Insulin Sensitization by PPARγ and GLUT-4 Overexpression/Translocation Mediates the Antidiabetic Effect of Plantago australis.

Plantago australis Lam. Subsp. hirtella (Kunth) Rahn is a medicinal plant used as a diuretic, anti-inflammatory, antibacterial, throat cancer treatment and for the control of diabetes. P. australis was collected in the state of Morelos, México. The hydroalcoholic extract (HAEPa) of P. australis was obtained by maceration and concentrated in vacuo. Once dry, it was evaluated through an oral glucose tolerance test (OGTT) in normoglycemic mice and in a non-insulin-dependent diabetic mice model. The expression of PPARγ and GLUT-4 mRNA was determined by rt-PCR, and GLUT-4 translocation was confirmed by confocal microscopy. The toxicological studies were conducted in accordance with the guidelines suggested by the OECD, sections 423 and 407, with some modifications. HAEPa significantly decreased glycemia in OGTT curves, as well as in the experimental diabetes model compared to the vehicle group. In vitro tests showed that HAEPa induced an α-glucosidase inhibition and increased PPARγ and GLUT-4 expression in cell culture. The LD50 of HAEPa was greater than 2000 mg/kg, and sub-chronic toxicity studies revealed that 100 mg/kg/day for 28 days did not generate toxicity. Finally, LC-MS analysis led to the identification of verbascoside, caffeic acid and geniposidic acid, and phytochemical approaches allowed for the isolation of ursolic acid, which showed significant PPARγ overexpression and augmented GLUT-4 translocation. In conclusion, HAEPa induced significant antidiabetic action by insulin sensitization through PPARγ/GLUT-4 overexpression.

Synthesis, In Vitro, In Vivo and In Silico Antidiabetic Bioassays of 4-Nitro(thio)phenoxyisobutyric Acids Acting as Unexpected PPARγ Modulators: An In Combo Study.

Four isobutyric acids (two nitro and two acetamido derivatives) were prepared in two steps and characterized using spectral analysis. The mRNA concentrations of PPARγ and GLUT-4 (two proteins documented as key diabetes targets) were increased by 3T3-L1 adipocytes treated with compounds 1-4, but an absence of in vitro expression of PPARα was observed. Docking and molecular dynamics studies revealed the plausible interaction between the synthesized compounds and PPARγ. In vivo studies established that compounds 1-4 have antihyperglycemic modes of action associated with insulin sensitization. Nitrocompound 2 was the most promising of the series, being orally active, and one of multiple modes of action could be selective PPARγ modulation due to its extra anchoring with Gln-286. In conclusion, we demonstrated that nitrocompound 2 showed strong in vitro and in vivo effects and can be considered as an experimental antidiabetic candidate.

Tracheal relaxation through calcium channel blockade of Achillea millefolium hexanic extract and its main bioactive compounds.

ETHNOPHARMACOLOGICAL IMPORTANCE: Achillea millefolium L. (Asteraceae) is used for the treatment of respiratory diseases, diabetes, and hypertension. AIM: to explore its tracheal relaxant properties and clarify its functional mechanism of action on smooth muscle cells, which allow us to propose it as a potential anti-asthmatic drug. MATERIAL AND METHODS: organic and hydro-alcoholic extracts from A. millefolium were obtained by macerations, then their relaxing effect on ex vivo isolated rat trachea rings was determined. Most active extract (hexanic extract, EHAm) was studied to determine its functional mechanism of action using synergic, antagonist and inhibitor agents related with the contraction/relaxation process of the smooth muscle. Also, EHAm was subjected to bio-guided fractionation by open-column chromatography (on silica gel) using cyclohexane-EtOAc (80:20) in an isocratic way to isolate main bioactive compounds. RESULTS: organic and hydro-alcoholic extracts showed relaxant effect in a concentration-response dependent manner, being EHAm the most active. The functional mechanism of action indicates that EHAm induced a non-competitive antagonism to the muscarinic receptors ; in addition, the NO/cGMP pathway is involved in the relaxation process of the tracheal smooth muscle. However, the most important mechanism of action showed by EHAm was related with the calcium channel blockade influx into the smooth muscle cells. On the other hand, epimeric sesquiterpene lactones leucodin (1) and achillin (2) were isolated and purified, which are responsible for the observed smooth muscle relaxant activity of the extract. CONCLUSION: hexanic extract of A. millefollium induced a significant relaxant effect on tracheal rat rings by calcium channel blockade and NO release.

Design, synthesis, in vitro, in vivo and in silico pharmacological characterization of antidiabetic N-Boc-l-tyrosine-based compounds.

