Theoretical Three-Dimensional Zinc Complexes with Glutathione, Amino Acids and Flavonoids

Article Theoretical Three-Dimensional Zinc Complexes with Glutathione, Amino Acids and Flavonoids

José Manuel Pérez de la Lastra 1,* , Celia Andrés-Juan 2, Francisco J. Plou 3 and Eduardo Pérez-Lebeña 4

1 Biotechnology of Macromolecules Research Group, Instituto de Productos Naturales y Agrobiología, IPNA, CSIC, 38206 San Cristóbal de la Laguna, Spain 2 CINQUIMA Institute and Department of Organic Chemistry, Facultad de Ciencias, University of Valladolid, 47011 Valladolid, Spain; [email protected] 3 Instituto de Catálisis y Petroleoquímica, CSIC, 28049 Madrid, Spain; [email protected] 4 Sistemas de Biotecnología y Recursos Naturales, 47625 Valladolid, Spain; [email protected] * Correspondence: [email protected]; Tel.: +34-922-260-112

Abstract: Zinc plays an important role in the regulation of many cellular functions; it is a signaling molecule involved in the transduction of several cascades in response to intra and extracellular stimuli. Labile zinc is a small fraction of total intracellular zinc, that is loosely bound to proteins and is easily interchangeable. At the cellular level, several molecules can bind labile zinc and promote its passage across lipophilic membranes. Such molecules are known as ionophores. Several of these compounds are known in the scientific literature, but most of them can be harmful to human health and are therefore not allowed for medical use. We here performed a theoretical three-dimensional study of known zinc ionophores, together with a computational energetic study and propose that some dietary flavonoids, glutathione and amino acids could form zinc complexes and facilitate the Citation: Pérez de la Lastra, J.M.; transport of zinc, with the possible biological implications and potential health benefits of these Andrés-Juan, C.; Plou, F.J.; natural compounds. The study is based on obtaining a molecular conformational structure of the Pérez-Lebeña, E. Theoretical zinc complexes with the lowest possible energy content. The discovery of novel substances that Three-Dimensional Zinc Complexes act as zinc ionophores is an attractive research topic that offers exciting opportunities in medicinal with Glutathione, Amino Acids and Flavonoids. Stresses 2021, 1, 123–141. chemistry. We propose that these novel complexes could be promising candidates for drug design to https://doi.org/10.3390/ provide new solutions for conditions and diseases related to zinc deficiency or impairment derived stresses1030011 from the dysregulation of this important metal.

Academic Editor: Keywords: Zinc ionophore; polyphenols; glutathione; dietary supplement; natural products Soisungwan Satarug

Received: 2 June 2021 Accepted: 22 July 2021 1. Introduction Published: 28 July 2021 All living forms are compartmentalized. Membranes surround the cells of all organisms and intracellular organelles, which act as a barrier to control the transfer of substances Publisher’s Note: MDPI stays neutral between the inside and outside of these structures. The lipophilic nature of the membranes with regard to jurisdictional claims in prevents the passage of charged particles, such as ions. Therefore, the transport of ions published maps and institutional affil- must be regulated by specific proteins that are an integral part of the membranes, acting iations. as channels allowing the entry and exit of ions [1]. These proteins maintain asymmetric concentrations of ions and facilitate their selective transport, which leads to the formation of energy that is eventually stored in the form of ATP [2]. Therefore, the control of ion transport and the homeostatic regulation of ion concentration levels across the membrane Copyright: © 2021 by the authors. plays a critical role in sustaining life. Licensee MDPI, Basel, Switzerland. Zinc plays an important role in protein function, being a structural element of many This article is an open access article macromolecules. Its importance has been recognized since 1869 when it was found to be distributed under the terms and necessary for the growth of Aspergillus niger [3] and later was found that it also affected the conditions of the Creative Commons growth of plants and animals. In humans, Ananda S Prasad first suspected zinc deficiency Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ in 1958 after he, at the request of James A. Halsted, evaluated a patient with iron deficiency. 4.0/).

Stresses 2021, 1, 123–141. https://doi.org/10.3390/stresses1030011 https://www.mdpi.com/journal/stresses Stresses 2021, 1 124

In addition In addition to iron deficiency, the patient appeared ~10 years older and was severely stunted and prepubertal age [4]. As a micronutrient, the importance of zinc has been reflected through the numerous diseases that are associated with its dietary deficiency. For example, zinc deficiency led to growth disorder, cognitive disorder and compromised immune function. Zinc deficiency occurs because of nutritional factors, but also in various disease states (malabsorption, Crohn’s disease, alcoholism, liver cirrhosis, chronic renal disease and other chronically debilitating diseases) [5–7]. Indeed, zinc supplementation improved these syndromes, as well as testicular and ovarian dysfunction or growth retardation [8]. Labile zinc is a small fraction of total intracellular zinc that is loosely linked to proteins and easily interchangeable. It is the pool sensitive to chelation and capable of detection by a variety of chemical and genetic sensors, participates in signal transduction pathways. Both free and labile zinc function as a second messenger molecule, modulating the activity of several enzymes and, as a result, signaling and metabolic pathways, as well as cellular activities. When cellular zinc levels are elevated, zinc transporter proteins (ZnT) contribute to modulating the cytoplasmic zinc balance by exporting zinc out to extracellular space or by sequestrating cytoplasmic zinc into intracellular compartments. On the contrary, when cellular zinc is depleted, zinc transporter (Zrt) and Irt-like proteins, known as ZIP proteins, work to increase cytoplasmic zinc concentrations [9–11]. At the cellular level, several natural molecules can bind labile zinc and promote its passage across lipophilic membranes. Such molecules are known as ionophores, from the ancient Greek, meaning “ion carriers”. Ionophores are membrane-active compounds that do not depend on active metabolism to exert their biological activity. They are chemical entities that bind reversibly with the ion, forming a supramolecular complex that can diffuse across hydrophobic membranes [12]. This capacity of ionophores to alter the normal ion balance of membranes can be used as a mechanism for inducing cell death, or to eliminate harmful microorganisms or cells with abnormal growth, like tumor cells [13]. Zinc chelators are able to transport zinc cations across the plasma membrane, independently of zinc transporters [14]. In chemistry, zinc is available in the form of inorganic and organic salts. Some inorganic zinc salts are zinc sulfate, zinc chloride, zinc oxide and zinc carbonate, while organic salts include zinc citrate, zinc lactate, zinc acetate, zinc gluconate, zinc stearate, zinc picolinate and zinc ascorbate [15,16]. The ion selectivity of ionophores is a combined function of the energy required for desolvation of the ion and the liganding energy obtained on complexation. For metal binding, thermodynamic stability of complexes is the measure of a metal ion’s tendency to selectively form a certain metal complex and is directly connected to metal-ligand bond energies, which would result in a decrease in the system’s free energy [17,18]. Several molecular characteristics can be correctly represented as a sum of individual bond properties, without the need to explicitly solve any wave equations. The molecular mechanics method refers to the study of such molecular characteristics in terms of non-quantum mechanical models (MM). In a nutshell, MM posits that the energy of a molecular system is made up of five additive, non-interacting components [19,20]. Numerous investigations have demonstrated that flavonoids influence the metabolism of zinc [21]. For example, rats fed with baicalin and rutin for long periods of time had lower liver levels of total zinc, iron and copper, suggesting that certain flavonoids, contain metal-binding motifs, like phytate, may retain these metals and render them inaccessible for absorption. On the other hand, polyphenols can transport zinc across the plasma membrane without relying on cell transport systems like zinc transporters or endocytosis. Dabbagh-Bazarbachi et al. investigated the interactions between a variety of polyphenols and polyphenol metabolites and zinc in mouse hepatocarcinoma Hepa 1–6 cells under physiological conditions [22]. After the addition of quercetin and epigallocatechin-gallate, the zinc-polyphenol chelation complex was formed and transported across the lipid bilayer. When given to cells, these complexes rapidly increase the intracellular zinc pool, which may be detected using various fluorophores such as Zinquin or FluoZin-3 [23]. They concluded Stresses 2021, 1 125

