Aleksandar Pavic

Synthesis and Evaluation of Series of Diazine-Bridged Dinuclear Platinum(II) Complexes through in Vitro Toxicity and Molecular Modeling: Correlation between Structure and Activity of Pt(II) Complexes

Polynuclear Pt(II) complexes are a novel class of promising anticancer agents with potential clinical significance. A series of pyrazine (pz) bridged dinuclear Pt(II) complexes with general formulas {[Pt(L)Cl]2(μ-pz)}(2+) (L, ethylenediamine, en; (±)-1,2-propylenediamine, 1,2-pn; isobutylenediamine, ibn; trans-(±)-1,2-diaminocyclohexane, dach; 1,3-propylenediamine, 1,3-pd; 2,2-dimethyl-1,3-propylenediamine, 2,2-diMe-1,3-pd) and one pyridazine (pydz) bridged {[Pt(en)Cl]2(μ-pydz)}(2+) complex were prepared. The anticancer potential of these complexes were determined through in vitro cytotoxicity assay in human fibroblasts (MRC5) and two carcinoma cell lines (A375 and HCT116), interaction with double stranded DNA through in vitro assay, and molecular docking study. All complexes inhibited cell proliferation with inhibitory concentrations in the 0.5-120 μM range. While {[Pt(1,3-pd)Cl]2(μ-pz)}(2+) showed improved activity and {[Pt(en)Cl]2(μ-pydz)}(2+) showed comparable activity to that of clinically relevant cisplatin, {[Pt(en)Cl]2(μ-pydz)}(2+) was less toxic in an assay with zebrafish (Danio rerio) embryos, causing no adverse developmental effects. The in vitro cytotoxicity of all diazine-bridged dinuclear Pt(II) complexes is discussed in correlation to their structural characteristics.

A comparative antimicrobial and toxicological study of gold(III) and silver(I) complexes with aromatic nitrogen-containing heterocycles: synergistic activity and improved selectivity index of Au(III)/Ag(I) complexes mixture

Five aromatic nitrogen-containing heterocycles, pyridazine (pydz, 1), pyrimidine (pm, 2), pyrazine (pz, 3), quinoxaline (qx, 4) and phenazine (phz, 5) have been used for the synthesis of gold(III) and silver(I) complexes. In contrast to the mononuclear Au1–5 complexes all having square-planar geometry, the corresponding Ag1–5 complexes have been found to be polynuclear and of different geometries. Complexes Au1–5 and Ag1–5, along with K[AuCl4], AgNO3 and N-heterocyclic ligands used for their synthesis, were evaluated by in vitro antimicrobial studies against a panel of microbial strains that lead to many skin and soft tissue, respiratory, wound and nosocomial infections. All tested complexes exhibited excellent to good antibacterial activity with minimal inhibitory (MIC) values in the range of 2.5 to 100 μg mL−1 against the investigated strains. The complexes were particularly efficient against pathogenic Pseudomonas aeruginosa (MIC = 2.5–30 μg mL−1) and had a marked ability to disrupt clinically relevant biofilms of strains with high inherent resistance to antibiotics. Moreover, the Au1–4 and Ag1–5 complexes exhibited pronounced ability to competitively intercalate double stranded genomic DNA of P. aeruginosa, which was demonstrated by gel electrophoresis techniques and supported by molecular docking into the DNA major groove. Antiproliferative effect on the normal human lung fibroblast cell line MRC5 has also been evaluated in order to determine therapeutic potential of Au1–5 and Ag1–5 complexes. Since the investigated gold(III) complexes showed much lower negative effects on the viability of the MRC5 cell line than their silver(I) analogues and slightly lower antimicrobial activity against the investigated strains, the combination approach to improve their pharmacological profiles was applied. Synergistic antimicrobial effect and the selectivity index of 10 were achieved for the selected gold(III)/silver(I) complexes mixtures, as well as higher P. aeruginosa PAO1 biofilm disruption activity, and improved toxicity profile towards zebrafish embryos, in comparison to the single complexes. To the best of our knowledge, this is the first report on synergistic activity of gold(III)/silver(I) complexes mixtures and it could have an impact on development of new combination therapy methods for the treatment of multi-resistant bacterial infections.

