Heme Modification Contributes to the Mechanism-Based Inactivation of Human Cytochrome P450 2J2 by Two Terminal Acetylenic Compounds

Abstract

The mechanism-based inactivation of human CYP2J2 by three terminal acetylenic compounds: N-(methylsulfonyl)-6-(2-propargyloxyphenyl)hexanamide (MS), 17-octadecynoic acid (OD), and danazol (DZ) was investigated. The loss of hydroxyebastine (OHEB) carboxylation activity in a reconstituted system was time- and concentration-dependent and required NADPH for MS and OD, but not DZ. The kinetic constants for the mechanism-based inactivation of OHEB carboxylation activity were: K_I of 6.1 μM and k_inact of 0.22 min^-1 for MS and K_I of 2.5 μM and k_inact of 0.05 min^-1 for OD. The partition ratios for MS and OD were ∼10 and ∼20, respectively. Inactivation of CYP2J2 by MS or OD resulted in a loss of the native heme spectrum and a similar decrease in the reduced CO difference spectrum. A heme adduct was observed in the MS-inactivated CYP2J2. The possible reactive metabolite which covalently modified the prosthetic heme was characterized by analysis of the glutathione conjugates formed by MS or OD following oxygenation of the ethynyl moiety. Liquid chromatography-mass spectrometry showed that inactivation by MS or OD did not lead to modification of apoprotein. Interaction of CYP2J2 with DZ produced a type II binding spectrum with a K_s of 2.8 μM and the IC₅₀ for loss of OHEB carboxylation activity was 0.18 μM. In conclusion, heme modification by MS and OD was responsible for the mechanism-based inactivation of CYP2J2. The results suggest that the ethynyl moiety of MS and OD faces the heme iron, whereas the isoxazole ring of DZ is preferentially oriented toward the heme iron of CYP2J2.

Fig. 1.

Chemical structures of compounds characterized in this study. (A) the three acetylenes: MS, OD and DZ. (B) prodrug EB and its two metabolites OHEB and CEB.

Fig. 2.

Metabolism of EB and OHEB by CYP2J2. (A) HPLC elution profile for the metabolites formed from EB in the CYP2J2 reconstituted system incubated at 37°C for 5 minute. (B) Kinetics for the formation of CEB from OHEB. The CYP2J2 reconstituted systems were incubated with increasing concentrations of OHEB from 0.625 to 40 μM at 37°C for 10 minute. The data for k_cat versus the OHEB concentrations were fitted to the Michaelis-Menten equation as described under Materials and Methods. (C) HPLC elution profile for the formation of CEB from OHEB by CYP2J2 or CYP3A4 in the reconstituted system following incubation at 37°C for 30 minute.

Fig. 3.

Time- and concentration-dependent loss of OHEB carboxylation activity and determination of the partition ratio for CYP2J2 during inactivation by MS. (A) CYP2J2 in the reconstituted system was incubated with 0 (●), 1 (○), 3 (▴), 5 (Δ), 10 μM (▪), 20 (□), or 100 μM (▾) MS and aliquots were removed at the times indicated and assayed for residual OHEB metabolism as described under Materials and Methods. The catalytic activity at time zero was used as the 100% control to calculate the initial rate constants for the inactivation (k_obs) for each concentration of MS. (B) Shows the fitting of the initial rate constants as a function of the MS concentrations to the Michaelis-Menten equation. The K_I and k_inact were determined to be 6.1 μM and 0.22 min⁻¹, respectively. (C) Determination of the partition ratio for the inactivation of CYP2J2 by MS. The percentage of catalytic activity remaining was determined as a function of the molar ratio of MS to CYP2J2 as described under Materials and Methods. The partition ratio (∼10) was estimated from the intercept of the linear regression line from the lower ratios of MS to CYP2J2 and the straight line obtained from higher ratios of MS to CYP2J2. The data represent the average of two separate experiments done in duplicate.

Fig. 4.

