Béla Gyurcsik

Dissociation of AfArsR from its target DNA sequence is promoted by the metalloids As ^III and Sb ^III , but not by Hg ^II and other soft metal ions, demonstrating the functional selectivity of the regulatory protein both in vitro and in vivo .

Zinc finger proteins are essential for the proper functioning of eukaryotic organisms, while their artificial variants can be used in cancer therapy or gene editing via their specific DNA recognition. Zn(II) binding is indispensable for the structural integrity of zinc fingers. While metal ion binding of zinc fingers has been extensively investigated in vitro, intracellular studies are rather scarce. Herein, we utilized the novel I‐Block assay, based on the regulation of β ‐galactosidase expression via the transcription inhibition ability of zinc finger proteins inside the modified ER1821ΔLacI Escherichia coli cells. The results indicated that the performance of the assay largely depends on the position of the DNA sequence targeted by the zinc finger protein, but once optimized, specific DNA binding of even the rather small three‐membered 1MEY# artificial zinc finger protein can be detected without large fusion tags. By fusion of 1MEY# with the nuclease domain of FokI restriction endonuclease leading to an artificial zinc finger nuclease, we demonstrated that the I‐Block system can distinguish between DNA binding and DNA cleavage inside the cells allowing for zinc finger nuclease screening. The investigation of metal ion/metalloid–zinc finger protein interactions within cellular conditions provided direct proof for the inhibitory effect of Cd(II), Hg(II), and As(III) on the DNA binding of a zinc finger protein at a molecular level.

Indoloquinolines are potent anticancer agents, but their poor aqueous solubility prevents clinical development. Indoloquinoline-based metal complexes offer an opportunity to circumvent this drawback. A series of new indolo[2,3-c]quinoline derivatives HL1–HL8 and their copper(II) complexes were synthesized, comprehensively characterized and tested for antiproliferative activity against MDA-MB-231, MCF-7, MCF-7 KCR, A549 and DU-145 cancer cells and compared to known isomeric indolo[3,2-c]quinolines (HL11–HL14 and 11–14). The Cu(II) complexes were generally as active, or slightly more so, than the proligands. Lead compounds HL8 and 8 showed superior anticancer activity compared to isomers HL14 and 14, respectively. Complex 8 was superior to HL8 in ROS generation in A549 cells, induced mitochondrial dysfunction as evidenced by JC-1 staining, inducted lactate dehydrogenase release in medium, inhibited DNA synthesis and triggered apoptosis. DNA-binding studies, supported by molecular docking calculations, showed strong affinity of the compounds for double stranded DNA, to which they bind by intercalation.

Monensin A transports Na ⁺ ions through the cell membrane, but it is also able to bind divalent metal ions potentially influencing the biological activity of the ionophore. In line with our previous studies, here we explored interaction of the essential Cu(II) with monensinate A and suggested the types of coordination compounds formed. Circular dichroism (VIS‐ and NIR‐CD), VIS light absorption, and electron paramagnetic resonance (EPR) spectroscopies, as well as electrospray ionization mass spectrometry (ESI‐MS) were utilized for identification and characterization of the complexes. Bis or mono Cu(II) complexes prevailed at ligand excess or comparable reactant concentrations, respectively. Excess Cu(II) salt, however, induced the formation of dinuclear coordination species, not observed previously with divalent metal cations as Mg(II), Ca(II), Co(II), Mn(II), Ni(II), Zn(II). The dinuclear complex is EPR‐active being a rare example of non‐EPR‐silent coordination compounds containing two Cu(II) centers and is the first case among the known metal(II) complexes of the antibiotic monensin A. The origin of the counterion did not influence the properties of the bis compound but contributed to the spectral behavior of the newly detected dinuclear Cu(II) species. This study revealed that the interactions of a drug molecule with metal(II) ions during its practical application may be significant and it should be considered in discussions about the mechanism of action.

