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Zinc is a crucial element in cellular processes, and its homeostasis has intricate relationships with the initiation, progression, and therapeutic intervention of cancer. Activation of the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway has been proven to be an effective strategy for cancer immunotherapy. Herein, we report four phosphorescent iridium complexes (Ir1–Ir4) with zinc chelating ligands. Among them, Ir1 can bind and image mitochondrial chelatable zinc ions via phosphorescence-lifetime responses, consequently modulating the expression of zinc-regulatory proteins. Furthermore, the in situ formed heteronuclear metal complex Ir1-Zn2 shows nuclease mimetic activities, capable of hydrolyzing mitochondrial DNA (mtDNA) to release mtDNA fragments for the activation of the cGAS-STING pathway. In conclusion, we designed a mitochondria-targeting phosphorescent Ir(III) complex with dual functions in dysregulation of zinc homeostasis and generation of nuclease in situ, which provides an innovative approach to stimulate the cGAS-STING pathway. Show less

Ran-binding domain-containing protein 2 (ZRANB2) is a zinc finger (ZF) protein that plays a key role in alternative splicing. ZRANB2 is composed of two ZF domains that contain four invariant cysteine residues per domain. ZRANB2 binds RNA targets that contain AGGUAA sequence motifs. Three constructs of ZRANB2, ZRANB2-ZF1 (first ZF domain), ZRANB2-ZF2 (second ZF domain), and ZRANB2-2D (both ZF domains), were isolated in the apo form and shown to bind Zn(II) via UV-visible-monitored competitive titrations with Co(II) as a spectroscopic probe. Zn binding to each construct led to the adoption of a limited secondary structure of each domain, as measured by circular dichroism (CD). Hydrogen-deuterium exchange coupled with mass spectrometry (HDX-MS) of the two-domain construct, ZRANB2-2D, revealed that both ZF domains adopt a more rigid structure upon Zn binding. Zn binding to the first ZF domain resulted in a greater decrease in the conformational dynamics than Zn binding to the second ZF domain. RNA binding to TRA2B pre-mRNA, a physiological splicing target, was measured by fluorescence anisotropy (FA), and high-affinity RNA binding was found to require Zn coordination to both domains. HDX-MS of ZRANB2-2D with TRA2B RNA as well as two optimized RNA sequences that contain a single and double AGGUAA hexamer revealed additional protection from H/D exchange for ZRANB2 in the presence of RNA. Here, greater protection was observed for the second ZF of ZRANB2-2D, suggesting a larger effect on conformational dynamics. A model for zinc-mediated RNA binding of ZRANB2 is proposed. Show less

Hydrogen sulfide (H2S) is an endogenous gasotransmitter that signals via persulfidation. There is evidence that the cysteine residues of certain zinc finger (ZF) proteins, a common type of cysteine rich protein, are modified to persulfides by H2S. To determine how frequently ZF persulfidation occurs in cells and identify the types of ZFs that are persulfidated, persulfide specific proteomics data were evaluated. 22 datasets from 16 studies were analyzed via a meta-analysis approach. Persulfidated ZFs were identified in a range of eukaryotic species, including Homo sapiens, Mus musculus, Rattus norvegicus, Arabidopsis thaliana, and Emiliania huxley (single-celled phytoplankton). The types of ZFs identified for each species encompassed all three common ZF ligand sets (4-cysteine, 3-cysteine-1-histidine, and 2-cysteine-2-hisitidine), indicating that persulfidation of ZFs is broad. Overlap analysis between different species identified several common ZFs. GO and KEGG analysis identified pathway enrichment for ubiquitin-dependent protein catabolic process and viral carcinogenesis. These collective findings support ZF persulfidation as a wide-ranging PTM that impacts all classes of ZFs. Show less