that flavonoids could generate water-insoluble membrane-permeant zinc complexes that penetrate the plasma membrane and serve as zinc ionophores. In order to ascertain whether other natural compounds, such as flavonoids, glutathione and plant amino acids, can serve as new zinc ionophores, we here provide a Stresses 2021, 1, FOR PEER REVIEW 3 computational energetic study of the forming zinc complexes. The study is based on obtaining a molecular conformational structure of the zinc complexes with the lowest possible energy content. We also discuss the relevance and biological implications of the formationconcluded of that such flavonoids complexes. could We generate hope that water-insoluble this preliminary membrane-permeant study could offer new zinc oppor- com- tunitiesplexes that to prevent penetrate zinc the deficiency plasma membrane by using nutraceutics, and serve as such zinc asionophores. amino acids, glutathione or flavonoids,In order to together ascertain with whether zinc supplementation. other natural compounds, such as flavonoids, glutathione and plant amino acids, can serve as new zinc ionophores, we here provide a compu- 2. Results tational energetic study of the forming zinc complexes. The study is based on obtaining a molecularFor initial conformational considerations structure and validation of the zinc of complexes our study, with we computedthe lowest thepossible energy energy and favorablecontent. We spatial also discuss orientation the relevance of some well-known and biological zinc implications ionophores of when the formation complexed of withsuch zinccomplexes. cations. We The hope differences that this inpreliminary their energetic study level, could derived offer new from opportunities molecular mechanics, to prevent iszinc summarized deficiency by in Tableusing1 nutraceutics,. For each complex, such as amino we studied acids, differentglutathione three-dimensional or flavonoids, to- structuralgether with conformations, zinc supplementation. considering those with the lowest possible energy and favorable geometry respect to the internal position of the zinc cation. 2. Results Table 1. Molecular mechanics computed energy E of free forms of zinc ionophores and when forming complexesFor initial with zinc considerations cations. and validation of our study, we computed the energy and favorable spatial orientation of some well-known zinc ionophores when complexed with zinc cations.Zinc Ionophore The differences in their energetic Free Form level, derived from Complexed molecular with mechanics, Zinc is summarizedPyrithione in Table 1. For each complex, 2.2 kcal/mol we studied different three-dimensional−37.2 kcal/mol structural conformations,PBT2 considering those 11.7.1 with kcal/mol the lowest possible energy−110.5 and kcal/mol favorable geometry respectTPEN to the internal position 38.9 of the kcal/mol zinc cation. −126.6 kcal/mol Free pyrithioneClioquinol has two structures 6.9 with kcal/mol different energies. The− left78.4 one kcal/mol has an energy − of E = 5.3 kcal/molHinokitiol and the right one is 18.1 E = kcal/mol 2.2 kcal/mol (the lower the125.6 energy, kcal/mol the higher the stability, so the balance is shifted to the right). The zinc-pyrithione complex has an energyFree of pyrithioneE = −37.2 kcal/mol, has two structureswhich is considerably with different more energies. stable The than left free one pyrithione has an energy (Fig- ofure E 1). = 5.3 The kcal/mol favorable and geometry the right of one the is comple E = 2.2x kcal/mol and spatial (the orientation lower the energy, of the themolecules, higher theshowed stability, that sothe the zinc balance cation iswas shifted in an tointernal the right). position The between zinc-pyrithione two adjacent complex pyrithione has an energymolecules, of E with = − 37.2the polar kcal/mol, groups which orienting is considerably towards the moreinside. stable than free pyrithione (FigureThe1). efficient The favorable shielding geometry of its polar of the core, complex which and delocalizes spatial orientation the cation of charge, the molecules, may ex- showedplain the that resultant the zinc complex’s cation was lipid in ansolubility, internal co positionnsistent between with the two role adjacent of pyrithione pyrithione as a molecules,zinc ionophore with (Figure the polar 1). groups orienting towards the inside.

FigureFigure 1. EnergyEnergy content content of of the the two two struct structuresures of free of free pyrithione pyrithione (a). Binding (a). Binding sites and sites spatial and spatial orien- orientationtation with withzinc cations zinc cations (b). Light (b). grey Light balls grey represent balls represent carbon carbonatoms. Blue atoms. balls Blue represent balls represent nitrogen nitrogenatoms. Non atoms. linked Non dark linked grey dark balls grey represent balls represent zinc cations. zinc cations.Red balls Red represent balls represent oxygen oxygenatoms. Yellow atoms. Yellowballs represent balls represent sulfur atoms. sulfur atoms.

TheSimilarly, efﬁcient the shieldingzinc ionophore of its polaradopted core, a disposition which delocalizes around the zinc cation cation charge, with may two explainmolecules the of resultant 5,7-Dichloro-2-((dimethylamino) complex’s lipid solubility, consistentmethyl) quinolin-8-ol with the role (PBT2) of pyrithione in opposite as a zincorientations ionophore (Figure (Figure 2).1 The). computed energy for the free form is E = 11.7 kcal/mol whereas for theSimilarly, zinc/PBT2 the complex zinc ionophore depicted adopted in Figure a disposition 2b was E = around−110.5 kcal/mol. the zinc cation with two moleculesTPEN of (N,N,N 5,7-Dichloro-2-((dimethylamino)′,N′-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine), methyl) quinolin-8-ol (PBT2) a zinc in ionophore opposite without oxygen atoms, has an E = 38.9 kcal/mol for the free form and of E = −126.6 kcal/mol for the complex zinc/TPEN. The spatial orientation of the most favorable complex also showed an internal zinc cation surrounded by four pyridine rings (Figure 3). Stresses 2021, 1 126

Stresses 2021, 1, FOR PEER REVIEW 4 orientations (Figure2). The computed energy for the free form is E = 11.7 kcal/mol whereas for the zinc/PBT2 complex depicted in Figure2b was E = −110.5 kcal/mol.

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Figure 2.2. StructureStructure ((aa)) andand three-dimensionalthree-dimensional studystudy ((bb)) ofof PBT2PBT2 (5,7-Dichloro-2-((dimethylamino)(5,7-Dichloro-2-((dimethylamino) methyl) quinolin-8-ol). Light grey balls represent carboncarbon atoms. Blue balls represent nitrogen atoms. Non linked dark grey balls represent zinc cations. Red balls represent oxygen atoms. Green balls Non linked dark grey balls represent zinc cations. Red balls represent oxygen atoms. Green balls represent chlorine atoms. represent chlorine atoms.

