Antonia T. do Amaral

Methods for the determination of partition coefficients based on the effect of solutes upon membrane structure

General procedures are presented for the determination of membrane-water partition coefficients (Kp) based on the measurement of effects that depend on the extent of partitioning. Two cases are deal with: (1) the general case when the effect is an unknown function of the total number of moles of partitioning solute (nT) and (2) the particular case when the effect is a linear function of nT. The procedure involves the evaluation of the effect as a function of nT for a variety of membrane volumes (VM), at constant volume of aqueous phase (VH2O), generating a family of curves. It is shown that when a given set of nT, VM produces the same effect, which indicates that the concentration of the solute in the membrane (nM/VM) is the same for the whole set, a plot of nT versus VM yields a straight line that allows the calculation of KP. In the particular case when the effect is linearly proportional to nT, the higher VM, the smaller the slope. In this case, these slopes can be used to calculate KP by plotting their inverse as a function of VM. These procedures were applied to the partitioning of a series of compounds, some of them with known KP, in egg phosphatidylcholine (EPC) membranes. The interaction of those compounds with the membrane was monitored either by fluorescence spectroscopy (by measuring either the excimer/monomer ratio or the quenching of intensity of an intercalated probe) or by ESR making use of a lipid spin probe. Compounds with known KP yielded values in good agreement with previously found ones. In principle, the procedures are applicable whenever it is possible to measure a property that depends on the extent of partitioning, allowing the determination of KP from raw data. In particular, it is very adequate for the use of spectroscopic techniques, one main advantage being that no separation of membrane and aqueous phases is required.

Cruzain inhibition by hydroxymethylnitrofurazone and nitrofurazone: investigation of a new target in Trypanosoma cruzi

Nitrofurazone (NF) and its derivative, hydroxymethylnitrofurazone (NFOH), have presented antichagasic activity. NFOH has higher activity and lower mutagenicity. The aim of this work was to assess whether NF and its derivative NFOH would also be inhibitors of cruzain, besides their trypanothione reductase inhibitory activity. In vitro cruzain inhibition tests were performed for both compounds, and the 50% inhibitory concentration (IC50) for NF and NFOH presented values of 22.83 ± 1.2 μM and 10.55 ± 0.81 μM, respectively. AM1 semi-empirical molecular modeling studies were performed to understand the activity of the compounds, corroborating the observed cruzain inhibitory activity.

Use of membrane spin label spectra to monitor rates of reaction of partitioning compounds: hydrolysis of a local anesthetic analog

ESR spectra of membrane spin probes are conventionally used to obtain structural information. Here we show, for the first time, that when a membrane-soluble compound undergoes a chemical reaction, time-dependent changes in the ESR spectra of membrane spin probes can yield information about the kinetics of reaction. A benzoic acid ester, analog of the local anesthetic tetracaine, partitions between aqueous and membrane phases, causing changes in membrane structure as monitored by the ESR spectra of a probe. At alkaline pH, the lineshapes are time-dependent and the spectra go back to that in the absence of drug. The changes follow pseudo-first order kinetics. This effect is due to drug hydrolysis leading to water-soluble products, as confirmed by direct spectrophotometric measurements of the reaction. The pseudo-first order rate constants found by the latter method are in very good agreement with those calculated by ESR. The rate of hydrolysis decreases with increasing membrane concentration. This phenomenon accounts in part for the increased potency and toxicity of the more membrane-soluble local anesthetics.

Nitroxides attenuate carrageenan-induced inflammation in rat paws by reducing neutrophil infiltration and the resulting myeloperoxidase-mediated damage

Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl) and other cyclic nitroxides have been shown to inhibit the chlorinating activity of myeloperoxidase (MPO) in vitro and in cells. To examine whether nitroxides inhibit MPO activity in vivo we selected acute carrageenan-induced inflammation on the rat paw as a model. Tempol and three more hydrophobic 4-substituted derivatives (4-azido, 4-benzenesulfonyl, and 4-(4-phenyl-1H-1,2,3-triazol-1-yl)) were synthesized, and their ability to inhibit the in vitro chlorinating activity of MPO and carrageenan-induced inflammation in rat paws was evaluated. All of the tested nitroxides inhibited the chlorinating activity of MPO in vitro with similar IC(50) values (between 1.5 and 1.8 μM). In vivo, the attenuation of carrageenan-induced inflammation showed some correlation with the lipophilicity of the nitroxide at early time points but the differences in the effects were small (<2-fold) compared with the differences in lipophilicity (>200-fold). No inhibition of MPO activity in vivo was evident because the levels of MPO activity in rat paws correlated with the levels of MPO protein. Likewise, paw edema, levels of nitrated and oxidized proteins, and levels of plasma exudation correlated with the levels of MPO protein in the paws of the animals that were untreated or treated with the nitroxides. The effects of the nitroxides in vivo were compared with those of 4-aminobenzoic hydrazide and of colchicine. Taken together, the results indicate that nitroxides attenuate carrageenan-induced inflammation mainly by reducing neutrophil migration and the resulting MPO-mediated damage. Accordingly, tempol was shown to inhibit rat neutrophil migration in vitro.