The gasotransmitter hydrogen sulfide (H2S) is thought to be involved in the post-translational modification of cysteine residues to produce reactive persulfides. A persulfide-specific chemoselective proteomics approach with mammalian cells has identified a broad range of zinc finger (ZF) proteins as targets of persulfidation. Parallel studies with isolated ZFs show that persulfidation is mediated by ZnII, O2, and H2S, with intermediates involving oxygen- and sulfur-based radicals detected by mass spectrometry and optical spectroscopies. A small molecule ZnII complex exhibits analogous reactivity with H2S and O2, giving a persulfidated product. These data show that ZnII is not just a biological structural element, but also plays a critical role in mediating H2S-dependent persulfidation. ZF persulfidation appears to be a general post-translational modification and a possible conduit for H2S signaling. This work has implications for our understanding of H2S-mediated signaling and the regulation of ZFs in cellular physiology and development. Show less

Understanding the functions of metal ions in biological systems is crucial for many aspects of research, including deciphering their roles in diseases and potential therapeutic use. Structural information about the molecular or atomic details of these interactions, generated by methods like X-ray crystallography, cryo-electron microscopy, or nucleic magnetic resonance, frequently provides details that no other method can. As with any experimental method, they have inherent limitations that sometimes lead to an erroneous interpretation. This manuscript highlights different aspects of structural data available for metal-protein complexes. We examine the quality of modeling metal ion binding sites across different structure determination methods, where different kinds of errors stem from, and how they can impact correct interpretations and conclusions.Many metalloproteins contain metal ions as integral components, while others bind them transiently in cellular processes like transport and signaling. Ions of metals like magnesium, iron, zinc, and copper are crucial components of enzymes, stabilizing their structure and providing their biological function, and each of them also plays multiple other roles in the body (Jomova et al., 2022). Calcium (Ca 2+ ) is the most abundant metal in the human body, most often associated with skeletal health, but it is also involved in muscle function, nerve transmission, and enzyme activity. Magnesium (Mg 2+ ) is also a cofactor in more than 300 enzymatic reactions and a multitude of cellular processes (Jahnen-Dechent & Ketteler, 2012). Working in concert, calcium and magnesium are essential for proper muscle contraction and relaxation (Potter et al., 1981), optimal nerve transmission and neuromuscular coordination (Kirkland et al., 2018), bone mineralization, and maintenance of normal bone (Rondanelli et al., 2021). It has been shown that stress can increase magnesium loss, and in turn, magnesium deficiency can further enhance susceptibility to stress, resulting in a magnesium and stress vicious circle (Pickering et al., 2020). Magnesium is also of interest for the potential prevention and treatment of numerous neurological disorders (Kirkland Show less

This study is based on the premise that the application of chemical synthesis strategies to structurally modify commercial drugs by complexation with biometals is a valid procedure to improve their biological effects. Our purpose is to synthesize a compound with greater efficacy than the original drug, able to enhance its antihypertensive and cardiac pharmacological activity. Herein, the structure of the coordination compound of Zn(II) and the antihypertensive drug olmesartan, [Zn(Olme)(H2O)2] (ZnOlme), is presented. After 8 weeks of treatment in SHR male rats, ZnOlme displayed a better blood pressure-lowering activity compared with olmesartan, with a noticeable effect even in the first weeks of treatment, while ZnCl2 showed similar results than the control. ZnOlme also reduced left ventricle (LV) weight and left ventricle/tibia length ratio (LV/TL), posterior wall thickness (PWT), and intraventricular septum in diastole (IVSd) suggesting its potential to prevent LV hypertrophy. Besides, ZnOlme reduced interstitial fibrosis (contents of collagen types I and III, responsible for giving rigidity and promoting vascular elasticity, respectively). The recovery of heart function was also evidenced by fractional shortening (diastolic left ventricular/systolic left ventricular) diameter determinations. Furthermore, ZnOlme increased the antioxidant capacity and prevented cardiac oxidative stress: it enhanced the reduction of reactive oxygen species generation, exerted a significant decrease in lipid peroxidation and enhanced glutathione contents in heart tissues compared to the control, Zn, and olmesartan treatments. Our results demonstrate that continuous oral administration of ZnOlme causes a better antihypertensive effect and grants enhancement of cardioprotection through antioxidant activity, in combination with hemodynamic improvement. Graphical Abstract Show less

