3. Materials and Methods

3.1. Computational Methods
Vitual screening on the DrugBank [23] was carried out using the Molecular Operating Environment (MOE) program [28], using four common points of the BK B1 and B2 pharmacophores described previously [25,26] that were used as a query. Previously, a 3D DrugBank database containing the 3D structure, together with a set of conformations of each molecule, was constructed as explained elsewhere [24]. Virtual screening was carried out on the subset of molecules with molecular weight between 200 and 600 (a total of 1703 molecules). Hits identified in the screening process were docked in the 3D models of the B1 and B2 bradykinin receptors, using a set of unique conformations resulting from thorough conformational searches for the diverse ligands studied and rank ordered by means of the MOE program [28].

3.2. In Vitro Assays

3.2.1. Ligand Displacement Assays
Raloxifene was tested for its ability to displace reference ligands on human recombinant bradykinin B1 or B2 receptors, respectively, expressed in CHO cells at 20 µM. For this purpose, first saturation isotherms were obtained with reference ligands ([3H] des-Arg10-BK (0.35 nM) in the case of B1, and [3H]-bradykinin (0.2 nM) for B2) incubated for 60 min at room temperature. Nonspecific binding was evaluated by adding a reference compound (desArg9[Leu8]-BK at 10 µM in the case of B1 and BK at 1 µM for B2). Antagonism of raloxifene was measured as a percent inhibition of specific binding of [3H]-bradykinin as control, obtained in the presence of raloxifene at 20 µM and using as reference compounds desArg10-KD for B1 and NPC-567 for B2.

3.2.2. Functional Efficacy Assays
Measurementent of the efficacy of the compound on the B2 receptor was carried out following the protocol described elsewhere [31]. The method is based on measuring differences in intracellular Ca2+ concentrations produced in diverse conditions on CHO cells expressing recombinant human B2 receptor by fluorometry. Agonism was measured through the capacity of the compound to increase Ca2+ concentration compared to BK (EC50 ~ 2.4 pM). Antagonism was measured through the capacity to antagonize BK based on the reduction of Ca2+ concentration.