Designing Novel Antitrypanosomal Agents from a Mixed Graph-Theoretical Substructural Approach

Chagas disease is nowadays the most serious parasitic health problem. This disease is caused by Trypanosoma cruzi. The great number of deaths and the insufficient effectiveness of drugs against this parasite have alarmed the scientific community worldwide. In an attempt to overcome this problem, a model for the design and prediction of new antitrypanosomal agents was obtained. This used a mixed approach, containing simple descriptors based on fragments and topological substructural molecular design descriptors. A data set was made up of 188 compounds, 99 of them characterized an antitrypanosomal activity and 88 compounds that belong to other pharmaceutical categories. The model showed sensitivity, specificity and accuracy values above 85%. Quantitative fragmental contributions were also calculated. Then, and to confirm the quality of the model, 15 structures of molecules tested as antitrypanosomal compounds (that we did not include in this study) were predicted, taking into account the information on the abovementioned calculated fragmental contributions. The model showed an accuracy of 100% which means that the ""in silico"" methodology developed by our team is promising for the rational design of new antitrypanosomal drugs. (C) 2009 Wiley Periodicals, Inc. J Comput Chem 31: 882-894. 2010

Docking and molecular dynamics simulation of quinone compounds with trypanocidal activity

In this work, two different docking programs were used, AutoDock and FlexX, which use different types of scoring functions and searching methods. The docking poses of all quinone compounds studied stayed in the same region in the trypanothione reductase. This region is a hydrophobic pocket near to Phe396, Pro398 and Leu399 amino acid residues. The compounds studied displays a higher affinity in trypanothione reductase (TR) than glutathione reductase (GR), since only two out of 28 quinone compounds presented more favorable docking energy in the site of human enzyme. The interaction of quinone compounds with the TR enzyme is in agreement with other studies, which showed different binding sites from the ones formed by cysteines 52 and 58. To verify the results obtained by docking, we carried out a molecular dynamics simulation with the compounds that presented the highest and lowest docking energies. The results showed that the root mean square deviation (RMSD) between the initial and final pose were very small. In addition, the hydrogen bond pattern was conserved along the simulation. In the parasite enzyme, the amino acid residues Leu399, Met400 and Lys402 are replaced in the human enzyme by Met406, Tyr407 and Ala409, respectively. In view of the fact that Leu399 is an amino acid of the Z site, this difference could be explored to design selective inhibitors of TR.

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

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Design, synthesis and biological evaluation of new aryl thiosemicarbazone as antichagasic candidates

The present work reports on the synthesis, biological assaying and docking studies of a series of 12 aryl thiosemicarbazones, which were planned to act over two main enzymes, cruzain and trypanothione reductase. These enzymes are used as targets of trypanocidal activity in Chagas disease control with a minimal mutagenic profile. Three p-nitroaromatic thiosemicarbazones showed high activity against Trypanosoma cruzi in in vitro assays (IC50 < 57 μM), and no mutagenic profile was observed in micronucleous tests. Although the in vitro inhibition test showed that 10-μM doses of eight compounds inhibited cruzain activity, no correlation was found between cruzain inhibition and trypanocidal activity. © 2013 Elsevier Masson SAS. All rights reserved.

Preparação e determinação da atividade toxicológica do pró-fármaco hidroximetilnitrofural, potencialmente antichagásico

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Planejamento, sintese e avaliação biológica de derivados furoxânicos e benzofuroxânicos potencialmente antichagásicos

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Structure- and ligand-based drug design approaches for neglected tropical diseases

Drug discovery has moved toward more rational strategies based on our increasing understanding of the fundamental principles of protein-ligand interactions. Structure( SBDD) and ligand-based drug design (LBDD) approaches bring together the most powerful concepts in modern chemistry and biology, linking medicinal chemistry with structural biology. The definition and assessment of both chemical and biological space have revitalized the importance of exploring the intrinsic complementary nature of experimental and computational methods in drug design. Major challenges in this field include the identification of promising hits and the development of high-quality leads for further development into clinical candidates. It becomes particularly important in the case of neglected tropical diseases (NTDs) that affect disproportionately poor people living in rural and remote regions worldwide, and for which there is an insufficient number of new chemical entities being evaluated owing to the lack of innovation and R&D investment by the pharmaceutical industry. This perspective paper outlines the utility and applications of SBDD and LBDD approaches for the identification and design of new small-molecule agents for NTDs.

