Development of a Fluorescent Quenching Based High Throughput Assay to Screen for Calcineurin Inhibitors
Fig 5
Approximately 1400 compounds were screened, in duplicate, against CaN using this assay format. 50% inhibition was set as the threshold to identify hits. Out of 16 hits identified, 4 were selected on the basis of Lipinski's rule and commercial availability. Two of the selected compounds produced a dose-dependent inhibition and at least more than 50% inhibition. The reaction was done in duplicate and the data point represents mean and standard error. The IC50 were determined using Prism software, using Eq 5, and the values were (a) 106.4 μM for LDN-0013905 (flunarizine hydrochloride), (b) 4.5 μM for LDN-0013906 (tri-fluoperazine dihydrochloride). The fact that tri-fluoperazine dihydrochloride (LDN-0013906) is a known inhibitor of CaN further validated our assay format.