Screening bioactive compounds with multi-targets from Rhodiola crenulata by a single column containing co-immobilized beta2-adrenergic receptor and voltage dependent anion channel isoform 1.

The pursuit of drugs having improved therapeutic efficacy necessitates increasing research on new assays for screening bioactive compounds with multi-targets. This work synthesized a chromatographic stationary phase containing co-immobilized beta2-adrenergic receptor (β2-AR) and voltage dependent anion channel isoform 1 (VDAC-1) to achieve such purpose. Specific ligands of the two receptors (e.g. salbutamol, methoxyphenamine, ATP and NADH) were utilized to characterize the specificity and bioactivity of the column. Validated application of the stationary phase was performed by screening multi-target compounds of Rhodiola crenulata using high performance affinity chromatography coupled with ESI-Q-TOF-MS. By zonal elution, we identified salidroside as a bioactive compound simultaneously binding to β2-AR and VDAC-1. The compound exhibited the binding sites of 1.0 × 10-7 and 4.0 × 10-7 M on the β2-AR and VDAC-1. On these sites, the association constants were calculated to be 3.3 × 104 and 1.0 × 104 M-1. Molecular docking indicated that the binding of salidroside to the two receptors occurred on Ser169 and Phe255of β2-AR, and the channel wall of VDAC-1. Taking together, we concluded that the column containing co-immobilized receptors has potential for screening bioactive compounds with multi-targets from complex matrices including traditional Chinese medicines.