TABLE 1

UGT enzyme assays for determination of IC50 values of HIV protease inhibitors

Enzymea
Substrates
[Subs]e
Analytical Standard
Detection
Control Inhibitors
[Inhibitor]
Percentage of inhibition
μM nm μM
UGT1A1b Bilirubin 5 Bilirubin 450 β-Estradiol 200 61.5
UGT1A3c β-Estradiol 40 β-Estradiol glucuronide 280 2-Hydroxy-estradiol 100 38.5
UGT1A4d Trifluoperazine 60 Trifluoperazine 256 Hecogenin 50 48.0
UGT1A6d HFC 50 HFC glucuronide 325 Naphthol 50 87.0
UGT1A9d HFC 10 HFC glucuronide 325 Propofol 50 37.0
UGT2B7d
HFC
50
HFC glucuronide
325
Eugenol
100
54.5

  • HFC, 7-hydroxy trifluoromethyl coumarin.

  • ↵ a Human cDNA-expressed UGT1A1 in baculovirus-infected insect cells.

  • ↵ b Buffer = 50 mM sodium citrate, pH 7.5; enzyme proteins = 0.2 mg/ml; time = 30 min.

  • ↵ c Buffer = 50 mM Tris-HCl, pH 7.5; enzyme proteins = 1 mg/ml; time = 30 min.

  • ↵ d Buffer = 50 mM Tris-HCl, pH 7.5; enzyme proteins = 0.4 mg/ml; time = 20 min (except 10 min for UGT1A6).

  • ↵ e Literature-apparent Km values are 5, 39, 61, 321, 2.3, and 161 μM, respectively, for these reactions.