Mononuclear gold(III) complexes with phenanthroline ligands as efficient inhibitors of angiogenesis: A comparative study with auranofin and sunitinib

Gold(III) complexes with 1,7- and 4,7-phenanthroline ligands, [AuCl3(1,7-phen-κN7)] (1) and [AuCl3(4,7-phen-κN4)] (2) were synthesized and structurally characterized by spectroscopic (NMR, IR and UV-vis) and single-crystal X-ray diffraction techniques. In these complexes, 1,7- and 4,7-phenanthrolines are monodentatedly coordinated to the Au(III) ion through the N7 and N4 nitrogen atoms, respectively. In comparison to the clinically relevant anti-angiogenic compounds auranofin and sunitinib, gold(III)-phenanthroline complexes showed from 1.5- to 20-fold higher anti-angiogenic potential, and 13- and 118-fold lower toxicity. Among the tested compounds, complex 1 was the most potent and may be an excellent anti-angiogenic drug candidate, since it showed strong anti-angiogenic activity in zebrafish embryos achieving IC50 value (concentration resulting in an anti-angiogenic phenotype at 50% of embryos) of 2.89μM, while had low toxicity with LC50 value (the concentration inducing the lethal effect of 50% embryos) of 128μM. Molecular docking study revealed that both complexes and ligands could suppress angiogenesis targeting the multiple major regulators of angiogenesis, such as the vascular endothelial growth factor receptor (VEGFR-2), the matrix metalloproteases (MMP-2 and MMP-9), and thioredoxin reductase (TrxR1), where the complexes showed higher binding affinity in comparison to ligands, and particularly to auranofin, but comparable to sunitinib, an anti-angiogenic drug of clinical relevance.

Fullerenol nanoparticles as a new delivery system for doxorubicin

Doxorubicin is a very potent chemotherapeutic drug, however its side effects limit its clinical use. The aim of this research was to investigate the properties of a fullerenol/doxorubicin nanocomposite, its potentially cytotoxic and genotoxic effects on malignant cell lines, as well as its toxicity towards zebra fish embryos. Chromatographic, NMR and mass spectral analysis of the nanocomposite imply that interactions between doxorubicin and fullerenol are non-covalent bonds. The stability of the nanocomposite was confirmed by the use of atomic force microscopy, dynamic light scattering and transmission electron microscopy. The nanocomposite, compared to the free doxorubicin at equivalent concentrations, significantly decreased the viability of MCF-7 and MDA-MB-231 cells. The flow cytometry results indicated that doxorubicin-loaded fullerenol could remarkably increase the uptake of doxorubicin suggesting that fullerenol might be a promising intracellular targeting carrier for the efficient delivery of antitumor drugs into tumor cells. The nanocomposite also affected cell cycle distribution. A genotoxicity test showed that the nanocomposite at all examined concentrations on MCF-7 and at lower concentrations on MDA-MB-231 cells caused DNA damage. Consequently, cell proliferation was notably reduced when compared with controls. Results of the zebrafish embryotoxicity assay showed a decreased overall toxicity, particularly cardiotoxicity and increased safety of the nanocomposite in comparison to doxorubicin alone, as manifested by a higher survival of embryos and less pericardial edema.

Silver(I) complexes with quinazoline and phthalazine: synthesis, structural characterization and evaluation of biological activities