0 0 FigureTPEN 2. Structure (N,N,N (a),N and-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine), three-dimensional study (b) of PBT2 (5,7-Dichloro-2-((dimethylamino) a zinc ionophore withoutmethyl) quinolin-8-ol). oxygen atoms, Light has angrey E balls = 38.9 represent kcal/mol carbon for the atoms. free formBlue balls and represent of E = −126.6 nitrogen kcal/mol atoms. forNon the linked complex dark grey zinc/TPEN. balls represent The spatialzinc cations. orientation Red balls of represent the most oxygen favorable atoms. complex Green alsoballs showedrepresent an chlorine internal atoms. zinc cation surrounded by four pyridine rings (Figure3).

Figure 3. Structure (a) and three-dimensional study (b) of TPEN (N,N,N′,N′-tetrakis (2-pyridinylmethyl)-1,2-ethanediamine). Light grey balls represent carbon atoms. Blue balls represent nitrogen atoms. Non linked dark grey balls represent zinc cations.

Clioquinol (5-Chloro-7-iodo-8-quinolinol) had an energy of E = 6.9 kcal/mol, whereas for the complex with zinc the energy was of E = −78.4 kcal/mol, indicating the favorable Figure 3.3. StructureStructure ( a()a and) and three-dimensional three-dimensional study study (b) of (b TPEN) of TPEN (N,N,N (N,N,N0,N0-tetrakis′,N′-tetrakis (2-pyridinylmethyl)- (2-pyridinyl- formation of the zinc. The geometry and spatial orientation of the complex showed the 1,2-ethanediamine).methyl)-1,2-ethanediamine). Light grey Light balls grey represent balls carbonrepresent atoms. carbon Blue atoms. balls represent Blue balls nitrogen represent atoms. nitrogen Non internal zinc cation between two adjacent hydroxyquinoline groups in opposite directions linkedatoms. darkNon greylinked balls dark represent grey balls zinc represent cations. zinc cations. and the zinc cation coordinated with oxygen and nitrogen atoms (Figure 4). Clioquinol (5-Chloro-7-iodo-8-quinolinol)(5-Chloro-7-iodo-8-quinolinol) had had an an energy energy of of E E = = 6.96.9 kcal/mol,kcal/mol, whereas for thethe complexcomplex withwith zinczinc thethe energyenergy was was of of E E = =− −78.478.4 kcal/mol,kcal/mol, indicating the favorablefavorable formation of thethe zinc.zinc. The geometry and spatialspatial orientationorientation ofof thethe complexcomplex showedshowed thethe internal zinc cation between two adjacent hy hydroxyquinolinedroxyquinoline groups in opposite directions and the zinc cation coordinated withwith oxygenoxygen andand nitrogennitrogen atomsatoms (Figure(Figure4 4).).

Figure 4. Structure (a) and three-dimensional study (b) of Clioquinol, 5-Chloro-7-iodo-8-quinolinol. Light grey balls represent carbon atoms. Blue balls represent nitrogen atoms. Non linked dark grey balls represent zinc cations. Red balls represent oxygen atoms. Purple balls represent iodine atoms. Green balls represent chlorine atoms.

Figure 2. Structure (a) and three-dimensional study (b) of PBT2 (5,7-Dichloro-2-((dimethylamino) methyl) quinolin-8-ol). Light grey balls represent carbon atoms. Blue balls represent nitrogen atoms. Non linked dark grey balls represent zinc cations. Red balls represent oxygen atoms. Green balls represent chlorine atoms.

Clioquinol (5-Chloro-7-iodo-8-quinolinol) had an energy of E = 6.9 kcal/mol, whereas for the complex with zinc the energy was of E = −78.4 kcal/mol, indicating the favorable Stresses 2021, 1 formation of the zinc. The geometry and spatial orientation of the complex showed 127the internal zinc cation between two adjacent hydroxyquinoline groups in opposite directions and the zinc cation coordinated with oxygen and nitrogen atoms (Figure 4).

FigureFigure 4. 4.Structure Structure ((aa)) andand three-dimensionalthree-dimensional studystudy ((bb)) ofof Clioquinol,Clioquinol, 5-Chloro-7-iodo-8-quinolinol.5-Chloro-7-iodo-8-quinolinol. LightLight grey grey balls balls represent represent carbon carbon atoms. atoms. Blue Blue balls balls represent represent nitrogen nitrogen atoms. atoms. Non Non linked linked dark dark grey grey Stresses 2021, 1, FOR PEER REVIEW 5 ballsballs represent represent zinc zinc cations. cations. RedRed ballsballs representrepresent oxygenoxygen atoms.atoms. PurplePurple ballsballs representrepresent iodineiodine atoms.atoms. GreenGreen balls balls represent represent chlorine chlorine atoms. atoms.

HinokitiolHinokitiol (2-hydroxycyclohepta-2,4,6-trien-1-o (2-hydroxycyclohepta-2,4,6-trien-1-one)ne) is structurally is structurally related related to tropolone to tropolone (a (awell-known well-known chelating chelating agents). agents). In our In computatio our computationalnal analysis, analysis, free hino freekitiol hinokitiol had an energy had an energyof E = 18.1 of E kcal/mol, = 18.1 kcal/mol, the dimer the form dimer had form an energy had an of energy E = 29.5 of kcal/mol, E = 29.5 kcal/mol,whereas the whereas com- theplex complex hinokitiol/zinc hinokitiol/zinc had an energy had an content energy of content E = −125.6 of E kcal/mol. = −125.6 The kcal/mol. spatial orientation The spatial orientationfor the lowest for energetic the lowest form energetic of hinokitiol/zinc form of hinokitiol/zinc showed an internal showed zinc an cation internal coordinated zinc cation coordinatedwith oxygen withatoms oxygen from the atoms hydroxyl from and the carbon hydroxylyl groups and carbonyl of the aromatic groups rings of the that aromatic were ringsorienting that their were hydrophobic orienting their isobutyl hydrophobic groups isobutylat opposite groups ends at(Figure opposite 5). ends (Figure5).

Figure 5.5. Structure (a) andand three-dimensionalthree-dimensional study study ( (bb)) of of Hinokitiol (2-hydroxycyclohepta- (2-hydroxycyclohepta-2,4,6- trien-1-one).2,4,6-trien-1-one). Light Light grey grey balls balls represent represent carbon carbon atoms. atoms. Non Non linked linked darkdark grey balls balls represent represent zinc cations.zinc cations. Red ballsRed balls represent represent oxygen oxygen atoms. atoms.

