Supramolecular interactions between ß-lapachone with cyclodextrins studied using isothermal titration calorimetry and molecular modeling.

Supramolecular interactions between ß-lapachone (ß-lap) and cyclodextrins (CDs) were investigated by isothermal titration calorimetry. The most favorable host: guest interaction was characterized using X-ray powder diffraction (XRD), differential scanning calorimetry and thermogravimetry (DSC/TG), spectroscopy (FT-IR), spectroscopy (2D ROESY) nuclear magnetic resonance (NMR), and molecular modeling. Phase solubility diagrams showed ß-, HP-ß-, SBE-ß-, γ-, and HP-γ-CDs at 1.5% (w/w) allowed an increase in apparent solubility of ß-lap with enhancement factors of 12.0, 10.1, 11.8, 2.4, and 2.2, respectively. ß-lap has a weak interaction with γ- and HP-γ-CDs and tends to interact more favorably with ß-CD and its derivatives, especially SBE-ß-CD (K = 4160 M-1 ; ΔG = -20.66 kJ·mol-1 ). Thermodynamic analysis suggests a hydrophobic interaction associated with the displacement of water from the cavity of the CD by the ß-lap. In addition, van der Waals forces and hydrogen bonds were responsible for the formation of complexes. Taken together, the results showed intermolecular interactions between ß-lap and SBE-ß-CD, thereby confirming the formation of the inclusion complex. Molecular docking results showed 2 main orientations in which the interaction of benzene moiety at the wider rim of the SBE-ß-CD is the most stable (average docking energy of -7.0 kcal/mol). In conclusion, ß-lap:SBE-ß-CD is proposed as an approach for use in drug delivery systems in cancer research.

Molecular modeling and cytotoxicity of diffractaic acid: HP-ß-CD inclusion complex encapsulated in microspheres.

In this pioneer study, 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CD) was used to improve the solubility of the diffractaic acid (DA) via inclusion complex (DA:HP-ß-CD). Subsequently, DA:HP-ß-CD was incorporated into poly-ε-caprolactone (PCL) microspheres (DA:HP-ß-CD-MS). Microspheres containing DA (DA-MS) or DA:HP-ß-CD (DA:HP-ß-CD-MS) were prepared using the multiple W/O/W emulsion-solvent evaporation technique. The phase-solubility diagram of DA in HP-ß-CD (10-50mM) showed an AL type curve with a stability constant K1:1=821M-1. 1H NMR, FTIR, X-ray diffraction and thermal analysis showed changes in the molecular environment of DA in DA:HP-ß-CD. The molecular modeling approach suggests a guest-host complex formation between the carboxylic moiety of both DA and the host (HP-ß-CD). The mean particle size of the microspheres were ∅DA-MS=5.23±1.65µm and ∅DA:HP-ß-CD-MS=4.11±1.39µm, respectively. The zeta potential values of the microspheres were ζDA-MS=-7.85±0.32mV and ζDA:HP-ß-CD-MS=-6.93±0.46mV. Moreover, the encapsulation of DA:HP-ß-CD into microspheres resulted in a more slower release (k2=0.042±0.001; r2=0.996) when compared with DA-MS (k2=0.183±0.005; r2=0.996). The encapsulation of DA or DA:HP-ß-CD into microspheres reduced the cytotoxicity of DA (IC50=43.29µM) against Vero cells (IC50 of DA-MS=108.48µM and IC50 of DA:HP-ß-CD-MS=142.63µM).

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.

Isolation, homology modeling and renal effects of a C-type natriuretic peptide from the venom of the Brazilian yellow scorpion (Tityus serrulatus).

Mammalian natriuretic peptides (NPs) have been extensively investigated for use as therapeutic agents in the treatment of cardiovascular diseases. Here, we describe the isolation, sequencing and tridimensional homology modeling of the first C-type natriuretic peptide isolated from scorpion venom. In addition, its effects on the renal function of rats and on the mRNA expression of natriuretic peptide receptors in the kidneys are delineated. Fractionation of Tityus serrulatus venom using chromatographic techniques yielded a peptide with a molecular mass of 2190.64 Da, which exhibited the pattern of disulfide bridges that is characteristic of a C-type NP (TsNP, T. serrulatus Natriuretic Peptide). In the isolated perfused rat kidney assay, treatment with two concentrations of TsNP (0.03 and 0.1 µg/mL) increased the perfusion pressure, glomerular filtration rate and urinary flow. After 60 min of treatment at both concentrations, the percentages of sodium, potassium and chloride transport were decreased, and the urinary cGMP concentration was elevated. Natriuretic peptide receptor-A (NPR-A) mRNA expression was down regulated in the kidneys treated with both concentrations of TsNP, whereas NPR-B, NPR-C and CG-C mRNAs were up regulated at the 0.1 µg/mL concentration. In conclusion, this work describes the isolation and modeling of the first natriuretic peptide isolated from scorpion venom. In addition, examinations of the renal actions of TsNP indicate that its effects may be related to the activation of NPR-B, NPR-C and GC-C.

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 PPARß/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-1ß) 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.

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-ß-cyclodextrin (HP-ß-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, (1)HNMR, scanning electron microscopy, and by molecular modeling approaches. In addition, we verified that HP-ß-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.

