Kinetics of cytochrome P450 3A4 inhibition by heterocyclic drugs defines a general sequential multistep binding process

Abstract

Cytochrome P450 (P450) 3A4 is the enzyme most involved in the metabolism of drugs and can also oxidize numerous steroids. This enzyme is also involved in one-half of pharmacokinetic drug–drug interactions, but details of the exact mechanisms of P450 3A4 inhibition are still unclear in many cases. Ketoconazole, clotrimazole, ritonavir, indinavir, and itraconazole are strong inhibitors; analysis of the kinetics of reversal of inhibition with the model substrate 7-benzoyl quinoline showed lag phases in several cases, consistent with multiple structures of P450 3A4 inhibitor complexes. Lags in the onset of inhibition were observed when inhibitors were added to P450 3A4 in 7-benzoyl quinoline O -debenzylation reactions, and similar patterns were observed for inhibition of testosterone 6β-hydroxylation by ritonavir and indinavir. Upon mixing with inhibitors, P450 3A4 showed rapid binding as judged by a spectral shift with at least partial high-spin iron character, followed by a slower conversion to a low-spin iron–nitrogen complex. The changes were best described by two intermediate complexes, one being a partial high-spin form and the second another intermediate, with half-lives of seconds. The kinetics could be modeled in a system involving initial loose binding of inhibitor, followed by a slow step leading to a tighter complex on a multisecond time scale. Although some more complex possibilities cannot be dismissed, these results describe a system in which conformationally distinct forms of P450 3A4 bind inhibitors rapidly and two distinct P450–inhibitor complexes exist en route to the final enzyme–inhibitor complex with full inhibitory activity.

Stepwise binding of inhibitors to human cytochrome P450 17A1 and rapid kinetics of inhibition of androgen biosynthesis

“…Both ketoconazole and clotrimazole, when mixed with P450 17A1, showed a rapid blue (hypsochromic) shift of the Soret band, followed by a slower red shift of the initial spectrum ( Figs. 4 and 5 ) to higher wavelength in the final “type II” complex ( 38 ), as reported for P450 3A4 ( 33 ). The completion of the changes required ∼20 s in the case of ketoconazole ( Fig.…”

“…A lag phase in the aforementioned rescue experiments would be consistent with the need for a conformational change associated with enzyme release and binding but would not necessarily prove its existence, as has been pointed out earlier ( 33 ). A very tightly bound inhibitor can also show such lag phases in simple models, for example, Figures 2 and 3 of Ref.…”

“…Three other P450 3A4 inhibitors that we studied previously ( 33 )—itraconazole, ritonavir, and indinavir—did not show strong enough spectral interaction with P450 17A1 to pursue these studies. (These were not tested for inhibition of enzyme activity.)…”

“…In recent work with P450 3A4 and five classic and clinically important inhibitor drugs, we demonstrated a stepwise process in which the inhibitors bound to the enzyme ( 33 ), expanding on some previous kinetic studies ( 34 ). In that work, we concluded that the inhibitors did not achieve maximum inhibition until the series of steps was completed.…”

“…We examined P450 17A1 reactions ( Fig. 1 ) with ketoconazole and clotrimazole, two of the drugs used in the P450 3A4 study ( 33 ), and abiraterone, in light of the report of Cheong et al. ( 30 ), which indicated a t 1/2 of ∼30 min for development of inhibition.…”