TheThe greatestgreatest differencedifference inin energyenergy was found for the TPEN molecules, in in free free form form waswas ofof 38.938.9 kcal/molkcal/mol and and comp complexedlexed with with zinc zinc was was of ofE = E − =126.6−126.6 kcal/mol. kcal/mol. The lowest The lowest shift shiftin the in computed the computed molecular molecular mechanical mechanical energy energy was found was for found pyrithione, for pyrithione, which in which free inform free was form of wasE = 2.2 of Ekcal/mol = 2.2 kcal/mol and and andin combination and in combination with zinc withcation, zinc was cation, of E = was −37.2 of Ekcal/mol = −37.2 (Table kcal/mol 1). (Table1). Next, an energetic study was carried out to analyze whether there may be energetically optimizedTable 1. Molecular ionophores mechanics that are computed able to chelateenergy E zinc, of free independently forms of zinc ionophores of their previous and when commer- form- ing complexes with zinc cations. cial forms (acetate, citrate, gluconate and zinc lactate), which are already on the market. In free forms,Zinc Ionophore the lowest energy (E = −358.3 Free kcal/mol)Form was found for Complexed zinc gluconate, with Zinc whereas the highestPyrithione energy for a free form of2.2 zinckcal/mol salts of organic acids (E−37.2 = − kcal/mol119.8 kcal/mol) correspondedPBT2 to zinc acetate. In all11.7.1 organic kcal/mol forms studied, the three-dimensional−110.5 kcal/mol analysis of the complexesTPEN showed an internal38.9 zinc kcal/mol cation surrounded by two−126.6 organic kcal/mol molecules in coordinationClioquinol with oxygen atoms6.9 from kcal/mol hydroxyl and carbonyl groups−78.4 kcal/mol of the organic molecules. (Figure6). Hinokitiol 18.1 kcal/mol −125.6 kcal/mol

Next, an energetic study was carried out to analyze whether there may be energetically optimized ionophores that are able to chelate zinc, independently of their previous commercial forms (acetate, citrate, gluconate and zinc lactate), which are already on the market. In free forms, the lowest energy (E = −358.3 kcal/mol) was found for zinc gluconate, whereas the highest energy for a free form of zinc salts of organic acids (E = −119.8 kcal/mol) corresponded to zinc acetate. In all organic forms studied, the three-dimensional analysis of the complexes showed an internal zinc cation surrounded by two organic molecules in coordination with oxygen atoms from hydroxyl and carbonyl groups of the organic molecules. (Figure 6). When combined with an amino acid (tryptophan) or a polyphenol (silybin), the corresponding complexes with zinc showed different energy. The lowest energy was found for the zinc gluconate, whereas the highest energy obtained corresponded to the amino acid and flavonoid complexes with zinc citrate (Table 1, Figure 6). Similar values were obtained for amino acid and flavonoid complexes formed with zinc acetate and zinc lactate (Table 1). Therefore, we decided to use zinc gluconate for further theoretical studies, as it was the most stable form of dietary zinc salts of organic acids, according to our molecular mechanics calculations (Figure 6). The complexes with zinc gluconate showed the lowest energy values, so the formation of these complexes with zinc gluconate were more stable than those formed with other forms of zinc salts of organic acids (Table 2). Stresses 2021, 1 128 Stresses 2021, 1, FOR PEER REVIEW 6

FigureFigure 6. Energy 6. Energy and andmost most favorable favorable spatial spatial orientation orientation of zinc of acetate, zinc acetate, zinc lact zincate, lactate, zinc citrate zinc citrateand zincand gluconate. zinc gluconate. Light grey Light balls grey represent balls represent carbon atoms. carbon Red atoms. balls Red represent balls represent oxygen atoms. oxygen Non- atoms. linkedNon-linked dark grey dark balls grey represent balls represent zinc cations. zinccations.

Table 2.When Molecular combined mechanics with computed an amino energy acid E (tryptophan)of free forms of or zinc a polyphenolsalts of organic (silybin), acids and the whencorresponding forming complexes complexes with withthe amino zinc showedacid tryptophan different and energy. the flavonoid The lowest silybin. energy was found for the zinc gluconate, whereas the highest energy obtained corresponded to the amino Zinc Salts of Complexed with acid and flavonoidFree complexes Form with zinc citrate (Table1, FigureComplexed6). Similar with values Silybin were Organicobtained Acids for amino acid and flavonoid complexesTryptophan formed with zinc acetate and zinc lactate Zinc(Table gluconate1). Therefore, −358.3 we decidedkcal/mol to use zinc−457.3 gluconate kcal/mol for further theoretical−320.9 kcal/mol studies, as it wasZinc the citrate most stable−277.4 form kcal/mol of dietary zinc− salts257.4 of kcal/mol organic acids, according−185.8 tokcal/mol our molecular mechanicsZinc acetate calculations −119.8 (Figurekcal/mol6). The complexes−419.6 kcal/mol with zinc gluconate−304 showed kcal/mol the lowest energyZinc lactate values, so− the188.9 formation kcal/mol of these− complexes412.9 kcal/mol with zinc gluconate−307.3 were kcal/mol more stable than those formed with other forms of zinc salts of organic acids (Table2). For this reason, we computed the energy and favorable spatial orientation of zinc gluconateTable 2. Molecularwhen complexed mechanics with computed natural energycompounds, E of free such forms as amino of zinc acids salts of (phenylalanine, organic acids and tryptophan,when forming histidine complexes and witharginine). the amino The acid most tryptophan favorable and forms the flavonoidof free amino silybin. acids were as dimers with different spatial orientation, according to our energetic calculations (Figure Zinc Salts of Complexed with Free Form Complexed with Silybin 7). TheOrganic dimer of Acids amino acid with lowest energy Tryptophanlevel was arginine (E = −16.7 kcal/mol) whereas the least stable and most energetic dimer of the amino acids studied corre- Zinc gluconate −358.3 kcal/mol −457.3 kcal/mol −320.9 kcal/mol sponded to tryptophan (E = −6.1 kcal/mol). The spatial orientations are shown in Figure 7. Zinc citrate −277.4 kcal/mol −257.4 kcal/mol −185.8 kcal/mol WhenZinc complexes acetate with− zinc119.8 gluconate, kcal/mol the −computed419.6 kcal/mol energy and spatial−304 kcal/mol orientation of the studiedZinc lactate amino acids− indicated188.9 kcal/mol the favorable−412.9 formation kcal/mol of the corresponding−307.3 kcal/mol ionophores of zinc cation surrounded by two molecules of amino acids and two molecules of gluconate, as shown in Figure 8. For this reason, we computed the energy and favorable spatial orientation of zinc gluconate when complexed with natural compounds, such as amino acids (phenylalanine, tryptophan, histidine and arginine). The most favorable forms of free amino acids were as dimers with different spatial orientation, according to our energetic calculations (Figure7) . Stresses 2021, 1 129

Stresses 2021, 1, FOR PEER REVIEW The dimer of amino acid with lowest energy level was arginine (E = −16.7 kcal/mol)7

whereas the least stable and most energetic dimer of the amino acids studied corresponded to tryptophan (E = −6.1 kcal/mol). The spatial orientations are shown in Figure7.

FigureFigure 7. Energy 7. Energy content content and andmost most favorable favorable spatial spatial orientation orientation of the of dimers the dimers of proteinogenic of proteinogenic aminoamino acids acids (phenylalanine, (phenylalanine, tryptophan, tryptophan, histidine histidine and arginine) and arginine) computed computed by molecular by molecular mechanics. mechanics. LightLight grey greyballs balls represent represent carbon carbon atoms. atoms. Blue Blueballs balls represent represent nitrogen nitrogen atoms. atoms. Red Redballs balls represent represent oxygenoxygen atoms. atoms.

