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In this paper, Cu(II), Mn(II) and Zn(II) complexes with N,N,N-trimethyl-2-oxo-2-(2-(1-(thiazol-2-yl)ethylidene)hydrazinyl)ethan-1-aminium chloride (HL$^1$Cl) were synthesized and characterized by single-crystal X-ray diffraction, IR spectroscopy, elemental analysis and DFT calculations. In all three complexes, a ligand (L$^1$) is coordinated in a deprotonated formally neutral zwitterionic form via NNO donor set atoms. Cu(II) and Zn(II) form mononuclear penta-coordinated complexes [CuL$^1$(N$_3$)(CH$_3$OH)]BF$_4$ and [ZnL$^1$(N$_3$)$_2$], respectively, while Mn(II) forms a binuclear [Mn$_2$L$^1$$_2$(μ-$_{1,1}$-N$_3$)$_2$(N$_3$)$_2$]·2CH$_3$OH complex, with unusual distorted trigonal-prismatic geometry around the metal centers. The antimicrobial activity of these complexes was tested against a panel of Gram-negative and Gram-positive bacteria, two yeasts and one fungal strain. The binuclear Mn(II) complex showed antifungal activity of similar intensity to amphotericin B. Based on the results of the brine shrimp test and DPPH radical scavenging activity, the most active Cu(II) and Mn(II) complexes were selected for evaluation of cytotoxic activity against five malignant cancer cell lines (HeLa, A375, MCF7, PC-3 and A549) and one normal cell line HaCaT. Both complexes showed significant activity. It should be pointed out that the activity of the Mn(II) complex against the MCF7 breast cancer cell line is only slightly weaker than that of cisplatin, but with selectivity to the tumor cell line in comparison to normal HaCaT cells, which is non-existent in the case of cisplatin.

Polyphenols are a large group of natural organic compounds mainly found in plants, in whom they have diverse protective and metabolic functions. It’s also known that phenolic compounds, especially tannins, interact with proteins in various significant and distinct ways. Tannins also complex proteins, which generally precipitates them and it’s their most important industrially utilised characteristic. This study, which was a continuation of our previous work, focused on interactions of aqueous tea plant extracts with laccase from Trametes 63ersicolour and β-amylase from Ipomoea batatas. Tea plants used in this study wereSaturejamontana, Menthapiperita, Salvia officinalis, Matricariachamomilla, Camellia cinensis and Arctostaphylosuva-ursi. Total phenolic content was determined using Folin-Ciocalteu reagent, which showed us that chosen plants vary considerably in their total phenolic content and the highest concentration was found to be in Arctostaphylosuva-ursi. Protein interactions between tea plant extracts were measured using spectrophotometric, spectrofluorimetric and electrophoretic methods. These methods showed that tea plant extracts lead to various structural changes within the protein, nature of which require further research. Finally, it was also found that all tea plant extracts, except Matricariachamomillaextract, reversibly precipitate β-amylase – whilst retaining most of its enzymatic activity after dissolving.Best results were obtained using Arctostaphylosuva-ursi extract, which precipitated the highest quantity of β-amylase, with highest activity retention. Although being an interesting phenomenon, furtherresearch is necessary to determine the nature andimportance of reversible tannic enzyme precipitation.

Few studies concerning antibacterial activity of ruthenium complexes have been published but all of them have been performed on small limited number of strains 1,2. In this study complex 1 ([(η6-toluene)Ru(ppf)Cl]PF6) and complex 2 ([(η6-p-cymene)Ru(ppf)Cl]PF6) where ppf is pyrido[2′,3′:5,6]pyrazino[2,3-f][1,10]phenanthrolinewere investigated as antibacterial agents. Previous study proved the cytotoxicity of these compounds 3. The structural difference between 1 and 2 reflected through the presence or absence of isopropyl group onto one of the ligand (toluene), resulted in significant different activity against melanoma cells 3. Five strains of Gram-positive bacteria (C. sporagenes, M. flavus, B. subtilis, S. lutea and S. aureus), and four strains of Gram-negative bacteria (S. enteritidis, P. vulgaris, P. aeruginosa and E. coli) were used for study of antibacterial activity of 1 and 2. While 2 did not show activity against most strains, complex 1 showed good results against all strains, but the best against Clostridium sporagenes and Proteus vulgaris. The obtained antibacterial activity of the complexes was in accordance with the nuclease activity obtained by plasmid DNA cleavage study. Complex 2 showed higher damaging effect to supercoiled DNA, than complex 1. Minor structural modifications of arene moiety resulted in major difference in activity of the complexes.

