Marcelo M. Rabello

Quercetin as an inhibitor of snake venom secretory phospholipase A2

As polyphenolic compounds isolated from plants extracts, flavonoids have been applied to various pharmaceutical uses in recent decades due to their anti-inflammatory, cancer preventive, and cardiovascular protective activities. In this study, we evaluated the effects of the flavonoid quercetin on Crotalus durissus terrificus secretory phospholipase A2 (sPLA2), an important protein involved in the release of arachidonic acid from phospholipid membranes. The protein was chemically modified by treatment with quercetin, which resulted in modifications in the secondary structure as evidenced through circular dichroism. In addition, quercetin was able to inhibit the enzymatic activity and some pharmacological activities of sPLA2, including its antibacterial activity, its ability to induce platelet aggregation, and its myotoxicity by approximately 40%, but was not able to reduce the inflammatory and neurotoxic activities of sPLA2. These results suggest the existence of two pharmacological sites in the protein, one that is correlated with the enzymatic site and another that is distinct from it. We also performed molecular docking to better understand the possible interactions between quercetin and sPLA2. Our docking data showed the existence of hydrogen-bonded, polar interactions and hydrophobic interactions, suggesting that other flavonoids with similar structures could bind to sPLA2. Further research is warranted to investigate the potential use of flavonoids as sPLA2 inhibitors.

The encapsulation of β-lapachone in 2-hydroxypropyl-β-cyclodextrin inclusion complex into liposomes: a physicochemical evaluation and molecular modeling approach.

The aim of this study was to encapsulate lapachone (β-lap) or inclusion complex (β-lap:HPβ-CD) in liposomes and to evaluate their physicochemical characteristics. In addition, the investigation of the main aspects of the interaction between β-lap and 2-hydroxypropyl-β-cyclodextrin (HPβ-CD), using both experimental and molecular modeling approaches was discussed. Furthermore, the in vitro drug release kinetics was evaluated. First, a phase solubility study of β-lap in HPβ-CD was performed and the β-lap:HPβ-CD was prepared by the freeze-drying technique. A 302-fold increase of solubility was achieved for β-lap in HPβ-CD solution with a constant of association K(1:1) of 961 M(-1) and a complexation efficiency of β-lap of 0.1538. (1)H NMR, TG, DSC, IR, Raman and SEM indicated a change in the molecular environment of β-lap in the inclusion complex. Molecular modeling confirms these results suggesting that β-lap was included in the cavity of HPβ-CD, with an intermolecular interaction energy of -23.67 kJ mol(-1). β-lap:HPβ-CD and β-lap-loaded liposomes presented encapsulation efficiencies of 93% and 97%, respectively. The kinetic rate constants of 183.95±1.82 μg/h and 216.25±2.34 μg/h were calculated for β-lap and β-lap:HPβ-CD-loaded liposomes, respectively. In conclusion, molecular modeling elucidates the formation of the inclusion complex, stabilized through hydrogen bonds, and the encapsulation of β-lap and β-lap:HPβ-CD into liposomes could provide an alternative means leading eventually to its use in cancer research.

2-Pyridyl thiazoles as novel anti-Trypanosoma cruzi agents: structural design, synthesis and pharmacological evaluation.

The present work reports on the synthesis, anti-Trypanosoma cruzi activities and docking studies of a novel series of 2-(pyridin-2-yl)-1,3-thiazoles derived from 2-pyridine thiosemicarbazone. The majority of these compounds are potent cruzain inhibitors and showed excellent inhibition on the trypomastigote form of the parasite, and the resulting structure-activity relationships are discussed. Together, these data present a novel series of thiazolyl hydrazones with potential effects against Chagas disease and they could be important leads in continuing development against Chagas disease.

Structural investigation of anti-Trypanosoma cruzi 2-iminothiazolidin-4-ones allows the identification of agents with efficacy in infected mice.

We modified the thiazolidinic ring at positions N3, C4, and C5, yielding compounds 6-24. Compounds with a phenyl at position N3, 15-19, 22-24, exhibited better inhibitory properties for cruzain and against the parasite than 2-iminothiazolidin-4-one 5. We were able to identify one high-efficacy trypanocidal compound, 2-minothiazolidin-4-one 18, which inhibited the activity of cruzain and the proliferation of epimastigotes and was cidal for trypomastigotes but was not toxic for splenocytes. Having located some of the structural determinants of the trypanocidal properties, we subsequently wished to determine if the exchange of the thiazolidine for a thiazole ring leaves the functional properties unaffected. We therefore tested thiazoles 26-45 and observed that they did not inhibit cruzain, but they exhibited trypanocidal effects. Parasite development was severely impaired when treated with 18, thus reinforcing the notion that this class of heterocycles can lead to useful cidal agents for Chagas disease.