Identifying roles for Z-DNA remains challenging given their dynamic nature. Here, we perform genome-wide interrogation with the DNABERT transformer algorithm trained on experimentally identified Z-DNA forming sequences (Z-flipons). The algorithm yields large performance enhancements (F1 = 0.83) over existing approaches and implements computational mutagenesis to assess the effects of base substitution on Z-DNA formation. We show Z-flipons are enriched in promoters and telomeres, overlapping quantitative trait loci for RNA expression, RNA editing, splicing, and disease-associated variants. We cross-validate across a number of orthogonal databases and define BZ junction motifs. Surprisingly, many effects we delineate are likely mediated through Z-RNA formation. A shared Z-RNA motif is identified in SCARF2, SMAD1, and CACNA1 transcripts, whereas other motifs are present in noncoding RNAs. We provide evidence for a Z-RNA fold that promotes adaptive immunity through alternative splicing of KRAB domain zinc finger proteins. An analysis of OMIM and presumptive gnomAD loss-of-function datasets reveals an overlap of Z-flipons with disease-causing variants in 8.6% and 2.9% of Mendelian disease genes, respectively, greatly extending the range of phenotypes mapped to Z-flipons. Show less

The complex [Zn(Phen)(H2O)L2] (I), where HL is 5-benzyltetrazole, Phen is 1,10-phenanthroline,was synthesized. The compound was characterized by standard physicochemical methods (elementalanalysis, powder X-ray diffraction, IR spectroscopy). According to X-ray diffraction data (CCDC no.2220597), zinc coordination environment in the crystal structure of I corresponds to a distorted trigonalbipyramid. The ligand HL is monodentate and is coordinated via tetrazolate ring nitrogen. The stability ofcomplex I was studied by NMR spectroscopy in DMSO. The cytotoxic properties of the compound wereassessed against HepG-2 (hepatocellular carcinoma) and MRC-5 (noncancerous human fibroblasts) cells.Complex I exhibits weak cytotoxic properties in the studied concentration range (1–100 μM). Show less

Abstract The complex [Zn(Phen)(H2O)L2] (I), where HL is 5-benzyltetrazole, Phen is 1,10-phenanthroline, was synthesized. The compound was characterized by standard physicochemical methods (elemental analysis, powder X-ray diffraction, IR spectroscopy). According to X-ray diffraction data (CCDC no. 2220597), zinc coordination environment in the crystal structure of I corresponds to a distorted trigonal bipyramid. The ligand HL is monodentate and is coordinated via tetrazolate ring nitrogen. The stability of complex I was studied by NMR spectroscopy in DMSO. The cytotoxic properties of the compound were assessed against HepG-2 (hepatocellular carcinoma) and MRC-5 (noncancerous human fibroblasts) cells. Complex I exhibits weak cytotoxic properties in the studied concentration range (1–100 µM). Show less

Transition metal coordination complexes have provided cancer treatment with new insights to overcome the limitations of current chemotherapeutic agents. Utilization of bifunctional tetrazole–carboxylate ligands with Zn(II) obtained two self-assembled complexes [Zn(HL1)(bipy)3/2(H2O)]·CH3OH·4(H2O) (1) (H3L1 = 1,3,5-tri(2-carboxymethyltetrazol-5-yl) benzene) and [Zn(L2)2(H2O)2]2·2H2O (2) (HL2 = (5-pyridin-3-yl-tetrazol-2-yl)-acetic acid). The X-ray diffraction results showed that the two complexes displayed a two-dimensional (2D) layer structure and a one-dimensional (1D) layer structure. Nanocoprecipitation with DSPE-PEG-2000 resulted in the formation of complex nanoparticles (NPS) with excellent water dispersion. In vitro CCK-8 assay indicated the two NPs exert high cytotoxicity and sensitivity and a low half-maximum inhibitory concentration (IC50) towards HeLa than HepG2 cells. In addition, the cytotoxicity was also confirmed by live/dead co-stained experiments. The presented experimental results showed the 1 and 2 NPs were capable of inhibiting cell proliferation in vitro and may help design coordination complex-based anticancer candidates for cancer cells. Show less

