| Assay Method Information | |
| | Biological Activity Assay |
| Description: | The measurements are carried out according to a protocol using two preparations of liposomes:The donor liposomes (A) contain a fluorescent sterol (DHE (dehydroergosterol));The acceptor liposomes (B) contain a fluorescent lipid (dansyl PE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(5-dimethylamino-1-naphthalenesulfonyl) (ammonium salt)) whose excitation spectrum covers the emission spectrum of DHE.Transport of DHE from liposomes A to liposomes B catalyzed by the ORD domain is accompanied by a FRET signal between DHE and Dansyl-PE. Based on this signal, the kinetics of transport can be measured in real time. This measurement of fluorescence is carried out on a microplate in a TECAN Infinite 1000 Pro instrument (temperature=37° C.). At the beginning, each measurement well contains liposomes B (130 μM), ORD domain (200 nM) and the test compound. At time t=5 min, liposomes A (130 μM) are added to start the exchange reaction. Each compound is tested in triplicate for final concentrations from 50 nM to 3 μM. The time constant (k) obtained for the kinetics in each case is then represented as a function of the concentration of the analog. From this representation, an inhibition constant is determined for each compound. The affinity constants Ki are classified as follows:Very strong affinity Ki<10 nMGood affinity Ki from 10 to 100 nMLow affinity Ki from 100 to 2000 nMThe compounds, other than the prodrugs, having a Ki<100 nM are regarded as active on OSBP.The lines U87-MG and A549 were obtained from the American Type Culture Collection (Rockville, MD, USA) and were cultured according to the supplier's instructions. The human glioblastoma cells U87-MG were cultured in Dulbecco minimum essential medium (DMEM) containing 10% of FCS and 1% of L-glutamine. The lung cancer cells A549 were cultured in RPMI1640 medium containing 10% of FCS and 1% of L-glutamine. The cell lines were maintained at 37° C. in a humidified atmosphere containing 5% CO2. The products were tested at 10 concentrations in triplicate and the cellular viability was evaluated after 72 h of treatment using the CellTiter Glo assay (Promega), which allows the number of live cells to be measured by luminescence (quantification of ATP). |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |