, vanadium is assimilated in the pentavalent state in the form of vanadates; it is reduced to the oxidovanadium(IV) species before then being subsequently reduced to the vanadium(III) species in the form of [V^III(H₂O)₅HSO₄]²⁺/[V^III(H₂O)₆]³⁺. These aquacomplexes are accumulated and stored in marine cells called vanadocytes. It is speculated that the function of vanadium in these organisms is to protect them against predators due to the possible toxicity of vanadium in high concentrations [9].

Some reducing agents can offer some type of understanding of how such redox processes can occur, such as glutathione, nicotinamide adenine dinucleotide phosphate (NADP) [16,17], tunichromes [18,19,20] and Vanabin 2 [21,22]. However, among the reducing agents described above, only a reduction from V⁵⁺ to V⁴⁺ occurs. The reduction to vanadium V³⁺ may be associated with reductions by organic sulfur species and sulfates [23]. This statement is based on the confirmation of the existence of high concentrations of sulfate ions and a pH close to 2 in the vacuoles of vanadocytes [23].

For humans, potable water that is rich in vanadate and diets that are rich in brown rice, beans, oats, seafood, seaweed, mushrooms, spinach, and some root vegetables, such as radishes, carrots, and beets, can lead to human body concentrations of vanadium of up to 100 mcg/day [24,25]. This daily intake increases the concentration of vanadate anions in the human bloodstream [26], and, due to the highly acidic stomach environment, there is the conversion to VO²⁺ and VO₂²⁺ [8,27,28]. These vanadium species heading to the intestinal environment suffer another chemical transformation to HVO₄²⁻ and V₂O₇⁴⁻ (vanadate dimer), even transforming into oligovanadates due to the slightly alkaline pH [29,30,31].

The availability and chemical transformations of vanadium in the biological medium favor its high affinity with biomacromolecules such as serum albumin [32,33], transferrin [34,35], and immunoglobulin [36]. In these cases, selective receptors are sensitized, resulting in endocytosis [32,37], allowing the vanadium species to enter the cell. On the other hand, when endocytosis does not occur, vanadium can enter the cell through phosphate and sulfate channels [38] or monocarboxylate, organic anion, and mitochondrial citrate transport proteins [39,40]. As can be noticed, there are numerous ways for vanadium species to enter the cell; however, all physiological alternatives associated with interaction and intercellular displacement are related to some proteins, e.g., dicarboxylate and tricarboxylate carriers, ATP-binding cassette (ABC) transporters, and divalent metal transporters and mitochondrial citrate transport proteins [40,41,42]. In each case, the strength of the vanadium–protein interaction will be closely related to which ligand ends up donating electron density or charge for its stabilization and subsequent availability. When there is an evaluation of external factors and not just the coordination of the metal center itself, pH and redox environment changes seem to be evident regarding the release of vanadium after binding to transport proteins.

2. Vanadium in Biological Systems

In the 1970s, the biological essentiality of vanadium was confirmed, and its physiological effects began to be better evaluated [43]. At the end of the 1970s, there was a shift in interest in the bioinorganic chemistry of vanadium driven by the discovery of structural and physiological similarities between phosphate and vanadate(V) groups (Figure 1A, such vanadium species strongly inhibit sodium and potassium pumps) [43,44]. In addition, the discovery of the presence of vanadium in two groups of enzymes (bromoperoxidases, belonging to haloperoxidase enzymes, and nitrogenase enzymes) increased scientific interest in understanding the role of vanadium in biological scenarios [43].

Haloperoxidase enzymes, also known as halogenating peroxidases, are enzymes that catalyze the oxidative halogenation reactions of substrates. Basically, these enzymes mediate the oxidation of halides (X⁻, being chlorides, bromides or iodides) by reaction with hydrogen peroxide (H₂O₂), in a two-electron process, to their respective hypohalides (OX⁻), halogenate species formed in situ. Such species are released when protonated, forming the respective acids (HOX) [45,46]. The reaction involved in the oxidation of halides by H₂O₂ catalyzed by haloperoxidases and the subsequent halogenation of substrates are shown in Equations (1) and (2), respectively [8,45].

X⁻ + H₂O₂ + H₃O⁺ → HOX + H₂O   where X⁻ = Cl⁻, Br⁻, or I⁻

(1)

R-H + HOX → R-X + H₂O               where X⁻ = Cl⁻, Br⁻, or I⁻

(2)

Haloperoxidases are classified according to two criteria, with one being based on the most electronegative species that suffer oxidation to their respective hypohalide and, consequently, the respective acid. On the other hand, the other classification is based on their prosthetic group and, consequently, their mechanism. Regarding the first criterion, there are chloroperoxidases, which can oxidize chlorides, bromides, and iodides; bromoperoxidases, which are able to oxidize bromides and iodides (and chlorides, to some extent); and, finally, iodoperoxidases, whose capacity is limited to iodine, the halogen with the highest oxidation potential and lowest electronegativity. Interestingly, there are still no reports of fluoroperoxidases due to the low oxidation potential and high electronegativity of fluorine [8,45]. Finally, regarding the second criterion, there are peroxidases that are dependent on the heme groups, i.e., porphyrin rings containing an iron metal center, and others that are dependent on vanadium.

Vanadium-dependent haloperoxidases, commonly abbreviated as VHPOs, are found in marine environments, bacteria, terrestrial fungi, and lichens [8,45]. Structurally, VHPOs present vanadium species in the form of vanadates [45,47,48]. When compared to heme-dependent ones, VHPOs exhibit greater stability with hydrogen peroxide. Curiously, chloroperoxidase obtained from the fungus Curvularia inaequalis is stable for days when exposed to at least 100 mM H₂O₂ and is resistant to temperatures of 70 °C, as well as the presence of organic cosolvents [45]. Thus, it can be noticed that VHPOs present structural characteristics like the enzymes from thermophilic organisms that resist high temperatures [49]; however, this chloroperoxidase is inactive at high concentrations of chloride ions [50]. The structural projection of the active site of the native chloroperoxidase of the fungus Curvularia inaequalis in a solid state obtained by single-crystal X-ray diffraction (SC-XRD) is shown in Figure 1B.

As can be seen in Figure 1B, in the active site of this native enzyme, the vanadium metal center (V) is coordinated to three non-protein oxygens in the equatorial plane and, apically, to a fourth oxygen in the position above (vanadate species). As the fifth donor atom, imidazole nitrogen from histidine residue (His-496) is coordinated underneath. Thus, in this case, vanadium appears as a trigonal bipyramid [47], a characteristic geometry of pentacoordinate vanadates in biological systems [44]. Overall, excess anionic charges, resulting from oxygen ligands, are stabilized by an extensive network of hydrogen bonds with amino acid residues in their protonated and cationic forms [51], since this enzyme works in an acidic pH range of 4.5 to 7, with 5.5 being the ideal value [49].

Chloroperoxidase from Curvularia inaequalis, as well as other enzymes belonging to this class, has the function of degrading and/or oxidizing the waxy protective layer of leaves and the cell wall of plant hosts to facilitate the microorganism access to nutrients [49]. The direct formation of halogenated metabolites, more specifically 5-chloro-4-hydroxy-3-methoxybenzaldehyde (5-chlorovanillin) and 2-chloro-4-hydroxy-3,5-dimethoxybenzaldehyde (2-chlorosyringaldehyde) (Figure 1C), from the degradation of wood lignin by the action of chloroperoxidase has already been detected and reported in the literature [49,52].