Polyphenols are a large group of natural organic compounds mainly found in plants with diverse protective and metabolic functions. Phenolic compounds are well known for their antioxidant properties and the name “antioxidant” is mostly associated with them1. It’s also known that phenolic compounds, especially tannins, interact with proteins in various significant and distinct ways. This study was focused on examining these characteristics on aqueous extracts of tea plants local to Serbia (freshly picked Satureja montana, Mentha spicata, Salvia officinalis and Matricaria chamomilla), which are known to have various amounts and types of phenolic compounds. We examined total concentration of phenolic compounds, tannins, flavonoids, antioxidant activity and interactions with bovine serum albumin and whey protein. It was concluded that S. montana extract had the highest concentration of polyphenols and tannins. Flavonoid concentration was measured using the aluminium-chloride method and it was concluded that S. officinalis had the highest flavonoid content. Antioxidant activities were measured using DPPH, FRAP and ABTS, which are Single Electron Transfer (SET) mechanism-based antioxidant tests and ORAC which is a Hydrogen Atom Transfer mechanism-based antioxidant test. It was concluded that SET mechanism-based antioxidant activities correspond to the total concentration of polyphenols and tannins, which meant that S. montana extract had the highest antioxidant activity. S. officinalis extract exhibited the highest antioxidant activity measured by the ORAC assay, which corresponded to the highest Flavonoid concentration. Interactions between plant extracts and bovine serum albumin were measured via spectrophotometric and spectrofluorimetric titrations. It was concluded that S. montana exhibited most pronounced interactions with the protein. Nature of such interactions is still unknown but using SDS-PAGE it was concluded that proteins exhibit significant structural changes after interacting with plant extracts.

A novel binuclear Cd complex (1) with hydrazone-based ligand was prepared and characterized by spectroscopy and single crystal X-ray diffraction techniques. Complex 1 reveals a strong pro-apoptotic activity in both human, mammary adenocarcinoma cells (MCF-7) and pancreatic AsPC-1 cancer stem cells (CSCs). While apoptosis undergoes mostly caspase-independent, 1 stimulates the activation of intrinsic pathway with noteworthy down regulation of caspase-8 activity in respect to non-treated controls. Distribution of cells over mitotic division indicates that 1 caused DNA damage in both cell lines, which is confirmed in DNA interaction studies. Compared to 1, cisplatin (CDDP) does not achieve cell death in 2D cultured AsPC-1 cells, while induces different pattern of cell cycle changes and caspase activation in 2D cultured MCF-7 cells, implying that these two compounds do not share similar mechanism of action. Additionally, 1 acts as a powerful inducer of mitochondrial superoxide production with dissipated trans-membrane potential in the majority of the treated cells already after 6 h of incubation. On 3D tumors, 1 displays a superior activity against CSC model, and at 100 μM induces disintegration of spheroids within 2 days of incubation. Fluorescence spectroscopy, along with molecular docking show that compound 1 binds to the minor groove of DNA. Compound 1 binds to the human serum albumin (HSA) showing that the HSA can effectively transport and store 1 in the human body. Thus, our current study strongly supports further investigations on antitumor activity of 1 as a drug candidate for the treatment of highly resistant pancreatic cancer. © 2018 Elsevier Inc. This is the peer-reviewed version of the article: Bjelogrlić, S., Todorović, T.R., Cvijetić, I., Rodić, M.V., Vujčić, M., Marković, S., Araškov, J., Janović, B., Emhemmed, F., Muller, C.D., Filipović, N.R., 2019. A novel binuclear hydrazone-based Cd(II) complex is a strong pro-apoptotic inducer with significant activity against 2D and 3D pancreatic cancer stem cells. Journal of Inorganic Biochemistry 190, 45–66. [https://doi.org/10.1016/j.jinorgbio.2018.10.002] Supplementary material: [http://cherry.chem.bg.ac.rs/handle/123456789/2975]

A new Ni(II) complex, [Ni(L)(H2O)] (1), with diethyl 3,3'-(2,2'-(1,1'-(pyridine-2,6-diyl)bis(ethan-1-yl-1-ylidene))bis(hydrazin-1-yl-2-ylidene))bis(3-oxopropanoate) ligand (H2L) was synthesized as a potential chemotherapeutic agent. Polidentate ligand was coordinated to Ni(II) NNN-tridentately, in dianionic form, while monodentate water coordination completed square-planar geometry around metal. Structure in the solution was determined by NMR spectroscopy and the same coordination mode was observed in the solid state using IR spectroscopy and further verified by DFT calculations and electrochemical studies. Thermal stability of 1 was determined in both air and nitrogen atmosphere. Anticancer activity of 1 was investigated on acute monocytic leukemia (THP-1) and pancreatic adenocarcinoma (AsPC-1) cell lines. On THP-1 cells 1 induced powerful apoptotic response (ED50 = 10 ± 3 µM), which was revealed to be only partially caspase-dependent, with activation of caspase-8 as the dominant course. This suggested that experimentally validated covalent binding of 1 to DNA is not the only mechanism responsible for programmed cell death. This was supported with experiments on AsPC-1 cells. Although treatment of those cells with 1 resulted in poor apoptotic response, cell cycle changes showed concentration-dependent shifts indicating a dual mechanism of activity. This study also reviews the results of preliminary biological screening, which demonstrates that 1 displays a unique pattern of anticancer activity with at least two mechanisms involved. The authors acknowledge networking support by the COST Action CM1106 StemChem – “Chemical Approaches to Targeting Drug Resistance in Cancer Stem Cells”. The work was funded by the Ministry of Education, Science and Technological Development of the Republic of Serbia (Grant 172055).

