Fluorescent Probes for Highly Selective and Sensitive Detection of Hydrogen Sulfide in living cells and Tissues

ABSTRACT: OBJECTIVE: To express recombinant human CYPs (2C8*1,*2,*3,*4, 2C9*1,*3,*13,*16, and 2D6*1,*10), determine their expression levels and activity. Then study their enzyme kinetic characteristics on stereoselective N-demethylation of fluoxetine (FLX). METHODS:. Recombinant human CYPs were prepared with the Spodopterafrugiperda Sf9 cell system. The recombinant human CYPs were subjected to Western blot analysis for determining the relative expression levels and their classical in vitro probe substrates were used to validate their metabolic activities. The kinetic parameters of different CYPs on R-, S-, and R/S-FLX N-demethylation were analysed with an LCMS/MS method. RESULTS: The recombinant CYPs were successfully expressed. The activity assays showed that all the CYPs were active towards their classical substrates. The metabolic profiles of FLX mediated by these CYPs appeared to behave in a classical Michaelis-Menten or a substrate inhibition fashion. For CYP2C8s, CYP2C8*3 showed a higher clearance compared to the wild type. The Km values of Rand S-FLX in CYP2C8*3 and CYP2C8*4 showed significant stereoselectivity. However, only CYP2C8*3 exhibited slight stereoselectivity between Rand S-FLX. For CYP2C9s, the Km value for S-FLX were ranked in order of CYP2C9*1 >> CYP2C9*16 > CYP2C9*3 > CYP2C9*13. All the 4 CYP2C9s showed a self-inhibitory effect only for R-FLX and R/S-FLX. The four allozymes of CYP2C9 were all predominantly toward the R-enantiomer, indicating significant stereoselectivity in CYP2C9-mediated N-demethylation metabolism of FLX. For CYP2D6s, the metabolic of FLX enantiomers obviously appeared stereoselectively catalyzed by CYP2D6*1,*2 and *39. The stereoselectively of CYP2D6*1 (R > S) was the reverse of CYP2D6*2/*39 (S > R). Additionally, CYP2D6*2 and *39 exhibited obvious self-inhibitory effect, which was not shown with CYP2D6*1 and *10. CONCLUSION: The recombinant CYPs were successfully expressed with good enzyme activities. Most of the variants evaluated showed lower catalytic activity towards FLX enantiomers and racemate. CYP2C9*1 and their variants, and CYP2D6*1 may affect the stereoselective N-demethylation to FLX.