The functions, purposes, and roles of metallothioneins have been the subject of speculations since the discovery of the protein over 60 years ago. This article guides through the history of investigations and resolves multiple contentions by providing new interpretations of the structure-stability-function relationship. It challenges the dogma that the biologically relevant structure of the mammalian proteins is only the one determined by X-ray diffraction and NMR spectroscopy. The terms metallothionein and thionein are ambiguous and insufficient to understand biological function. The proteins need to be seen in their biological context, which limits and defines the chemistry possible. They exist in multiple forms with different degrees of metalation and types of metal ions. The homoleptic thiolate coordination of mammalian metallothioneins is important for their molecular mechanism. It endows the proteins with redox activity and a specific pH dependence of their metal affinities. The proteins, therefore, also exist in different redox states of the sulfur donor ligands. Their coordination dynamics allows a vast conformational landscape for interactions with other proteins and ligands. Many fundamental signal transduction pathways regulate the expression of the dozen of human metallothionein genes. Recent advances in understanding the control of cellular zinc and copper homeostasis are the foundation for suggesting that mammalian metallothioneins provide a highly dynamic, regulated, and uniquely biological metal buffer to control the availability, fluctuations, and signaling transients of the most competitive Zn(II) and Cu(I) ions in cellular space and time. Show less

Background: Angiotensin II receptor blockers were designed as therapeutic agents to block the binding site of the angiotensin II receptor type 1 (AT1R). Methodology: The structure of telmisartan was modified by coordination to the biometal Zn(II), resulting in the compound ZnTelm. Its antihypertensive activity and cellular mechanisms in comparison to telmisartan were studied. Results: Compared with telmisartan, ZnTelm displayed stronger binding to AT1R (binding studies on AT1R-transfected human embryonic kidney cells) and a greater reduction of reactive oxygen species and cytosolic calcium concentration induced by angiotensin II. The antihypertensive activity of the complex (assessed in an N(G)-Nitro-L-arginine methyl ester-induced hypertension model) was significantly higher. ZnTelm also reduced hypertrophy in aortic artery rings and tubular collagen deposition. Conclusion: ZnTelm enhances the AT1R blockade and consequently its antihypertensive effect. Show less

Cellular oxidative stress is considered an inducer of carcinogenesis but the association of reactive oxygen species (ROS) with cancer is sometimes contradictory. The antihypertensive drugs candesartan and valsartan were reported to behave as antioxidant agents. In the present study, we prepared their Zn(II) coordination complexes, [ZnCand(H2O)2]·2H2O (ZnCand) and [ZnVals(H2O)2] (ZnVals), and determined that they also depleted ROS by the induction of a reductive state in response to glutathione (GSH) generation and decreased lung cancer cell viability (IC50 = 175 and 220 µM, respectively), while being non-cytotoxic for normal lung fibroblasts (MRC5). The Zn complexes affected the mitochondria membrane, increased the pro- and anti-apoptotic protein ratio, Bax/Bcl-XL, and caspase-9 activation, by late apoptosis. Their co-incubation with N-acetylcysteine (NAC) exacerbated ROS reduction and increased cell death, whereas the H2O2 co-treatment restored the ROS values and normal cell growth. These data suggest that the excess reducing equivalents and low levels of ROS are also critical for the functioning of A549 cells. Show less

