Alexandre A. de Castro

Computational Enzymology and Organophosphorus Degrading Enzymes: Promising Approaches Toward Remediation Technologies of Warfare Agents and Pesticides

The re-emergence of chemical weapons as a global threat in hands of terrorist groups, together with an increasing number of pesticides intoxications and environmental contaminations worldwide, has called the attention of the scientific community for the need of improvement in the technologies for detoxification of organophosphorus (OP) compounds. A compelling strategy is the use of bioremediation by enzymes that are able to hydrolyze these molecules to harmless chemical species. Several enzymes have been studied and engineered for this purpose. However, their mechanisms of action are not well understood. Theoretical investigations may help elucidate important aspects of these mechanisms and help in the development of more efficient bio-remediators. In this review, we point out the major contributions of computational methodologies applied to enzyme based detoxification of OPs. Furthermore, we highlight the use of PTE, PON, DFP, and BuChE as enzymes used in OP detoxification process and how computational tools such as molecular docking, molecular dynamics simulations and combined quantum mechanical/molecular mechanics have and will continue to contribute to this very important area of research.

Combined experimental and theoretical study on the removal of pollutant compounds by peroxidases: affinity and reactivity toward a bioremediation catalyst

Water pollution is a significant and growing problem throughout the world, especially in developing countries. In order to minimize environmental problems, catalysts have increasingly been designed to remove pollutants from the water. In an attempt to innovate by the creation of new low-cost alternatives to efficiently remove pollutants, the enzymatic treatment has been intensely studied for this purpose. Reactions catalyzed by enzymes are able to perform specific treatments, commonly with high rates of the final products. With this, the enzyme, peroxidase, is a promising candidate as a bioremediation catalyst. The efficiency of oxidoreductive enzymes, such as horseradish peroxidase (HRP) and soybean peroxidase (SP) have been studied, given that their performance depends on the substrate. In this investigation, experimental techniques and theoretical calculations have been employed in order to investigate the oxidative process for the ferulic acid and bromophenol blue dyes, performed by HRP and SP. Both enzymes showed a comparable behavior with respect to ferulic acid substrate. On the other hand, by utilizing bromophenol blue dye as a substrate, the behavior of the employed catalysts was significantly different. Experimental data have shown that HRP was more active toward bromophenol blue when compared to ferulic acid, being more rapidly degraded by the HRP enzyme. This tendency was confirmed by our theoretical docking, PM6 semi-empirical method, and DFT calculation results, in which the interaction, binding energies, and transition states were determined.

Molecular insight into the inhibition mechanism of plant and rat 4-hydroxyphenylpyruvate dioxygenase by molecular docking and DFT calculations

The 4-hydroxyphenylpyruvate dioxygenase (HPPD) is a relevant target protein for therapeutic and agrochemical research. It is an iron-dependent enzyme, and its inhibition has very different effects on plants and animals. In animals, the enzyme has an important role in the catabolism of tyrosine, and in the plant, it operates in the cascade of photosynthesis. Potent HPPD inhibitors have been described, and all contain the 1,3-diketone group in its shape. In this research, we carried out a study of the interaction modes of HPPD enzymes from plant and rat with selective and non-selective herbicides which already available with their structures to identify the molecule groups which are essential to their activity and those that are likely to changes, mediated by molecular computations. In this theoretical investigation, methods of molecular docking, reaction mechanism (QM/MM) and AIM calculations were employed, aiming the search for new more active and selective herbicides. Modifications were performed for DAS 645 and DAS 869 inhibitors. DAS 645 presented a good selectivity for the inhibition of the plant enzyme, and the modifications to the analogs design done increased its activity. For this compound, π–π* stacking interactions seem to be important, and this fact was proven by using AIM calculations. The other prototype compound, DAS 869, a potent inhibitor for both enzymes, had its increased activity in the plant and rat enzyme after added groups capable of performing π–π* stacking interactions.