The Whencalculated complexes energy with of free zinc zinc gluconate, gluconate the computedwas of E = energy −358.3 and Kcal/mol spatial (Table orientation 1). of However,the studied when amino complexed acids indicatedwith the amino the favorable acids, the formation computed of the energy corresponding was lower ionophores in all casesof studied, zinc cation indicating surrounded that bythere two are molecules favorable of mechanical amino acids forces and two that molecules make these of gluconate, com- plexesas shownmore stable in Figure and less8. energetic than in their corresponding free forms. For the amino acids studied,The calculated the lowest energyvalue of of computed free zinc gluconatemechanical was energy of E was = − obtained358.3 Kcal/mol for the com-(Table1 ). plexHowever, of tryptophan when with complexed zinc gluconate with the (Table amino 3). acids,The lowest the computed energy of energyall compounds was lower is in obtainedall cases for the studied, glutathione/zi indicatingnc thatgluconate there arecomplex, favorable with mechanical −641 kcal/mol. forces that make these complexesThe calculated more energy stable andof free less zinc energetic gluconate than inwas their of E corresponding = −358.3 kcal/mol free forms.(Table For1). the However,amino when acids studied,complexed the lowestwith the value amino of computedacids, the mechanicalcomputed energy energy was obtainedlower in forall the casescomplex studied, of indicating tryptophan that with there zinc are gluconate favorable (Table mechanical3). The lowestforces that energy make of allthese compounds com- plexesis obtainedmore stable for and the glutathione/zincless energetic than gluconate in their corresponding complex, with free−641 forms. kcal/mol. For the amino acids studied, the lowest value of computed mechanical energy was obtained for the com- plexTable of tryptophan 3. Molecular with mechanics zinc gluconate computed (Table energy E3). of The free formslowest of energy natural compounds,of all compounds such as aminois obtainedacids, for glutathione the glutathione/zi and polyphenolsnc gluconate and when complex, forming complexeswith −641 withkcal/mol. zinc gluconate. Compound Free Form Complexed with Zinc Gluconate Table 3. Molecular mechanics computed energy E of free forms of natural compounds, such as amino acids,Phenylalanine glutathione and polyphenols−16.2 and kcal/mol when forming complexes −with414.1 zinc kcal/mol gluconate. Tryptophan −6.1 kcal/mol −457.3 kcal/mol CompoundHistidine Free Form 7.2 kcal/mol Complexed with−450.5 Zinc kcal/mol Gluconate PhenylalanineArginine −16.2 kcal/mol−16.7 kcal/mol −414.1−403.2 kcal/mol kcal/mol Glutathione −15.4 kcal/mol −641 kcal/mol Tryptophan −6.1 kcal/mol −457.3 kcal/mol Silybin 15.8 kcal/mol −320.9 kcal/mol HistidineHesperidin 7.2 kcal/mol 39 kcal/mol −450.5−309.8 kcal/mol kcal/mol ArginineXeractinol −16.7 kcal/mol 15.5 kcal/mol −403.2−324.2 kcal/mol kcal/mol Glutathione −15.4 kcal/mol −641 kcal/mol Silybin 15.8 kcal/mol −320.9 kcal/mol Hesperidin 39 kcal/mol −309.8 kcal/mol Xeractinol 15.5 kcal/mol −324.2 kcal/mol

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FigureFigure 8. Energy 8. Energy content content and andmost most favorable favorable spatial spatial orientation orientation of zinc of gluconate zinc gluconate complexes complexes with with proteinogenicproteinogenic amino amino acids acids (phenylalanine, (phenylalanine, tryptoph tryptophan,an, histidine histidine and arginine). and arginine). Light grey Light balls grey balls represent carbon atoms. Blue balls represent nitrogen atoms. Non linked dark grey balls represent zinc cations. Red balls represent oxygen atoms. Stresses 2021, 1, FOR PEER REVIEW 9

Stresses 2021, 1 represent carbon atoms. Blue balls represent nitrogen atoms. Non linked dark grey balls represent131 zinc cations. Red balls represent oxygen atoms.

The computed energy value of glutathione and the spatial orientation of both free form andThe when computed complexed energy with value zinc of gluconate glutathione is shown and the in spatial Figureorientation 9. of both free form and when complexed with zinc gluconate is shown in Figure9.

Figure 9. Energy content and most favorable spatial orientation of free glutathione (a) and when Figure 9. Energy content and most favorable spatial orientation of free glutathione (a) and when complexed with zinc gluconate (b). Light grey balls represent carbon atoms. Blue balls represent complexed with zinc gluconate (b). Light grey balls represent carbon atoms. Blue balls represent nitrogen atoms. Non linked dark grey balls represent zinc cations. Red balls represent oxygen atoms nitrogen atoms. Non linked dark grey balls represent zinc cations. Red balls represent oxygen atoms andand yellow yellow balls balls represent represent sulfur sulfur atoms. atoms. The complex of glutathione with zinc gluconate showed the least energetic value The complex of glutathione with zinc gluconate showed the least energetic value (E (E = −641 kcal/mol) obtained from the compounds studied (Table2). The most favorable = −641 kcal/mol) obtained from the compounds studied (Table 2). The most favorable spa- spatial orientation corresponded to the zinc cation surrounded by four molecules: two tial orientation corresponded to the zinc cation surrounded by four molecules: two outer outer antiparallel mirrored images of GSH molecules at opposite ends and two inner antiparallel mirrored images of GSH molecules at opposite ends and two inner gluconate gluconate molecules with same orientation (Figure9b). molecules with same orientation (Figure 9b). Finally, we performed a computational analysis of the complex of three well known Finally, we performed a computational analysis of the complex of three well known polyphenols (hesperidin, silybin and xeractinol) with zinc gluconate, with the assumption polyphenols (hesperidin, silybin and xeractinol) with zinc gluconate, with the assumption that the high stability of the complexes was due to the formation of a greater number of that the high stability of the complexes was due to the formation of a greater number of intramolecular hydrogen bonds between the oxygenated functions of the polyphenol and intramolecularthe zinc cation. hydrogen The zinc bonds binding between sites can the be ox formedygenated through functions the hydroxylof the polyphenol and carbonyl and thegroups zinc cation. of the polyphenols The zinc binding (Figure sites 10 ).can be formed through the hydroxyl and carbonyl groupsBoth of the in polyphenols free form and (Figure in combination 10). with zinc gluconate, the computed energy contentBoth and in free spatial form orientation and in combination of dietary polyphenolswith zinc gluconate, (hesperidin, the computed silybin and energy xeractinol), con- tentasrepresentatives and spatial orientation of the ﬂavonoid of dietary family, polyphenols is shown (hesperidin, in in Table 2silybin and Figure and xeractinol), 11. as representatives of the flavonoid family, is shown in in Table 2 and Figure 11. The lowest energy value, as computed by molecular mechanics and thus most stable form of these complexes was found for the xeractinol/zinc gluconate (E = −324.2 kcal/mol) and the higher value of computed energy (E = −309.81 kcal/mol) was fond for the hesperidin/zinc gluconate complex (Figure 11). In all flavonoid studies, the complexes with zinc gluconate consisted in an internal zinc cation surrounded by two molecules of gluconate and one molecules of flavonoid with their carbonyl or hydroxyl groups facing internally to facilitate the coordination with the internal zinc (Figure 11). Stresses 2021, 1 132 Stresses 2021, 1, FOR PEER REVIEW 10

Stresses 2021, 1, FOR PEER REVIEW 10

FigureFigureFigure 10. 10.Intramolecular10. IntramolecularIntramolecular hydrogen hydrogenhydrogen bonds bondsbetweenbonds betweenbetween the oxygenated thethe oxygenatedoxygenated functions of functionsfunctions the polyphenols ofof thethe polyphenols polyphenols silybin,silybin,silybin, hesperidin hesperidinhesperidin and xeractinol andand xeractinolxeractinol in combination inin combinationcombination with thewith withzinc cation.the thezinc zinc cation. cation.