Furthermore, the same biodegradation of lignin by VHPOs leads to the formation of trichloroacetylated compounds which subsequently decay through other environmental processes (at more basic pH values), to chloroform (CHCl₃). The releases of CHCl₃ in atmosphere might form reactive chlorine species by photolysis, affecting the atmospheric ozone negatively [49,53]. On the other hand, VHPOs from algae and other marine organisms have the functions of regulating the amounts of cellular H₂O₂ and, with the formation of halogenated compounds, defense against infection by microorganisms [53].

According to both experimental and computational studies [54], Figure 1D depicts the synthesis of the HOX species by haloperoxidase. As evidenced in this figure, the mechanism of hypochlorous acid formation begins with the resting enzyme complex A, a dioxidodihydroxydovanadium(V) species. Studies, including density functional theory (DFT), suggest that hydrogen peroxide coordinates to the metallic center, forming complex B, an unstable hydroperoxidovanadium(V) dioxide(hydroxide) species. This species, in turn, reacts with the halide to form HOX acid (OH⁺ transfer) [54,55]. During the catalytic cycle, the oxidation state of the metal center constantly remains pentavalent (5+), with no electron transfer from/to the vanadium metal center [54,55]. Previously, through different studies, especially those from crystallographic [47], it was understood that there was the formation of an intermediate species oxidoperoxovanadium(V) (PDB code 1IDU) [47]; however, by DFT calculations, it was shown that this peroxospecies would be a stable secondary product of an alternative proton transfer channel [54,55].

Thus, the final substrate halogenation step has not yet been fully characterized, with it being suggested that the forming HOX species does not diffuse into the solution for subsequent reaction with substrate, but with the protein participating in the substrate halogenation step. Positively charged amino acid residues present below the His-496 residue and negatively charged residues on the other side of the substrate lead to the formation of a dipole moment. Positively charged amino acid residues carry a positive field, generating a gradient between vanadate and a substrate binding site. Thus, the HOX formed in the active site is diffused along the electric field vectors to the site containing the substrate. Furthermore, such vectors are aligned to the C–X bond formation. Overall, due to the presence of electrostatic interactions, dipole moments, and electric fields with favorable vectors, there is a decrease in the energetic values for the halogenation of the substrate [54].

The study, development, and application of compounds containing vanadium metal centers or other oxophilic transition metals that, inspired by VHPOs, can function as biomimetics of the catalytic activity of these enzymes are increasing [56].

Figure 1. (A) Chemical structures for phosphate, vanadate, and pervanadate. In pervanadate, the exact coordination geometry and the number and arrangement of water ligands is uncertain, and other structures may exist in the solution. (B) A 3D structure with a corresponding zoom representation of the active site of native chloroperoxidase from the fungus Curvularia inaequalis (PDB code 1IDQ) [47]. In the zoom representation, the polar amino acid residues are represented in the form of a yellow stick while vanadate (VO₄) is represented as a sphere. Elements’ colors: oxygen, nitrogen, and vanadium in red, blue, and gray, respectively. For better interpretation, hydrogen atoms were omitted. (C) Chemical structure for the main products from the biodegradation of lignin by chloroperoxidase. (D) Simplified mechanism of the formation of hypochlorous/hypobromous acid by haloperoxidase-containing vanadium. Elements’ colors: oxygen, nitrogen, vanadium, chlorine, phosphorus, and bromine in red, blue, gray, green, orange, and purple, respectively.

Figure 1. (A) Chemical structures for phosphate, vanadate, and pervanadate. In pervanadate, the exact coordination geometry and the number and arrangement of water ligands is uncertain, and other structures may exist in the solution. (B) A 3D structure with a corresponding zoom representation of the active site of native chloroperoxidase from the fungus Curvularia inaequalis (PDB code 1IDQ) [47]. In the zoom representation, the polar amino acid residues are represented in the form of a yellow stick while vanadate (VO₄) is represented as a sphere. Elements’ colors: oxygen, nitrogen, and vanadium in red, blue, and gray, respectively. For better interpretation, hydrogen atoms were omitted. (C) Chemical structure for the main products from the biodegradation of lignin by chloroperoxidase. (D) Simplified mechanism of the formation of hypochlorous/hypobromous acid by haloperoxidase-containing vanadium. Elements’ colors: oxygen, nitrogen, vanadium, chlorine, phosphorus, and bromine in red, blue, gray, green, orange, and purple, respectively.

3. Natural Enzymes with Vanadium in Their Catalytic Core

The biomimetic process of the industrial Haber–Bosch synthesis—a method used to synthesize ammonia from hydrogen and nitrogen—involves nitrogenase enzymes in diazotrophic microorganisms. These enzymes are responsible for nitrogen fixation in the soil, i.e., by the transformation of an inert and biologically unusable species of nitrogen, such as dinitrogen (N₂) to ammonium (NH₄⁺), which is used in the life cycle of microorganisms [8,57,58]. Among nitrogenases, three types of isozymes are well known: Fe-nitrogenases, dependent only on iron; the Mo-nitrogenases, dependent on iron and molybdenum, and, finally, V-nitrogenases, dependent on both iron and vanadium [57,58]. Although Mo-nitrogenases are the most abundant, several diazotrophic microorganisms have genes for the expression of Fe-nitrogenases or V-nitrogenases, or even both. In this context, factors such as the low environmental concentration of molybdenum or inefficiency in its transport can lead to greater expression of these two other nitrogenases, known as “alternative nitrogenases” [57].

V-nitrogenases are found together with other nitrogenases in the soil bacteria of the genus Azobacter, in the cyanobacteria of the genera Anabaena and Nostoc, and in the lichens of the genus Peltigera [8]. As an example, the reaction involved in nitrogen fixation catalyzed by V-nitrogenases of bacterium Azobacter chroococcum is shown in Equation (3) [58].

N₂ + 14H⁺ + 12e⁻ + 40 MgATP → 2NH₄⁺ + 3H₂ + 40MgADP + 40P_i

(3)

The biotransformation of N₂ to NH₄⁺ is a reduction that is conducted through an essential electrochemical process, using protons as a source of hydrogen and employing electrons. To achieve this, the necessary energy comes from the hydrolysis of adenosine triphosphate to its diphosphate form (ATP and ADP, respectively) [57]. Furthermore, the activity of V-nitrogenases in the activation and reductive hydrogenation of carbon dioxide (CO₂) to ethylene (C₂H₄) and other alkenes is of particular interest to the industry [53].

V-nitrogenases have two components: one Fe-protein and one FeV-protein. The Fe-protein consists of α₂-dimer containing a [4Fe-4S] cluster and two sites for interaction with ATP. The second component, the FeV-protein, consists of α₂β₂γ₂-heterohexamer with a P-cluster [8Fe-7S] and a cluster of vanadium and iron as the cofactor (FeV-co) [57,58]. From a mechanistic point of view, the electrons necessary for the reduction reactions migrate from the [4Fe-4S] cluster (Fe-protein), where the hydrolysis of ATP to ADP occurs, towards the P-cluster and, finally, to the FeV-co (VFe-protein), where electrons and protons are accumulated for substrate reduction, which occurs in this same cofactor [57,58]. It is important to highlight that the catalytic cofactor FeV-co is a cluster consisting of [V-7Fe-8S-C-(R)-homocitrate], with a bridged carbonate anion coordinated to two iron atoms [57,58]. The structural projections of the FeV-cofactor from the bacterium Azobacter vinelandii in a solid state obtained by SC-XRD, in a non-catalytic process and under catalysis, are shown in Figure 2.

