Conformational dynamics of CYP3A4 demonstrate the important role of Arg212 coupled with the opening of ingress, egress and solvent channels to dehydrogenation of 4-hydroxy-tamoxifen

Abstract

Background: Structure-based methods for P450 substrates are commonly used during drug development to identify sites of metabolism. However, docking studies using available X-ray structures for the major drug-metabolizing P450, CYP3A4, do not always identify binding modes supportive of the production of high-energy toxic metabolites. Minor pathways such as P450-catalyzed dehydrogenation have been experimentally shown to produce reactive products capable of forming biomolecular adducts which can lead to increased risk toxicities. 4-Hydroxy-tamoxifen (4OHT) is metabolized by CYP3A4 via competing hydroxylation and dehydrogenation reactions.

Methods: Ab initio gas-phase electronic structural characterization of 4OHT was used to develop a docking scoring scheme. Conformational sampling of CYP3A4 with molecular dynamics simulations along multiple trajectories were used to generate representative structures for docking studies using recently published heme parameters. A key predicted binding mode was tested experimentally using site-directed mutagenesis of CYP3A4 and liquid chromatography-mass spectroscopy analysis.

Results: Docking with MD-refined CYP3A4 structures incorporating hexa-coordinate heme parameters identifies a unique binding mode involving ARG212 and channel 4, unobserved in the starting PDB ID: 1TQN X-ray structure. The models supporting dehydrogenation are consistent with results from in vitro incubations.

General significance: Our models indicate that coupled structural contributions of the ingress, egress and solvent channels to the CYP3A4 active site geometries play key roles in the observed 4OHT binding modes. Thus adequate sampling of the conformational space of these drug-metabolizing promiscuous enzymes is important for substrates that may bind in malleable regions of the enzyme active-site.

Figure 1

Scheme for one putative pathway of P450 mediated bioactivation of tamoxifen (TAMX) to estrogen-receptor (ER) active 4-hydroxy-tamoxifen via P450-catalyzed hydroxylation, followed by conversion to 4-hydroxy-tamoxifen quinone methide (4OHT-qm) which then forms an adduct with a biomolecule (X) via an electrophilic attack.

Figure 2

Atomic numbering and dihedral labeling scheme for 4-hydroxy-tamoxifen.

Figure 3

Four (A–D) different energy minimized geometries of 4-hydroxy tamoxifen identified at the B3LYP/6–31G* level of theory.

Figure 4

Scheme for alternate hydrogen abstraction pathways for 4OHT intermediates along the dehydrogenation reaction coordinate. Differences in the sum of electronic and thermal free energies for each species are shown relative to that of the lowest energy equivalent intermediate in black. Electron affinities are shown where applicable as relative energy to previous neutral intermediate in the same pathway in parentheses in red. All energies given in units of kcal/mol.

Figure 5

Final bond dissociation energies of carbon-hydrogen and oxygen-hydrogen bonds involved in the dehydrogenation of 4OHT at 4 Å extension for all intermediates on the dehydrogenation reaction coordinate: (A) first hydrogen abstraction, (B) second hydrogen abstraction. The legend indicates the hydrogen abstracted and for (B), the intermediate and the hydrogen abstracted.

Figure 6

Ratio of Autodock3 binding modes supportive of observed metabolism of 4-hydroxy-tamoxifen reaction mechanisms alpha-hydroxylation (α-OH) dehydrogenation modes 1 and 2 (dH1 & dH2), N-demethylation (NdM) and nonproductive and/or ambiguous modes (Non) with CYP3A4 experimentally derived x-ray (A) PDB ID: 1W0E, (B) PDB ID: 1TQN and (C) molecular dynamics refined m2 refined with quantum mechanics based heme parameters for resting high-spin (ic6) and Compound-I (cpdi) with (wq) and without (00) RESP charges assigned to the heme. Statistically significant differences are indicated with symbols: †, ‡ and *.

Figure 7

Active site and major channels (red grid) for structure m2-cpdi (cyan) and w0e-cpdi (yellow) shown with orientation of representative poses scored for different reaction mechanisms (A) α-hydroxylation [αOH], (B) dehydrogenation [dH2], (C) N-demethylation [NdM] and (D) dehydrogenation [dH1]. Volumes defining active-site and ingress/egress channels are shown as red mesh and labeled S (solvent), 2b, 2e and 4 channels. The 1w0e-cpdi and m2-cpdi model are shown at slightly different angles, and the peptide backbone in the foreground for all pictures has been hidden to allow the differences in the structure and orientation of channels and substrate to be seen more clearly. Active-site mapping performed with UCSF HOLLOW, figures generated with PYMOL.

Figure 8

Differences in channel 4 and position of ARG212. (A) Volume and placement of channel 4 as defined by CAVER shown as a surface for each model m2 (lime green), m3 (purple), m5 (orange) and 2V0M (yellow). The CYP3A4 backbone is shown for the m2 model as a grey ribbon, the F–G loop (which includes the F’ and G’ helices) is shown in red. (B) Position of ARG212 and heme for m2 (lime green), m3 (purple), m5 (orange) and 2V0M (yellow) shown in reference to docked conformation of 4OHT (cyan) in dH2 pose. Hydrogen atoms have been hidden for clarity. Neighboring residues to ARG212 are shown only as ribbon for the back bone and atoms hidden also for clarity. Image generated (A) with PYMOL, and (B) with UCSF CHIMERA.

Figure 9

4OHT relative dehydrogenation and hydroxylation 4OHT products of Wild-Type (WT) versus Mutant (R212A) CYP3A4. Statistical significance indicated with (*).