Analyses of a huge number of crystallographic structures of the PPAR ligand-binding site bound to an agonist have revealed that this isotype has two binding modes in a single pocket. 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. = 6)/*** < 0.001; ** < 0.01; * < 0.05 compared with control group. 2.5. Molecular Docking Studies Based on the in vitro biological assays and the preliminary enzyme inhibition evaluations, the most active compounds were selected to explain the experimental activities on these relevant targets. A preliminary molecular docking simulation was performed to assess the presumed binding mode of 1C5 into the receptors GPR40, PPAR and the enzyme AKR1B1. A pilot in silico calculation was done using DIA-DB [27], a web server for the prediction of antidiabetic drugs via inverse virtual screening of the input molecules 1C5 against a set of 18 protein targets identified as key elements in diabetes, within which are included PPAR, GPR40 and AKR1B1, among others [28]. Subsequently, a more specific and refined analysis was carried out for the most active compounds (1C3). Refined molecular docking reveals that compounds 2 and 3 internalize into the ligand binding site of PPAR and interact by electrostatic and hydrogen bonds with Ser-289, His-323, His-449 and Tyr-473, all of them essential for the activation of this receptor. However, compound OSI-027 3 (the most active in vitro) showed an additional interaction with Ser-342, characteristic of PPAR partial agonists (Figure 3). Analyses of a huge number of crystallographic structures of the PPAR ligand-binding site bound to an agonist have revealed that this isotype has two binding modes in a single pocket. These two binding modes correspond to full and partial agonists [29]. Side effects of glitazones, including weight gain, edema, congestive heart failure, and the recently reported increased risk of bone fracture are major undesired effects associated with the use of PPAR full agonists [30]. On the other hand, partial agonists interact mainly through a hydrogen bond with Ser342. This interaction corresponds to several carboxylic ligands present in the majority of the PPAR partial agonists that forms a hydrogen bond with the Ser342, such as showed by compound 3. Open in a separate window Figure 3 (A) 3D binding model of compounds 1C3 into the ligand binding site of PPAR. Compounds are presented as stick models: 1 (green), 2 (cyan) and 3 (magenta), and aminoacids as lines. Dashed line signifies polar interactions; (B) 2D interaction map of the OSI-027 most active compound 3 and PPAR. For GPR40, binding poses depicted in Figure 4 suggest that the in vitro active compounds OSI-027 1, 2 and 3 interact through electrostatic bonds with residues of Arg-183 and Arg-2258, and by hydrogen bonds Rabbit Polyclonal to DGAT2L6 with Tyr-91, Asn-2244, Tyr-2240, all of them showed by well-known GPR40 allosteric agonists (such as TAK-875). On the other hand, the disposition of the biphenyl ring in 1, which was the most potent in the in vitro screening, fits into the GPR40 ligand-binding-better than the other compounds generating – interactions with Phe-142 (Figure 4B). The docking score for compound 1 was the highest (?G = ?10.63 kcal/mol), in comparison with compounds 2 and 3 (?G = ?10.31 and ?9.96 kcal/mol, respectively). Open in a separate window Figure 4 (A) 3D binding model of compounds 1C3 into the allosteric ligand binding site of GPR40. Compounds are presented as stick models: 1 (green), 2 (cyan) and 3 (magenta). (B) 2D interaction map of the most active compound 1 and GPR40. In the case of AKR1B1, solutions of molecular docking into the catalytic site of this enzyme show that acid moieties of compounds 1, 2 and 3 interact with Tyr-48, His-110 and Trp-111 showed in several currently inhibitors of this enzyme, such as zopolrestat and tolrestat. Also, the naphthyl ring of 2 conserves an interaction with Trp-111 through – stacking (Figure 5). All compounds showed moderate in vitro inhibition of this enzyme. Open in a separate window Figure 5 (A) 3D binding model of compounds 1C3 into the active site of Aldose reductase (AKR1B1). Compounds are presented as stick models: 1 (green), 2 (cyan) and 3 (magenta); (B) 2D interaction map of the second most active compound OSI-027 2 and AKR1B1. 2.6. In Vivo Antidiabetic Effect of Compounds = 6). * < 0.05 versus Tween 80 (10%) group. In the in vivo assay, compound 1 exhibited potential differences.