In this study, we synthesized five N-Boc-L-tyrosine-based analogues to glitazars. The in vitro effects of compounds 1-5 on protein tyrosine phosphatase 1B (PTP-1B), peroxisome proliferator-activated receptor alpha and gamma (PPARα/γ), glucose transporter type-4 (GLUT-4) and fatty acid transport protein-1 (FATP-1) activation are reported in this paper. Compounds 1 and 3 were the most active in the in vitro PTP-1B inhibition assay, showing IC50s of approximately 44 µM. Treatment of adipocytes with compound 1 increased the mRNA expression of PPARγ and GLUT-4 by 8- and 3-fold, respectively. Moreover, both compounds (1 and 3) also increased the relative mRNA expression of PPARα (by 8-fold) and FATP-1 (by 15-fold). Molecular docking studies were performed in order to elucidate the polypharmacological binding mode of the most active compounds on these targets. Finally, a murine model of hyperglycemia was used to evaluate the in vivo effectiveness of compounds 1 and 3. We found that both compounds are orally active using an exploratory dose of 100 mg/kg, decreasing the blood glucose concentration in an oral glucose tolerance test and a non-insulin-dependent diabetes mellitus murine model. In conclusion, we demonstrated that both molecules showed strong in vitro and in vivo effects and can be considered polypharmacological antidiabetic candidates.

Design, Synthesis and in Combo Antidiabetic Bioevaluation of Multitarget Phenylpropanoic Acids.

We have synthesized a small series of five 3-[4-arylmethoxy)phenyl]propanoic acids employing an easy and short synthetic pathway. The compounds were tested in vitro against a set of four protein targets identified as key elements in diabetes: G protein-coupled receptor 40 (GPR40), aldose reductase (AKR1B1), peroxisome proliferator-activated receptor gama (PPARγ) and solute carrier family 2 (facilitated glucose transporter), member 4 (GLUT-4). Compound 1 displayed an EC50 value of 0.075 µM against GPR40 and was an AKR1B1 inhibitor, showing IC50 = 7.4 µM. Compounds 2 and 3 act as slightly AKR1B1 inhibitors, potent GPR40 agonists and showed an increase of 2 to 4-times in the mRNA expression of PPARγ, as well as the GLUT-4 levels. Docking studies were conducted in order to explain the polypharmacological mode of action and the interaction binding mode of the most active molecules on these targets, showing several coincidences with co-crystal ligands. Compounds 1-3 were tested in vivo at an explorative 100 mg/kg dose, being 2 and 3 orally actives, reducing glucose levels in a non-insulin-dependent diabetes mice model. Compounds 2 and 3 displayed robust in vitro potency and in vivo efficacy, and could be considered as promising multitarget antidiabetic candidates. This is the first report of a single molecule with these four polypharmacological target action.

Antidiabetic effect of Achillea millefollium through multitarget interactions: α-glucosidases inhibition, insulin sensitization and insulin secretagogue activities.

ETHNOPHARMACOLOGICAL IMPORTANCE: Achillea millefolium L. (Asteraceae) is a perennial herb used in Mexican folk medicine for treatment of several pathologies, including inflammatory and spasmodic gastrointestinal disorders, hepatobiliary complaints, overactive cardiovascular, respiratory ailments and diabetes. AIM OF THE STUDY: To evaluate the potential antidiabetic effect in vivo and to establish the potential mode of action through in vitro approaches of Achillea millefolium. MATERIALS AND METHODS: The antidiabetic effect of hydroalcoholic extract of Achillea millefolium (HAEAm) was evaluated on the oral glucose tolerance tests, in normoglycemic and experimental Type 2 diabetic mice models. In addition, we evaluated the possible mode of action in in vitro assays to determine α-glucosidases inhibition, the insulin secretion and calcium mobilization in RINm5F cells and PPARγ and GLUT4 expression in 3T3-L1 cells. RESULTS: HAEAm showed significant glucose diminution on oral glucose tolerance test and in acute experimental Type 2 diabetic assay with respect to the control (p < 0.05). In addition, HAEAm promoted the α-glucosidases inhibition by 55% at 1mg/ml respect to control. On the other hand, HAEAm increased the PPARγ (five-times) and GLUT4 (two-fold) relative expression than control (p < 0.05). Finally, HAEAm significantly increased the insulin secretion and [Ca2+]i compared with control. CONCLUSION: The HAEAm possesses in vivo antidiabetic effect, having such effect through multitarget modes of action that involve antihyperglycemic (α-glucosidases inhibition), hypoglycemic (insulin secretion) and potential insulin sensitizer (PPARγ/GLUT4 overexpression) actions.

