Measuring Binding Kinetics of Ligands with Tethered Receptors by Fluorescence Polarization and Total Internal Reflection Fluorescence

摘要

Binding kinetics of nuclear receptors and their specificnligands was measured using polarization anisotropyncomplemented with total internal reflection fluorescence.nBinding affinities of tethered full length human estrogennreceptor alpha (ER ) with 17u0001-estradiol, diethylstil-nbestrol, raloxifene, 4-hydroxytamoxifen, tamoxifen, andngenistein were measured to be 100 (as reference), 100,n35, 21, 8, and 1.5, respectively. They agreed withnpublished results. For the first time, rate constants werenmeasured, and off rates were 1.5, 1.5, 1.3, 1.6, 1.7, andn2.3 × 10 3 s 1 while on rates were 11, 10, 3.3, 2.4,n1.0, and 0.26 × 105 M 1s 1, respectively. For thenantiestrogen drugs, their comparable off-rates cor-nrelated well with their equally similar potency. Elevennginsenosides were screened as potential ligands. Nonenwere found to bind to ER , but Rb1(S) and 20(S)-Rg3nwere shown to bind to peroxisome proliferator-activat-ned receptor gamma. The latter finding corroboratednstrongly with the therapeutic effects of ginsenosides onndiabetic mice observed in a separate study. Ournmethod would complement surface plasmon resonancenassay for small ligands in the mass range of tens tonhundreds of Daltons.