β‐Lactamases grant resistance to bacteria against β‐lactam antibiotics. The active center of TEM‐1 β‐lactamase accommodates a Ser‐Xaa‐Xaa‐Lys motif. TEM‐1 β‐lactamase is not a metalloenzyme but it possesses several putative metal ion binding sites. The sites composed of His residue pairs chelate borderline transition metal ions such as Ni(II). In addition, there are many sulfur‐containing donor groups that can coordinate soft metal ions such as Hg(II). Cd(II) may bind to both types of the above listed donor groups. No significant change was observed in the circular dichroism spectra of TEM‐1 β‐lactamase on increasing the metal ion content of the samples, with the exception of Hg(II) inducing a small change in the secondary structure of the protein. A weak nonspecific binding of Hg(II) was proven by mass spectrometry and ^119mHg perturbed angular correlation spectroscopy. The hydrolytic process of ampicillin catalyzed by TEM‐1 β‐lactamase was described by the kinetic analysis of the set of full catalytic progress curves, where the slow, yet observable conversion of the primary reaction product into a second one, identified as ampilloic acid by mass spectrometry, needed also to be considered in the applied model. Ni(II) and Cd(II) slightly promoted the catalytic activity of the enzyme while Hg(II) exerted a noticeable inhibitory effect. Hg(II) and Ni(II), applied at 10 μM concentration, inhibited the growth of E. coli BL21(DE3) in M9 minimal medium in the absence of ampicillin, but addition of the antibiotic could neutralize this toxic effect by complexing the metal ions.

The chirality of the polyether ionophore monensic acid A can be successfully used to study its coordination ability in solution. A complementary approach to gain new insights into the complexation chemistry of the antibiotic (studied previously by circular dichroism (CD) spectroscopy in the ultraviolet range (UV-CD)) is presented. (1) Methods: The CD spectroscopy in the visible (VIS-CD) and near-infrared (NIR-CD) range is applied to evaluate the affinity of deprotonated monensic acid A (monensinate A) towards Ni(II) or Co(II) cations in methanolic solution. Competition experiments between a variety of colorless divalent metal ions for binding the ligand anion were also performed. (2) Results: The stability constants of the species observed in binary Ni(II)/Co(II)-monensinate systems and their distribution were reevaluated with the VIS- and NIR-CD techniques. The data confirmed the formation of mono and bis complexes depending on the metal-to-ligand molar ratio. The studies on the systems containing two competing divalent metal cations exclude the formation of ternary complex species but provide an opportunity to also calculate the stability constants of Zn(II), Mg(II), and Ca(II) monensinates. (3) Conclusions: The advantages of CD spectroscopy in the VIS-NIR range (“invisible” ligand and metal salts, “visible” chiral complex species) simplify the experimental dataset evaluation and increase the reliability of computed results.

The nuclease domain of colicin E7 cleaves double-strand DNA non-specifically. Zn²⁺ ion was shown to be coordinated by the purified NColE7 as its native metal ion. Here, we study the structural and catalytic aspects of the interaction with Ni²⁺, Cu²⁺ and Cd²⁺ non-endogenous metal ions and the consequences of their competition with Zn²⁺ ions, using circular dichroism spectroscopy and intact protein mass spectrometry. An R447G mutant exerting decreased activity allowed for the detection of nuclease action against pUC119 plasmid DNA via agarose gel electrophoresis in the presence of comparable metal ion concentrations. It was shown that all of the added metal ions could bind to the apoprotein, resulting in a minor secondary structure change, but drastically shifting the charge distribution of the protein. Zn²⁺ ions could not be replaced by Ni²⁺, Cu²⁺ and Cd²⁺. The nuclease activity of the Ni²⁺-bound enzyme was extremely high in comparison with the other metal-bound forms, and could not be inhibited by the excess of Ni²⁺ ions. At the same time, this activity was significantly decreased in the presence of equivalent Zn²⁺, independent of the order of addition of each component of the mixture. We concluded that the Ni²⁺ ions promoted the DNA cleavage of the enzyme through a more efficient mechanism than the native Zn²⁺ ions, as they directly generate the nucleophilic OH⁻ ion.