Modification of the heme prosthetic group of CYP2J2 by MS. (A) HPLC elution profiles monitored at 400 nm for prosthetic heme in the absence or presence of NADPH. Native heme loss and heme adduct formation were observed in the +NADPH sample. (B) Photodiode-array spectrum of the native heme eluting at 26 minute. (C) Photodiode-array spectrum of the heme adduct eluting at 30 minute. (D) The reduced CO difference spectrum of the reconstituted system incubated with MS in the absence and presence of NADPH. The experimental procedures are described under Materials and Methods.

Fig. 5.

Time- and concentration-dependent loss of OHEB carboxylation and determination of the partition ratio for CYP2J2 during inactivation by OD. (A) CYP2J2 in the reconstituted system was incubated with 0 (●), 1 (○), 3 (▴), 20 (Δ), 50 (▪), and 100 μM (□) OD and aliquots were removed at the times indicated and assayed for residual OHEB metabolism as described under Materials and Methods. The catalytic activity at time zero was used as the 100% control to calculate the initial rate constants for the inactivation (k_obs) for each concentration of OD. (B) Shows the fitting of the initial rate constants for the inactivation as a function of the OD concentrations to the Michaelis-Menten equation. The K_I and k_inact values were determined to be 2.5 μM and 0.05 min⁻¹, respectively. (C) Determination of the partition ratio for the inactivation of CYP2J2 by OD. The percentage of catalytic activity remaining was determined as a function of the molar ratio of OD to CYP2J2 as described under Materials and Methods. The partition ratio (∼20) was estimated from the intercept of the linear regression line from the lower ratios of OD to CYP2J2 and the straight line obtained from higher ratios of OD to CYP2J2. The data represent the average of two separate experiments.

Fig. 6.

Change in the CYP2J2 native heme due to the inactivation by OD. (A) HPLC elution profiles monitored at 400 nm for the prosthetic heme in the −NADPH and +NADPH samples. (B) The reduced CO difference spectra of the reconstituted system incubated with OD in the absence and presence of NADPH. The experimental procedures are described under Materials and Methods.

Fig. 7.

LC-MS/MS analysis of the GSH conjugate formed during the metabolism of MS. A reaction mixture containing CYP2J2 was incubated with MS in the presence of NADPH and the reactive intermediate was trapped with 10 mM GSH. The GSH conjugate was analyzed by LC-MS as described under Materials and Methods. (A) The full mass spectrum of the MS-GSH conjugate with the MH⁺ ion at m/z 647. (B) The MS/MS spectrum of the MS-GSH conjugate. (C) The proposed structure of the MS-GSH conjugate. The dashed lines indicate the sites of fragmentation. The MS/MS spectrum was obtained in the positive mode and analyzed using the Xcalibur software package.

Fig. 8.

LC-MS/MS analysis of the GSH conjugate formed during the metabolism of OD. A reaction mixture containing CYP2J2 was incubated with OD in the presence of NADPH and the reactive intermediate was trapped with 2 mM GSH. The GSH conjugate was analyzed by LC-MS as described under Materials and Methods. (A) The full mass spectrum of the OD-GSH conjugate with the MH⁺ ion at m/z 603. (B) The MS/MS spectrum of the OD-GSH conjugate. (C) The proposed structure of the OD-GSH conjugate. The dashed lines indicate the sites of fragmentation. The MS/MS spectrum was obtained in the positive mode and analyzed using the Xcalibur software package.

Fig. 9.

Optical spectra for the binding of DZ to CYP2J2. (A) DZ binding to CYP2J2 was performed as described in Materials and Methods and resulted in a type II binding spectrum as shown here. Inset, the difference in the absorbance between the peak and the trough of each spectrum plotted versus the corresponding DZ concentration. The data points were fitted to the Michaelis-Menten equation. The K_s was estimated to be 2.8 μM DZ. (B) Determination of the IC₅₀ for DZ inhibition of the carboxylation of 20 μM OHEB as described in Materials and Methods. The data were fitted to the dose-response equation as described under Materials and Methods.