Studies toward the structural optimization of novel thiazolylhydrazone-based potent antitrypanosomal agents

In previous studies, we identified promising anti-Trypanosoma cruzi cruzain inhibitors based on thiazolylhydrazones. To optimize this series, a number of medicinal chemistry directions were explored and new thiazolylhydrazones and thiosemicarbazones were thus synthesized. Potent cruzain inhibitors were identified, such as thiazolylhydrazones 3b and 3j, which exhibited IC(50) of 200-400nM. Furthermore, molecular docking studies showed concordance with experimentally derived structure-activity relationships (SAR) data. In the course of this work, lead compounds exhibiting in vitro activity against both the epimastigote and trypomastigote forms of T. cruzi were identified and in vivo general toxicity analysis was subsequently performed. Novel SAR were documented, including the importance of the thiocarbonyl carbon attached to the thiazolyl ring and the direct comparison between thiosemicarbazones and thiazolylhydrazones.

Structural Design, Synthesis and Structure–Activity Relationships of Thiazolidinones with Enhanced Anti‐Trypanosoma cruzi Activity

Pharmacological treatment of Chagas disease is based on benznidazole, which displays poor efficacy when administered during the chronic phase of infection. Therefore, the development of new therapeutic options is needed. This study reports on the structural design and synthesis of a new class of anti‐Trypanosoma cruzi thiazolidinones (4 a–p). (2‐[2‐Phenoxy‐1‐(4‐bromophenyl)ethylidene)hydrazono]‐5‐ethylthiazolidin‐4‐one (4 h) and (2‐[2‐phenoxy‐1‐(4‐phenylphenyl)ethylidene)hydrazono]‐5‐ethylthiazolidin‐4‐one (4 l) were the most potent compounds, resulting in reduced epimastigote proliferation and were toxic for trypomastigotes at concentrations below 10 μM, while they did not display host cell toxicity up to 200 μM. Thiazolidinone 4 h was able to reduce the in vitro parasite burden and the blood parasitemia in mice with similar potency to benznidazole. More importantly, T. cruzi infection reduction was achieved without exhibiting mouse toxicity. Regarding the molecular mechanism of action, these thiazolidinones did not inhibit cruzain activity, which is the major trypanosomal protease. However, investigating the cellular mechanism of action, thiazolidinones altered Golgi complex and endoplasmic reticulum (ER) morphology, produced atypical cytosolic vacuoles, as well as induced necrotic parasite death. This structural design employed for the new anti‐T. cruzi thiazolidinones (4 a–p) led to the identification of compounds with enhanced potency and selectivity compared to first‐generation thiazolidinones. These compounds did not inhibit cruzain activity, but exhibited strong antiparasitic activity by acting as parasiticidal agents and inducing a necrotic parasite cell death.

Enhanced Antiproliferative Activity of the New Anticancer Candidate LPSF/AC04 in Cyclodextrin Inclusion Complexes Encapsulated into Liposomes

LPSF/AC04 (5Z)-[5-acridin-9-ylmethylene-3-(4-methyl-benzyl)-thiazolidine-2,4-dione] is an acridine-based derivative, part of a series of new anticancer agents synthesized for the purpose of developing more effective and less toxic anticancer drugs. However, the use of LPSF/AC04 is limited due to its low solubility in aqueous solutions. To overcome this problem, we investigated the interaction of LPSF/AC04 with hydroxypropyl-β-cyclodextrin (HP-β-CyD) and hydroxypropyl-γ-cyclodextrin (HP-γ-CyD) in inclusion complexes and determine which of the complexes formed presents the most significant interactions. In this paper, we report the physical characterization of the LPSF/AC04–HP-CyD inclusion complexes by thermogravimetric analysis, differential scanning calorimetry, infrared spectroscopy absorption, Raman spectroscopy, 1HNMR, scanning electron microscopy, and by molecular modeling approaches. In addition, we verified that HP-β-CyD complexation enhances the aqueous solubility of LPSF/AC04, and a significant increase in the antiproliferative activity of LPSF/AC04 against cell lines can be achieved by the encapsulation into liposomes. These findings showed that the nanoencapsulation of LPSF/AC04 and LPSF/AC04–HP-CyD inclusion complexes in liposomes leads to improved drug penetration into the cells and, as a result, an enhancement of cytotoxic activity. Further in vivo studies comparing free and encapsulated LPSF/AC04 will be undertaken to support this investigation.