(a)

Figure 11. Cont.

(a) Stresses 2021, 1 133 Stresses 2021, 1, FOR PEER REVIEW 11

(b)

FigureFigure 11. 11. EnergyEnergy content content and andspatial spatial orientation orientation of poly ofphenols polyphenols silybin, xeractinol silybin, xeractinol and hesperidin and hesperidin both in free forms (a) or in complexes with zinc gluconate (b). Light grey balls represent carbon atoms.both Red in free balls forms represent (a) oroxygen in complexes atoms. Non with linked zinc dark gluconate grey balls ( brepresent). Light zinc grey cations. balls represent carbon atoms. Red balls represent oxygen atoms. Non linked dark grey balls represent zinc cations. 3. Discussion The lowest energy value, as computed by molecular mechanics and thus most stable We here provide a computational energetic analysis of the forming zinc complexes − toform determine of these whether complexes natural was substances, found for such the as xeractinol/zinc flavonoids, glutathione gluconate and (E plant = amino324.2 kcal/mol) acids,and can the act higher as novel value zinc of ionophores. computed Our energy research (E =is −focused309.81 on kcal/mol) the study wasof molecular fond for the hes- conformationalperidin/zinc structure gluconate of complexthe zinc complexe (Figure 11s with). In the all ﬂavonoidlowest amount studies, of energy. the complexes This with favorablezinc gluconate energy content consisted indicates in an that internal polyphenols, zinc cation glutathione surrounded and amino by two acids molecules may of glu- formconate zinc and complexes, one molecules which ofmight ﬂavonoid function with as theirnovel carbonyl ionophores. or hydroxylThese natural groups facing internally to facilitate the coordination with the internal zinc (Figure 11).

3. Discussion We here provide a computational energetic analysis of the forming zinc complexes to determine whether natural substances, such as ﬂavonoids, glutathione and plant amino acids, can act as novel zinc ionophores. Our research is focused on the study of molecular Stresses 2021, 1 134

conformational structure of the zinc complexes with the lowest amount of energy. This favorable energy content indicates that polyphenols, glutathione and amino acids may form zinc complexes, which might function as novel ionophores. These natural compounds might be attractive candidates for the treatment of diseases caused by zinc deficiency. The chemistry of ionophore molecules is an important area of supramolecular chemistry [24]. The notion of a metal ionophore consist of a ligand attached to a metal cation in a reversible manner. In the bonded form, the metal complex is generally charge neutral as well as sufficiently hydrophobic to facilitate membrane passage across lipid membranes. Metal cation release can then be activated by either a low local concentration of the metal cation or the presence of a stimulus that favors metal cation release. Due to the reversibility of the metal binding process, the ionophore may repeatedly transport metal cations across membranes [25]. Experimental determinations of the stability constants of ionophore metal-complexes and the corresponding free energies of complexation reactions are time-consuming and expensive tasks. Stable and favorable complexation with metals may improve ionophore bioavailability to cells, being a major critical step in the process of a chemical acting as zinc ionophores [26]. As a result, a theoretical quantitative assessment of complex stability, together with the analysis of the geometry and spatial orientation around the metal cation, may prove to be a valuable complement to experimental investigations, allowing researchers to decrease the number of tests performed and to identify the strategy of “optimization” of known metal binders, among other advantages. The zinc ion is stereo-chemically inactive. Zinc conformational structure is not important in our study, only as a chelating agent. The zinc ion is highly flexible in terms of coordination numbers, exhibiting coordination dynamics that do not conform to the stere- ochemical restrictions common to transition metal ions. Additionally, it is distinguished from magnesium (Mg2+) and calcium (Ca2+) because it forms considerably stronger complexes with water and a range of anions and ligands [26]. These features are critical for the proper functioning of its biological activities. To validate our molecular mechanics approach, we first computed the energy content of several known zinc ionophores (pyrithione, PBT2, TPEN, clioquinol and hinokitiol) both as free molecules and when forming complexes with zinc cation. In all cases studied, the values obtained for the free molecules had positive values and negative values when complexed with zinc cation. In some complex up to 100 kcal/mol lower. This reduc- tion in conformational energy implies the favorable formation of the zinc complexes, as corresponding for zinc ionophores. Another aspect of potential ionophore molecules is the geometry of the most stable form of the ionophore-metal cation complex [25]. Ionophores are flexible, the final spatial orientation of the complexes includes configurational or conformational remodeling of the ionophore to accommodate the internalization of the metal ion. Ionophores, in general, are molecules having backbones of a range of different topologies that include oxygen or nitrogen atoms that are strategically spaced [25]. The liganding atoms include a variety of functional groups, including ether, alcohol, carboxyl and amide, among others. It is through ion-dipole interaction that the neutral oxygen or nitrogen function as a ligand of cations, which is comparable to the solvation of ions in highly dielectric fluids. For the permeation through the lipophilic environment of the membrane, the polar groups of ionophore complexes must face inward, while the outside of the complex is covered with different hydrocarbon hydrophobic groupings. In our analysis, the most favorable geometry of the complex zinc pyrithione showed the zinc cation in an internal position between two adjacent pyrithione molecules, with their polar groups orienting towards the inside [27]. Similarly, for the zinc ionophore TPEN, the spatial orientation of the most favorable complex with zinc showed an internal zinc cation surrounded by four pyridine rings. The resultant complex’s lipid solubility of ionophores can be explained in part by the efficient shielding of its polar core, which delocalizes the cation charge and in part by the complex’s external compatibility with low-dielectric solvents [28]. Several articles have Stresses 2021, 1 135