Harpalycin 2 inhibits the enzymatic and platelet aggregation activities of PrTX-III, a D49 phospholipase A2 from Bothrops pirajai venom.

BACKGROUND: Harpalycin 2 (HP-2) is an isoflavone isolated from the leaves of Harpalyce brasiliana Benth., a snakeroot found in northeast region of Brazil and used in folk medicine to treat snakebite. Its leaves are said to be anti-inflammatory. Secretory phospholipases A2 are important toxins found in snake venom and are structurally related to those found in inflammatory conditions in mammals, as in arthritis and atherosclerosis, and for this reason can be valuable tools for searching new anti-phospholipase A2 drugs. METHODS: HP-2 and piratoxin-III (PrTX-III) were purified through chromatographic techniques. The effect of HP-2 in the enzymatic activity of PrTX-III was carried out using 4-nitro-3-octanoyloxy-benzoic acid as the substrate. PrTX-III induced platelet aggregation was inhibited by HP-2 when compared to aristolochic acid and p-bromophenacyl bromide (p-BPB). In an attempt to elucidate how HP-2 interacts with PrTX-III, mass spectrometry, circular dichroism and intrinsic fluorescence analysis were performed. Docking scores of the ligands (HP-2, aristolochic acid and p-BPB) using PrTX-III as target were also calculated. RESULTS: HP-2 inhibited the enzymatic activity of PrTX-III (IC50 11.34 ± 0.28 µg/mL) although it did not form a stable chemical complex in the active site, since mass spectrometry measurements showed no difference between native (13,837.34 Da) and HP-2 treated PrTX-III (13,856.12 Da). A structural analysis of PrTX-III after treatment with HP-2 showed a decrease in dimerization and a slight protein unfolding. In the platelet aggregation assay, HP-2 previously incubated with PrTX-III inhibited the aggregation when compared with untreated protein. PrTX-III chemical treated with aristolochic acid and p-BPB, two standard PLA2 inhibitors, showed low inhibitory effects when compared with the HP-2 treatment. Docking scores corroborated these results, showing higher affinity of HP-2 for the PrTX-III target (PDB code: 1GMZ) than aristolochic acid and p-BPB. HP-2 previous incubated with the platelets inhibits the aggregation induced by untreated PrTX-III as well as arachidonic acid. CONCLUSION: HP-2 changes the structure of PrTX-III, inhibiting the enzymatic activity of this enzyme. In addition, PrTX-III platelet aggregant activity was inhibited by treatment with HP-2, p-BPB and aristolochic acid, and these results were corroborated by docking scores.

Inhibition of Neurotoxic Secretory Phospholipases A(2) Enzymatic, Edematogenic, and Myotoxic Activities by Harpalycin 2, an Isoflavone Isolated from Harpalyce brasiliana Benth.

Secretory phospholipases A(2) (sPLA(2)) exert proinflammatory actions through lipid mediators. These enzymes have been found to be elevated in many inflammatory disorders such as rheumatoid arthritis, sepsis, and atherosclerosis. The aim of this study was to evaluate the effect of harpalycin 2 (Har2), an isoflavone isolated from Harpalyce brasiliana Benth., in the enzymatic, edematogenic, and myotoxic activities of sPLA(2) from Bothrops pirajai, Crotalus durissus terrificus, Apis mellifera, and Naja naja venoms. Har2 inhibits all sPLA(2) tested. PrTX-III (B. pirajai venom) was inhibited at about 58.7%, Cdt F15 (C. d. terrificus venom) at 78.8%, Apis (from bee venom) at 87.7%, and Naja (N. naja venom) at 88.1%. Edema induced by exogenous sPLA(2) administration performed in mice paws showed significant inhibition by Har2 at the initial step. In addition, Har2 also inhibited the myotoxic activity of these sPLA(2)s. In order to understand how Har2 interacts with these enzymes, docking calculations were made, indicating that the residues His48 and Asp49 in the active site of these enzymes interacted powerfully with Har2 through hydrogen bonds. These data pointed to a possible anti-inflammatory activity of Har2 through sPLA(2) inhibition.

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

The aim of this study was to encapsulate lapachone (ß-lap) or inclusion complex (ß-lap:HPß-CD) in liposomes and to evaluate their physicochemical characteristics. In addition, the investigation of the main aspects of the interaction between ß-lap and 2-hydroxypropyl-ß-cyclodextrin (HPß-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 ß-lap in HPß-CD was performed and the ß-lap:HPß-CD was prepared by the freeze-drying technique. A 302-fold increase of solubility was achieved for ß-lap in HPß-CD solution with a constant of association K(1:1) of 961 M(-1) and a complexation efficiency of ß-lap of 0.1538. (1)H NMR, TG, DSC, IR, Raman and SEM indicated a change in the molecular environment of ß-lap in the inclusion complex. Molecular modeling confirms these results suggesting that ß-lap was included in the cavity of HPß-CD, with an intermolecular interaction energy of -23.67 kJ mol(-1). ß-lap:HPß-CD and ß-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 ß-lap and ß-lap:HPß-CD-loaded liposomes, respectively. In conclusion, molecular modeling elucidates the formation of the inclusion complex, stabilized through hydrogen bonds, and the encapsulation of ß-lap and ß-lap:HPß-CD into liposomes could provide an alternative means leading eventually to its use in cancer research.