A new Zn(II)-based potential chemotherapeutic agent was synthesized from the ligand 2-quinolinecarboxaldehyde selenosemicarbazone (Hqasesc). Single crystal X-ray diffraction analysis showed that the Zn(II) complex consists of a cation [Zn(Hqasesc)(2)](2+), two perchlorate anions and one ethanol solvent molecule. The interaction of calf thymus (CT) DNA and human serum albumin (HSA) with the Zn(II) complex was explored using absorption and emission spectral methods, and also has been supported by molecular docking studies. The complex has more affinity to minor DNA groove than major, with no significant intercalation. The HSA interaction studies of the complex revealed the quenching of the intrinsic fluorescence of the HSA through a static quenching mechanism. The antitumor activity of the ligand and the complex against pancreatic adenocarcinoma cell line (AsPC-1) and acute monocytic leukemia (THP-1) cells was evaluated. Both compounds are strong concentration-dependent apoptosis inducers in THP-1 cells. While Hqasesc in AsPC-1 cells induces apoptosis only at the highest concentration, treatment with the Zn complex shows a concentration-dependent apoptotic response, where the treated cells are arrested in the G1-to-S phase accompanied with extensive activation of caspase-8 and -9. These results indicate that the ligand and Zn(II) complex display cell phenotype specific activity. Supplementary material: [http://cherry.chem.bg.ac.rs/handle/123456789/3461]

Phenols containing halogens, which tend to deactivate the aromatic nuclei, constitute a significant category of highly toxic and difficult-to-degrade pollutants, which arise from a wide variety of industries. The main purpose of this study was to obtain an inexpensive immobilized enzyme for the removal of phenols. Partially purified potato polyphenol oxidase (PPO) was immobilized onto different commercial and laboratory produced carriers. Three of the obtained biocatalysts, with the highest PPO activities, namely Eupergit C250L-PPO; Celite-PPO and CelluloseM-PPO, were tested in a batch reactor for the removal of phenol, 4-chlorophenol and 4-bromophenol. In the case of 2.5 mM substrates with Eupergit C250L-PPO, an around 45% removal of 4-bromophenol was achieved, while the removals 4-chlorophenol and phenol were 35 and 20%, respectively. The reusability of Eupergit C250L-PPO for the removal of 4-chlorophenol was tested. After eight repeated tests, the efficiency of 4-chlorophenol removal by Eupergit C250L-PPO immobilisate had decreased to 55%. Halogenovani fenoli imaju dezaktivirano aromatično jezgro i čine značajnu kategoriju veoma toksičnih i teško razgradivih zagađivača u raznim industrijskim granama. Glavni cilj ovog rada je bio dobijanje jeftinog imobilizovanog enzima za uklanjanje fenola. Delimično prečišćena polifenol-oksidaza (PFO) iz krompira je imobilizovana na različitim komercijalnim i laboratorijski sintetizovanim nosačima. Od dobijenih biokatalizatora, tri sa najvećim aktivnostima PPO, nazvani Eupergit C250L-PFO; Celit-PFO i CelulozaM-PFO, testirani su u reaktoru za uklanjanje fenola, 4-hlorfenola i 4-bromfenola. U slučaju 2,5 mM supstrata sa Eupergit C250L-PFO, postignuto je oko 45 % razgradnje 4-bromfenola, dok su 4-hlorfenol i fenol razgrađeni 35, odnosno 20 %. Testirana je i sposobnost višestruke upotrebe Eupergit C250L-PFO imobilizata za uklanjanje 4-hlorfenola. Nakon osam ponovljenih ciklusa efikasnost Eupergit C250L-PFO imobilizata za uklanjanje 4-hlorfenola je pala na 55%.