New silver(I) complexes with quinazoline (qz) and phthalazine (phtz), [Ag(NO3)(qz)]n (1) and {[Ag(CH3CN)]2(μ-phtz)2}[BF4]2 (2), have been synthesized and structurally characterized by using different spectroscopic and single-crystal X-ray diffraction techniques. The obtained results revealed that the reaction of AgNO3 with qz at room temperature in a 2:1 molar ratio led to the formation of the polynuclear complex 1. However, the reaction of AgBF4 with phtz under the same experimental conditions resulted in the formation of the dinuclear complex 2. The solution behaviour and air/light stability of these silver(I) complexes have been investigated. The complexes 1 and 2, along with the silver(I) salts used for their synthesis, were evaluated by in vitro antimicrobial studies against a panel of microbial strains that lead to many skin and soft tissue, respiratory, wound, and nosocomial infections. The obtained results indicate that all tested silver(I) compounds have good antibacterial activity with MIC values in the range from 1.5 to 15.6 μg mL−1 against the investigated strains. On the other hand, their antifungal activity against Candida albicans was moderate. In order to determine the therapeutic potential of 1 and 2, their antiproliferative effect on the normal human lung fibroblast cell line MRC5, hemolytic effect on red blood cells and embryotoxicity on zebrafish (Danio rerio) have also been evaluated.

Investigation of the microbial diversity of an extremely acidic, metal-rich water body (Lake Robule, Bor, Serbia)

An investigation of the microbial diversity in the extremely acidic, metal-rich Lake Robule was performed using culture-dependant and culture- independent (T-RFLP) methods. In addition, the ability of the indigenous bac- teria from the lake water to leach copper from a mineral concentrate was tested. T-RFLP analysis revealed that the dominant bacteria in the lake water samples were the obligate heterotroph Acidiphilium cryptum (≈50 % of the total bacte- ria) and the iron-oxidizing autotroph Leptospirillum ferrooxidans (≈40 %) The iron/sulfur-oxidizing autotroph Acidithiobacillus ferrooxidans was reported to be the most abundant bacteria in the Lake in an earlier study, but it was not detected in the present study using T-RFLP, although it was isolated on solid media and detected in enrichment (bioleaching) cultures. The presence of the two bacterial species detected by T-RFLP (L. ferrooxidans and A. cryptum) was also confirmed by cultivation on solid media. The presence and relative abundance of the bacteria inhabiting Lake Robule was explained by the physio- logical characteristics of the bacteria and the physico-chemical characteristics of the lake water.

Synthesis and anti-Candida activity of novel benzothiepino[3,2-c]pyridine derivatives.

A novel series of thiepine derivatives were synthesized and evaluated as potential antimicrobials. All the synthesized compounds were evaluated for their antimicrobial activities in vitro against the fungi Candida albicans (ATCC 10231), C. parapsilosis (clinical isolate), Gram‐negative bacterium Pseudomonas aeruginosa (ATCC 44752), and Gram‐positive bacterium Staphylococcus aureus (ATCC 25923). Synthesized compounds showed higher antifungal activity than antibacterial activity, indicating that they could be used as selective antimicrobials. Selected thiepines efficiently inhibited Candida hyphae formation, a trait necessary for their pathogenicity. Thiepine 8‐phenyl[1]benzothiepino[3,2‐c]pyridine (16) efficiently killed Candida albicans at 15.6 μg/mL and showed no embryotoxicity at 75 μg/mL. Derivative 8‐[4‐(4,5‐dihydro‐1H‐imidazol‐2‐yl)phenyl][1]benzothiepino[3,2‐c]pyridine (23) caused significant hemolysis and in vitro DNA interaction. The position of the phenyl ring was essential for the antifungal activity, while the electronic effects of the substituents did not significantly influence activity. Results obtained from in vivo embryotoxicity on zebrafish (Danio rerio) encourage further structure optimizations.