Asymmetric biocatalysis of the nerve agent VX by human serum paraoxonase 1: molecular docking and reaction mechanism calculations

Organophosphorus compounds have been employed in agricultural activity for a long time, causing serious public health problems. Due to their toxic properties, these compounds have also been used as chemical weapons. In view of this scenario, the catalytic degradation and the development of bioremediation processes of organophosphorus compounds have been of wide interest. Among several enzymes capable of degrading organophosphorus compounds, the human serum paraoxonase 1 has shown good potential for this purpose. To evaluate the interaction mode between the human serum paraoxonase 1 (wild-type and mutants) enzymes and the VX compound, one of the most toxic organophosphorus compounds known, molecular docking calculations were conducted. In addition, seeking to analyze the reaction pathway and the stereochemistry preference by human serum paraoxonase 1 and the Rp and Sp enantiomers of VX, quantum mechanical/molecular mechanics calculations were performed. Our theoretical findings put in evidence that the wild-type and mutant human serum paraoxonase 1 enzymes strongly interact with VX. Moreover, with the quantum mechanical/molecular mechanics study, we observed that the human serum paraoxonase 1 preferentially degrades one enantiomer in relation to the other. The current results indicate key points for designing new, more efficient mutant human serum paraoxonase 1 enzymes for VX degradation.

Identification of biotransformation products of disperse dyes with rat liver microsomes by LC-MS/MS and theoretical studies with DNA: Structure-mutagenicity relationship using Salmonella/microsome assay

Azo dyes are known as a group of substances with DNA damage potential that depend on the nature and number of azo groups connected to aromatic rings (benzene and naphthalene), chemical properties, e.g. solubility and reactive functional groups, which significantly affect their toxicological and ecological risks. In this paper, we used in vitro models to evaluate the metabolism of selected textile dyes: Disperse Red 73 (DR 73), Disperse Red 78 (DR 78) and Disperse Red 167 (DR 167). To evaluate the mutagenic potential of the textile dyes, the Salmonella mutagenicity assay (Ames test) with strains TA 98 and TA 100 in the presence and absence of the exogenous metabolic system (S9) was used. DR73 was considered the most mutagenic compound, inducing both replacement base pairs (TA 100) and also changing frameshift (TA 98) mutations that are reduced in the presence of the S9 mixture. Furthermore, we used rat liver microsomes in the same experimental conditions of the S9 mixture to metabolize the dyes and the resultant solutions were analyzed using a liquid chromatography coupled to a quadrupole linear ion trap mass spectrometry (LC-MS/MS) to investigate the metabolites formed by the in vitro biotransformation. Based on this experiment, we detected and identified two biotransformation products for each textile dye substrate analyzed. Furthermore, to evaluate the interaction and reactivity of these compounds with DNA, theoretical calculations were also carried out. The results showed that the chemical reaction occurred preferentially at the azo group and the nitro group, indicating that there was a reduction in these groups by the CYP P450 enzymes presented in the rat microsomal medium. Our results clearly demonstrated that the reduction of these dyes by biological systems is a great environmental concern due to increased genotoxicity for the body of living beings, especially for humans.

Oxime K074 – in vitro and in silico reactivation of acetylcholinesterase inhibited by nerve agents and pesticides

Abstract Oxime K074 was formerly considered to be a lead structure for design of novel oximes for reactivation of tabun-inhibited acetylcholinesterase (AChE). In this study, we are summarizing its reactivation activity in case of other nerve agents (sarin, cyclosarin, VX and Russian VX) and pesticides (chlorpyrifos, methylchlorpyrifos and DDVP). For this purpose, standard in vitro method using rat brain homogenate was used. As resulted, oxime K074 was able to reactivate brain ChE (cholinesterases) inhibited by all used nerve agents and pesticides excluding cyclosarin-inhibited ChE. Only slight modification in structure of sarin (isopropyl moiety) and cyclosarin (cyclohexyl moiety) caused extraordinary differences in the reactivation of acetylcholinesterase inhibited by these nerve agents. Obtained molecular docking results suggest that the oxime K074 interacts very well with the inhibitors addressed in this work, and the data obtained by the QM/MM approach showed a good correlation with our experimental results of reactivation rate (%) by the oxime K074.

