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Drug Metab Dispos. 2020 Mar 19. pii: dmd.119.089813. doi: 10.1124/dmd.119.089813. [Epub ahead of print]

Mechanistic Studies of Cytochrome P450 3A4 Time Dependent Inhibition Using Two Cysteine- Targeting Electrophiles.

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Experiments designed to identify the mechanism of cytochromes P450 inactivation are critical to the drug discovery. Small molecules irreversible inhibit P450 enzymatic activity via two primary mechanisms: apoprotein adduct formation or heme modification. Understanding the interplay between chemical structures of reactive electrophiles and the impact on CYP3A4 structure and function can ultimately provide insights into drug design to minimize P450 inactivation. In a previous study, raloxifene and N-(1-pyrene) iodoacetamide (PIA) alkylated cytochrome P450 3A4 (CYP3A4) in vitro, however only raloxifene influenced enzyme activity. Here, two alkylating agents with cysteine selectivity, PIA and pyrene maleimide (PM), were utilized to investigate protein alkylation and CYP3A4 activity. The compound's effect on 1) enzymatic activity, 2) carbon monoxide (CO) binding capacity, 3) intact heme content, and 4) protein conformation were measured. Results showed that PM had a large time-dependent loss of enzyme activity, whereas PIA did not. The differential effect on enzymatic activity between PM and PIA was mirrored in the CO binding data. Despite disruption of CO binding, neither compound affected the heme concentrations inferring there was no destruction or alkylation of the heme. Lastly, differential scanning fluorescence showed PM-treated CYP3A4 caused a shift in the onset temperature required to induce protein aggregation which was not observed for CYP3A4 treated with PIA. In conclusion, alkylation of CYP3A4 apoprotein can have a variable impact on catalytic activity, CO binding, and protein conformation that may be compound-dependent. These results highlight the need for careful interpretation of experimental results aimed at characterizing the nature of P450 enzyme inactivation with UV spectroscopy. SIGNIFICANCE STATEMENT: Understanding the mechanism of CYP3A4 time dependent inhibition is critical to the drug discovery. In this study, we use two cysteine-targeting electrophiles to probe how subtle variation in inhibitor structure may impact the mechanism of CYP3A4 time dependent inhibition and confound interpretation of traditional MBI diagnostic experiments. Ultimately, this simplified system was used to reveal insights into CYP3A4 biochemical behavior. The insights may have implications that aid in understanding the susceptibility of CYP enzymes to the effects of electrophilic intermediates generated via bioactivation.


cytochrome P450; drug design; drug development/discovery; enzyme inhibitors; enzyme mechanism; structure-activity relationships; translation/protein synthesis

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