As can be seen from Figure 2, the vanadium metal center appears as hexacoordinated, being coordinated with three sulfur atoms, namely the imidazole nitrogen of histidine residue and two oxygens of a 3-hydroxy-3-carboxy-adipic acid ((R)-homocitrate, HCA) molecule, one of which being alcoholate and the other being carboxylate [58]. From studies with FeV-co reduced by dithionate, it is assumed that, in a resting state, metallic atoms present the oxidation state [V^III, 4Fe^III, 3Fe^II]. It is also suggested that the vanadium center presents a trivalent oxidation state that is unchanged during the catalytic cycle [60]. In the center of this cofactor, there is a carbide linked to six iron atoms [58,61].

4. Bioaccumulation of Vanadium by Terrestrial Organisms

Despite vanadium having an essential role in the biological function of the enzymes haloperoxidases and nitrogenases, some organisms might accumulate vanadium for non-obvious reasons that have not yet been fully comprehended, e.g., Polychaeta marine annelids and marine ascidians, such as Ascidia gemmata. Vanadium is concentrated (bioaccumulation) by mushrooms of the genus Amanita (A.), e.g., A. regalis, A. velatipes, and A. muscaria. In the last species cited, the content of this metal is higher than four hundred times the contents found in mushrooms of the same genus. In 1972, the biomolecule in the form of which vanadium is accumulated by these mushrooms was isolated and identified as amavadin [62]. In 1999, the solid-state structure of amavadin complexed with calcium(II) coordinated to the ligand was elucidated by SC-XRD (Figure 3) [63,64].

The amavadin complex has the formula [Δ-V^IV(S,S)-HIDPA)₂]²⁻, which comprehends two units of the pro-ligand (S,S)-N-hydroxyimino-(2,2′)-dipropionic acid in a trianionic form coordinated to a non-oxido vanadium(IV) metal center, i.e., without the presence of a coordinated oxido ligand. It results in the coordination of vanadium(IV) with two nitrogens (1.982(8) Å and 1.999(8) Å) and two oxygens (1.940(7) Å and 1.956(7) Å) of the hydroxyimino functions (C –N(O)–C), as well as four carboxylate oxygens (2.028(7) Å, 2.028(9) Å, 2.042(8) Å and 2.070(8) Å) [62,63,64]. The geometry of the coordination sphere of the vanadium metal center in amavadin leads to a chirality in the metal center. Only the delta (Δ) species were obtained and evaluated crystallographically [64]. From a chemical point of view, this complex is stable in air, having a bluish color in aqueous solution, while, in some organic solvents, e.g., dimethylsulfoxide, N,N-dimethylformamide, and acetone, its metal center is oxidized to vanadium(V), resulting in a reddish solution [63].

5. Vanadium Salts, Complexes, Prodrugs, and Metallodrugs

The discovery of the cytotoxicity of cis-diamindichloretoplatinum(II) (cisplatin) to solid tumors by Rosenberg at the end of 1960s resulted in increasing interest in the field of inorganic medicinal chemistry—the discovery of metallodrugs. Metallodrugs are often prodrugs, in some cases pharmaceutical agents, that contain metal ions [65]. In the form of prodrugs, the metallodrugs might be converted into an active compound by ligand substitution and/or redox reactions in the biological matrix. Mainly due to their inorganic compounds, which might form different geometries with different coordination numbers, their unique mechanism of action has been explored in the treatment of a variety of diseases, disorders, and infections, with anticancer, anti-inflammatory, antibacterial, antifungal, and antiviral elements being subjects of interest [66,67].

Recently, several reports have highlighted the importance of vanadium-based compounds as novel prodrugs and metallodrugs with antiprotozoal, antibacterial, antiviral, and antineoplastic therapeutic actions based on in vitro and in vivo assays [68,69,70,71]. In this sense, high importance is given to the elucidation of the mechanisms that vanadium-based compounds suffer until they reach the targets, e.g., the exchange of ligands and the study of redox chemistry under physiological conditions, as well as their mechanisms of action and interactive profiles with biomacromolecules [43].

Over the past few years, the study of vanadium(III), oxidovanadium(IV), and dioxidovanadium(V) complexes with ligands containing oxygen, nitrogen, and sulfur as electron donor atoms have been explored as antineoplastic elements that have prompted advances and perspectives in regard to clinical applications [43]. Additionally, different authors have been reporting the interactive profile between vanadium salts or vanadium complexes and deoxyribonucleic acid (DNA), as well as, in some cases, extending these biophysical characterizations to the serum albumins [43,72,73].

It is important to highlight that most of the biological reports of vanadium-based compounds focus on the way that they might have mimetic activity in terms of biologically active molecules or ions such as phosphate. Vanadium derivatives, e.g., vanadates, have a high biological similarity to phosphate, mainly due to their similarity in the acidity constant (pK_a). The pK_a values for the phosphate species H₂PO₄⁻, HPO₄²⁻, and PO₄³⁻ are 2.1, 7.2, and 12.5, respectively [74], while for the vanadates H₂VO₄⁻, HVO₄²⁻, and VO₄³⁻, they are 3.5, 7.8, and 12.7, respectively [75,76]. These values influence the solubility, bioavailability, and pharmacokinetics of the cited ions in the biological environment.

The mimetic activity of vanadium-based compounds can be harmful when the stability of the interaction between enzymes and vanadium is very high [73,77,78,79]. On the other hand, when the biologically active compounds are enzymes with vanadium in their catalytic core, a strong affinity, mainly promoted by hydrogen bond interactions between the inorganic complex and the amino acid residues, is required (driven by the electronic characteristics of the ligands coordinated at the metallic center) [73,80,81,82].

Several vanadium complexes (especially vanadium(IV) complexes, vanadate, oxidovanadium sulfate, and metavanadate) have insulin-mimicking actions [83,84,85,86,87]. Despite these actions having been known for more than a century [88,89], the reasons for them are not quite clear and involve several possibilities, including the phosphorylation of the insulin receptor [19,90,91,92] and activation of the PI3K/AKT pathway [68,93,94,95], which promotes the translocation of glucose transporters (GLUT4) to the cell membrane. This might increase the glucose uptake by muscle and adipose cells via mitogen-activated protein kinases (MAPK), influencing gene expression and cell therapy in increasing expressions of phosphoenolpyruvate carboxykinase (PEPCK) [95,96] and glucose-6-phosphatase (G6Pase) [96,97,98], which are critical for hepatic gluconeogenesis.

A highly relevant factor that must be highlighted is the extreme complexity that vanadium metallodrugs have demonstrated for in vivo assays, e.g., the potential action of reversing peripheral insulin resistance was reported, with it being observed that is possible to activate the MAPK/ERK signaling pathway [99,100,101]. It might generate disorder and differentiation of osteoblasts, which promotes the activity of these cells, resulting in the synthesis of the bone matrix and mineralization.

6. Vanadium Complexes with Antidiabetic Activities

Diabetes mellitus, one of the most common metabolic disorders, is characterized by problems in the metabolism of lipids, proteins, and carbohydrates, leading to a high concentration of sugars in the bloodstream. This disorder results from the autoimmune destruction of β-cells in the pancreas, which leads to little/no production of the hormone insulin (type I) or resistance of the cells to the action of secreted insulin (type II) [102]. Diabetes type II accounts for about 90% to 95% of all diagnosed cases of diabetes [103].