7-Hydroxycoumarin modulates the oxidative metabolism, degranulation and microbial killing of human neutrophils

In the present study, we assessed whether 7-hydroxycoumarin (umbelliferone), 7-hydroxy-4-methylcoumarin, and their acetylated analogs modulate some of the effector functions of human neutrophils and display antioxidant activity. These compounds decreased the ability of neutrophils to generate superoxide anion, release primary granule enzymes, and kill Candida albicans. Cytotoxicity did not mediate their inhibitory effect, at least under the assessed conditions. These coumarins scavenged hypochlorous acid and protected ascorbic acid from electrochemical oxidation in cell-free systems. On the other hand, the four coumarins increased the luminol-enhanced chemiluminescence of human neutrophils stimulated with phorbol-12-myristate-13-acetate and serum-opsonized zymosan. Oxidation of the hydroxylated coumarins by the neutrophil myeloperoxidase produced highly reactive coumarin radical intermediates, which mediated the prooxidant effect observed in the luminol-enhanced chemiluminescence assay. These species also oxidized ascorbic acid and the spin traps α-(4-pyridyl-1-oxide)-N-tert-butylnitrone and 5-dimethyl-1-pyrroline-N-oxide. Therefore, 7-hydroxycoumarin and the derivatives investigated here were able to modulate the effector functions of human neutrophils and scavenge reactive oxidizing species; they also generated reactive coumarin derivatives in the presence of myeloperoxidase. Acetylation of the free hydroxyl group, but not addition of the 4-methyl group, suppressed the biological effects of 7-hydroxycoumarin. These findings help clarify how 7-hydroxycoumarin acts on neutrophils to produce relevant anti-inflammatory effects.

MPO Inhibitors Selected by Virtual Screening

The hemeprotein myeloperoxidase (MPO) participates in innate immune defense through its ability to generate potent microbicidal oxidants. However, these oxidants are also key mediators of the tissue damage associated with many inflammatory diseases. Thus, there is considerable interest in developing therapeutically useful MPO inhibitors. Here, we used structure‐based drug design (SBDD) and ligand‐based drug design (LBDD) to select for potentially new and selective MPO inhibitors. A pharmacophore model was developed based on the crystal structure of human MPO in complex with salicylhydroxamic acid (SHA), a known inhibitor of the enzyme. The pharmacophore model was used to screen the ZINC database for potential ligands, which were further filtered on the basis of their physical‐chemical properties and docking score. The filtered compounds were visually inspected, and nine were purchased for experimental studies. Surprisingly, almost all of the selected compounds belonged to the aromatic hydrazide class, which had been previously described as MPO inhibitors. The compounds selected by virtual screening were shown to inhibit the chlorinating activity of MPO; the top four compounds displayed IC50 values ranging from 1.0 to 2.8 µM. MPO inactivation by the most effective compound was shown to be irreversible. Overall, our results show that SBDD and LBDD may be useful for the rational development of new MPO inhibitors.

The cysteine-rich protein thimet oligopeptidase as a model of the structural requirements for S-glutathiolation and oxidative oligomerization.