Chemical synthesis, docking studies and biological effects of a pan peroxisome proliferator-activated receptor agonist and cyclooxygenase inhibitor

The compound (5Z)-5-[(5-bromo-1H-indol-3-yl)methylene]-3-(4-chlorobenzyl)-thiazolidine-2,4-dione (LYSO-7) was synthesised in order to obtain a new type of anti-inflammatory drug, designed with hybrid features to inhibit cyclooxygenase (COX) and also to activate peroxisome proliferator-activated receptor (PPAR). Results obtained from docking (in silico) studies corroborated with experimental data, showing the potential affinity between the studied ligand and targets. The specificity of LYSO-7 for COX-enzymes was detected by the inhibition of COX-1 and COX-2 activities by 30% and 20%, respectively. In transactivation reporter gene assays LYSO-07 showed a pan partial agonist effect on the three PPAR subtypes (PPARγ, PPARα and PPARβ/δ). The agonist action on PPARγ was also observed by a pharmacological approach, as the reduction in the Escherichia coli lipopolysaccharide (LPS)-induced interleukin 1 beta (IL-1β) secretion and nitric oxide (NO) production by mouse neutrophils was blocked by GW9962, a specific PPARγ antagonist. Additionally, the in vivo effect was measured by reduced carrageenan-induced neutrophil influx into the subcutaneous tissue of mice. Taken together, these data show that LYSO-7 displays a potent in vivo anti-inflammatory effect during the innate acute response, which is dependent on its associated COX inhibitory activities and PPAR activation.

Design, synthesis and structure-activity relationship of phthalimides endowed with dual antiproliferative and immunomodulatory activities

The present work reports the synthesis and evaluation of the antitumour and immunomodulatory properties of new phthalimides derivatives designed to explore molecular hybridization and bioisosterism approaches between thalidomide, thiosemicarbazone, thiazolidinone and thiazole series. Twenty-seven new molecules were assessed for their immunosuppressive effect toward TNFα, IFNγ, IL-2 and IL-6 production and antiproliferative activity. The best activity profile was observed for the (6a-f) series, which presents phthalyl and thiazolidinone groups.

New PPARγ partial agonist improves obesity-induced metabolic alterations and atherosclerosis in LDLr(-/-) mice.

Peroxisome proliferator-activated receptor gamma (PPARγ) regulates multiple pathways involved in the pathogenesis of obesity and atherosclerosis. Here, we evaluated the therapeutic potential of GQ-177, a new thiazolidinedione, on diet-induced obesity and atherosclerosis. The intermolecular interaction between PPARγ and GQ-177 was examined by virtual docking and PPAR activation was determined by reporter gene assay identifying GQ-177 as a partial and selective PPARγ agonist. For the evaluation of biological activity of GQ-177, low-density lipoprotein receptor-deficient (LDLr(-/-)) C57/BL6 mice were fed either a high fat diabetogenic diet (diet-induced obesity), or a high fat atherogenic diet, and treated with vehicle, GQ-177 (20mg/kg/day), pioglitazone (20mg/kg/day, diet-induced obesity model) or rosiglitazone (15mg/kg/day, atherosclerosis model) for 28 days. In diet-induced obesity mice, GQ-177 improved insulin sensitivity and lipid profile, increased plasma adiponectin and GLUT4 mRNA in adipose tissue, without affecting body weight, food consumption, fat accumulation and bone density. Moreover, GQ-177 enhanced hepatic mRNA levels of proteins involved in lipid metabolism. In the atherosclerosis mice, GQ-177 inhibited atherosclerotic lesion progression, increased plasma HDL and mRNA levels of PPARγ and ATP-binding cassette A1 in atherosclerotic lesions. GQ-177 acts as a partial PPARγ agonist that improves obesity-associated insulin resistance and dyslipidemia with atheroprotective effects in LDLr(-/-) mice.