Several vanadium complexes have insulin-mimicking and insulin-like actions [83,84,85,86,87]. An advantage of the application of vanadium salts or complexes is their capacity to decrease glucose levels in hyperglycemic conditions rather than in normoglycemic conditions, making, in this case, hypoglycemic conditions not a concern [104]. In 1899, there was a report by Lyonnet and coworkers [105] about the administration of sodium metavanadate (NaV^VO₃, Figure 4) in patients with different health problems, including three with diabetes, who reported a slight reduction in glycemic levels [89,106]. In 1985, scientific interest in vanadium compounds for treating diabetes was boosted by an in vivo study reported by Heyliger and coworkers [107]. In this case, the authors orally administered sodium orthovanadate (Na₃V^VO₄, Figure 4) in female Wistar rats in the presence of streptozotocin (STZ, a widely used to induce diabetes). The NaV^VO₄ administration led to a reduction in the high serum glycemic levels.

Regarding vanadium derivatives, a widely evaluated possibility was oxidovanadium(IV) sulfate, commonly also known as vanadyl sulfate (V^IVOSO₄, Figure 4). Several tests on the capacity of V^IVOSO₄ to decrease glucose levels in humans have been carried out. Such vanadium compounds have been shown to decrease glycemic levels as well as increase the sensitivity of insulin receptors. Thus, V^IVOSO₄ can increase the effectiveness of administered insulin [38,108,109], and its toxicity was reported to be 6- to 10-fold lower than that of vanadate [109], although it is poorly absorbed in the gastrointestinal tract (1–10%) [110]. Unfortunately, V^IVOSO₄ leads to undesirable gastrointestinal side effects [109,110].

Compared to vanadium salts, vanadium-based complexes, whose vanadium species are coordinated with appropriate organic ligands, may present better efficacy and tissue absorption, as well as a reduction in side effects caused by such metals [109]. Among the complexes with insulin mimetic activity, bis(maltolato)oxidovanadium(IV) (BMOV, Figure 4) [111] stands out—a neutral and water-soluble complex that presents two monoanionic units of the maltol ligand (O,O donor atom set) coordinates to the oxidovanadium(IV) center. Maltol, a food additive used in confectionery and bakeries [112], does not raise major concerns regarding its toxicity in this context [113]. An in vivo study using male Wistar rats with STZ-induced diabetes orally administered BMOV normalized blood glycemic levels without increasing insulin levels. Furthermore, when compared to V^IVOSO₄ [110] evaluated in a previous study [114], the reduction in glycemia occurred at a lower dosage and time (24 h vs. 1 to 2 weeks) [110,113].

Two inorganic complexes analogs to BMOV with variation in the aliphatic chain of the maltolate ligand: bis(ethylmaltolato)oxidovanadium(IV) (BEOV, Figure 4) and bis(isopropylmaltolato)oxidovanadium(IV) (BIOV, Figure 4), were administered orally and by intraperitoneal injection, respectively. These two analogs had similar insulin mimetic activities compared with BMOV (methyl derivative) [115]; however, BEOV had lower toxicity than BMOV [113]. It is important to highlight that BEOV had a positive assessment in the initial clinical phase (phase I—assess toxicity in healthy humans) and was accepted for the continuity of clinical assays to the intermediate phase (phase II—minimum effective dose assessment). In phase I, pharmacokinetic, bioavailability, and, especially, safety and tolerability parameters were evaluated in 40 volunteers without diabetes who were administered increasing doses of 10–90 mg orally per day. At this stage, no adverse health problems were observed in any of the volunteers, and better uptake and bioavailability were reported than in the positive control (V^IVOSO₄) [89,115]. In phase II, in 2007, seven patients with diabetes type II received 20 mg of BEOV orally per day, reducing blood glycemic levels. However, in 2009, the trials were suspended due to the reported kidney problems in patients undergoing treatment [38,88].

There are several discussions in the literature about whether it is vanadium in its form (V) or form (IV) (or both) that are responsible for the bioactivity as an insulin mimic. Shechter and coworkers [116] suggest that both forms present bioactivity, with the mechanism varying between them, i.e., tetravalent species are active in the plasma membrane, facilitating glucose uptake and, possibly, inhibiting lipolysis, while pentavalent species act only in the cytosol, promoting increased glucose levels and fat metabolism [116,117]. Furthermore, tetracoordinated vanadium acts as a substrate for enzymes that convert organic phosphates due to the similarities between both species, following the same rule re-ported with vanadates. However, pentacoordinated vanadium acts as a transition-state analog for phosphoester hydrolytic enzymes [104].

The biotransformations and chemical speciation of V^VOSO₄ and BMOV were studied by X-ray absorption near-edge structure (XANES), a X-ray absorption spectroscopy (XAS) mode, in cell cultures of human hepatoma (HepG1), human lung carcinoma (A549), and mouse adipocytes and preadipocytes (3T3-L1), as well as the corresponding cell culture media. It was evidenced that, in the cell culture media, both compounds undergo changes, resulting in similar mixtures of predominantly five- and six coordinate vanadium(V) species. These formed species were metabolized by HepG2 cells or mature 3T3-L1 adipocytes, resulting in the reduction of pentavalent to tetravalent vanadium as well as oxidation in the reverse direction. In that work, the capacity of the evaluated mammalian cells to generate and maintain V^V species when treated with V^IV complexes (like BMOV) was highlighted. Furthermore, the subcellular fractionation of A549 cells shows that the reduction in vanadium(V) species occurs mainly in the cell cytoplasm [118].

Despite the insulin mimic capacity of vanadium salts and vanadium-based complexes having been known for more than a century [88,89], the reasons for it are not quite clear and involve some possibilities, including the phosphorylation of the insulin receptor [19,90,91,92] and activation of the PI3K/AKT pathway [68,93,94,95] that promotes the translocation of glucose transporters (GLUT4) to the cell membrane. This might increase the glucose uptake by muscle and adipose cells via MAPK, influencing gene expression and cell therapy in increasing expressions of phosphoenolpyruvate carboxykinase (PEPCK) [95,96] and glucose-6-phosphatase (G6Pase) [96,97,98], which are critical for hepatic gluconeogenesis.

7. Vanadium Complexes with Anti-Inflammatory Action in Neurodegenerative Diseases

Several vanadium complexes that can act as metallodrugs have increasingly demonstrated action on different molecular targets, representing new advances for medicinal chemistry [40]. Several articles in recent years have demonstrated that vanadium complexes play a role in modulating biological processes such as enzyme inhibition [119], modulation of oxidative stress [120,121,122], and regulation of the immune response [123]. These complexes have potential as inhibitors of specific enzymes involved in disease progression, activators, or suppressors of signaling pathways for cellular function, and even as agents that mimic or modulate natural biochemical processes [124].

The therapeutic repertoire of vanadium complexes is dependent on biologically active targets, as, in addition to increasing efficacy and specificity, they also need to evaluate the potential for toxicity and side effects when compared to traditional therapeutic approaches [83,84,85,86,87]. As new vanadium complexes are synthesized and biological tests are evaluated, possible applications in specific therapies are being revealed; however, the optimization of the chemical structure of vanadium complexes (labile groups, organic ligands, and redox potentials) is essential in regard to improving its bioavailability, and possible toxic effects or multiple actions become crucial for efficiency and increased therapeutic results [125,126,127,128,129,130,131,132,133,134,135].