Fluorescence imaging is a powerful tool in biomedical research. It has been frequently used to uncover or better understand physiological mechanisms in disease-related processes such as cancer. The majority of chromophores used for imaging are based on a 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) scaffold. However, their applications are limited due to their poor water solubility as well as poor cancer cell selectivity. To circumvent these drawbacks, we present herein the use of bis(dipyrrinato)zinc(II) complexes. As this class of compounds is associated with a quenching effect of the excited state in water, the lead compound of this study (3) was encapsulated in a polymer matrix with biotin as a targeting moiety (3-NP). This encapsulation improved the water solubility, overcame the quenching effects in water, as well as allowed selective accumulation in the lysosomes with a bright fluorescence signal in monolayer cells as well as 3D multicellular tumor spheroids (MCTS). As a benefit from the biotin targeting moiety, the nanoparticles were majorly taken up by the sodium dependent multivitamin transporter (SMVT) which is overexpressed in various cancers cells and selectively accumulated in cancerous cells over noncancerous cells. Show less

AbstractCoordination‐driven self‐assembly has been established as an effective strategy for the efficient construction of intricate architectures in both natural and artificial systems, for applications ranging from gene regulation to metal–organic frameworks. Central to these systems is the need for carefully designed organic ligands, generally with rigid components, that can undergo self‐assembly with metal ions in a predictable manner. Herein, we report the synthesis and study of three novel organic ligands that feature 3,6‐disubstituted acridine as a rigid spacer connected to two 2‐(1,2,3‐triazol‐4‐yl)pyridine “click” chelates through hinges of the same length but differing flexibility. The flexibility of these “three‐atom” hinges was modulated by i) moving from secondary to tertiary amide functional groups and ii) replacing an sp2 amide carbon with an sp3 methylene carbon. In an effort to understand the role of hinge flexibility in directing self‐assembly into mononuclear loops or dinuclear cylinders, the impact of these changes on self‐assembly outcomes with zinc(II), iron(II), and copper(II) ions is described. Show less

AbstractThe 2‐(1,2,3‐triazol‐4‐yl)pyridine motif, with its facile “click” synthesis and remarkable coordinative properties, is an attractive chelate for applications in the metal‐directed self‐assembly of intricate three‐dimensional structures. Organic ligands that bear two such chelates bridged by flexible hinge moieties readily undergo self‐assembly with metal ions of different coordination geometries to generate a series of topologically diverse metallomacrocycles that can be used for numerous applications. Herein, the synthesis and self‐assembly of one such ligand with zinc(II), copper(II), and palladium(II) ions is reported, and the stability of the resulting metallomacrocycles described. An investigation into the use of these metallomacrocycles for the recognition of both small‐molecule substrates, such as deoxyguanosine monophosphate, and larger biological assemblies, such as DNA and RNA guanine quadruplexes, is also described. Show less

Cell death triggered by photodynamic therapy can occur through different mechanisms: apoptosis, necrosis or autophagy. However, recent studies have demonstrated the existence of other mechanisms with characteristics of both necrosis and apoptosis. These new cell death pathways, collectively termed regulated necrosis, include a variety of processes triggered by different stimuli. In this study, we evaluated the cell death mechanism induced by photodynamic treatments with two photosensitizers, meso-tetrakis (4-carboxyphenyl) porphyrin sodium salt (Na-H2TCPP) and its zinc derivative Na-ZnTCPP, in two human breast epithelial cell lines, a non-tumoral (MCF-10A) and a tumoral one (SKBR-3). Viability assays showed that photodynamic treatments with both photosensitizers induced a reduction in cell viability in a concentration-dependent manner and no dark toxicity was observed. The cell death mechanisms triggered were evaluated by several assays and cell line-dependent results were found. Most SKBR-3 cells died by either necrosis or apoptosis. By contrast, in MCF-10A cells, necrotic cells and another cell population with characteristics of both necrosis and apoptosis were predominant. In this latter population, cell death was PARP-dependent and translocation of AIF to the nucleus was observed in some cells. These characteristics are related with parthanatos, being the first evidence of this type of regulated necrosis in the field of photodynamic therapy. Show less