Thimet oligopeptidase (EP24.15) is a cysteine-rich metallopeptidase containing fifteen Cys residues and no intra-protein disulfide bonds. Previous work on this enzyme revealed that the oxidative oligomerization of EP24.15 is triggered by S-glutathiolation at physiological GSSG levels (10–50 µM) via a mechanism based on thiol-disulfide exchange. In the present work, our aim was to identify EP24.15 Cys residues that are prone to S-glutathiolation and to determine which structural features in the cysteinyl bulk are responsible for the formation of mixed disulfides through the reaction with GSSG and, in this particular case, the Cys residues within EP24.15 that favor either S-glutathiolation or inter-protein thiol-disulfide exchange. These studies were conducted by in silico structural analyses and simulations as well as site-specific mutation. S-glutathiolation was determined by mass spectrometric analyses and western blotting with anti-glutathione antibody. The results indicated that the stabilization of a thiolate sulfhydryl and the solvent accessibility of the cysteines are necessary for S-thiolation. The Solvent Access Surface analysis of the Cys residues prone to glutathione modification showed that the S-glutathiolated Cys residues are located inside pockets where the sulfur atom comes into contact with the solvent and that the positively charged amino acids are directed toward these Cys residues. The simulation of a covalent glutathione docking onto the same Cys residues allowed for perfect glutathione posing. A mutation of the Arg residue 263 that forms a saline bridge to the Cys residue 175 significantly decreased the overall S-glutathiolation and oligomerization of EP24.15. The present results show for the first time the structural requirements for protein S-glutathiolation by GSSG and are consistent with our previous hypothesis that EP24.15 oligomerization is dependent on the electron transfer from specific protonated Cys residues of one molecule to previously S-glutathionylated Cys residues of another one.

Synthesis of L-cysteine-based boron compounds and their evaluation as proteasome inhibitors

Amides, boronic acids and ester derivatives from L-cysteine were synthesized via a simple, short and inexpensive synthetic route, in order to achieve a novel class of inhibitors for the 20S proteasome. IC50 values of up to 52 μM were obtained with L-cysteine boronic ester derivatives, which also exhibited reversible inhibition. Assays with lysates and whole cell of NIH/3T3 fibroblast NIH Swiss mice were also performed using the boron compound S-benzyl-cysteine-(3-phenyl)boronic ester. This study revealed that for whole cells and lysed cells, the boron compound inhibited proteasome with the same level of efficiency as for the free enzyme (residual enzyme activity for whole cells: 31.5%; lysed cells: 43%; and free enzyme: 43%). Molecular modeling studies were also applied to understand the interactions between the synthesized compounds and the 3-D proteasome structure.

Química Medicinal: 25 anos de planejamento racional de fármacos

The Sociedade Brasileira de Quimica is commemorating its 25th anniversary, and this paper is intended to draw an overview of the Brazilian Medicinal Chemistry over all these years. In 1977 Brazil had almost no activities at all in the field, albeit many efforts were already on the way for encouraging Brazilian Scientists to enter the area. Among many different endeavours to help medicinal chemists to fulfil their proposals and the establishment of an on-going research with the help of networks, the Sociedade Brasileira de Quimica created, in 1991, its own Division on Structure and Activity Relationship, which became the Division of Medicinal Chemistry, in 1997.

Aminoacylase 1-catalysed deacetylation of bioactives epoxides mycotoxin-derived mercapturates; 3,4-epoxyprecocenes as models of cytotoxic epoxides

The mycotoxin aflatoxin B1 (AFB1) is a carcinogenic food contaminant which is metabolically activated by epoxydation. The metabolism of mycotoxins via the mercapturate metabolic pathway was shown, in general, to lead to their detoxication. Mercapturic acids thus formed (S-substitued-N-acetyl-l-cysteines) may be accumulated in the kidney and either excreted in the urine or desacetylated by Acylase 1 (ACY1) to yield cysteine S-conjugates. To be toxic, the N-acetyl-l-cysteine-S-conjugates first have to undergo deacetylation by ACY 1. The specificity and rate of mercapturic acid deacetylation may determine the toxicity, however the exact deacetylation processes involved are not well known. The aim of this study was to investigate the role of ACY1 in the toxicity of some bioactive epoxides from Aflatoxin B1. We characterized the kinetic parameters of porcine kidney and human recombinant aminoacylase-1 towards some aromatic and aliphatic-derived mercapturates analogue of mycotoxin-mercapturic acids and 3,4-epoxyprecocene, a bioactive epoxide derivated from aflatoxin. The deacetylation of mercapturated substrates was followed both by reverse phase HPLC and by TNBS method. Catalytic activity was discussed in a structure-function relationship. Ours results indicate for the first time that aminoacylase-1 could play an important role in deacetylating mercapturate metabolites of aflatoxin analogues and this process may be in relation with their cyto- and nephrotoxicity in human.