Indomethacin

To induce adipocyte differentiation by BMPs in the absence of induction cocktails, both WT brown preadipocytes and 3T3-L1 white preadipocytes were grown in regular growth medium supplemented with combination of rhBMPs (3.3 to 8.3 nM), insulin (20 nM) and T3 (1 nM) or vehicle as indicated in the text and figure legends for 7–13 days. To stimulate thermogenic program, differentiated cells were incubated with 500 µM dibutyrul cyclic AMP for 4 hrs. Cells were grown in growth medium without hormonal supplements for 18 hrs prior to cAMP stimulation.
C3H10T1/2 cells were grown in the presence and absence of 8.3 nM rhBMP-7 for 3 days to reach confluence (day 3). These cells were then induced to adipocyte differentiation using protocols described below for additional 7 days (day 10). Adipocyte differentiation was done by treating confluent cells for 48 hours in medium supplemented with 20 nM insulin and 1 nM triiodothyronine (T3), 0.5 mM isobutylmethylxanthine (IBMX), 5 µM dexamethasone, and 0.125 mM indomethacin. Cells were placed back to growth medium supplemented with insulin and T3, which was then changed every second day. After four to five more days in this medium, cells exhibited a fully differentiated phenotype with massive lipid accumulation.

A collection of on-hand 656 FDA-approved drugs was computationally screened against a panel of 339 molecular targets representing the species-specific expansion of 73 target assays used in Novartis safety panels. Each of the 339 target proteins was represented by its set of known ligands, as extracted from the ChEMBL_2 database. The two-dimensional structural similarity of a drug to a target’s ligand set was quantified as an E-value using the SEA, then subjected to a molecular charge filter. Predictions were tested retrospectively using proprietary databases including GeneGo Metabase, Thompson Reuters Integrity, Drugbank and GVKBio; novel predictions were tested prospectively in Novartis in vitro assays. Binding assays, and, when available, functional assays were performed including scintillation proximity, fluorometric imaging, filtration, fluorescence polarization, patch clamp, time-resolved fluorescence resonance energy transfer (TR-FRET), and homogenous time-resolved fluorescence (HTRF) assays. Concentration-response curves were calculated using XLfit (v.2 or v.4, IDBS, Guildford, UK) or a corresponding in-house software. All curves were redrawn using GraphPad PRISM v.5. Adverse drug reaction data were extracted from the world drug index (WDI) and encoded using the Medicinal dictionary for regulatory affairs (MedDRA). Using target annotations from GeneGo Metabase, Integrity, Drugbank, ChEMBL, and GVKBio, target-ADR pairs for all drugs were enumerated. Disproportionality analysis in conjunction with a Chi-squared test for association was carried out for all drug-target pairs. False discovery rate was controlled using the Benjamini-Hochberg correction for multiple hypothesis testing. Pharmacokinetic data were extracted from Integrity. For target and drug promiscuity analysis, combined external and internal target annotations were used. The computational workflow apart from SEA was implemented in Pipeline Pilot version 8 and statistical analyses were performed in R. Platelet aggregometry was performed in human blood for chlorotrianisene and indomethacin with acetylsalicylic acid as a positive control.