Multiparameter analysis of a screen for progesterone receptor ligands: comparing fluorescence lifetime and fluorescence polarization measurements.
Direct measurement of the fluorescence lifetime (FLT) of a fluorescent label is an emerging method for high-throughput screening. Changes in the fluorescence lifetime can be correlated to changes in the non-radiative relaxation pathway(s) for the excited state of the label. These pathways can be environmentally sensitive, such as when a labeled analyte is free in solution versus bound to a receptor. Because lifetime is an intrinsic property of a fluorophore, it is not concentration dependent, and therefore has advantages similar to those of ratiometric fluorescent techniques such as fluorescence resonance energy transfer or fluorescence polarization. We have applied the FLT measurement technique to a screen of a small compound library in order to identify compounds that bind to the progesterone receptor, and compared the results to those obtained by performing the assay in fluorescence polarization mode. Each readout modality showed excellent Z'; values, with the FLT readout performing slightly better in this respect. Interfering compounds could be rapidly identified for either assay format by comparing the results between the two formats.
Marks
Bryan D
BD
Invitrogen Corporation, Madison, WI, USA.
Qadir
Naveeda
N
Eliason
Hildegard C
HC
Shekhani
Mohammed Saleh
MS
Doering
Klaus
K
Vogel
Kurt W
KW
D019326:17-alpha-Hydroxyprogesterone / Q000378:metabolism; D001667:Binding, Competitive; D004353:Drug Evaluation, Preclinical / Q000379:methods; D005454:Fluorescence Polarization / Q000379:methods; D005456:Fluorescent Dyes / Q000737:chemistry; D006727:Hormone Antagonists / Q000378:metabolism; D006801:Humans; D008024:Ligands; D015735:Mifepristone / Q000378:metabolism; D011374:Progesterone / Q000378:metabolism; D011980:Receptors, Progesterone / Q000737:chemistry / Q000378:metabolism*; D015203:Reproducibility of Results
D005456:Fluorescent Dyes; D006727:Hormone Antagonists; D008024:Ligands; D011980:Receptors, Progesterone; D015735:Mifepristone; D011374:Progesterone; D019326:17-alpha-Hydroxyprogesterone