In addition to the best-known activities, such as insulin mimetic [83,84], DNA/HSA interaction [125,135], and anticarcinogenic activity [124], some targets of vanadium-based complexes have been studied with the aim of preventing chronic neuroinflammation, which is directly related to the progression of Alzheimer’s disease (AD) [136,137] as well as with amyloidosis-related diseases. In the last case, Xu and coworkers [136] reported the synthesis and characterization of three oxidovanadium complexes ((NH₄)[VO(O₂)₂(bipy)]·4H₂O, bis(ethylmaltolato)oxidovanadium(IV), and (bipyH₂)H₂[O{VO(O₂)(bipy)}₂]·5H₂O, Figure 5), identifying them as potential inhibitors of aggregation of human islet amyloid polypeptide (hIAPP) with distinct thermodynamic behavior and reductions in the production of oligomers (corresponding IC₅₀ values of 33.0, 14.7, and 8.50 μM). Xu and coworkers [121] reported the replacement of the bromo to nitro group in the corresponding ligands 2-(5-bromo-2-hydroxylbenzylideneamino) with benzoic acid (H₂bhbb) and 2-(5-nitro-2-hydroxylbenzylideneamino) benzoic acid (H₂nhbb) complexed with oxidovanadium(IV) (VO(bhbb)·H₂O and VO(nhbb)·H₂O, respectively, Figure 5) that might decrease the inhibitory effect on the aggregation. Interestingly, comparing BEOV with BMOV (Figure 4), the replacement of ethyl with methyl moieties [90,136] increases the inhibitory capacity of aggregation about 3-fold, while the replacement of 2,2′-bipyridine (bipy) with 1,10-phenanthroline (phen, in (NH₄)[VO(O₂)₂(phen)]·2H₂O, Figure 5) also increased the inhibitory capacity of aggregation 5-fold, indicating that not only is the nature of the vanadium species important in the development of metallodrugs for amyloidosis-related diseases; the chemical groups in the ligand complexed with the metallic center are significant as well.

In AD, it is necessary to highlight the immunological cells of the central nervous system (CNS), called microglia [138], which are responsible for maintaining cerebral homeostasis and also for responding to injuries or diseases, allowing for assessment and/or responses to disturbances in the brain, such as cellular changes, infections or anomalous concentrations of proteins. One of the most peculiar characteristics of the microglia is the possibility of switching between pro-inflammatory (M1) [139,140] and anti-inflammatory or reparative (M2) [139] phenotypes which, in promoting tissue repair, the resolution of inflammation, or under pathological conditions, can be closely related to neurodegenerative diseases (Alzheimer’s, Parkinson’s) and CNS injuries (stroke) [139,140,141].

Specifically in AD, microglia [142,143,144,145,146] undergo overactivation, releasing pro-inflammatory cytokines, such as interleukin-1 beta (IL-1β) [147], interleukin-6 (IL-6) [148,149] and tumor necrosis factor alpha (TNF-α) [150,151,152,153], increasing neuroinflammation and further damaging neurons. Reports in the literature show that vanadium complexes and new materials derived from vanadium have the capacity to modulate microglia, inhibiting overactivation and decreasing the production and release of harmful pro-inflammatory cytokines. This modulation is most likely associated with signaling pathways or transcription factors that regulate microglial activation states or promote a change towards a more neuroprotective phenotype.

The use of vanadium-based complexes and other metallodrugs can affect a specific point or a series of distinct functions, so, in addition to microglia, the nuclear factor-kappa B (NF-κB) [154,155] pathway is a target. This factor is linked to inhibitory proteins known as inhibitors of kappa B (IκBs), which, after activation by various stimuli (cytokines, pathogens or stress) trigger phosphorylation.

Another group that may be associated with degenerative diseases are the MAPKs [68,154]; however, this is another large family of kinases that involves those regulated by extracellular signal (ERK), N-terminal c-Jun (JNK), and p38 MAPK, which are activated in response to various stimuli such as cytokines, growth factors, and cellular stressors [38,156,157,158].

8. Vanadium Complexes as Inhibitors of Protein Tyrosine Phosphatases (PTPs)

The protein tyrosine phosphatases (PTPs) have a rich and wide range of functions, especially catalytic hydrolysis of the phosphate function of tyrosine residues in proteins [159,160]. This process is vital for metabolism and immunological responses [159,160,161]. Structurally, these phosphatases have catalytic sites containing active sites called PTP loops [162,163,164], which present cysteine amino acid residues involved in the catalytic activity [165]. The amino acid residue cysteine is important because it allows for reversible oxidation–reduction cycles, leading to the formation of sulfenic acid as an intermediate and, subsequently, the return to the thiol form [164,165]. The catalytic mechanism involves the nucleophilic attack of this cysteine residue on the phosphate group of a tyrosine-phosphorylated substrate, resulting in the formation of a covalent enzyme–phosphate intermediate [165,166]. Unwantedly, irreversible oxidation of this sulfenic acid leads to the production of sulfinic and sulfonic acids [167].

Although PTPs demonstrate great specificity for substrates, deregulation (leading to phosphorylation) of their activity, interrupting normal cellular signaling pathways, either due to the mutations in regulatory domains or altered interactions with substrates, contributes to diseases such as cancer, metabolic diseases, and even autoimmune diseases [168]. Regarding these issues, where the control and/or inhibition of PTPs [169,170,171,172] is necessary, vanadium complexes have shown numerous possibilities for future applications. This is due to their structural conditions (vanadium cations in the presence of specific organic molecules) [169,170,171,172,173], the geometry of the inorganic complex (it is believed that the protein stabilizes the vanadium geometry more effectively, generally the trigonal bipyramidal) [168] and redox potential (common oxidation number variation of V³⁺, V⁴⁺, and V⁵⁺) to mimic the transition state [173,174,175,176,177] of substrate binding or to selectively disrupt interactions within the catalytic site. Thus, the vanadium complexes act as inhibitors and can compete with the phosphate substrate or bind allosterically [169,177,178] to regulatory domains that control enzymatic activity.

A vanadium complex evaluated for its interactions with PTPs was the potential antidiabetic compound BMOV. Using the crystal-soaking technique of protein tyrosine phosphatase 1B (PTP1B) C215S to trap mutant crystals in BMOV solution, it was possible to evaluate such interactions by SC-XRD. The presence of vanadium in the form of vanadate in the enzyme was observed through this technique, which allowed for the conclusion that this uncoordinated vanadium species is the active form of the BMOV complex. This vanadium species presents itself as a trigonal bipyramid containing a hydroxy group of serine residue in an apical position and participates in a complex network of hydrogen bonds for its stabilization [179].

Some of the most investigated derivatives of vanadium are the simple salts sodium orthovanadate (Na₃V^VO₄, Figure 4) and sodium metavanadate (NaV^VO₃, Figure 4), which have been found to competitively inhibit tyrosinase phosphatases by mimicking the transition state of phosphate hydrolysis, disrupting oncogenic signaling pathways that depend on anomalous tyrosine phosphorylation and offering, as an example, potential therapeutic benefits in the treatment of cancer [169].

One of the great successes in vanadium chemistry is the structure–activity characteristic that the complexes present, since the coordination with the Lewis bases (ligands) is quite varied, depending on their oxidation state, allowing for coordination to hard, intermediate centers and soft centers, such as bases containing oxygen, nitrogen, and sulfur, respectively [170,173,176,177]. This rich variation in coordination with ligands associated with oxidation–reduction potential allows the complexes to have high inhibitory potency and selectivity for specific phosphatase isoforms [180]. In addition, they also exhibit antioxidant properties, which would allow for the treatment of diseases associated with oxidative stress [169,170,171,172,176,177].

9. Vanadium Complexes as Anticarcinogenic Action

As briefly described in Section 5, metallodrugs and their biological activity compounds, especially anticarcinogens, remind us essentially of drugs and prodrugs derived from platinum, such as cisplatin. Cisplatin was the first drug derived from a platinum(II) used for antineoplastic purposes, receiving approval from the United States Food and Drug Administration (US FDA) in 1978 [181].

