Article Figures & Data
Figures
- Fig. 1.
Structures of PM and PIA. PM (left) and PIA (right) differ by the electrophilic group attached to a pyrene core. Arrows indicate the flow of electrons from an attacking cysteine thiolate anion nucleophile.
- Fig. 2.
Inactivation of CYP3A4 by PM and PIA. A solution containing CYP3A4 Supersomes in phosphate buffer was preincubated with PM or PIA (50 µM) for various times. After preincubation, mixture solutions were 20-fold diluted into a secondary incubation containing 10 µM midazolam. 1-hydroxymidazolam formation was monitored, and the activity was calculated as a percentage of the product formation observed at the 0-minute preincubation time point. The mean of transformed data for relative reaction rate of triplicate determinations ± S.E. is shown. Circles and squares denote treatment by PM and PIA, respectively.
- Fig. 3.
Alkylation of CYP3A4 apo-protein by PM and PIA. Purified recombinant CYP3A4 (0.5 µM) was reacted with of PIA or PM (5 µM) in separate tubes for 10 minutes. The samples were analyzed by high-resolution mass spectrometry, and resultant deconvoluted spectra are shown in the chromatogram for PM (top panel) and PIA (bottom panel).
- Fig. 4.
Effect of small molecule treatment on CYP3A4 CO binding capacity. (A) Incubations containing CYP3A4 Supersomes were treated with PM (50 µM). After incubation of CYP3A4 with PM for 0, 0.5, 2, and 5 minutes at 37°C, CO binding was measured using UV-visible difference spectra as described in Materials and Methods. (B) Incubation CYP3A4 Supersomes with PIA (50 µM). After incubation of CYP3A4 with PM for 0, 5, 10, and 20 minutes, CO binding was measured using UV-visible difference spectra as described in Materials and Methods. (C) Incubation CYP3A4 Supersomes with either ABT or raloxifene in the presence of NADPH. Reaction was initiated by the addition of NADPH and incubated for 30 minutes at 37°C.
- Fig. 5.
Quantitative assessment of intact heme loss after CYP3A4 treatment with PM and PIA. CYP3A4 Supersomes (4 pmol) were incubated with vehicle (0.5% DMSO), PM (50 µM), PIA (50 µM), ABT (200 µM), and raloxifene (100 µM) as described in the Materials and Methods section. After an incubation of 0, 1, 2, 5, 10, and 20 minutes, aliquots were quenched and extracted for LC-UV-MS/MS analysis. The percentage of intact heme remaining after treatment is shown. Points and error bars represent the mean of triplicate experiments ± S.E.
- Fig. 6.
Free heme release from the CYP3A4 after PM and PIA treatment. CYP3A4 Supersomes (40 pmol/ml, without b5) were incubated with vehicle (0.5% DMSO), PM (50 µM), and PIA (50 µM) in separate incubations. The incubations were quenched, and the total and free heme were differentially extracted as described in Materials and Methods. Left panel shows the time course of total intact heme, whereas the right panel shows the percentage of heme released into solution over time. Points and error bars represent the mean of triplicate experiments ± S.E.
- Fig. 7.
Effect of PIA and PM pretreatment on recombinant 3A5 activity and carbon monoxide binding spectra. (A) A solution containing CYP3A5 Supersomes in phosphate buffer was preincubated with PM or PIA (50 µM) for various incubation times. After preincubation, mixture solutions were 20-fold diluted into a secondary incubation containing 10 µM midazolam. Formation of 1-hydroxymidazolam was monitored, and the activity was calculated as a percentage of the product formation observed relative to vehicle-treated control at each time point. Transformed data for relative reaction rate of triplicate determinations ± S.E. is shown. Circles and squares denote treatment by PM and PIA, respectively. (B) Incubations containing CYP3A5 Supersomes were treated with PM (50 µM, red line), PIA (50 µM, blue line), or DMSO (green line) for 5 minutes at 37°C. CO binding was measured using UV-visible spectrometer difference spectra scanning from 400 to 500 nm as described in Materials and Methods.
Tables
- TABLE 1
Effect of PIA and PM pretreatment on recombinant CYP3A4 onset Tagg
Values represent means of duplicate determinations.
Treatment Onset Tagg (°C) DMSO 48.8 Pyrene 48.6 Pyrene iodoacetamide 48.3 Pyrene maleimide 36.0
Additional Files
Data Supplement
- Supplemental Figure -
Supplementary Figure 1 - Representative aggregation curves for four treatments of recombinant CYP3A4 (as measured by scattering intensity at 266 nm) over a thermal ramp.
- Supplemental Figure -
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