AbstractComplexation studies of the dinucleating ligand H3L (H3L=2‐{[bis(pyridin‐2‐ylmethyl)amino]methyl}‐6‐{[bis(6‐pivaloylamidopyridin‐2‐ylmethyl)amino]methyl}‐4‐methylphenol), with metal‐binding sites A and B, which both provide four donors to a metal ion; a tertiary amine; two pyridines (substituted with amide hydrogen‐bond donors in site B), and a bridging phenolate, with ZnII, CuII, and GaIII are reported. The titration of H3L with the three metal ions in solution was monitored by NMR spectroscopy or EPR and UV/Vis/near‐IR spectroscopy, as well as by ESI‐MS to analyze the selectivity of the two metal‐ion sites A and B of this model ligand for metallophosphatases; the spectroscopic assignments are supported by X‐ray crystallography results. The first ZnII ion coordinates to site A with unsubstituted pyridine donors and, upon addition of a second equivalent of ZnII, this coordinates to the sterically less accessible site B. From a similar titration with GaIII, it emerges that only a mononuclear complex is obtained, with the GaIII center coordinated to site A. When one equivalent of GaIII is reacted with the mononuclear ZnII complex, ZnII is forced by GaIII to exchange the site; this results in a dinuclear complex with GaIII in site A and ZnII in site B. With CuII, two isomers are observed: one with and the other without a bridging phenolate; these differ significantly in their spectroscopic and magnetic properties. Show less

Natural metalloenzymes are often the most proficient catalysts in terms of their activity, selectivity, and ability to operate at mild conditions. However, metalloenzymes are occasionally surprising in their selection of catalytic metals, and in their responses to metal substitution. Indeed, from the isolated standpoint of producing the best catalyst, a chemist designing from first-principles would likely choose a different metal. For example, some enzymes employ a redox active metal where a simple Lewis acid is needed. Such are several hydrolases. In other cases, substitution of a non-native metal leads to radical improvements in reactivity. For example, histone deacetylase 8 naturally operates with Zn(2+) in the active site but becomes much more active with Fe(2+). For β-lactamases, the replacement of the native Zn(2+) with Ni(2+) was suggested to lead to higher activity as predicted computationally. There are also intriguing cases, such as Fe(2+)- and Mn(2+)-dependent ribonucleotide reductases and W(4+)- and Mo(4+)-dependent DMSO reductases, where organisms manage to circumvent the scarcity of one metal (e.g., Fe(2+)) by creating protein structures that utilize another metal (e.g., Mn(2+)) for the catalysis of the same reaction. Naturally, even though both metal forms are active, one of the metals is preferred in every-day life, and the other metal variant remains dormant until an emergency strikes in the cell. These examples lead to certain questions. When are catalytic metals selected purely for electronic or structural reasons, implying that enzymatic catalysis is optimized to its maximum? When are metal selections a manifestation of competing evolutionary pressures, where choices are dictated not just by catalytic efficiency but also by other factors in the cell? In other words, how can enzymes be improved as catalysts merely through the use of common biological building blocks available to cells? Addressing these questions is highly relevant to the enzyme design community, where the goal is to prepare maximally efficient quasi-natural enzymes for the catalysis of reactions that interest humankind. Due to competing evolutionary pressures, many natural enzymes may not have evolved to be ideal catalysts and can be improved for the isolated purpose of catalysis in vitro when the competing factors are removed. The goal of this Account is not to cover all the possible stories but rather to highlight how variable enzymatic catalysis can be. We want to bring up possible factors affecting the evolution of enzyme structure, and the large- and intermediate-scale structural and electronic effects that metals can induce in the protein, and most importantly, the opportunities for optimization of these enzymes for catalysis in vitro. Show less