Cisplatin is still widely used as a first option for the treatment of carcinomas and sarcomas or as a second option when the tumor is resistant to the cocktail initially used [182]. More specifically, cisplatin is used mainly in the treatment of ovarian carcinomas and testicular teratomas and is also used in various types of head and neck, colon, bladder, esophageal, and gastric neoplasms [183]. Cisplatin and its derivatives, also based on platinum(II), cause collateral damage and resistance processes, even though they are still used today [181,183]. For this reason, research with vanadium complexes has gained attention, as they decrease the side effects and increase the diversification of targets that the inorganic complexes might use.

Vanadium complexes can affect several cells signaling pathways that are frequently deregulated in cancer cells, especially the PI3K/AKT and MAPK pathways [183,184], which are involved in cell proliferation and differentiation. It is also known that vanadium derivatives have the potential to alter the expression of proteins that regulate apoptosis through the activation of caspases such as caspase-3 and caspase-9 [93,185] or through increasing the level of some proteins, such as apoptotic Bax [186,187]. Modulation of the increase in Bax causes an inversion of the concentration of Bcl-2, which is an antiapoptotic protein, favoring cell death.

In addition to specific points of vanadium action, such as those mentioned above, the classical approach of interactions with DNA also appears to be quite effective; however, it is known that vanadium complexes can inhibit the proliferation of tumor cells by interfering in the cell cycle, causing arrest in several specific phases, such as the G1 phase and the G2/M phase [93,124,188]. This means that vanadium in its biologically active form, in the G1 phase, acts directly at the restriction point (R), interfering with the verification and presence of essential nutrients for growth signals and DNA integrity, thus interfering with the p53 protein [189], which can induce the expression of CDK inhibitors (especially p21) [189,190], preventing the repair cycle.

In a similar way, vanadium complexes act in the G2 phase [93,124,188,191] and in the M phase (mitosis), where there is the synthesis of proteins and organelles necessary for cell division, specifically acting on regulatory proteins, including cyclin B [93] and cyclin-dependent kinase 1 (CDK1) [68,69]. In 2019, Lu and coworkers [192] reported a series of octahedral vanadium(III) complexes containing [V^III(Cl)₂THF] cores and imine ligands derived from indene moieties and S-alkyl/phenyl anilines ([V^III(Cl)₂L′L″THF], Figure 6). These vanadium complexes had antiproliferative activities for the human cancer cells: gastric (MGC803), esophageal (EC109), breast (MCF7), and liver (HepG2). The vanadium(III) complex with the best antiproliferative activity was capable of inducing apoptosis in MGC803 cells in a dose-dependent manner and through caspase-mediated mechanisms. This mechanism occurs due to changes in the expression of the pro- and anti-apoptotic proteins Bax and Bcl-2, respectively [192].

In vitro antineoplastic activity has been reported for different oxidation states of vanadium coordinated with imine ligands derived from S-substituted dithiocarbazates, e.g., Sarhan and coworkers [193] reported the synthesis and characterization of the octahedral oxidovanadium(IV) complex ([V^IVO(smdha)(bpy)], Figure 6), where smdha and bpy is dehydroacetic acid Schiff base of S-methyldithiocarbazate and 2,2′-bipyridine, respectively) with a half-maximal inhibitory concentration (IC₅₀) value of 29.30 and 59.16 μM for HepG2 and MCF7, respectively. These IC₅₀ values are better than those corresponding to H₂smdha (81.64 and 151.58 μM) and cisplatin (31.60 and 73.30 μM).

Additionally, Banerjee and coworkers [194] synthesized three oxidovanadium(IV) complexes based on 1,10-phenanthroline (phen) and 2,2′-bipyridine (bipy): [V^IVOL¹(phen)], [V^IVOL²(phen)], and [V^IVOL¹(bipy)] (Figure 6) with IC₅₀ values in the range of 16.60–29.86 μM against cervical cancer cells (HeLa). On the other hand, in a more recent article, Banerjee and coworkers [195] reported a series of mononuclear non-oxido vanadium(IV)complexes ([V^IV(L¹⁻⁴)₂], Figure 6) featuring tridentate bi-negative ONS chelating S-alkyl/aryl-substituted dithiocarbazate ligands (H₂L¹⁻⁴) with an improvement in the IC₅₀ values, being in the range of 6.3–17.1 μM. These vanadium-based complexes showed comparable cytotoxic potential with some chemotherapeutic drugs, e.g., cyclophosphamide (IC₅₀ = 21.5 μM) and cisplatin (IC₅₀ = 70 μM), as well as against the HeLa cells [194].

Interestingly, Sahu and coworkers [196] reported IC₅₀ values in the range of 6.73–18.21 μM for one oxidomethoxidovanadium(V) ([V^VO(L)(OMe)], Figure 6) and two mixed-ligand oxidovanadium(IV) ([V^IVO(L)(phen)] and [V^IVO(L)(bipy)]) (Figure 6) against MCF7 cells. The obtained IC₅₀ values for the vanadium-based complexes are better than those obtained for cisplatin (73.55 μM) and for the ligands 1,10-phenanthroline (phen), 2,2′-bipyridine (bipy), and S-benzyl-3-(2-hydroxy-3-ethoxyphenyl)methylenedithiocarbazate (H₂L) (25.02–38.67 μM range, Figure 6). The same trend was also identified by Yekke-Ghasemi and coworkers [197] for mono- or dinucleated oxidovanadium(V) complexes with imine ligands also derived from S-substituted dithiocarbazates (Figure 6, IC₅₀ = 0.6692 μM). Overall, these data reinforce the importance of vanadium in the discovery of novel anticarcinogenic candidates.

10. Serum Albumin in Metallodrug Discovery

Serum albumin is the main globular protein found in the blood being synthesized in the liver. Among its several important functions are its buffering pH capacity, neutralization of radicals, and its reducing of oxidative stress, maintaining of oncotic pressure, and binding and transporting of different endogenous and exogenous compounds (including hormones, fatty acids, calcium, magnesium, and drugs) [198,199,200,201,202,203,204,205]. In the drug discovery field, there is one important pre-clinical step about the biophysical characterization of potential drugs with serum albumin. More precisely, bovine serum albumin (BSA) is one of the most studied protein models in this step mainly due to its lowest cost compared with the human serum albumin (HSA) and the high percentage of identity and similarity (76 and 88%, respectively) among them [205,206,207,208]. Thus, the drug-binding affinities of BSA can be comparable with HSA.

The α-helix motifs comprise most of the structural content of the heart-shaped serum albumin. The molecular weight for BSA and HSA is 66,463 and 66,439 Da, respectively, with corresponding total amino acid residues of 583 and 585 [209,210]. The root mean square deviation (RMSD) value for their non-bound structure is around 1.634 dimensionless (Figure 7A), reinforcing their high three-dimensional (3D) structure similarity. For HSA (Figure 7B), residues 1–195, 196–383, and 384–585 form the α-helical domains I, II, and III, respectively, while, for BSA, slight differences can be evidenced (Figure 7C) [211,212]. Compounds with different chemical natures might bind with serum albumin, mainly due to the existence of two subdomains (A and B) for each domain. Overall, long and medium fatty acids can bind into seven possible sites, while four additional sites were reported for short fatty acids. Additionally, there is one binding site to bilirubin and three main sites for drugs located in subdomains IIA, IIIA, and IB, known as sites I, II, and III, respectively (Figure 7) [206,210]. These three subdomains are the main ones explored in drug-displacement assays [213,214,215,216,217].

