Repozytorium

Induced Fit Describes Ligand Binding to Membrane-Associated Cytochrome P450 3A4

Autorzy

David Tyler Sweeney

Francisco Zárate-Pérez

Kamila Stokowa-Sołtys

John C. Hackett

Rok wydania

2023

Czasopismo

Molecular Pharmacology

Numer woluminu

104

Strony

154-163

DOI

10.1124/molpharm.123.000698

Kolekcja

Naukowa

Język

Angielski

Typ publikacji

Artykuł

Streszczenie

Cytochrome P450 3A4 (CYP3A4) is the dominant P450 involved in human xenobiotic metabolism. Competition for CYP3A4 therefore underlies several adverse drug–drug interactions. Despite its clinical significance, the mechanisms CYP3A4 uses to bind diverse ligands remain poorly understood. Highly monodisperse CYP3A4 embedded in anionic lipoprotein nanodiscs containing an equal mixture of 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1’-rac-glycerol) were used to determine which of the limiting kinetic schemes that include protein conformational change, conformational selection (CS) or induced fit (IF), best described the binding of four known irreversible inhibitors. Azamulin, retapamulin, pleuromutilin, and mibrefadil binding to CYP3A4 nanodiscs conformed to a single-site binding model. Exponential fits of stopped-flow UV-visible absorption spectroscopy data supported multiple-step binding mechanisms. Trends in the rates of relaxation to equilibrium with increasing ligand concentrations were ambiguous as to whether IF or CS was involved; however, global fitting and consideration of the rate constants favored an IF mechanism. In the case of mibrefadil, a transient complex was observed in the stopped-flow UV-visible experiment, definitively assigning the presence of IF in ligand binding. While these studies only consider a small region of CYP3A4’s vast ligand space, they provide kinetic evidence that CYP3A4 can use an IF mechanism. CYP3A4 is capable of oxidizing numerous xenobiotics, including many drugs. Such promiscuity could not be achieved without conformational changes to accommodate diverse substrates. It is unknown whether conformational heterogeneity is present before (conformational selection) or after (induced fit) ligand binding. Stopped-flow measurements of suicide inhibitors binding to nanodisc-embedded CYP3A4 combined with sophisticated numerical analyses support that induced fit better describes ligand binding to this important enzyme.

Słowa kluczowe

AZM, azamulin, BRC, bromocriptine, CYP3A4, cytochrome P450 3A4, MIB, mibefradil, MDZ, midazolam, MSP1D1, membrane scaffoid protein 1D1, PLM, pleuromutilin, POPC, 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine, POPG, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol), radius of gyration, RPM, retapamulin, TST, testosterone

Adres publiczny

http://dx.doi.org/10.1124/molpharm.123.000698

Strona internetowa wydawcy

http://molpharm.aspetjournals.org

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