Redox behavior and biological properties of ferrocene bearing porphyrins

In order to improve antimicrobial effects of previously studied meso-tetrakis(4-ferrocenylphenyl)porphyrin 1, we have modified its structure by replacing two trans-positioned ferrocenylphenyl moieties with methoxy methylene substituted tert-butylphenyl moieties. Newly synthesized 54,154-bis-(ferrocenyl)-104,204-bis-(tert-butyl)-102,106,202,206-tetrakis-(methoxy-methylene)-5,10,15,20-tetraphenylporphyrin 4 was chemically characterized in detail (by NMR, UV/Vis, IR, MALDI-TOF and ESI MS spectrometry, cyclic voltammetry, prediction of the relative lipophilicity as well as computational methods) and its biological effects were studied in terms of its antibacterial and antifungal activity (both with and without photoactivation), cytotoxicity, hemolysis and DNA cleavage. New ferrocene bearing porphyrin 4 has demonstrated a broader antimicrobial spectrum and modified effects on eukaryotic cells compared to 1. This was discussed in terms of its i) increased lipophilicity, while exhibiting lower toxicity, and ii) the redox potential of a two-electron process that is shifted to lower values, in comparison to ferrocene, thus, entering the physiologically available range and being activated towards redox interactions with biomolecules.

Potent anti-melanogenic activity and favorable toxicity profile of selected 4-phenyl hydroxycoumarins in the zebrafish model and the computational molecular modeling studies

7-Hydroxy-4-phenylcoumarin (7C) and 5,7-dihydroxy-4-phenylcoumarin (5,7C) have been evaluated as potential anti-melanogenic agents in the zebrafish (Danio rerio) model in comparison to commercially utilized depigmenting agents hydroquinone and kojic acid. 7C and 5,7C decreased the body pigmentation at 5 µg/mL, while did not affect the embryos development and survival at doses ≤50 µg/mL and ≤25 µg/mL. Unlike hydroquinone and kojic acid, 4-phenyl hydroxycoumarins were no melanocytotoxic, showed no cardiotoxic side effects, neither caused neutropenia in zebrafish embryos, suggesting these compounds may present novel skin-whitening agents with improved pharmacological properties. Inhibition of tyrosinase was identified as the possible mode of anti-melanogenic action. Molecular docking studies using the homology model of human tyrosinase as well as adenylate cyclase revealed excellent correlation with experimentally obtained results.

Anti-quorum sensing activity, toxicity in zebrafish (Danio rerio) embryos and phytochemical characterization of Trapa natans leaf extracts

ETHNOPHARMACOLOGICAL RELEVANCE Trapa natans L. (water chestnut or water caltrop) is a widespread aquatic plant, which has been cultivated for food and traditional medicine since ancient times. Pharmacological studies showed that water chestnut exhibits the wide range of biological activities, such as antimicrobial, antioxidative, analgesic, anti-inflammatory, as well as antiulcer. AIM OF THE STUDY Evaluation of anti-virulence potential and toxicity of T. natans methanol (TnM), acetone (TnA) and ethyl acetate (TnEA) leaf extracts. MATERIALS AND METHODS The anti-quorum sensing activity of Tn extracts was addressed by measuring their effects on biofilm formation, swarming motility and pyocyanin and elastase production in Pseudomonas aeruginosa. Specific P. aeruginosa biosensors were used to identify which of the signaling pathways were affected. The lethal and developmental toxicity of extracts were addressed in vivo using the zebrafish (Danio rerio) model system. The phenolic composition of T. natans leafs extracts was analyzed by a linear ion trap-OrbiTrap hybrid mass spectrometer (LTQ OrbiTrapMS) and UHPLC system configured with a diode array detector (DAD) hyphenated with the triple quadrupole mass spectrometer. RESULTS Subinhibitory concentrations of Tn leaf extracts (0.2 MIC) inhibited pyocyanin and elastase production up to 50% and 60%, respectively, and reduced swarming zones, comparing to non-treated P. aeruginosa. TnA inhibited biofilm formation by 15%, TnM showed a stimulatory effect on biofilm formation up to 20%, while TnEA showed no effect. The bioactive concentrations of TnM and TnA were not toxic in the zebrafish model system. Twenty-two phenolic compounds were tentatively identified in TnM, where thirteen of them were identified in T. natans for the first time. Tn extracts, as well as their major components, ellagic and ferulic acids, demonstrated the ability to interfere with P. aeruginosa Las and PQS signaling pathways. CONCLUSIONS This study demonstrates anti-virulence potential of Tn leaf extracts against medically important pathogen P. aeruginosa and confirms the ethnopharmacological application of this plant against microbial infections.