Structure and bonding in NbX5 X = (F, Cl, Br and I) complexes: a molecular orbital perspective in the C–H bond activation

In the present work, theoretical studies of the reactivity and stability of the NbX5 complexes (X = F, Cl, Br and I) were carried out in the methane C–H bond activation. To study the chemical bonds formation of these complexes, an energy decomposition analysis was performed together with QTAIM calculations. The main results indicated that the interaction and binding energies are higher for NbF5 in relation to the halogen series. The niobium complexes gaps are influenced by the electronegativity of the halogens and the Nb–X bonding lengths. According to the energy diagram, the electrons less connected to the bond are σNb–I; on the other hand, the best electron acceptor is σ*Nb–F. The QTAIM calculations confirmed stronger Nb–X chemical bonds in NbF5 complexes. Regarding the reactivity of the niobium complexes, the overall results indicate better thermodynamic and kinetic conditions for the NbF5 complex.

Peroxoniobium inhibits leukemia cell growth

A new class of polyoxoniobate complex has been synthesized and characterized as a novel anticancer agent for photodynamic therapy. The complex inhibits the growth of chronic myelogenous leukemia cells with an IC50 value of 30 μM, in the dark. However, upon exposure to light (365 nm) there is a fivefold increase in the cytotoxic activity. Light radiation activate the complex with the formation of radical species capable of interacting with DNA according to our experimental and theoretical data.

Insights into the pharmaceuticals and mechanisms of neurological orphan diseases: Current Status and future expectations

ABSTRACT Several rare or orphan diseases have been characterized that singly affect low numbers of people, but cumulatively reach ˜6%–10% of the population in Europe and in the United States. Human genetics has shown to be broadly effective when evaluating subjacent genetic defects such as orphan genetic diseases, but on the other hand, a modest progress has been achieved toward comprehending the molecular pathologies and designing new therapies. Chemical genetics, placed at the interface of chemistry and genetics, could be employed to understand the molecular mechanisms of subjacent illnesses and for the discovery of new remediation processes. This review debates current progress in chemical genetics, and how a variety of compounds and reaction mechanisms can be used to study and ultimately treat rare genetic diseases. We focus here on a study involving Amyotrophic lateral sclerosis (ALS), Duchenne Muscular Dystrophy (DMD), Spinal muscular atrophy (SMA) and Familial Amyloid Polyneuropathy (FAP), approaching different treatment methods and the reaction mechanisms of several compounds, trying to elucidate new routes capable of assisting in the treatment profile.

Interactions of cantharidin-like inhibitors with human protein phosphatase-5 in a Mg2+ system: molecular dynamics and quantum calculations

AbstractThe serine/threonine protein phosphatase type 5 (PP5) is a promising target for designing new antitumor drugs. This enzyme is a member of the PPP phosphatases gene family, which catalyzes a dephosphorylation reaction: a regulatory process in the signal transduction pathway that controls various biological processes. The aim of this work is to study and compare the inhibition of PP5 by ten cantharidin-like inhibitors in order to bring about contributions relevant to the better comprehension of their inhibitory activity. In this theoretical investigation, we used molecular dynamics techniques to understand the role of key interactions that occur in the protein active site; QM calculations were employed to study the interaction mode of these inhibitors in the enzyme. In addition, atoms in molecules (AIM) calculations were carried out to characterize the chemical bonds among the atoms involved and investigate the orbital interactions with their respective energy values. The obtained results suggest that the Arg275, Asn303, His304, His352, Arg400, His427, Glu428, Val429, Tyr451, and Phe446 residues favorably contribute to the interactions between inhibitors and PP5. However, the Asp271 and Asp244 amino acid residues do not favor such interactions for some inhibitors. Through the QM calculations, we can suggest that the reactional energy of the coordination mechanism of these inhibitors in the PP5 active site is quite important and is responsible for the inhibitory activity. The AIM technique employed in this work was essential to get a better comprehension of the transition states acquired from the mechanism simulation. This work offers insights of how cantharidin-like inhibitors interact with human PP5, potentially allowing the design of more specific and even less cytotoxic drugs for cancer treatments. Graphical AbstractInteractions of cantharidin-like inhibitors with human protein phosphatase-5 in a Mg2+ system