Zinc finger proteins specifically recognize DNA sequences and, therefore, play a crucial role in living organisms. In this study the Zn(II)-, and DNA-binding of 1MEY#, an artificial zinc finger protein consisting of three finger units was characterized by multiple methods. Fluorimetric, circular dichroism and isothermal calorimetric titrations were applied to determine the accurate stability constant of a zinc finger protein. Assuming that all three zinc finger subunits behave identically, the obtained thermodynamic data for the Zn(II) binding were ΔH_{binding site} = − (23.5 − 28.0) kcal/mol (depending on the applied protonation state of the cysteines) and logβ’_pH 7.4 = 12.2 ± 0.1, being similar to those of the CP1 consensus zinc finger peptide. The specific DNA binding of the protein can be characterized by logβ’_{pH 7.4} = 8.20 ± 0.08, which is comparable to the affinity of the natural zinc finger proteins (Sp1, WT1, TFIIIA) toward DNA. This value is ~ 1.9 logβ’ unit higher than those determined for semi- or nonspecific DNA binding. Competitive circular dichroism and electrophoretic mobility shift measurements revealed that the conditional stability constant characteristic for Zn(II) binding of 1MEY# protein increased by 3.4 orders of magnitude in the presence of its target DNA sequence. Graphical

Cys2His2 zinc finger proteins are important for living organisms, as they—among other functions—specifically recognise DNA when Zn(II) is coordinated to the proteins, stabilising their ββα secondary structure. Therefore, competition with other metal ions may alter their original function. Toxic metal ions such as Cd(II) or Hg(II) might be especially dangerous because of their similar chemical properties to Zn(II). Most competition studies carried out so far have involved small zinc finger peptides. Therefore, we have investigated the interactions of toxic metal ions with a zinc finger proteins consisting of three finger units and the consequences on the DNA binding properties of the protein. Binding of one Cd(II) per finger subunit of the protein was shown by circular dichroism spectroscopy, fluorimetry and electrospray ionisation mass spectrometry. Cd(II) stabilised a similar secondary structure to that of the Zn(II)-bound protein but with a slightly lower affinity. In contrast, Hg(II) could displace Zn(II) quantitatively (logβ′ ≥ 16.7), demolishing the secondary structure, and further Hg(II) binding was also observed. Based on electrophoretic gel mobility shift assays, the Cd(II)-bound zinc finger protein could recognise the specific DNA target sequence similarly to the Zn(II)-loaded form but with a ~0.6 log units lower stability constant, while Hg(II) could destroy DNA binding completely.

A series of novel estradiol-based salicylaldehyde (thio)semicarbazones ((T)SCs) bearing (O,N,S) and (O,N,O) donor sets and their Cu(II) complexes were developed and characterized in detail by ¹H and ¹³C nuclear magnetic resonance spectroscopy, UV–visible and electron paramagnetic resonance spectroscopy, electrospray ionization mass spectrometry and elemental analysis. The structure of the Cu(II)-estradiol-semicarbazone complex was revealed by X-ray crystallography. Proton dissociation constants of the ligands and stability constants of the metal complexes were determined in 30% (v/v) DMSO/H₂O. Estradiol-(T)SCs form mono-ligand complexes with Cu(II) ions and exhibit high stability with the exception of estradiol-SC. The Cu(II) complexes of estradiol-TSC and its N,N-dimethyl derivative displayed the highest cytotoxicity among the tested compounds in MCF-7, MCF-7 KCR, DU-145, and A549 cancer cells. The complexes do not damage DNA according to both in vitro cell-free and cellular assays. All the Cu(II)-TSC complexes revealed significant activity against the Gram-positive Staphylococcus aureus bacteria strain. Estradiol-TSCs showed efficient antioxidant activity, which was decreased by complexation with Cu(II) ions. The exchange of estrone moiety to estradiol did not result in significant changes to physico-chemical and biological properties.