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Metal ions are important for many biological processes and are steadily available in the human body. Metal concentrations can be extremely high in diseased areas of various pathological conditions. Some synthetic and natural drugs need to be activated by metal ions as prodrugs. In this review, we provide a few examples to illustrate how metal ions activate and mediate drug targeting in the body. This knowledge may be helpful for the development of more effective drugs and pharmaceutical formulations.
Metal ions play essential roles in many biological processes such as cellular respiration (1), signal transduction (2), memory storage (3,4), and metabolism (5–8). They serve as active centers in metalloenzymes or maintain protein structures for catalytic functions (9). For example, two Zn ions are found in the catalytic domains of matrix metalloproteinases (10). The catalytic Zn(ii) is co-ordinated by three conserved histidine residues from the HEXXHXXGXXH motif, and the structural zinc is co-ordinated by two imidazole nitrogens from the sheet, as well as a histidine and aspartate from the long S-shaped loop.
Metal ions have many other functions in biological systems. In respiratory chain reactions, electrons are passed along a series of enzyme complexes: complex I (NADH/ubiquinone oxidoreductase), complex II (succinate/ubiquinone oxidoreductase), complex III (cytochrome bc1 complex), and complex IV (cytochrome c oxidase) (1). The electrons are generated from NADH, produced by oxidation of nutrients such as glucose, and eventually transferred to molecular oxygen (11). The redox chain reactions generate energy that is stored in adenosine triphosphate (ATP), originated from ADP and phosphate (11). Metal ions with variable oxidation states, for example, iron and copper, are involved in redox reactions (12). They serve as electron donors or acceptors and initiate the reduction-oxidation reactions.
Many metalloenzymes are also used for electrophilic catalysis (13,14). For example, carbonic anhydrase is a metalloenzyme containing zinc in its active center (15). This enzyme catalyzes the interconversion of carbon dioxide and bicarbonate. After co-ordination with H2O, Zn(ii) facilitates deprotonation of the water molecule to form a Zn(ii)-OH- fragment (15). This newly produced nucleophilic hydroxide ion can further attack the carbonyl group of carbon dioxide to convert it into bicarbonate. Many biochemical reactions require metal ions, which act as metal centers for enzymes and therefore facilitate biochemical reactions (9).
However, both deficiency and excess of metal ions in the human body result in various dieases (16–21). Menkes disease is caused by mutations of the ATP7A gene (22–24). ATP7A is a transmembrane protein that transports copper to secreted copper enzymes or exports surplus copper from cells (22–24). Mutations of the ATP7A gene lead to poor distribution of copper in the body, reduced activity of copper enzymes, and Menkes syndrome. Wilson disease is caused by copper accumulation in the liver, brain, and other organs (25). Normally, ATP7B exports copper from the liver into the bile. However, mutated ATP7B induces copper release into the bloodstream and its migration to other organs including the brain, kidneys, and eyes (26,27). Dyshomeostasis of copper also causes Parkinson’s (28) and Alzheimer’s diseases (29). In Alzheimer’s, excess copper induces protein misfolding and accelerates protein aggregation to form fibrillar plaques that characterize the diseases.
Metals have been used in medicine for several thousands of years (30), and metallodrugs are currently being clinically used for the treatment of various diseases (30,31). The discovery of the antitumor activity of cisplatin is considered a breakthrough for metallodrugs. Cisplatin exhibits a broad spectrum of antitumor activities that can be used to treat a variety of cancers (32,33). It was approved as an anticancer drug for testicular and ovarian cancers by the US Food and Drug Administration in 1978 (33). The successful application of cisplatin in patient treatment stimulated great interest in the development of new metallodrugs for therapy and diagnosis.
A large number of synthetic and natural drugs require activation by metal ions in the body as prodrugs (34). Metal ion co-ordination with organic and natural drugs provides advantages over the drugs themselves. There are many new properties generated from metal incorporation, such as reduction and oxidation ability, bio-catalytic ability, and a wide structural diversity (35). Metal ions act as binding sites on drugs for biomacromolecules, redox centers to generate reactive species, catalytic centers to facilitate binding, and triggers to induce structure refinement. Knowledge of metal activation of synthetic and natural drugs may be helpful for the development of more effective drugs and pharmaceutical formulations. We illustrate how drugs can be modified by the incorporation of metal ions including iron, copper, and zinc in the body.
