Interactions of Isoquinoline Alkaloids with Transition Metals Iron and Copper.

Data on alkaloid interactions with the physiologically important transition metals, iron and copper, are mostly lacking in the literature. However, these interactions can have important consequences in the treatment of both Alzheimer's disease and cancer. As isoquinoline alkaloids include galanthamine, an approved drug for Alzheimer's disease, as well as some potentially useful compounds with cytostatic potential, 28 members from this category of alkaloids were selected for a complex screening of interactions with iron and copper at four pathophysiologically relevant pH and in non-buffered conditions (dimethyl sulfoxide) by spectrophotometric methods in vitro. With the exception of the salts, all the alkaloids were able to chelate ferrous and ferric ions in non-buffered conditions, but only five of them (galanthine, glaucine, corydine, corydaline and tetrahydropalmatine) evoked some significant chelation at pH 7.5 and only the first two were also active at pH 6.8. By contrast, none of the tested alkaloids chelated cuprous or cupric ions. All the alkaloids, with the exception of the protopines, significantly reduced the ferric and cupric ions, with stronger effects on the latter. These effects were mostly dependent on the number of free aromatic hydroxyls, but not other hydroxyl groups. The most potent reductant was boldine. As most of the alkaloids chelated and reduced the ferric ions, additional experimental studies are needed to elucidate the biological relevance of these results, as chelation is expected to block reactive oxygen species formation, while reduction could have the opposite effect.

Amaryllidaceae Alkaloids of Norbelladine-Type as Inspiration for Development of Highly Selective Butyrylcholinesterase Inhibitors: Synthesis, Biological Activity Evaluation, and Docking Studies.

Alzheimer's disease (AD) is a multifactorial neurodegenerative condition of the central nervous system (CNS) that is currently treated by cholinesterase inhibitors and the N-methyl-d-aspartate receptor antagonist, memantine. Emerging evidence strongly supports the relevance of targeting butyrylcholinesterase (BuChE) in the more advanced stages of AD. Within this study, we have generated a pilot series of compounds (1-20) structurally inspired from belladine-type Amaryllidaceae alkaloids, namely carltonine A and B, and evaluated their acetylcholinesterase (AChE) and BuChE inhibition properties. Some of the compounds exhibited intriguing inhibition activity for human BuChE (hBuChE), with a preference for BuChE over AChE. Seven compounds were found to possess a hBuChE inhibition profile, with IC50 values below 1 µM. The most potent one, compound 6, showed nanomolar range activity with an IC50 value of 72 nM and an excellent selectivity pattern over AChE, reaching a selectivity index of almost 1400. Compound 6 was further studied by enzyme kinetics, along with in-silico techniques, to reveal the mode of inhibition. The prediction of CNS availability estimates that all the compounds in this survey can pass through the blood-brain barrier (BBB), as disclosed by the BBB score.

Chelation of Iron and Copper by Quercetin B-Ring Methyl Metabolites, Isorhamnetin and Tamarixetin, and Their Effect on Metal-Based Fenton Chemistry.

Quercetin, a common flavonoid from human diet, is extensively metabolized. Its two metabolites with the preserved flavonoid core were tested in detail for their interactions with transition metals, iron and copper. Both compounds chelated both metals; however, there were some significant differences between them notwithstanding that the major chelation site (3-hydroxy-4-keto) was the same. The complex stoichiometries were also determined under different pH conditions and in both oxidation states. Mostly, complexes 2:1, flavonoid to metal, were observed. Both compounds reduced iron and copper in a bell-shaped manner with tamarixetin being less potent in general. Both metabolites potentiated the Fenton reaction triggered by iron, while they were able to decrease the copper-based Fenton reaction under acidic conditions. In cellular experiments, both metabolites attenuated the copper-triggered hemolysis with isorhamnetin being more potent. In conclusion, there are differences between methylated metabolites of quercetin in relation to their interactions with biologically relevant transition metals.

Probing the Structure and Function of the Cytosolic Domain of the Human Zinc Transporter ZnT8 with Nickel(II) Ions.