recently been published that report the utility of theoretical calculations on these kind of chemical interactions [29,30]. In human nutrition, zinc deficiency is a global nutritional problem. Zinc supplementation has been used for the treatment of several diseases, but mainly for its potential ability to attenuate the accumulation of free radicals [31]. The total zinc content of eukaryotic cells is quite high, approximately 200 µM. However, the majority of cellular zinc is firmly bound to proteins [32]. A weak-binding fraction, capable of rapidly triggering ligand exchanges, is represented by the free (bioavailable, rapidly exchangeable, or labile) zinc ion. This labile zinc comprises the major mobile reactive pool of cellular zinc. This pool is not a viable source for incorporation into proteins, as it is loosely bound to low molecular ligands (such as amino acids and citrate). When labile zinc combines with a low-molecular-weight ligands or chelators to be absorbed, solubility is increased [33]. As a result, the chemical form in which this labile zinc is made available is of critical consideration. Amino acids (e.g., histidine, tryptophan) and organic acids (e.g., citrate, gluconate) have been used to improve zinc absorption and have been utilized in zinc supplements to improve zinc bioavailability. In most of these studies usually only one type of Zn2+salt was used rising the question whether different counterions and ligands of Zn2+ may influence its uptake and antioxidative function. Zinc supplementation has been proposed as an efficacious chemo-preventive agent for some cancers [34]. Zinc can restrict the growth of cancer cells via cell cycle arrest, as well as induction of apoptosis and necrosis [35]. In clinical studies oral supplementation with zinc gluconate resulted in a decreased frequency of infections, production of tumor necrosis factor alpha (TNF-) and oxidative stress in elderly subjects, compared to placebo [36]. Therefore, any strategies that can increase zinc levels in the cancer cells are likely to be effective cancer treatment strategies, having minimal toxicity to normal cells. Accordingly, various complexes between zinc and bioactive molecules have been prepared and explored in the development of new antitumor drugs [25]. Although zinc sulfate is the most widely utilized zinc form, zinc gluconate stabilized with glycine has the same bioavailability and metabolic behavior as zinc sulphate in aqueous solution with the advantages of good solubility, mild flavor and low cost of production [37]. Our theoretical results suggest that zinc gluconate forms the most favorable complex of zinc among four zinc salts of organic acids: zinc gluconate, zinc citrate, zinc acetate and zinc lactate. These organic salts are already available on the market as zinc food supplements. All salt studied have negative energy values of up to E = −358.3 kcal/mol, as in the case of zinc gluconate, indicating that they were acting as zinc complexes. Therefore, zinc gluconate was used as a starting point for its complexation with amino acids, glutathione and flavonoids. Tryptophan and histidine-rich sites exposed to the solution could be the most likely targets for the first phase of the metallization process by zinc [38]. Zinc complexes with proteinogenic amino acids have been recently proposed as potential candidates for der- matological treatments in the cosmetic and pharmaceutical industries. Theoretically, these derivatives should be better absorbed and safer due to their non-ionic structure and weak influence on the skin pH, which will make them excellent ingredients for dermal products [39]. Our theoretical tree-dimensional study shows that histidine and tryptophan formed stable complexes with zinc gluconate. The complexes obtained with tryptophan or histidine have lower energy values, up to 300 kcal/mol less than in the case of zinc salts of organic acids, indicating the formation of a more stable basal complex than the individual compounds. We observed that more ligands are formed between the zinc cation of the primary ionophores with the amino acids and polyphenols. The decrease in the total energy of the complex might be due to the formation of hydrogen bonds between the hydroxyl and amino groups present in final complex, which results in greater energy stability of the final complex. This is consistent with the finding that, in clinical studies, orally administered zinc-histidine displayed a 3-fold bioavailability, as compared to zinc sulphate [40]. Zinc salts of amino acids may exert neuroprotection. In the brain, zinc-histidine was revealed to be the preferred substrate for membrane transport, suggesting that amino acids play a role Stresses 2021, 1 136

in biological zinc complexation [26]. Zinc-histidine has been studied as the most effective yet least toxic form of zinc for the treatment of central nervous system disorders associated with zinc deficiency. The neuroprotective role of zinc supplementation with histidine is related to the attenuation of oxidative stress [41]. Glutathione is a key antioxidant peptide found in plants, animals, fungi and some bacteria and archaea, capable of preventing damage to important cellular components caused by reactive oxygen species (ROS), such as free radicals, peroxides, lipid peroxides, as well as heavy metals [42]. Reduced glutathione GSH is considered a chelating ligand for zinc, as they form various complexes [26]. It is a peptide formed between the carboxyl group of the glutamate side chain and cysteine, where the carboxyl group of this residue is attached by peptide linkage to glycine. Our theoretical result also showed that the complexes obtained with glutathione have much lower energy contents than zinc gluconate. Flavonoids are antioxidants present in food and play an important role in the defense against oxidative stress [43], which is present in various physiological and pathophysiological processes. The antioxidant activity of phenolic compounds results from a combination of their transition metal chelating and free radical scavenging properties. In addition, they act by inhibiting enzymes such as oxidases, lipoxygenase, myeloperoxidase, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and xanthine oxidase, preventing the generation of ROS in vivo, as well as organic hydroperoxides [44,45]. For example, zinc and polyphenols play an important antioxidant role in the immune response against SARS- CoV-2 [46]. We first studied the potential interaction of the flavonoids silybin, with zinc salts of organic acids. The energy of silybin, when forming complexes with zinc salts of organic acids showed lower values for zinc lactate and zinc acetate, but slightly higher for zinc gluconate and zinc citrate. This is consistent with previous reports showing that the flavones morin and quercetin interfered with zinc accumulation, which resulted in growth improvement. However, supplemental zinc turned the initially benefic action of morin and quercetin into potential toxicity, which may increase the anticancer potency of these drugs [47]. The complexation with Zn2+ also improved the biological activity of the free rutin. Rutin–zinc has shown cytotoxicity against leukemia, multiple myeloma and melanoma cell lines, but did not show any cytotoxicity against normal cells in vitro. One explanation is that Rutin–zinc complex is more efficient as an antioxidant agent than free rutin [48]. In humans, diabetic polyneuropathy has been documented to improve by taking supplements of zinc/polyphenol [49]. When we consume zinc without any food, iron inhibits the absorption of zinc in our body, with a molar ratio of iron:zinc of 25:1 [16]. The formation of poorly soluble complexes with natural chelating agents, such as inositol hexaphosphate and pentaphosphates (phytic acid), leads to reduced assimilation of zinc. Phytates, present in foods such as cereals, corn and rice, has a very negative effect on zinc absorption [50]. These metal-polyphenol complexes are reported to exhibit better pharmaceutical properties. Some studies indicate that certain individuals should anticipate their zinc status if they routinely eat procyanidin-containing food [51]. Ionophores are molecules that alter the permeability of biological membranes in response to certain metal ions for which they have an affinity and selectivity. Ionophores form a strong bond with a specific metal ion, shielding it from the effects of the surrounding environment. As a result, the metal ion is more easily transported into the hydrophobic core of the lipid membrane. Thus, ionophores function as a mobile carrier, transporting ions through the hydrophobic environment of cell membranes. We can illustrate the capacity of flavonoids, for example, as zinc ionophores, with a motorcyclist/motorbike interaction model. The biker would represent the zinc cation, which by itself cannot cross the membrane without the help of the motorbike, which, in this case, would be the flavonoid acting as an ionophore (Figure 12). Stresses 2021, 1 137 Stresses 2021, 1, FOR PEER REVIEW 15