Bleomycin is a glycopeptide antibiotic made from Streptomyces verticillus that contains bleomycin A2 (see structural formula 1) and B2 (see structural formula 2) as the major constituents (36). It has been used clinically for the treatment of various cancers including Hodgkin’s lymphoma (37), non-Hodgkin lymphoma (38), and testicular cancer (39). Bleomycin comprises four regions with different functions: a metal-binding domain, a DNA binding domain, a linker region, and a carbohydrate region (40). The N-terminus consists of pyrimidoblamic acid and a β-hydroxyhistidine that constitute the metal-binding domain (41). Bleomycin is enriched with nitrogen atoms that act as donors for co-ordination to metal ions. A five co-ordination mode was observed in the crystal structure of bleomycin-Co(III)-peroxy (42). The four equatorial ligands are an imidazole nitrogen, a deprotonated amide, a pyrimidine nitrogen, and the secondary amine of the β-aminoalanine. The axial position is occupied by the primary amine of the β-aminoalanine. The DNA binding domain is composed of bithiazole and a substituent group that is positively charged at the C-terminus. The orientation of two rings in bithiazole is an interesting topic that has been discussed in several papers (43–47). In the solid state, the two thiazolium rings adopt the trans-conformation, whereas in solution, they adopt the the cis-conformation in the bound state with DNA (43–47). Bleomycin derivatives vary mainly in the bithiazole tail moiety. The N- and C-terminal domains are connected via a linker that contains an unusual (3S,4R)-4-amino-3-hydroxy-2-methyl-pentanoic acid (40).
Each part of bleomycin makes an important contribution to its anticancer activity. For example, variation of the linker affects DNA cleavage efficiency and the ratio of double to single strand DNA cleavage (48). The carbohydrate domain containing l-gulose and 3-O-carbamoyl-d-mannose is connected to the β-hydroxyhistidine residue (40). The glycosylation contributes to cellular uptake and co-ordination to metal ions. However, the non-ribosomal peptide backbone is difficult to recognize due to the extensive modifications of the peptide.
As an anticancer drug, bleomycin causes both single- and double-strand DNA breaks (49). The cleavage process is highly dependent on O2 and metal ions, for example, iron (49,50). Bleomycin co-ordinates to Fe(ii) to generate a F(ii) complex with bleomycin that can be further activated by the reaction with oxygen to produce bleomycin-Fe(III)-OOH (51) as shown below.
DNA cleavage by the active species occurs through a multi-step process. Metallobleomycin recognizes the guanosine base at 5′ G-C and 5′ T-G sequences (52). Bleomycin-Fe(III)-OOH binds to DNA first (Figure 2A). The binding details were revealed by the crystal structure of DNA-bound Co(III)-bleomycin B2 (42). The bithiazole moiety is inserted into the space between two base pairs and stacked with their nitrogen-containing heterocycles. This binding mode is stabilized by water-mediated interactions. The metal-binding domain and disaccharide moieties partially stack against each other. This orientation of the two bleomycin regions is stabilized by a net of hydrogen bonds to each respective wall of the DNA minor groove. The sugar moieties perform fine adjustment of the position of the metal-binding domain relative to the target C4′-H (42). In a model of Fe(III) complex with DNA, the distance between the distal peroxide oxygen and H4′ on C4′ is shorter than those in the other metal complexes with DNA (40). The closer distance accounts for the higher activity of the Fe(III) complex with bleomycin and also supports the direct abstraction of the hydrogen atom from the adjacent deoxyribose sugar.
The active species further cleaves DNA with site-specificity. The DNA cleavage starts with the removal of a 4′-hydrogen atom from C4′ of deoxyribose by the metal-bound peroxide oxygen group of the activated bleomycin-Fe(III)-OOH, leading to the formation of bleomycin-Fe(IV)=O, water and the sugar-centered radical (53,54). The newly produced free radical initiates the cleavage of both DNA single and double strands. There are two cleavage pathways depending on the availability of oxygen (55) (Figure 1). In the oxygen-dependent pathway, the transient C4′-radical reacts with O2 first (56–58). Further reduction and rearrangement of the peroxide radical leads to direct DNA strand breaks, forming 3′-phosphoglycolate and 5′-phosphate product and a base propenal. In the oxygen-depleted pathway, oxidation at the C4′-position after the hydrogen abstraction initiates a series of further chemical reactions resulting in the formation of propenal (59). The intermediate is decomposed to 4′-keto abasic sites and free nucleic acid bases in the presence of a base. However, the double-strand DNA cleavage is only observed in the oxygen-dependent pathway (60).
