Sonia R.W. Louro

Atypical fluoroquinolone gold(III) chelates as potential anticancer agents: Relevance of DNA and protein interactions for their mechanism of action

Quinolones are known for their antimicrobial and antitumor activities. Gold(III) compounds constitute an emerging class of biologically active substances, of special interest as potential anticancer agents. In this work three gold(III) complexes of the fluoroquinolones antimicrobial agents norfloxacin (NOR), levofloxacin (LEVO) and sparfloxacin (SPAR) were prepared and characterized with physicochemical and spectroscopic techniques. In these complexes, NOR, LEVO and SPAR act as bidentate neutral ligands bound to gold(III) through the nitrogen atoms of the piperazine ring, which is an unusual mode of coordination for this class of compounds. Two chloride ions occupy the remaining coordination sites. The cytotoxic activity of the fluoroquinolones and their gold(III) complexes was tested against the A20 (murine lymphoma), B16-F10 (murine melanoma) and K562 (human myeloid leukemia) tumor cell lines as well as the L919 (murine lung fibroblasts) and MCR-5 (human lung fibroblasts) normal cells lines. All complexes were more active than their corresponding free ligands. Complex [AuCl(2)(LEVO)]Cl was selected for DNA fragmentation and cell cycle analysis. Spectroscopic titration with calf-thymus DNA (CT DNA) showed that the complexes can bind weakly to CT DNA, probably by an external contact (electrostatic or groove binding). The complexes exhibit good binding propensity to bovine serum albumin (BSA) having relatively high binding constant values.

Dynamics of heme complexed with human serum albumin: a theoretical approach

Human serum albumin (HSA) is the most abundant protein in the blood serum. It binds several ligands and has an especially strong affinity for heme, hence becoming a natural candidate for oxygen transport. In order to analyze the interaction of HSA-heme, molecular dynamics simulations of HSA with bound heme were performed. Based on the results of X-ray diffraction, the binding site of the heme, localized in subdomain IB, was considered. We analyzed the fluctuations and their correlations along trajectories to detect collective motions. The role of H bonds and salt bridges in the stabilization of heme in its pocket was also investigated. Complementarily, the localization of water molecules in the hydrophobic pocket and the interaction with heme were discussed.

Carboxyl groups at the membrane interface as molecular targets for local anesthetics

The interaction of the tertiary amine drugs chlorpromazine and dibucaine in their cationic form with carboxyl groups at the membrane surface is studied at concentrations relevant to anesthesia. Spin-labeled stearic acid is used both to provide the carboxyl groups and to monitor binding and ionization behavior in egg lecithin liposomes. Membrane anesthetic concentrations are spectrophotometrically obtained. They are shown to determine the drug influence on carboxyl groups at the membrane surface, independently of aqueous concentrations. The intramembrane association constants (related to the usual aqueous phase ones through the partition coefficient) of the drugs with fatty acids are determined. The same value (10(2) M-1) is obtained for both drugs, suggesting that it is approximately the same for all tertiary amine local anesthetics. pH titrations of anesthetic-treated spin-labeled membranes are performed. The observed shifts in the fatty acid pK are higher than can be produced assuming uniform distribution of the drug in the membrane surface, implying that there is an increased affinity of local anesthetics for superficial carboxyl. This affinity could account for the resting block of voltage-gated Na+ channels. Under these considerations, local anesthetic binding sites at voltage-gated Na+ channels and at sarcoplasmic reticulum Ca(2+)-ATPase are proposed.

Norfloxacin Zn(II)-based complexes: acid base ionization constant determination, DNA and albumin binding properties and the biological effect against Trypanosoma cruzi.

