Human cytochrome P450 enzymes bind drugs and other substrates mainly through conformational-selection modes

Guengerich, F. Peter; Wilkey, Clayton J.; Phan, Thanh T.N.

doi:10.1074/jbc.ra119.009305

Cited by 44 publications

(37 citation statements)

References 84 publications

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“…An initial phase to recognize the tunnel entrance followed by a temporary superficial association, as we observed it, stands in agreement with a model describing a two-step binding process allowing kinetically efficient ligand uptake. This would enable the ligand to efficiently minimize the, otherwise even longer, recognition phase and is in accordance with recent observations regarding proteins with similarly buried active sites such as CYP101A1 and nuclear receptors 11,21,27–30 . In the active site, eight out of ten accessing ligands adopted a pose which would allow an oxidation reaction to proceed at a site of metabolism (SOM) that would ultimately result in a metabolite in agreement with experiment 31 .…”

Section: Resultssupporting

confidence: 90%

“…This points towards an induced-fit mechanism as opposed to conformational selection 33 . Only recently the latter was proposed to be the main mechanism for multiple CYPs including CYP2D6 30 , even though for several other CYPs induced-fit scenarios were not ruled out. Besides movements of the FG loop, we observed helix A, the BC loop, the HI loop, and helix B to be involved in conformational changes (Table S8).…”

Section: Resultsmentioning

confidence: 99%

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Spontaneous Ligand Access Events to Membrane-Bound Cytochrome P450 2D6 Sampled at Atomic Resolution

Fischer

Smieško

2019

Sci Rep

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The membrane-anchored enzyme Cytochrome P450 2D6 (CYP2D6) is involved in the metabolism of around 25% of marketed drugs and its metabolic performance shows a high interindividual variation. While it was suggested that ligands access the buried active site of the enzyme from the membrane, no proof from unbiased simulations has been provided to support this hypothesis. Laboratory experiments fail to capture the access process which is suspected to influence binding kinetics. Here, we applied unbiased molecular dynamics (MD) simulations to investigate the access of ligands to wild-type CYP2D6, as well as the allelic variant CYP2D6*53. In multiple simulations, substrates accessed the active site of the enzyme from the protein-membrane interface to ultimately adopt a conformation that would allow a metabolic reaction. We propose the necessary steps for ligand access and the results suggest that the increased metabolic activity of CYP2D6*53 might be caused by a facilitated ligand uptake.

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Section: Resultssupporting

confidence: 90%

Section: Resultsmentioning

confidence: 99%

Spontaneous Ligand Access Events to Membrane-Bound Cytochrome P450 2D6 Sampled at Atomic Resolution

Fischer

Smieško

2019

Sci Rep

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“…In contrast to several other P450 3A4 substrates ( 33 , 38 ), the binding of 7-OBz quinoline was rapid, with >90% of the change finished in the first 200 ms ( Fig. S1 C ).…”

Section: Resultsmentioning

confidence: 88%

“…Although the kinetics of the spectral changes of inhibitor binding could be modeled in terms of three steps ( 34 ), there are deficiencies in the system. We subsequently showed that substrate binding to P450 3A4 is dominated by a “conformational selection” model, as opposed to induced fit ( 38 ), as in the case of P450 17A1 ( 39 ). The results indicate that multiple species of P450 3A4 are in equilibrium in the absence of ligand, and one (or more) of these conformations then binds the ligand.…”

mentioning

confidence: 96%

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

Guengerich

McCarty

Chapman

2021

Journal of Biological Chemistry

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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.

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“…Recent kinetic data indicate that several promiscuous drug‐metabolizing CYPs and some substrate‐specific CYPs utilize CF . Included in this work are CYP–ligand combinations for which the ligand‐bound crystal structure is conformationally distinct from the ligand‐free CYP structure.…”

Section: Mechanisms Of Promiscuitymentioning

confidence: 99%

Mechanisms of promiscuity among drug metabolizing enzymes and drug transporters

Atkins

2019

The FEBS Journal

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Detoxication, or ‘drug‐metabolizing’, enzymes and drug transporters exhibit remarkable substrate promiscuity and catalytic promiscuity. In contrast to substrate‐specific enzymes that participate in defined metabolic pathways, individual detoxication enzymes must cope with substrates of vast structural diversity, including previously unencountered environmental toxins. Presumably, evolution selects for a balance of ‘adequate’ kcat/KM values for a wide range of substrates, rather than optimizing kcat/KM for any individual substrate. However, the structural, energetic, and metabolic properties that achieve this balance, and hence optimize detoxication, are not well understood. Two features of detoxication enzymes that are frequently cited as contributions to promiscuity include the exploitation of highly reactive versatile cofactors, or cosubstrates, and a high degree of flexibility within the protein structure. This review examines these intuitive mechanisms in detail and clarifies the contributions of the classic ligand binding models ‘induced fit’ (IF) and ‘conformational selection’ (CS) to substrate promiscuity. The available literature data for drug metabolizing enzymes and transporters suggest that IF is exploited by these promiscuous detoxication enzymes, as it is with substrate‐specific enzymes, but the detoxication enzymes uniquely exploit ‘IFs’ to retain a wide range of substrates at their active sites. In contrast, whereas CS provides no catalytic advantage to substrate‐specific enzymes, promiscuous enzymes may uniquely exploit it to recruit a wide range of substrates. The combination of CS and IF, for recruitment and retention of substrates, can potentially optimize the promiscuity of drug metabolizing enzymes and drug transporters.

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Human cytochrome P450 enzymes bind drugs and other substrates mainly through conformational-selection modes

Cited by 44 publications

References 84 publications

Spontaneous Ligand Access Events to Membrane-Bound Cytochrome P450 2D6 Sampled at Atomic Resolution

Spontaneous Ligand Access Events to Membrane-Bound Cytochrome P450 2D6 Sampled at Atomic Resolution

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

Mechanisms of promiscuity among drug metabolizing enzymes and drug transporters

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