The iron ion is essential for bleomycin anticancer activity. It activates bleomycin through co-ordination and O2 oxidation (49,50) and further acts as the active center for DNA cleavage (51).
Non-steroidal anti-inflammatory drugs (NSAIDs) comprise a large group of medications used to treat inflammation as well as pain and fever (61). Some examples of NSAIDs include aspirin, indomethacin, ibuprofen, naproxen, piroxicam, and nabumetone. Most NSAIDs non-selectively inhibit both cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2). Cyclooxygenases promote the bis-dioxygeneration and subsequent reduction of arachidonic acid to prostaglandins (62). Prostaglandins are messenger molecules in the generation of the inflammatory response (63). Aspirin (see structural formula 3) is the only clinically used NSAID that covalently modifies the COX proteins, whereas all other NSAIDs are non-covalent binders (62). They act as either rapidly reversible and competitive inhibitors, or slow- and tight-binding inhibitors. However, toxicity is a major side-effect due to their inhibition of gastrointestinal prostaglandins produced via the COX-1 pathway (64). Therefore, more efforts are focused on the design and development of selective COX-2 inhibitors with reduced adverse effects compared to the traditional NSAIDs.
Knowledge of two target proteins, COX-1 and COX-2, may help us to understand the mechanisms of action of their inhibitors and aid the design of more effective drugs. Human COX-1 and COX-2 exist as homodimers of 576 and 581 amino acids, respectively (62). They share 60% sequence similarity and show structural homology. Each monomer consists of three domains: the epidermal growth factor domain, the membrane-binding domain, and the catalytic domain. In this last domain, the COX and peroxidase active sites are located on either side of the heme center. A hydrophobic channel forms the COX active site. The helices of the membrane-binding domain create the entrance to the hydrophobic channel. Selectivity is achieved through substitution of isoleucine at position 523 in COX-1 with a valine in COX-2. The mutation of the amino acid increases the size of the active site by approximately 25% in COX-2 (65). The enlarged binding pocket can accommodate larger molecules. Therefore, COX-2 selective inhibitors, such as celecoxib and rofecoxib, which have a large sulfanilamide side chain, can fit into the COX-2 but not the COX-1 binding pocket.
Copper is an essential trace element, which is incorporated in many metalloenzymes (66). Its distribution and homeostasis are tightly regulated (67). Cu(ii) forms complexes with NSAIDs to enhance anti-inflammatory activity and reduce gastrointestinal toxicity compared to their uncomplexed parent drugs (68,69). Cu(ii) complexes with NSAIDs are typically monomeric or dimeric through co-ordination with the carboxylate groups (68). In the dimers, the co-ordination geometry of the metal ion is octahedral, with the carboxylate groups bridging the two structural units through co-ordination to the two metal ions. In the monomers, the co-ordination number for Cu(ii) ranges from 4 to 6. The crystal structures of monomeric complexes typically display a trans unidentate bis (carboxylate) binding.
Cu(ii)-complexed aspirin is sevenfold more selective than aspirin itself in the inhibition of COX-2 (70). The mode of anti-inflammatory action of the Cu complexes may be similar to their parent NSAIDs, by acetylating serine 530 (70,71). Aspirin can easily fit into the binding pockets of both COX-1 and COX-2 due to its smaller size. However, copper-complexed aspirin may form a covalent dimer that no longer fits into the smaller binding pocket of COX-1 (72). The larger dimer no longer fits into the smaller binding pocket of COX-1. The larger cavity of COX-2 can more readily accommodate the dimer of Cu(ii) with aspirin. The positively charged Cu(ii) also balances the negative charge of carboxylates in the aspirin structure that also allows the aspirin dimer to more easily fit into the hydrophobic pocket of COX-2. It is also explains why the other Cu(ii)-complexed NSAIDs are more potent inhibitors than their uncomplexed parent drugs.
