Complete Reaction Phenotyping of Propranolol and 4-Hydroxypropranolol with the 19 Enzymes of the Human UGT1 and UGT2 Families

Abstract

Propranolol is a competitive non-selective beta-receptor antagonist that is available on the market as a racemic mixture. In the present study, glucuronidation of propranolol and its equipotent phase I metabolite 4-hydroxypropranolol by all 19 members of the human UGT1 and UGT2 families was monitored. UGT1A7, UGT1A9, UGT1A10 and UGT2A1 were found to glucuronidate propranolol, with UGT1A7, UGT1A9 and UGT2A1 mainly acting on (S)-propranolol, while UGT1A10 displays the opposite stereoselectivity. UGT1A7, UGT1A9 and UGT2A1 were also found to glucuronidate 4-hydroxypropranolol. In contrast to propranolol, 4-hydroxypropranolol was found to be glucuronidated by UGT1A8 but not by UGT1A10. Additional biotransformations with 4-methoxypropanolol demonstrated different regioselectivities of these UGTs with respect to the aliphatic and aromatic hydroxy groups of the substrate. Modeling and molecular docking studies were performed to explain the stereoselective glucuronidation of the substrates under study.

Mutual Modulation of the Activities of Human CYP2D6 and Four UGTs during the Metabolism of Propranolol

“…Furthermore, the aqueous residue obtained from the combined incubations with SAN300 and one of the four UGT strains was found to contain two propranolol glucuronides at similar production rates to those previously reported [ 30 ], indicating that the activity of UGTs in single-expression yeast strains remained unaffected even if used in this mixedstrain incubation. No hydroxypropranolol glucuronides were detected in any of the reactions, which was expected as CYP2D6 and UGTs are not physically proximate in this assay, making subsequent glucuronidation after hydroxylation unlikely.…”

“…In our previous studies, different reaction times were used in the enzyme bag method for UGT-catalyzed glucuronidation and P450-catalyzed hydroxylation [ 30 , 34 ]. The diploid yeast used in this study co-expressed both CYP2D6 and one of the four different UGTs ( Table 2 ).…”

“…The optimized enzyme bag method for diploid yeasts was used for the biotransformation of propranolol by four diploid yeast strains which co-express CYP2D6 and one of four UGTs (UGT1A7, UGT1A8, UGT1A9, or UGT2A1). Those four human UGTs were previously shown to catalyze the glucuronidation of propranolol and/or 4-hydroxypropranolol [ 30 ]. The diploid yeast strain SAN300 that only expresses CYP2D6 and human CPR was used as a control.…”

“…Tris buffer, potassium chloride, glycerol and Triton X-100 were sourced from Carl Roth GmbH & Co. KG (Karlsruhe, Germany); UDPGA, (±)-propranolol, ( R )-propranolol, and (±)-4-hydroxypropranolol were obtained from Sigma-Aldrich (Taufkirchen, Germany); (±)-5-hydroxypropranolol was purchased from Cayman Chemical Company (Ann Arbor, MI, USA); the NADPH regeneration system was sourced from Promega Corporation (Madison, WI, USA); and 4-methoxypropranolol was synthesized in-house as described previously [ 30 , 31 , 32 ]. Ammonium hydrogen carbonate was purchased from VWR International GmbH (Darmstadt, Germany); and phosphate buffered saline (PBS, pH 7.4) was purchased from VWR Chemicals LLC (Solon, OH, USA).…”

“…However, the latter reaction is mainly catalyzed by CYP1A2, which also possesses a certain ability for ring hydroxylation [ 24 ]. In our previous study, propranolol was found to be glucuronidated by UGT1A7, UGT1A9, UGT1A10, and UGT2A1, while its metabolite 4-hydroxypropranolol was found to be converted by UGT1A7, UGT1A8, UGT1A9, and UGT2A1 [ 30 ]. The possible pathways of propranolol metabolism by CYP2D6 and these UGTs are illustrated in Figure 1 .…”