Probing the effect of sildenafil on Progesterone and Testosterone production by an intracellullar FRET/BRET combined approach

Forster Resonance Energy Transfer (FRET)-based biosensors have been recently applied to the study of biological pathways. In this study, a new biosensor was validated for the first time in live HEK293 and steroidogenic MLTC-1 cell line for studying the PDE5 inhibitor effect on hCG/LH-induced steroidogenic pathway. The sensor benefits of FRET between, a donor (D), the fluorescein-like diarsenical probe able to covalently bind a tetracysteine motif fused to the PDE5 catalytic domain and, an acceptor (A), the rhodamine probe conjugated to the pseudosubstrate cGMPS. Affinity constant values (Kd) of 5.63.2 M and 13.70.8 M were obtained from HEK293 and MLTC-1 cells, respectively. The detection was based on the competitive displacement of the cGMPS-rhodamine conjugate by sildenafil; Ki values were 3.60.3 nM (IC50= 2.3 nM) in HEK293 cells and 101.0 nM (IC50=3.9 nM) in MLTC-1 cells. The monitoring of both cAMP and cGMP by BRET allowed the exploitation of PDE5i effects on steroidogenesis, indicating that sild...