In a model of the copper complex with 3-benzoyl-α methyl benzene acetic acid (see structural formula 4) with COX-2, the orientation of the Cu(ii) complex resembles a selective COX-2 inhibitor (Figure 2B) (71). The carboxylates are critical for hydrogen bond interactions with Arg 106 and Tyr 341 through hydrogen bonds. Hydrophobic interactions with phenyl and benzoyl groups also contribute to stabilization of the Cu(ii) complex binding to its target COX-2.
In summary, copper is an essential trace element for many biological functions. Copper accumulates at sites of inflammation (69), and its concentration in serum increases significantly under the inflammatory conditions (73,74). Salicylates are even considered as carriers to transport copper to target cells in the body (75–77). The pharmacological activities of Cu(ii) complexes depend on the physico-chemical properties of the complexes themselves rather than those of their uncomplexed parent drugs (75).
The drug, AMD3100 (see structural formula 5), was originally developed as a potential candidate for the treatment of HIV (78). Further development was terminated after the phase II clinical trials due to lack of oral availability and significant cardiac side-effects (79). To enter a cell, the envelope glycoprotein GP120 of the HIV virus interacts with the primary receptor CD4 and then with a coreceptor CCR5 or CXCR4. This binding leads to membrane fusion and viral uptake (80–82). Therefore, both CCR5 and CXCR4 can serve as targets for HIV inhibitors. Further studies led to a new application for AMD3100 in the mobilization of stem cells for autologous transplantation in non-Hodgkin’s lymphoma and multiple myeloma patients (83). AMD3100 is currently in clinical use under the trade name Mozobil.
AMD3100 contains two cyclam (1,4,8,11-tetraazacyclotetradecane) units (see structural formula 6) that are bridged by the aromatic phenylenebis(methylene) linker. It prevents HIV-1 entry by binding and blocking the coreceptor CXCR4 (84). Cyclams bind strongly and relatively rapidly to Zn(ii) at micromolar concentrations, close to those in the blood (85). AMD3100 may therefore acquire Zn(ii) in the blood. The affinity of AMD3100 for the coreceptor CXCR4 is enhanced 7–50-fold by co-ordination to the transition metal ions Cu(ii), Zn(ii) or Ni(ii) (86). However, only the Zn(ii) complex with AMD3100 is more active than AMD3100 itself in the anti-HIV activity. This observation led to the suggestion that the Zn(ii) complex is an important antagonist in vivo (87).
In theory, there are five possible configurations for each cyclam complex with metal ions depending on the alignment of NH protons (Figure 3). Only three of them are stable enough and detectable in solution: trans-I and -III, and cis-V (88,89). Zn(ii), together with side-chain carboxylate binding, induces the unique folded configuration for AMD3100, cis-V (88,90). The cis-V configuration may be very important for the anti-HIV activity of cyclams. It is stabilized by binding to specific Asp and Glu carboxylate side chains of the coreceptor CXCR4 via metal co-ordination and H-bonding. Hydrophobic interactions between the periphery of the macrocycle and protein side chains may also be important for entry into the hydrophobic cavities of the membrane protein. In a model of human CXCR4, there is a pocket in which the trans and cis configurations of metallobicyclam can bind by direct metal co-ordination to (Asp and Glu) carboxylate side chains, by cyclam-NH···carboxylate H-bonding, and also by hydrophobic interactions with tryptophan residues (11) (Figure 2C). These studies suggest that configurationally restricted metallomacrocycles may be highly effective CXCR4 inhibitors.
In addition, AMD3100 is being used for the treatment of cancer patients with the same mode of action as for anti-HIV. CXCR4 binding to its natural ligand CXCL12 leads to the retention of stem cells in the bone marrow (91). Therefore, blocking CXCR4 mobilizes hematopoietic stem cells and progenitors into blood. AMD3100 binding to CXCR4 enables stem cells to detach from the bone marrow niche and migrate into the peripheral blood. AMD3100 is very effective in increasing stem cell mobilization and harvest.