The human zinc transporter ZnT8 provides the granules of pancreatic ß-cells with zinc (II) ions for assembly of insulin hexamers for storage. Until recently, the structure and function of human ZnTs have been modelled on the basis of the 3D structures of bacterial zinc exporters, which form homodimers with each monomer having six transmembrane α-helices harbouring the zinc transport site and a cytosolic domain with an α,ß structure and additional zinc-binding sites. However, there are important differences in function as the bacterial proteins export an excess of zinc ions from the bacterial cytoplasm, whereas ZnT8 exports zinc ions into subcellular vesicles when there is no apparent excess of cytosolic zinc ions. Indeed, recent structural investigations of human ZnT8 show differences in metal binding in the cytosolic domain when compared to the bacterial proteins. Two common variants, one with tryptophan (W) and the other with arginine (R) at position 325, have generated considerable interest as the R-variant is associated with a higher risk of developing type 2 diabetes. Since the mutation is at the apex of the cytosolic domain facing towards the cytosol, it is not clear how it can affect zinc transport through the transmembrane domain. We expressed the cytosolic domain of both variants of human ZnT8 and have begun structural and functional studies. We found that (i) the metal binding of the human protein is different from that of the bacterial proteins, (ii) the human protein has a C-terminal extension with three cysteine residues that bind a zinc(II) ion, and (iii) there are small differences in stability between the two variants. In this investigation, we employed nickel(II) ions as a probe for the spectroscopically silent Zn(II) ions and utilised colorimetric and fluorimetric indicators for Ni(II) ions to investigate metal binding. We established Ni(II) coordination to the C-terminal cysteines and found differences in metal affinity and coordination in the two ZnT8 variants. These structural differences are thought to be critical for the functional differences regarding the diabetes risk. Further insight into the assembly of the metal centres in the cytosolic domain was gained from potentiometric investigations of zinc binding to synthetic peptides corresponding to N-terminal and C-terminal sequences of ZnT8 bearing the metal-coordinating ligands. Our work suggests the involvement of the C-terminal cysteines, which are part of the cytosolic domain, in a metal chelation and/or acquisition mechanism and, as now supported by the high-resolution structural work, provides the first example of metal-thiolate coordination chemistry in zinc transporters.

The influence of microbial isoflavonoid specific metabolites on platelets and transition metals iron and copper.

BACKGROUND: Isoflavonoids seem to possess positive cardiovascular and other beneficial effects in humans. HYPOTHESIS: Their low bioavailability, however, indicates that small isoflavonoid metabolites formed by human microflora can significantly contribute to these activities. STUDY DESIGN: Testing antiplatelet activity ex vivo in human blood and interaction with transition metals in vitro. METHODS: The effect on platelet aggregation induced by different triggers (arachidonic acid, collagen, ADP, TRAP-6), and interactions with transition metals (iron and copper chelation/reduction) were evaluated against four isoflavonoid-specific metabolites: S-equol; O-desmethylangolensin; 2-(4-hydroxyphenyl) propionic acid (HPPA); and 4-ethylphenol. RESULTS: S-equol, 4-ethylphenol and O-desmethylangolensin blocked platelet aggregation induced by arachidonic acid and collagen. S-equol even matched the potency of acetylsalicylic acid in the case of collagen, which is the most physiological inducer of aggregation. Moreover, their effects in general seemed to be biologically relevant and attainable at achievable plasma concentrations, with the exception of HPPA which was ineffective. While only O-desmethylangolensin mildly chelated iron and copper, all four compounds markedly reduced cupric ions. Their direct free radical scavenging effects seem to have little clinical relevance. CONCLUSION: This study has shown that S-equol, O-desmethylangolensin and 4-ethylphenol, arising from isoflavonoid intake, can have biologically relevant effects on platelet aggregation.

Interaction of isolated silymarin flavonolignans with iron and copper.

Silymarin, the standardized extract from the milk thistle (Silybum marianum), is composed mostly of flavonolignans and is approved in the EU for the adjuvant therapy of alcoholic liver disease. It is also used for other purported effects in miscellaneous nutraceuticals. Due to polyhydroxylated structures and low systemic bioavailability, these flavonolignans are likely to interact with transition metals in the gastrointestinal tract. The aim of this study was to analyze the interactions of pure silymarin flavonolignans with copper and iron. Both competitive and non-competitive methods at various physiologically relevant pH levels ranging from 4.5 to 7.5 were tested. Only 2,3­dehydrosilybin was found to be a potent or moderately active iron and copper chelator. Silybin A, silybin B and silychristin A were less potent or inactive chelators. Both 2,3­dehydrosilybin enantiomers (A and B) were equally active iron and copper chelators, and the preferred stoichiometries were mainly 2:1 and 3:1 (2,3­dehydrosilybin:metal). Additional experiments showed that silychristin was the most potent iron and copper reductant. Comparison with their structural precursors taxifolin and quercetin is included as well. Based on these results, silymarin administration most probably affects the kinetics of copper and iron in the gastrointestinal tract, however, due to the different interactions of individual components of silymarin with these transition metals, the biological effects need to be evaluated in the future in a much more complex study.

The Stoichiometry of Isoquercitrin Complex with Iron or Copper Is Highly Dependent on Experimental Conditions.

Interaction of flavonoids with transition metals can be partially responsible for their impact on humans. Stoichiometry of the iron/copper complex with a flavonoid glycoside isoquercitrin, a frequent component of food supplements, was assessed using competitive and non-competitive methods in four (patho)physiologically-relevant pH values (4.5. 5.5, 6.8, and 7.5). Isoquercitrin chelated all tested ions (Fe2+, Fe3+, Cu2+, and Cu⁺) but its affinity for Cu⁺ ions proved to be very low. In general, the chelation potency dropped with pH lowering. Metal complexes of 1:1 stoichiometry were mostly formed, however, they were not stable and the stoichiometry changed depending on conditions. Isoquercitrin was able to reduce both Cu2+ and Fe3+ ions at low ratios, but its reducing potential was diminished at higher ratios (isoquercitrin to metal) due to the metal chelation. In conclusion, this study emphasizes the need of using multiple different methods for the assessment of chelation potential in moderately-active metal chelators, like flavonoids.