Antiprotozoal Nitazoxanide Derivatives: Synthesis, Bioassays and QSAR Study Combined with Docking for Mechanistic Insight.

In view of the serious health problems concerning infectious diseases in heavily populated areas, we followed the strategy of lead compound diversification to evaluate the near-by chemical space for new organic compounds. To this end, twenty derivatives of nitazoxanide (NTZ) were synthesized and tested for activity against Entamoeba histolytica parasites. To ensure drug-likeliness and activity relatedness of the new compounds, the synthetic work was assisted by a quantitative structure-activity relationships study (QSAR). Many of the inherent downsides - well-known to QSAR practitioners - we circumvented thanks to workarounds which we proposed in prior QSAR publication. To gain further mechanistic insight on a molecular level, ligand-enzyme docking simulations were carried out since NTZ is known to inhibit the protozoal pyruvate ferredoxin oxidoreductase (PFOR) enzyme as its biomolecular target.

Synthesis of nitro(benzo)thiazole acetamides and in vitro antiprotozoal effect against amitochondriate parasites Giardia intestinalis and Trichomonas vaginalis.

We synthesized four 5-nitrothiazole (1-4) and four 6-nitrobenzothiazole acetamides (5-8) using an easy two step synthetic route. All compounds were tested in vitro against amitochondriate parasites Giardia intestinalis and Trichomonas vaginalis, showing excellent antiprotozoal effects. IC50's of the most potent compounds range from nanomolar to low micromolar order, being more active than their drugs of choice. Compound 1 (IC50=122 nM), was 44-times more active than Metronidazole, and 10-fold more effective than Nitazoxanide against G. intestinalis and showed good trichomonicidal activity (IC50=2.24 µM). This compound did not display in vitro cytotoxicity against VERO cells. The in vitro inhibitory effect of compounds 1-8 and Nitazoxanide against G. intestinalis fructose-1,6-biphosphate aldolase (GiFBPA) was evaluated as potential drug target, showing a clear inhibitory effect over the enzyme activity. Molecular docking of compounds 1, 4 and Nitazoxanide into the ligand binding pocket of GiFBPA, revealed contacts with the active site residues of the enzyme. Ligand efficiency metrics of 1 revealed optimal combinations of physicochemical and antiprotozoal properties, better than Nitazoxanide.

2-acylamino-5-nitro-1,3-thiazoles: preparation and in vitro bioevaluation against four neglected protozoan parasites.

The 2-acylamino-5-nitro-1,3-thiazole derivatives (1-14) were prepared using a one step reaction. All compounds were tested in vitro against four neglected protozoan parasites (Giardia intestinalis, Trichomonas vaginalis, Leishmania amazonensis and Trypanosoma cruzi). Acetamide (9), valeroylamide (10), benzamide (12), methylcarbamate (13) and ethyloxamate (14) derivatives were the most active compounds against G. intestinalis and T. vaginalis, showing nanomolar inhibition. Compound 13 (IC50=10nM), was 536-times more active than metronidazole, and 121-fold more effective than nitazoxanide against G. intestinalis. Compound 14 was 29-times more active than metronidazole and 6.5-fold more potent than nitazoxanide against T. vaginalis. Ureic derivatives 2, 3 and 5 showed moderate activity against L. amazonensis. None of them were active against T. cruzi. Ligand efficiency indexes analysis revealed higher intrinsic quality of the most active 2-acylamino derivatives than nitazoxanide and metronidazole. In silico toxicity profile was also computed for the most active compounds. A very low in vitro mammalian cytotoxicity was obtained for 13 and 14, showing selectivity indexes (SI) of 246,300 and 141,500, respectively. Nitazoxanide showed an excellent leishmanicidal and trypanocidal effect, repurposing this drug as potential new antikinetoplastid parasite compound.

Synthesis of benzologues of Nitazoxanide and Tizoxanide: a comparative study of their in vitro broad-spectrum antiprotozoal activity.

We have synthesized two new benzologues of Nitazoxanide (NIT) and Tizoxanide (TIZ), using a short synthetic route. Both compounds were tested in vitro against six protozoa (Giardia intestinalis, Trichomonas vaginalis, Entamoeba histolytica, Plasmodium berghei, Leishmania mexicana and Trypanosoma cruzi). Compound 1 (benzologue of NIT) showed broad antiprotozoal effect against all parasites tested, showing IC(50)'s<5 µM. This compound was five-times more active than NIT, and 18-times more potent than metronidazole against G. intestinalis. It was 10-times more active than pentamidine against L. mexicana, and it was sevenfold more potent than benznidazole versus T. cruzi. This compound could be considered as a new broad spectrum antiprotozoal agent.