Acyclic Nucleoside Phosphonates
The acyclic nucleoside phosphonates [(S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine] (see structural formula 7, HPMPC, cidofovir), [9-(2-phosphonylmethoxyethyl)adenine] (see structural formula 8, PMEA, adefovir), and [(R)-9-(2-phosphonylmethoxypropyl)adenine] (see structural formula 9, PMPA, tenofovir) are effective against a wide spectrum of DNA viruses and retrovirus infection (92). Adefovir was approved for the treatment of chronic hepatitis B by the United States Food and Drug Administration in 2002 (93). Cidofovir is an injectable antiviral medication for the treatment of cytomegalovirus retinitis for patients with AIDS (94). Tenofovir was approved by the United States Food and Drug Administration for the treatment of HIV and chronic hepatitis B (95). Due to poor bioavailability, adefovir and tenofovir have been modified for use as oral drugs, bis(pivaloyloxymethyl)-PMEA [bis(POM)-PMEA, adefovir dipivoxil] and bis(isopropyloxycarbonyloxymethyl)-PMPA [bis(POC)-PMPA, tenofovir disoproxil], respectively (92). Both adefovir and tenofovir have demonstrated activity against a wide range of retroviruses, that is, simian immunodeficiency virus, feline immunodeficiency virus, visna-maedi virus, and murine leukemia/sarcoma viruses (96).
As there is a phosphate in their structure, acyclic nucleoside phosphonates only require two more phosphates through phosphorylation to become active (97). The triphosphates then participate the DNA assembly under bio-catalysis by competing with the natural substrate deoxyadenosine triphosphate. However, the enzyme for the incorporation of cidofovir is viral DNA-dependent DNA polymerase, whereas for tenofovir and adefovir, it is human immunodeficiency virus or hepatitis B virus RNA-dependent DNA polymerase (reverse transcriptase). Incorporation of the drug during DNA synthesis interrupts further chain elongation (98). Addition of one molecule of adefovir is enough to terminate the polymerization reaction (96). However, cidofovir requires an exact duplication to efficiently terminate the chain reaction (99).
DNA polymerases require metal ions for catalysis of the phosphoryl transfer (100,101).
Two metal ions, typically Mg2+, are found in the active site (Figures 2D and 4) (102–104). One of the metals binds to the α-phosphate hydroxyl groups of the deoxynucleoside triphosphate (dNTP), for example, ATP (see structural formula 10) and a primer, whereas the other interacts with the hydroxyl groups of both β- and γ-phosphates (102) (Figure 4). The two metal centers are bridged by two aspartate residue carboxylate groups of the polymerase and the α-phosphate of the dNTP. The metal ion bound to the primer alters the electron distribution of the 3′-hydroxyl group of the primer and therefore promotes its attack on the α-phosphate of the dNTP substrate at the active site (102). The surrounding aspartates balance the positive charge of the metal ions and stabilize the pentacovalent transition state during the course of the reaction. The metal ion bound to the hydroxyl groups of the β- and γ-phosphates also facilitates the removal of the β- and γ-phosphates.
Duocarmycins (duocarmycin A (see structural formula 11) as an example) are a class of anticancer antibiotics first isolated from Streptomyces bacteria in 1988 (105,106). As their discovery, many additional effective analogs have been synthesized. Among them, Bizelesin and KW-2189 have been in phase II clinical trials against several different types of cancers (107). Modification of duocarmycin analoges can convert highly toxic DNA-alkylating agents to highly selective antitumor compounds, which are also highly active against the most resistant hypoxic tumor cells in vivo (108).
Duocarmycins exert their anticancer activities through a sequence-selective alkylation of DNA (109,110). The reversible and stereoelectronically controlled adenine-N3 addition to the least substituted cyclopropane carbon occurs in the selected AT rich region in the minor groove of DNA. In addition to the covalent bond, the non-covalent binding including hydrophobic interactions and van der Waals contacts also contributes significantly to the alkylation reaction. The non-covalent interactions further stabilize the newly produced adduct and make the alkylation reaction less reversible or completely irreversible. Selectivity is achieved due to more accessible space adjacent to an A-T base pair, which is occupied by an amino substituent on a G-C base pair. Duocarmycins can insert into this narrow AT-rich minor groove of DNA and selectively alkylate the nucleobase adenine at the N3 position. DNA binding further induces the conformational change of duocarmycins, disrupting the vinylogous amide stabilization, and activating them for nucleophilic attack (111).