The interaction between metallodrugs and serum albumin is of significant interest because it can influence the pharmacokinetic, bioavailable, and therapeutic efficacy of these drugs. Generally, metallodrugs bind to serum albumin with high affinity due to the interaction between the metal ions and amino acid residues, impacting their metabolism, distribution, and excretion [219,220,221,222,223]. High affinity might lead to a longer half-life and prolonged circulation time in the bloodstream, as well as facilitate the solubilization of metallodrugs in a complex biological matrix and protect them from premature degradation or inactivation (minimizing potential toxicity) [224]. As an example, cisplatin and its derivatives, carboplatin and oxaliplatin (Figure 8), bind to serum albumin, affecting not only their distribution but also decreasing the renal toxicity and limiting the concentration of free drugs in the bloodstream that might cause damage to the kidney [225,226,227,228,229]. Additionally, in regard to auranofin (Figure 8), a gold-based prodrug used in the treatment of rheumatoid arthritis, it was reported that the binding to albumin affects its anti-inflammatory activity [224,230,231] as well as the interaction between albumin with gallium nitrate (Figure 8, used for treating hypercalcemia and certain cancers) [232] and ruthenium-based compounds (mainly investigated as anticancer, e.g., NAMI-A, NKP1339, RM175, and RAPTA-C, Figure 8) [233], which is crucial for their stability and in reducing potential side effects.

11. Biophysical Characterization on the Interactions Between Albumin and Vanadium-Based Compounds

Biophysical techniques, e.g., ultraviolet-visible (UV-Vis) absorption, isothermal titration calorimetry, steady-state and time-resolved fluorescence, combined with in silico calculations, are widely used to evaluate the interactions between albumin and different metallodrugs or prodrugs, including those which are vanadium-based [125,234,235,236,237]. This characterization offers a crucial role regarding design, efficacy, and safety [202]. As an example, vanadium has antidiabetic properties, and its binding to albumin may influence its availability and efficacy in glucose metabolism regulation, as well as vanadium compounds that can affect enzyme activity, which their interaction with albumin might modulate [126,127,128]. This same interaction might serve as a detoxification pathway for vanadium, reducing its free concentration in the blood and mitigating its potential toxic effects [129].

Albumin has specific binding sites for metals and other small molecules. The vanadium ions vanadate (VO₄³⁻) and oxidovanadium (VO²⁺) interact specifically in the binding sites for metals [130], e.g., Cobbina and coworkers [131] reported that V^IVO₂⁺ occupies at least two types of binding sites, specifically a strong vanadium binding site (known as VBS1, without indicating its specific location) and the weak vanadium binding site (known as VBS2, also without indicating its specific location). This evidence was obtained by the combination of circular dichroism (CD), electron paramagnetic resonance (EPR), and UV-Vis absorption data. Interestingly, VBS1 binds with the 1 mol equivalent of V^IVO⁺₂ while VBS2 binds with several mol equivalents of V^IVO via non-specific interactions involving carboxylate or imidazole side chains from the amino acid residues [131,132]. The presence of 1,2-dimethyl-3-hydroxy-4(1H)-pyridinone (maltol) may enhance the binding capacity of V^IVO to albumin, while fatted albumins decreased the binding capacity of V^IVO due to the structural change on the biomacromolecule induced by the fatty acids [33].

Unfortunately, a few reports have discussed the structural characterization of the pose of vanadium-based compounds into albumin. Generally, in silico calculations are utilized to suggest it, as depicted in Figure 9. Chaves and coworkers [238] identified subdomain IIA (site I), where the fluorophore Trp-214 residue can be found, as the main region for the interaction between HSA and dioxidovanadium(V) complexes coordinated with pyridoxal and/or resorcinol (complexes C1–C3 in Figure 9), being stabilized mainly by hydrogen bonding and van der Waals forces. However, in a more recent report, Martins and coworkers [125] identified that dioxidovanadium(V) complexes coordinated with hydrazone-type iminic ligands derived from (3-formyl-4-hydroxybenzyl)triphenylphosphonium chloride and aromatic hydrazides (complexes C4–C8 in Figure 9) bind into an external pocket, known as site III, probably due to the high steric volume of the ligands coordinated with vanadium(V) species. Hydrophobic, hydrogen bond, and π-cation interactions were identified as the main forces responsible for the stability of the complex HSA:C4–C8. In this case, potential π-cation interactions were identified due to the high number of positive charged amino acid residues located into subdomain IB.

Interestingly, Fioravanço and coworkers [135] identified that dioxidovanadium(V) complexes coordinated with p-substituted benzohydrazides condensed with salicylaldehyde (complexes C9–C12 in Figure 9) or pyridoxal hydrochloride (complexes C13–C16 in Figure 9) interacts into subdomain IIA (site I) independently of being positive charged or neutral complexes, being stabilized by the same intermolecular forces reported by Chaves and coworkers [238]. Finally, Dias and coworkers [126] also identified site I as the main binding pocket to some vanadium complexes, i.e., [V^VO₂(maltol)₂]⁻, [V^VO₂(dmpp)(OH)(H₂O)]⁻, and [V^VO₂(dmpp)₂]⁻, where maltol and dmpp are 3-hydroxy-2-methyl-4-pyrone and 1,2-dimethyl-3-hydroxy-4(1H)-pyridinone, respectively. In this case, the interaction between the inorganic complexes with the protein surface is mainly driven by hydrogen bonding and/or hydrophobic forces.

Since we summarized the binding sites for vanadium(V) complexes exclusively, there are insights indicating that the main binding site for vanadium-based compounds is more dependent on the nature of the coordinated ligands than on the charge of the metal center. The binding site and type of interactions that lead to the stability of the complex albumin:vanadium-based compounds impact the thermodynamic trend that drives the association.

Generally, the time-resolved fluorescence decays to non-bound albumin are better fitted in a bi-exponential function with the measured corresponding to one shorter (τ₁) and other longer lifetimes (τ₂), e.g., for HSA 1.76 ± 0.09 and 5.75 ± 0.09 ns, respectively [133]. If the fluorescence lifetimes for albumin did not significantly change in the presence of vanadium-based compounds, it can be stated that the quenching process occurred through a purely static mechanism, indicating that the Stern–Volmer approach is the best mathematical approximation to estimate the binding affinity [134]. It was detected via steady-state and/or time-resolved fluorescence measurements that the binding vanadium-based compounds and albumin occur by ground state association (static fluorescence quenching mechanisms), e.g., for dioxidovanadium(V) complexes with a triphenylphosphonium moiety [125] or the mixture of the ground state (static) and collisional phenomenon (dynamic), e.g., vanadium(V) complexed with salicylaldehyde or pyridoxal hydrochloride [135], indicating that the ligands complexed with vanadium ions might interfere in the main fluorescence quenching mechanisms for albumin. A purely dynamic mechanism for albumin induced by vanadium-based compounds was not reported.

In a mixture of static and dynamic quenching mechanisms, the Stern–Volmer quenching constant (K_SV) is not used to determine the binding affinity; therefore, the binding constant (K_a or K_b) obtained mainly by modified Stern–Volmer or double logarithmic approaches [134] can be used to estimate the binding capacity between albumin and vanadium-based compounds. Drug absorption is described as the initial pharmacokinetic step which can be negatively affected by the weak binding capacity of metallodrugs to albumin; however, both the distribution and absorption of metallodrugs to different tissues (targets) is feasible when the binding is moderate, indicating some differences in cases of high binding affinity–feasible absorption, but its distribution to the tissues is limited mainly due to the stability of the complex [202,239]. In this sense, high and moderate binding affinity are detected when K_SV, K_a, or K_b values are higher than 10⁶ M⁻¹ and in the range of 10⁴–10⁶ M⁻¹, respectively. The reported data indicated that most of the vanadium-based complexes bind moderately to strongly with albumin [125,126,127,128,129,130,132,135,238], meaning that these potential metallodrugs might be carried out by HSA in the human bloodstream with a favorable binding capacity.