FigureFigure 12. 12. Biker/motorcycleBiker/motorcycle analogy analogy model model of flavonoids of flavonoids acting acting as zinc as zincionoph ionophores.ores. The metal The metalbind- ingbinding event event permits permits the ionophore the ionophore to traffic to traffic metal metalcations cations acrossacross the hydropho the hydrophobicbic environment environment of cell membranes.of cell membranes. In the bound In the state, bound the state,zinc cations the zinc (biker) cations and (biker) flavonoids and flavonoids(motorbikes) (motorbikes) are sufficiently are hydrophobic to permit passage across lipid membranes, where the zinc ions would not be soluble. sufficiently hydrophobic to permit passage across lipid membranes, where the zinc ions would not be soluble. 4. Materials and Methods 4. MaterialsIn this conformational and Methods energy study, the following steps were considered: In this conformational energy study, the following steps were considered: 4.1. Known Zinc Ionophores as Models for the Study 4.1. Known Zinc Ionophores as Models for the Study The following compounds were chosen as known zinc ionophores able to modulate intracellularThe following labile compoundszinc concentrations were chosen [52] as: pyrithione known zinc (PT) ionophores (an antimicrobial able to modulate effective in- againsttracellular several labile pathogens zinc concentrations of the genera [52]: pyrithione Streptococcus (PT) (anand antimicrobial Staphylococcus effective) [27], against5,7-Di- chloro-2-((dimethylamino)several pathogens of the generamethyl)Streptococcus quinolin-8-oland (PBT2),Staphylococcus N,N,N′,N)[′-tetrakis(2-pyridinyl-27], 5,7-Dichloro-2- 0 0 methyl)-1,2-ethanediamine((dimethylamino) methyl) quinolin-8-ol (TPEN), 2-hydroxyc (PBT2), N,N,Nyclohepta-2,4,6-trien-1-one,N -tetrakis(2-pyridinylmethyl)-1,2- (hinokitiol) andethanediamine 5-Chloro-7-iodo-8-quinolinol (TPEN), 2-hydroxycyclohepta-2,4,6-trien-1-one (clioquinol). (hinokitiol) and 5-Chloro-7- iodo-8-quinolinol (clioquinol). 4.2. Molecular Mechanics Calculations 4.2. Molecular Mechanics Calculations Energy content of different molecules and zinc complexes with polyphenols and Energy content of different molecules and zinc complexes with polyphenols and amino acids was calculated using ChemBio3D Ultra 16,0 (Perkin-Elmer Inc., Waltham, amino acids was calculated using ChemBio3D Ultra 16,0 (Perkin-Elmer Inc., Waltham, MA, USA) and Molecular Mechanics calculation method (MM2) optimization method. MA, USA) and Molecular Mechanics calculation method (MM2) optimization method. Calculations were performed to obtain the energy of different conformations of the same Calculations were performed to obtain the energy of different conformations of the same 3D-complex,3D-complex, analyzing analyzing and and exposing exposing those with different energy of formation. Different spatialspatial conformations conformations were were estimated estimated for for each each complex. complex. The The lower lower the the energy, energy, the higher thethe stability.stability. Therefore,Therefore, the the lowest lowest possible possible energy energy content content was thenwas selectedthen selected and expressed and ex- pressedin kcal/mol. in kcal/mol. ToTo calculate the potential energy of the molecule based on force fields,fields, the MM2 methodmethod was was used. used. This This type type of ofmethod method consid consideredered the the atoms atoms as a as ball aball containing containing the nu- the cleusnucleus and and the thefused fused electrons electrons as a solid as a solidsphere sphere without without considering considering any electronic any electronic energy term.energy term. TheThe total total energy energy of of a a molecule molecule was was described described by by the the sum sum of of the the following following interactions: interactions:

ETotal = E Stretching + E Bending + E Torsion + E Non-bonded inte ETotal = EStretching+ EBending+ ETorsion+ ENon−bonded inte MM2 was chosen as a rapid method for determining the geometry, molecular ener- MM2 was chosen as a rapid method for determining the geometry, molecular energies, gies,vibrational vibrational spectra spectra and enthalpies and enthalpies of formation of formation of stable of moleculesstable molecules in their in basal their state. basal It state.was primarily It was primarily used to determineused to determine geometries geom in largeetries molecules, in large molecules, such as those such of as biological those of biologicaland pharmaceutical and pharmaceutical importance, importance, which were currentlywhich were beyond currently the reach beyond of more the intensivereach of moremolecular intensive orbital-based molecular methods.orbital-based For methods. this reason, For MM2this reason, was not MM2 useful was fornot modelling useful for Stresses 2021, 1 138

transition state of chemical processes that involved a large spectrum of experimental parameters, such as force ﬁeld parameters.

4.3. Compounds under Theoretical Analysis For theoretical considerations, we studied different zinc salts of organic acids, com- monly used for dietary supplements already on the market, such as zinc gluconate, zinc citrate, zinc acetate and zinc lactate. The proteinogenic amino acids: phenylalanine, tryptophan, histidine and arginine; the peptide glutathione (GSH) and the flavonoids: silybin, hesperidin and xeractinol were also studied. To ascertain whether theoretical computation of the zinc complexes was compatible with their potential roles as zinc ionophores, we studied different three-dimensional structural conformations for each complex, considering those with the lowest possible energy and favorable geometry respect to the internal position of the zinc cation. This energy is understood to be derived exclusively from molecular mechanics. The type, strength and specificity of metal ion interaction with ionophores complexing groups should provide a good estimate of the range of lowest energy configurations of free ionophores that can easily complex with metals.

5. Conclusions Our energetic and three-dimensional analysis of the zinc complexes with amino acids, glutathione and flavonoids studied was compatible with the considerations required for acting as potential zinc ionophores. The study of energy content, binding orientation of zinc cations and three-dimensional disposition of the molecules might be useful for initial theoretical studies of novel molecules to be considered as potential zinc ionophores. Traditional zinc ionophores, such as pyrithione, PBT2, TPEN and clioquinol, are associated with problems of cell toxicity [53–55], so their use is very limited or non-existent. Moreover, these molecules are expensive to synthesize, so the use of new ionophores that meet the conditions of safety and ease of obtaining and use are always welcome in the world of medicine. The energetic study suggests that polyphenol, glutathione and amino acids may form zinc complexes, likely acting as new ionophores. We propose that these novel complexes could be promising candidates for drug design to provide new solutions for conditions and diseases related to zinc deficiency or impairment derived from the dysregulation of this important metal. This preliminary study provides new opportunities to prevent zinc deficiency by using polyphenol nutraceutics, together with zinc supplementation. The regulation of intracellular zinc levels is fundamental to the survival of living organisms including humans. Zinc deficiency, particularly in developing countries, is still a widespread nutritional deficiency, due not only to a low intake of this essential trace metal, but also to poor bioavailability. These chelators form complexes that are transported to the cells and, once at the inside or even at the outside, zinc complexes must dissociate into single compounds due to the low intracellular concentration of free and labile zinc, joining to proteins or peptides. This electrochemical potential depends on the concentration of ions on either side of the membrane, as well as the chemical potential which depends on the concentration of ionophores on either side of the membrane. Further experimental studies are necessary to conclude that the substances, such as amino acids, glutathione and polyphenols, that are harmless and even beneficial to human health, are really acting as zinc ionophores. We are hoping to stimulate collaborative research interests from medicinal chemistry to develop new strategies and experiments to reinforce this idea.

Author Contributions: Conceptualization, C.A.-J. and E.P.-L.; investigation, C.A.-J. and E.P.-L.; writing—review and editing, C.A.-J.; J.M.P.d.l.L.; E.P.-L. and F.J.P.; supervision, C.A.-J. and J.M.P.d.l.L. All authors have read and agreed to the published version of the manuscript. Stresses 2021, 1 139

Funding: This research was funded by “Junta de Castilla y León”, projects FEDER-VA115P17 and VA149G18); by projects APOGEO (Cooperation Program INTERREG-MAC 2014–2020, with European Funds for Regional Development-FEDER, “Agencia Canaria de Investigación, Innovación y Sociedad de la Información (ACIISI) del Gobierno de Canarias” (project ProID2020010134), CajaCanarias (project 2019SP43); and Spanish Ministry of Economy and Competitiveness (Grant PID2019-105838RB-C31). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Conﬂicts of Interest: The authors declare no conﬂict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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