Methyl 1,2,9,9a-tetrahydrocyclopropa[c]pyrido[3,2-e]indol-4-one-7-carboxylate (see structural formula 12) is a fully synthetic analoge of duocarmycins with a unique 8-ketoquinoline structure on the pharmacophore, which can co-ordinate to metal ions, for example, Zn2+, for activation (Figure 5) (112). This activation induces its binding to the nucleobase adenine at the N3 position in the minor groove of DNA, which is similar to its parent duocarmycin (113). The DNA alkylation rate and efficiency have been increased 104-fold, which allows DNA alkylation to occur at Zn2+ concentrations as low as 10−5m (113,114). The enhancement in the rate of DNA alkylation originates from enhanced DNA binding affinity and selectivity and a contribution to catalysis (114,115). However, the metal-ion enhancement of the rate and efficiency of DNA alkylation is not observed for duocarmycins, indicating this behavior is unique to the 8-ketoquinoline core structure (12) (113). The local Zn2+ concentration was found to be 700% higher in breast carcinoma than in normal cells. This analoge could exhibit an enhanced activity against breast carcinoma attributable to the difference in Zn levels.
Conclusion and Perspective
Some organic and natural drugs are inactive or less active after administration. They become active or more active through metal binding in the body. The examples in this review illustrate several different methods of drug targeting mediated by metal ions through metal co-ordination, reduction-oxidation, and structure conversion. For example, bleomycin acquires a redox center after binding to Fe(ii) (51). The newly produced species can be further activated by the reaction with oxygen to generate a reactive center, bleomycin-Fe(III)-OOH (51). This redox center initiates further oxidation–reduction reaction to cause direct abstraction of the hydrogen atom from the adjacent deoxyribose sugar (53,54). Eventually, a series of sequential redox chain reactions results in DNA strand breaks (51). In this example, both metal co-ordination and reduction–oxidation are combined together to activate bleomycin and mediate DNA cleavage.
Non-steroidal anti-inflammatory drugs may form a covalent dimer through co-ordination to Cu(ii) (68). Non-steroidal anti-inflammatory drugs acquire an additional binding site after binding to Cu(ii) (70,71). The positively charged Cu(ii) also balances the negative charge of carboxylates in some NSAIDs, for example, aspirin, and makes the NSAID dimers more stable in the hydrophobic pocket of COX-2 (70,71). Thus, NSAIDs can achieve higher binding affinities with their target COX. The 8-ketoquinoline structure in the analoge of duocarmycins can co-ordinate to metal ions, for example, Zn2+ for activation (112). This activation induces its binding to the nucleobase adenine at the N3 position in the minor groove of DNA (113). Metal binding is a key for the high DNA alkylation rate and efficiency. The increase in the DNA alkylation rate originates from enhanced DNA-binding affinity and selectivity (114,115). In another example, Mg(ii) are required by DNA polymerases for catalysis of the phosphoryl transfer (100,101). After incorporation into the polymer chains, acyclic nucleoside phosphonates terminate the chain reactions.
Zn(ii), together with side-chain carboxylate binding, induces the unique folded configuration for AMD3100, cis-V (85,90). The cis-V configuration may be very important for the anti-HIV activity of cyclams and is stabilized by binding to specific Asp and Glu carboxylate side chains of the coreceptor CXCR4 via metal co-ordination and H-bonding (88,90). Zn binding leads to refinement of the AMD3100 structure in solution and also provides an extra binding site for AMD3100 (88,90).
Metal ions play a crucial role, acting as mediators for drug targeting in the body. However, many important issues need to be addressed. To explore various factors that affect uptake and release of metals in the body is an important topic in the future. Thus, more effective pharmaceutical activity will be obtained by recruiting metals to drugs at optimal conditions.
We thank Dan Fryxell at R&D Systems Inc, for his critical review and comments on the manuscript.
Xun Tang received her Ph.D. in chemical engineering from Northwest University, China in 2008. Currently, she is a Lecturer at Xi'an Shiyou University. Xiangyang Liang is Scientist at R&D Systems, Inc. He received his Ph.D. in bioinorganic chemistry from the University of Edinburgh in 2002, under the supervision of Professor Peter Sadler. His research has been involved in protein folding, dynamics, protein -protein interactions, drug binding, drug synthesis and structure-based drug design. He is particularly interested in understanding the role of metal ions in biology and medicine..