Article Figures & Data
Figures
- Fig. 1.
Comparative effect of acolbifene, bazedoxifene, lasofoxifene, tamoxifen, toremifene, ospemifene, droloxifene, clomifene, raloxifene, and arzoxifene on carbazeran 4-oxidation catalyzed by human liver cytosol. A SERM (25 μM), valproic acid (50 μM; negative control), or DMSO (1% v/v; vehicle) was coincubated with carbazeran (3 μM) and pooled liver cytosol (20 μg protein) at 37°C for 5 minutes. Data are expressed as percentage of activity in the vehicle-treated control group and expressed as mean ± S.E.M. of three independent experiments conducted in duplicate. *Significantly different from the vehicle-treated control group (P < 0.05). The rate of reaction in the vehicle-treated control group was 567 ± 18 pmol/min per milligram protein.
- Fig. 2.
Concentration–response relationship in the inhibition of human liver cytosolic AOX1-mediated carbazeran 4-oxidation by acolbifene, bazedoxifene, lasofoxifene, tamoxifen, and raloxifene. Pooled liver cytosol (20 μg protein) was incubated with carbazeran (3 μM) and varying concentrations of (A) acolbifene (0.3–300 μM), (B) bazedoxifene (0.0003–25 μM), (C) lasofoxifene (0.0003–30 μM), (D) tamoxifen (0.003–100 μM), (E) raloxifene (0.00001–1 μM), or DMSO (1% v/v; vehicle) at 37°C for 5 minutes. Data are expressed as percentage of activity in the vehicle-treated control group and expressed as mean ± S.E.M. of three or four independent experiments conducted in duplicate or triplicate. *Significantly different from the vehicle-treated control group (P < 0.05).
- Fig. 3.
Lineweaver–Burk plots for inhibition of human liver cytosolic AOX1-mediated carbazeran 4-oxidation by bazedoxifene, lasofoxifene, tamoxifen, and raloxifene. Pooled liver cytosol (20 μg protein) was incubated with carbazeran (0.5, 1, 2, or 4 μM) and varying concentrations of (A) bazedoxifene (0, 0.1, 0.3, or 1 μM), (B) lasofoxifene (0, 0.1, 0.3, or 1 μM), (C) tamoxifen (0, 1, 3, or 10 μM), or (D) raloxifene (0, 0.03, 0.06, or 0.1 μM) at 37°C for 5 minutes. Data are expressed as percentage of activity in the vehicle-treated control group and expressed as mean ± S.E.M. of four to five independent experiments conducted in duplicate.
- Fig. 4.
Inhibition of human liver cytosolic AOX1-mediated carbazeran 4-oxidation by metabolite/structural analogs of bazedoxifene. (A) Pooled liver cytosol (20 μg protein) was incubated with carbazeran (3 μM) and bazedoxifene, bazedoxifene N-oxide, des(1-azepanyl)ethylbazedoxifene (each at 25 μM), valproic acid (50 μM; negative control), or DMSO (1% v/v; vehicle) at 37°C for 5 minutes. (B and C) Pooled liver cytosol (20 μg protein) was incubated with carbazeran (3 μM) and varying concentrations of (B) bazedoxifene N-oxide (0.0003–25 μM), (C) des(1-azepanyl)ethylbazedoxifene (0.0003–25 μM), or DMSO (1% v/v; vehicle) at 37°C for 5 minutes. Data are expressed as percentage of activity in the vehicle-treated control group and expressed as mean ± S.E.M. of three to five independent experiments conducted in duplicate. *Significantly different from the vehicle-treated control group (P < 0.05).
- Fig. 5.
Inhibition of human liver cytosolic AOX1-mediated carbazeran 4-oxidation by the tetrahydronaphthalene class of SERMs. (A) Pooled liver cytosol (20 μg protein) was incubated with carbazeran (3 μM) and lasofoxifene, 7-methoxylasofoxifene, cis-4-(1,2,3,4-tetrahydro-6-methoxy-2-phenyl-1-naphthalenyl)phenol, nafoxidine (each at 25 μM), valproic acid (50 μM; negative control), or DMSO (1% v/v; vehicle) at 37°C for 5 minutes. (B–D) Pooled liver cytosol (20 μg protein) was incubated with carbazeran (3 μM) and varying concentrations of (B) 7-methoxylasofoxifene (0.003–100 μM), (C) cis-4-(1,2,3,4-tetrahydro-6-methoxy-2-phenyl-1-naphthalenyl)phenol (0.03–30 μM), (D) nafoxidine (0.003–100 μM), or DMSO (1% v/v; vehicle) at 37°C for 5 minutes. Data are expressed as percentage of activity in the vehicle-treated control group and expressed as mean ± S.E.M. of three to seven independent experiments conducted in duplicate. *Significantly different from the vehicle-treated control group (P < 0.05). #Significantly different from the vehicle-treated control group and the lasofoxifene-treated group.
- Fig. 6.
Molecular docking of SERMs and their structural analogs to the active site of human AOX1. The predicted binding of the 10 compounds is shown with the molybdenum cofactor [MOS, dioxothiomolybdenum (VI) ion] visible toward the center of each frame and the distance between it and the ligand central oxygen atom shown in pink. The key residues are shown as labeled. Computed hydrogen bonds are shown in light blue.
- Fig. 7.
Correlation analysis of the experimentally derived IC50 values in the inhibition of human AOX1 by SERM and the in silico generated values in human AOX1-binding efficiency by SERM. Experimentally derived IC50 values are shown in Table 1. AOX1-binding efficiency values are shown in Table 4.