The quinone avarone, isolated from the marine sponge Dysidea avara, possesses the ability to chemically modify proteins. In this work, modification of lectin isolated from the coral Gerardia savaglia by avarone was examined. The techniques used for studying the modification were: SDS PAGE, isoelectric focusing and hemagglutination testing. The results of the SDS PAGE indicate dimerization of the protein. A shift of the pI toward lower value occurs upon modification. The change of the hemagglutination activity of the protein confirms that chemical modification of G. savaglia lectin by avarone changes its ability to interact with the membrane of erythrocytes. Avaron, hinon izolovan iz morskog sunđera Dysidea avara, poseduje sposobnost da hemijski modifikuje proteine. U ovom radu ispitivana je modifikacija lektina izolovanog iz korala Gerardia savaglia avaronom. Tehnike za praćenje hemijske modifikacije bile su: SDS PAGE, izoelektrično fokusiranje i hemaglutinacioni test. Rezultati SDS PAGE upućuju na dimerizaciju proteina. Dolazi do pomeranja pI vrednosti proteina. Promena hemaglutinacione aktivnosti G. savaglia lektina avaronom uticala je na njegovu sposobnost interakcije sa membranom eritrocita.

Changes in electrophoresis pattern after interaction of supercoiled plasmid pBR322 DNA with avarol was studied at a micromolar concentration of reactants under mild reaction conditions. Interactions of avarol with linear high-molecular CT-DNA at millimolar concentrations were analyzed by electrophoresis and UV spectrophotometry. It was observed that avarol is capable of quenching ethidium bromide fluorescence in DNA bands. An increase in the absorbance of DNA was detected. The results indicate the binding of avarol to DNA and/or modification of nucleotide bases. Proučavane su promene elektroforetskog ponašanja DNA posle interakcija superhelikoidalnog plazmida pBR322 s avarolom pri mikromolarnim koncentracijama reaktanata pod blagim reakcionim uslovima. Interakcije avarola sa linearnom visokomolekulskom CT-DNA pri milimolarnim koncentracijama analizirane su elektroforezom i UV spektrofotometrijom. Uočeno je da je avarol u stanju da gasi fluorescenciju etidijum-bromida u trakama koje potiču od DNA. Detektovan je porast apsorbancije DNA. Rezultati ukazuju na vezivanje avarona za DNA i/ili modifikaciju nukleotidnih baza.

In order to contribute to the understanding of mechanisms by which regulatory proteins recognize genetic information stored in DNA, analyses of their interaction with specific nucleotides are usually performed. In this study, the electrophoretic mobility shift assay (EMSA) was applied to analyze the interaction of nuclear proteins from the liver of rats of different age i.e., young (3-month-old), middle- aged (12-month-old) and aged (24-month-old), with radioactively labelled synthetic oligonucleotide analogues, corresponding to GRE. The levels of GRE binding activity were assessed by quantitative densitometric scanning of the autoradiograms. The results showed statistically significant decreasing values of up to 78% and 49% in middle aged and old animals, respectively, compared to young animals (p LT 0.05). The specificity of the nuclear proteins-GRE interaction was demonstrated by competition experiments with unlabelled GRE. In a supershift assay, using the antibody BuGR2, it was shown that the GR proteins present in nuclear extracts have a high affinity for the GRE probe. The stabilities of the protein-DNA complexes were analysed and it was concluded that they changed during ageing. U cilju doprinosa razumevanju mehanizama pomoću kojih regulatorni proteini prepoznaju genetičku informaciju koju nosi DNK, analiziraju se njihove interakcije sa specifičnim nukleotidima. U ovom radu je metodom EMSA analizirana interakcija jedarnih proteina iz jetri pacova iz tri starosne grupe (mladi - 3 meseca, srednje doba – 12 meseci i stari – 24 meseca) sa sintetičkim, radioaktivno obeleženim, oligonukleotidnim analogom GRE. Nivo vezujuće aktivnosti GRE je određivan kvantitativno denzitometrijskom autoradiografijom. Rezultati su pokazali da postoji statistički značajan pad vrednosti GRE-vezujuće aktivnosti do 78% kod životinja srednjeg starosnog doba i do 49 % kod starih životinja, u poređenju sa vrednostima dobijenim za mlade životinje (p LT 0.05). Specifičnost interakcije jedarnih proteina i GRE je određena eksperimentima kompeticije sa neobeleženim GRE. Korišćenjem antitela BuGR2 pokazano je da je glukokortikoidni receptor protein koji u jedarnom ekstraktu ima najveći afinitet za GRE probu. Analizirana je stabilnost kompleksa protein-DNK i zaključeno je da se menja tokom starenja.

13th Balkan biochemical Biophysical Days and Meeting on Metabolic Disorders, October 12.-15., 2003, Kusadasi, Turkey