It is important to highlight that, since the binding between albumin and vanadium-based complexes is mainly characterized by in vitro assays without the complexity of a biological medium, it is difficult to extrapolate the biophysical data for in vivo assays. However, some reports [224,240] indicate an increase in the aqueous solubility and residence time, as well as a decrease in the toxicity of inorganic complexes after binding with albumin, leading to a favorable environment on the pharmacological behavior of metallodrugs. Additionally, when metallodrugs bind to albumin, they are often less available to interact with their intended target, potentially reducing efficacy but also potentially reducing off-target effects [223,241,242]. In other words, albumin also can be used in drug delivery systems to improve the pharmacokinetics of vanadium-based compounds, potentially reducing off-target effects.

12. Structural Evaluation of the Interaction Between Model Proteins and Vanadium-Based Compounds

Some proteins have been used as model proteins to evaluate and better understand the interactions between vanadium complexes and proteins by several techniques. Such proteins have structural characteristics that justify their use, such as a small number of binding sites, moderate size, high conformational stability, and high purity grades with commercial availability [82]. Hen egg white lysozyme is a small antimicrobial glycoside hydrolase which has been used as a model protein for X-ray crystallography and is able to produce robust and well-diffracting crystals [243,244]. Bovine pancreatic trypsin is one of the most typical serine proteases. From a crystallographic point of view, both these model proteins produce well-diffracting crystals under different experimental conditions [244]. Some vanadium(IV) complexes, including bis(maltolato)oxidovanadium(IV) (BMOV), had their interactions with hen egg white lysozyme and bovine trypsin evaluated by different techniques, e.g., electrospray ionization mass spectrometry (ESI-MS), electron paramagnetic resonance (EPR), and even X-ray crystallography [245,246,247,248]. Despite the advances of this last technique, there are still a few studies that have reported interactions between oxidovanadium(IV) ions and proteins using this resource [82,245].

Regarding the maltolato-derived complex, ESI-MS(+) spectra were recorded. In the raw spectrum, for each HEWL peak, other signals at higher m/z values are visible, indicating the formation of HEWL−V^IVO−maltolate adducts. In the deconvoluted spectrum, interactions and multiple binding of the “initial” V^IVO(maltolato)₂ specie and [V^IVO(maltolato)]⁺ specie are evidenced at both pH values, as well as the binding of the [V^IVO]²⁺ ions at the more acidic pH. With increasing pH, the adducts with the V^IVO(maltolate)₂ species also increase. By means of anisotropic EPR, it is patterns that suggest the coordination of oxidovanadium(IV) metal centers to oxygens of aspartic acid/glutamic acid-COO⁻ or asparagine/glutamine-CO side chains which contradict the preference of cisplatin (with a softer Pt^II center) for the histidine residue of the evaluated protein [245].

By means of soaking technique, HEWL single crystals obtained at pH 7.5 were immersed in BMOV solution, obtaining two distinct single crystals, although both have three equivalent binding sites. In both the crystals, there is evidence of different vanadium(IV) species interacting by non-covalent bindings with the protein surface (cis-[V^IVO(maltolato)₂(H₂O)] and [V^IVO(maltolato)(H₂O)₃] in the first crystal and two molecules of [V^IVO(maltolato)₂(H₂O)] in the second) and covalent bindings ([V^IVO(H₂O)₄]²⁺ in the first crystal and cis-[V^IVO(maltolato)₂] in the second, both binding to the same asparagine residue). By changing the growth medium of the HEWL single crystal, including reducing the pH to 4.0, which was subsequently immersed in BMOV solution, a different single crystal was obtained. The structure of this one now presents covalent interactions of three species [V^IVO(H₂O)₃]²⁺ together with two additional vanadium atoms [245].

Recently, by SC-XRD technique, single crystals of the same model protein but obtained at 1.1 M sodium chloride and 0.1 M sodium acetate at pH 4.0 immersed in BMOV solution had their solid-state structure elucidated, evidencing four distinct V binding sites. Between them, an unexpected bis(vanadium)-containing fragment is localized. This species is composed of two vanadium metal centers, both fully coordinated with oxygen species. Also, a modification on the side chain of a lysine residue, corresponding to the formation of an iminic double bond between the keto group of the maltolate moiety and the N-terminal group of this amino acid, has been noted. The two involved maltolate ligands suffered cleavage, both being coordinated to one vanadium center. In addition to the lysine residue, the fragment is coordinated to an aspartate residue from a symmetry related molecule [246]. The presence of this fragment containing two vanadium metal centers is also confirmed by gel electrophoresis under denaturing conditions of solubilized soaked crystals and by mass spectroscopies [246].

Co-crystallization of bovine trypsin in the presence of V^IVO/picolinic acid allows crystallographic results showing only a single adduct between vanadium and the model protein, with the ion being elucidated as an oxidovanadium(IV) with a distorted octahedral geometry. The vanadium coordination polyhedra is completed by a bond with a serine residue (alkoxide donor atom) and two picolinate anionic ligands by its N, O donor atoms. Co-crystallization of the same enzyme with vanadium(IV) and phenanthroline (phen) in the presence of imidazole (crystallization conditions in this case) afforded a structure with a single V-enzyme adduct. The metallic center is coordinated to a serine residue by its alkoxide atom, to a bidentate phenanthroline ligand, and to an imidazole. The distorted octahedral geometry is completed by two oxygen donor atoms, which could be interpreted as two oxido or an oxido–hydroxy couple and, consequently, as a dioxidovanadium(V) or an oxidovanadium(IV) species, respectively [247].

The trypsin crystals with vanadium species did not show EPR signals at 77 K, probably indicating a diamagnetic vanadium(V) species. However, fresh solutions of lyophilized trypsin and V^IVOSO₄ in the presence of phenanthroline/bipyridine (molar ratio 1:1 and 1:2) at pH 7.4 showed an EPR signal, indicating a probable oxidation of the vanadium species in the crystallization process. [247].

13. Future Perspectives

The future of metallodrugs lies in leveraging their unique properties for targeted, effective, and personalized medical treatments. Advances in targeted delivery systems, mechanistic understanding, combination therapies, and personalized medicine will drive the development of next generation metallodrugs. As research continues to uncover new therapeutic applications and optimize existing ones, metallodrugs will play an increasingly important role in modern medicine. Based on the biological importance of vanadium in different organisms, mainly targeting some specific enzymes with a good biophysical characterization of the interaction with serum albumin (in vitro pharmacokinetic profile), the vanadium-based compounds have significant promise regarding advanced medical treatments. These potential drugs leverage the unique properties of metals, such as redox activity, coordination chemistry, and electronic structure, to target diseases in ways that traditional organic molecules cannot.

The design and synthesis of novel vanadium-based compounds with low toxicity and greater biocompatibility are still crucial. Based on this review, the importance of the relationship between the chemical structure of potential metallodrugs and their biological activity to optimize their therapeutic properties was noticed. In addition, the understanding of how vanadium-based compounds interact with biological molecules, e.g., with serum albumin at the molecular level (biophysical characterization), can lead to the design of more effective and selective agents to decrease the toxicity, such as off-target effects, and improve the therapeutic aspects with good residence time in the bloodstream.