Zn(II) complexes with norfloxacin (NOR) in the absence or in the presence of 1,10-phenanthroline (phen) were obtained and characterized. In both complexes, the ligand NOR was coordinated through a keto and a carboxyl oxygen. Tetrahedral and octahedral geometries were proposed for [ZnCl2(NOR)]·H2O (1) and [ZnCl2(NOR)(phen)]·2H2O (2), respectively. Since the biological activity of the chemicals depends on the pH value, pH titrations of the Zn(II) complexes were performed. UV spectroscopic studies of the interaction of the complexes with calf-thymus DNA (CT DNA) have suggested that they can bind to CT DNA with moderate affinity in an intercalative mode. The interactions between the Zn(II) complexes and bovine serum albumin (BSA) were investigated by steady-state and time-resolved fluorescence spectroscopy at pH 7.4. The experimental data showed static quenching of BSA fluorescence, indicating that both complexes bind to BSA. A modified Stern–Volmer plot for the quenching by complex 2 demonstrated preferential binding near one of the two tryptophan residues of BSA. The binding constants obtained (Kb) showed that BSA had a two orders of magnitude higher affinity for complex 2 than for 1. The results also showed that the affinity of both complexes for BSA was much higher than for DNA. This preferential interaction with protein sites could be important to their biological mechanisms of action. The analysis in vitro of the Zn(II) complexes and corresponding ligand were assayed against Trypanosoma cruzi, the causative agent of Chagas disease and the data showed that complex 2 was the most active against bloodstream trypomastigotes.

Hydration of hydrophobic biological porphyrins

Explicit solvent, single solute molecular dynamics simulations of protoporphyrin IX and its Fe(2+) complex (heme) in water were performed. The relation of solute-solvent was examined through the spatial distribution functions of water molecules around the centroid of the porphyrin ring. A detailed description of the time-averaged structure of water surrounding the solutes as well as of its fluctuations is presented.

Norfloxacin and N-Donor Mixed-Ligand Copper(II) Complexes: Synthesis, Albumin Interaction, and Anti-Trypanosoma cruzi Activity

Copper(II) complexes with the first-generation quinolone antibacterial agent norfloxacin containing a nitrogen donor heterocyclic ligand 2,2′-bipyridine (bipy) or 1,10-phenanthroline (phen) were prepared and characterized by IR, EPR spectra, molar conductivity, and elemental analyses. The experimental data suggest that norfloxacin was coordinated to copper(II) through the carboxylato and ketone oxygen atoms. The interaction of the copper(II) complexes with bovine serum albumin (BSA) and human serum albumin (HSA) was investigated using fluorescence quenching of the tryptophan residues and copper(II) EPR spectroscopy. The results of fluorescence titration revealed that copper(II) complexes have a moderate ability to quench the intrinsic fluorescence of the albumins through a static quenching mechanism. EPR experiments showed that BSA and HSA Cu(II) sites compete with NOR for Cu(II)-bipy and Cu(II)-phen to form protein mixed-ligand complexes. Copper(II) complexes, together with the corresponding ligands, were evaluated for their trypanocidal activity in vitro against Trypanosoma cruzi, the causative agent of Chagas disease. The tests performed using bloodstream trypomastigotes showed that the Cu(II)-N-donor precursors and the metal complexes were more active than the free fluoroquinolone.

Fluorescence Studies of gold(III)-Norfloxacin Complexes in Aqueous Solutions

Formation of gold(III) complexes with the synthetic antibiotic norfloxacin (NF) was investigated in aqueous solution at pH 4.0, 7.5 and 10.6, with the ligand in cationic, zwitterionic and anionic forms, respectively. UV-Visible spectroscopy, steady state and time-resolved fluorometry were used to characterize the complexes. Binding sites, association constants and fluorescence lifetimes of the complexes were obtained. Au3+ binding to zwitterionic NF produced a fluorescence decrease and a small red shift. Fluorescence changes as a function of Au3+ concentration were fitted using a one-site binding model and the association constant was obtained,

Interaction of alkanols and local anesthetics with spin-labeled Ca2+-ATPase of sarcoplasmic reticulum vesicles

K_b^{{zw}} = {1}.{7} \times {1}{0^{{5}}}\;{{\text{M}}^{{1}}}