The leishmanicidal flavonols quercetin and quercitrin target Leishmania (Leishmania) amazonensis arginase

Polyamine biosynthesis enzymes are promising drug targets for the treatment of leishmaniasis, Chagas' disease and African sleeping sickness. Arginase, which is a metallohydrolase, is the first enzyme involved in polyamine biosynthesis and converts arginine into ornithine and urea. Ornithine is used in the polyamine pathway that is essential for cell proliferation and ROS detoxification by trypanothione. The flavonols quercetin and quercitrin have been described as antitrypanosomal and antileishmanial compounds, and their ability to inhibit arginase was tested in this work. We characterized the inhibition of recombinant arginase from Leishmania (Leishmania) amazonensis by quercetin, quercitrin and isoquercitrin. The IC50 values for quercetin, quercitrin and isoquercitrin were estimated to be 3.8, 10 and 4.3 mu M, respectively. Quercetin is a mixed inhibitor, whereas quercitrin and isoquercitrin are uncompetitive inhibitors of L. (L.) amazonensis arginase. Quercetin interacts with the substrate L-arginine and the cofactor Mn2+ at pH 9.6, whereas quercitrin and isoquercitrin do not interact with the enzyme's cofactor or substrate. Docking analysis of these flavonols suggests that the cathecol group of the three compounds interact with Asp129, which is involved in metal bridge formation for the cofactors Mn-A(2+) and Mn-B(2+) in the active site of arginase. These results help to elucidate the mechanism of action of leishmanicidal flavonols and offer new perspectives for drug design against Leishmania infection based on interactions between arginase and flavones. (C) 2012 Elsevier Inc. All rights reserved.

Increasing the activity of copper(II) complexes against Leishmania through lipophilicity and pro-oxidant ability

Copper complexes with fluorinated beta-diketones were synthesized and characterized in terms of lipophilicity and peroxide-assisted oxidation of dihydrorhodamine as an indicator of redox activity. The biological activity of the complexes was tested against promastigotes of Leishmania amazonensis. Inhibition of trypanosomatid-specific trypanothione reductase was also tested. It was found that the highly lipophilic and redox-active bis(trifluoroacetylacetonate) derivative had increased toxicity towards promastigotes. These results indicate that it is possible to modulate the activity of metallodrugs based on redox-active metals through the appropriate choice of lipophilic chelators in order to design new antileishmanials. Further work will be necessary to improve selectivity of these compounds against the parasite.

1,4-Diamino-2-butanone, a putrescine analogue, promotes redox imbalance in Trypanosoma cruzi and mammalian cells

The putrescine analogue 1,4-diamino-2-butanone (DAB) is highly toxic to various microorganisms, including Trypanosoma cruzi. Similar to other a-aminocarbonyl metabolites. DAB exhibits pro-oxidant properties. DAB undergoes metal-catalyzed oxidation yielding H2O2, NH4+ ion, and a highly toxic alpha-oxoaldehyde. In vitro. DAB decreases mammalian cell viability associated with changes in redox balance. Here, we aim to clarify the DAB pro-oxidant effects on trypomastigotes and on intracellular T. cruzi amastigotes. DAB (0.05-5 mM) exposure in trypomastigotes, the infective stage of T. cruzi, leads to a decline in parasite viability (IC50 c.a. 0.2 mM DAB; 4 h incubation), changes in morphology, thiol redox imbalance, and increased TcSOD activity. Medium supplementation with catalase (2.5 mu M) protects trypomastigotes against DAB toxicity, while host cell invasion by trypomastigotes is hampered by DAB. Additionally, intracellular amastigotes are susceptible to DAB toxicity. Furthermore, pre-treatment with 100-500 mu M buthionine sulfoximine (BSO) of LLC-MK2 potentiates DAB cytotoxicity, whereas 5 mM N-acetyl-cysteine (NAC) protects cells from oxidative stress. Together, these data support the hypothesis that redox imbalance contributes to DAB cytotoxicity in both T. cruzi and mammalian host cells. (C) 2012 Elsevier Inc. All rights reserved.

New chemical modalities for leishmaniasis drug discovery

Leishmaniasis is one of the major parasitic diseases among neglected tropical diseases with a high rate of morbidity and mortality. Human migration and climate change have spread the disease from limited endemic areas all over the world, also reaching regions in Southern Europe, and causing significant health and economic burden. The currently available treatments are far from ideal due to host toxicity, elevated cost, and increasing rates of drug resistance. Safer and more effective drugs are thus urgently required. Nevertheless, the identification of new chemical entities for leishmaniasis has proven to be incredibly hard and exacerbated by the scarcity of well-validated targets. Trypanothione reductase (TR) represents one robustly validated target in Leishmania that fulfils most of the requirements for a good drug target. However, due to the large and featureless active site, TR is considered extremely challenging and almost undruggable by small molecules. This scenario advocates the development of new chemical entities by unlocking new modalities for leishmaniasis drug discovery. The classical toolbox for drug discovery has enormously expanded in the last decade, and medicinal chemists can now strategize across a variety of new chemical modalities and a vast chemical space, to efficiently modulate challenging targets and provide effective treatments. Beyond others, Targeted p Protein Degradation (TPD) is an emerging strategy that uses small molecules to hijack endogenous proteolysis systems to degrade disease-relevant proteins and thus reduce their abundance in the cell. Based on these considerations, this thesis aimed to develop new strategies for leishmaniasis drug discovery while embracing novel chemical modalities and navigating the chemical space by chasing unprecedented chemotypes. This has been achieved by four complementary projects. We believe that these next-generation chemical modalities for leishmaniasis will play an important role in what was previously thought to be a drug discovery landscape dominated by small molecules.