Abstract
Human constitutive androstane receptor (hCAR) regulates xenobiotic metabolism. Its large and flexible ligand binding pocket can accommodate structurally diverse compounds. An assay for characterizing the binding of ligands to hCAR is needed but has not been reported. Here, we first discovered the promiscuous kinase inhibitor SNS-032 and its derivative THAL-SNS-032 as binders of hCAR, then developed BODIPY FL SNS 032 (14) as a high-affinity hCAR fluorescent probe (K (d): 300 ± 30 nM) in a TR-FRET binding assay and used it to characterize hCAR ligands for their competitive binding activities. BODIPY FL SNS 032 also displayed high binding affinities to multiple kinases, such as hGSK3A (K (d): 4.5 ± 0.2 nM), hCDK9/CycT1 (K (d): 5.1 ± 0.6 nM), hMAPK15 (K (d): 340 ± 20 nM), hCASK (K (d): 550 ± 30 nM), and hCAMKK2 (K (d): 530 ± 40 nM). BODIPY FL SNS 032 is therefore a versatile probe for hCAR and multiple kinases.