- Fig. 8.
Effect of estrone and estrone sulfate on inhibition of human liver cytosolic AOX1-mediated carbazeran 4-oxidation by bazedoxifene. (A) Pooled liver cytosol (20 μg protein) was incubated with carbazeran (3 μM) and varying concentrations of estrone (0.0003–30 μM), estrone sulfate (1–3000 μM), or DMSO (1% v/v; vehicle) at 37°C for 5 minutes. (B) Pooled liver cytosol (20 μg protein) was incubated with carbazeran (3 μM) and the combination of bazedoxifene (0.1 μM) with varying concentrations of estrone sulfate (30–1000 μM), estrone (0.03–10 μM), or DMSO (1% v/v; vehicle) at 37°C for 5 minutes. Data are expressed as percentage of activity in the vehicle-treated control group and expressed as mean ± S.E.M. of three or four independent experiments conducted in duplicate. *Significantly different from the vehicle-treated control group (P < 0.05). #Significantly different from the bazedoxifene-treated group (P < 0.05). The bazedoxifene (0.1 μM) group alone inhibited carbazeran 4-oxidation by 41% ± 3% in the estrone experiment and 38% ± 4% in the estrone sulfate experiment.
Tables
- TABLE 1
IC50 values and minimum inhibitory concentration in the inhibition of human liver cytosolic AOX1-mediated carbazeran 4-oxidation by SERMs and the structural analogs of bazedoxifene and lasofoxifene
Data are expressed as mean ± S.E.M. of three to seven independent experiments conducted in duplicate or triplicate.
Chemical Class Chemical IC50 (µM) Minimum Inhibitory Concentration (µM) Benzothiophene Raloxifene 0.028 ± 0.004 0.01 Indole Bazedoxifene 0.19 ± 0.02 0.03 Tetrahydronaphthalene Lasofoxifene 0.30 ± 0.02 0.03 Triphenylethylene Tamoxifen 7.30 ± 0.72 10 Benzopyran Acolbifene 29.5 ± 4.6a 30 Bazedoxifene and metabolites/analogs Indole Des(1-azepanyl)ethylbazedoxifene 0.10 ± 0.01b 0.003 Indole Bazedoxifene 0.19 ± 0.02 0.03 Indole Bazedoxifene N-oxide 0.29 ± 0.04b 0.003 Lasofoxifene and analogs Tetrahydronaphthalene Lasofoxifene 0.30 ± 0.02 0.03 Tetrahydronaphthalene 7-Methoxylasofoxifene 1.56 ± 0.21c 0.003 Tetrahydronaphthalene Cis-4-(1,2,3,4-tetrahydro-6-methoxy-2-phenyl-1-naphthalenyl)phenol 2.35 ± 0.24c 3 Tetrahydronaphthalene Nafoxidine 2.50 ± 0.23c 1 - TABLE 2
Apparent Ki values and mode of inhibition of human liver cytosolic AOX1-mediated carbazeran 4-oxidation by bazedoxifene, lasofoxifene, tamoxifen, and raloxifene
Data are expressed as mean ± S.E.M. for four or five independent experiments conducted in duplicate.
Chemical Class Chemical Apparent Ki (µM) Mode of Inhibition Ratio of Apparent Ki to Apparent Km Benzothiophene Raloxifene 0.028 ± 0.002 Noncompetitive (full) 0.0044 Tetrahydronaphthalene Lasofoxifene 0.14 ± 0.02 Competitive (full) 0.02 Indole Bazedoxifene 0.14 ± 0.02 Competitive (full) 0.02 Triphenylethylene Tamoxifen 2.78 ± 0.47a Competitive (full) 0.44 ↵a Significantly different from the raloxifene group (P < 0.05).
Experimental Rank (Based on IC50) Compound O-Mo Distancea(Å) Key Hydrogen Bonds Key Rings Key van der Waals Interactions 2 Bazedoxifene 4.453 Glu-888 Asn-1084 MOS Phe-885 Ala-919 Ile-1085 3 Lasofoxifene 4.729 Asn-1084 MOS — Ala-919 Ile-1085 4 Tamoxifen 6.143 — — Ile-1085 5 Acolbifene 7.667 Asn-1084 — Ile-1085 1 Raloxifene 10.039 Lys-893 Glu-1270 — — MOS, dioxothiomolybdenum (VI) ion.
↵a Distance between the ligand central oxygen atom and the molybdenum cofactor.
- TABLE 4
Docking analysis in the molecular interaction between human AOX1 and bazedoxifene, lasofoxifene, and their structural analogs
Experimental Rank (Based on IC50) Parent Drug and Analogs Log [IC50 (M)] Binding Efficiency Bazedoxifene and analogs 1 Des(1-azepanyl)ethylbazedoxifene −7.00 −0.938 2 Bazedoxifene −6.72 −0.897 3 Bazedoxifene N-oxide −6.54 −0.848 Lasofoxifene and analogs 1 Lasofoxifene −6.52 −0.797 2 7-Methoxylasofoxifene −5.81 −0.790 3 Cis-4-(1,2,3,4-tetrahydro-6-methoxy-2-phenyl-1-naphthalenyl)phenol −5.63 −0.761 4 Nafoxidine −5.60 −0.787
Data Supplement
- Supplemental Data -
Figures S1-S7 and Tables S1-S2.
- Supplemental Data -
