Characterization of the active site properties of CYP4F12

Article abstract of DOI:10.1124/dmd.114.059626

Cytochrome P450 4F12 is a drug-metabolizing enzyme that is primarily expressed in the liver, kidney, colon, small intestine, and heart. The properties of CYP4F12 that may impart an increased catalytic selectivity (decreased promiscuity) were explored through in vitro metabolite elucidation, kinetic isotope effect experiments, and computational modeling of the CYP4F12 active site. By using astemizole as a probe substrate for CYP4F12 and CYP3A4, it was observed that although CYP4F12 favored astemizole O-demethylation as the primary route of metabolism, CYP3A4 was capable of metabolizing astemizole at multiple sites on the molecule. Deuteration of astemizole at the site of O-demethylation resulted in an isotope effect of 7.1 as well as an 8.3-fold decrease in the rate of clearance for astemizole by CYP4F12. Conversely, although an isotope effect of 3.8 was observed for the formation of the O-desmethyl metabolite when deuterated astemizole was metabolized by CYP3A4, there was no decrease in the clearance of astemizole. Development of a homology model of CYP4F12 based on the crystal structure of cytochrome P450 BM3 predicted an active site volume for CYP4F12 that was approximately 76% of the active site volume of CYP3A4. As predicted, multiple favorable binding orientations were available for astemizole docked into the active site of CYP3A4, but only a single binding orientation with the site of O-demethylation oriented toward the heme was identified for CYP4F12. Overall, it appears that although CYP4F12 may be capable of binding similar ligands to other cytochrome P450 enzymes such as CYP3A4, the ability to achieve catalytically favorable orientations may be inherently more difficult because of the increased steric constraints of the CYP4F12 active site. Copyright

Full text of DOI:10.1124/dmd.114.059626

DMD Fast Forward. Published on July 29, 2014 as doi:10.1124/dmd.114.059626
Title Page
Characterization of the Active Site Properties of CYP4F12
John Eksterowicz, Dan A. Rock, Brooke M. Rock, Larry C. Wienkers and Robert S. Foti
Amgen Molecular Structure and Characterization, South San Francisco, CA, 94080
(J.E.)
Amgen Pharmacokinetics and Drug Metabolism, Seattle, WA, 98119
(D.A.R., B.M.R., L.C.W., R.S.F.)
Copyright 2014 by the American Society for Pharmacology and Experimental Therapeutics.

Running Title Page:
CYP4F12 Active Site Characterization
Corresponding author:
Robert S. Foti
Pharmacokinetics and Drug Metabolism
Amgen, Inc.
1201 Amgen Court West
Mail Stop AW2/D3205
Seattle, WA 98119
Phone: (206)265-7164
FAX: (206)217-2651
rfoti@amgen.com
Manuscript metrics:
Text pages: 19
Tables: 1 (+1 supplemental)
Figures: 7
References: 64
Words in the abstract: 266
Words in the introduction: 483
Words in the discussion: 1575
Abbreviations: P450, cytochrome P450; NADPH, nicotinamide adenine dinucleotide
phosphate, reduced; fm, fraction metabolized.

Abstract
Cytochrome P450 4F12 is a drug metabolizing enzyme that is primarily expressed in the
liver, kidney, colon, small intestine and heart. The properties of CYP4F12 that may impart an
increased catalytic selectivity (decreased promiscuity) were explored through in vitro metabolite
elucidation, kinetic isotope effect experiments and computational modeling of the CYP4F12
active site. Using astemizole as a probe substrate for CYP4F12 and CYP3A4, it was observed
that while CYP4F12 favored astemizole O-demethylation as the primary route of metabolism,
CYP3A4 was capable of metabolizing astemizole at multiple sites on the molecule. Deuteration
of astemizole at the site of O-demethylation resulted in an isotope effect of 7.1 as well as an
8.3-fold decrease in the rate of clearance for astemizole by CYP4F12. Conversely, while an
isotope effect of 3.8 was observed for the formation of the O-desmethyl metabolite when
deuterated astemizole was metabolized by CYP3A4, there was no decrease in the clearance of
astemizole. Development of a homology model of CYP4F12 based on the crystal structure of
cytochrome P450 BM3 predicted an active site volume for CYP4F12 that was approximately
76% of the active site volume of CYP3A4. As predicted, multiple favorable binding orientations
were available for astemizole docked into the active site of CYP3A4, but only a single binding
orientation with the site of O-demethylation oriented towards the heme was identified for
CYP4F12. Overall, it appears that while CYP4F12 may be capable of binding similar ligands to
other cytochrome P450 enzymes such as CYP3A4, the ability to achieve catalytically favorable
orientations may be inherently more difficult due to the increased steric constraints of the
CYP4F12 active site.

Introduction
CYP4F12 is a member of the cytochrome P450 (P450) family of drug metabolizing
enzymes that is primarily expressed in the liver, small intestine, colon, kidney and heart (Bylund
et al., 2001; Guengerich, 2005; Hashizume et al., 2001). Currently, there is limited information
available on the relative protein expression levels of CYP4F12, however initial analyses would
suggest that it is a widely expressed but relatively minor P450 isoform (Stark et al., 2004).
Sequence alignment of the amino acid sequence of CYP4F12 indicates it is approximately 78 –
83% homologous with the other members of the CYP4F family. To date, seven allelic variants of
the enzyme have been identified, with two of the seven exhibiting altered kinetics (Bylund et al.,
2001; Cauffiez et al., 2004). CYP4F12 expression has been shown to be inducible through
activation of the p53 pathway, a sequence-specific transcription factor known to regulate the
expression of multiple P450 isoforms, and also exhibits an increased binding to the pregnane-X
receptor in the presence of rifampicin (Goldstein et al., 2013; Hariparsad et al., 2009).
The P450 family of drug metabolizing enzymes is capable of catalyzing the metabolism
of substrates with a wide array of structural and physical chemical attributes (Johnson and
Stout, 2005; Lewis et al., 1999; Lewis et al., 1998). CYP4F12 exhibits similar pharmacophore
attributes to other CYP4 isoforms as well as P450 BM3, with known endogenous substrates
including arachidonic acid, leukotriene B4 and prostaglandins (Bylund et al., 2001; Kikuta et al.,
2007; Stark et al., 2004). CYP4F12 also catalyzes the metabolism of a number of xenobiotics
including astemizole, terfenadine and ebastine and is known to metabolize a number of the
same substrates as CYP3A4 and CYP2J2 (Hashizume et al., 2002; Lee et al., 2010; Parikh et
al., 2003). The comparison of the relative catalytic and inhibitory promiscuities of P450 isoforms
identified a unique ligand profile for CYP4F12, with its susceptibility to inhibition being similar to
P450 isoforms such as CYP3A4 but having a catalytic selectivity similar to that of substrate-

specific homeostatic isoforms such as CYP26A1 (Foti et al., 2011).
The objective of the work presented here was to characterize the active site properties of
CYP4F12 that result in a decreased catalytic promiscuity through detailed kinetic analyses and
molecular modeling. A thorough structural assessment was conducted in order to determine
which physicochemical properties result in an increased inhibitory potency for CYP4F12.
Metabolite identification assays were carried out to identify the sites of metabolism for
astemizole with CYP4F12 as compared to CYP3A4, a P450 isoform with a well-documented
ability to catalyze a wide range of substrates. Using kinetic isotope experiments, the ability of
CYP4F12 to allow for metabolic switching for a given substrate in its active site was probed.
Finally, a homology model of CYP4F12 was designed and compared to crystal structure data of
CYP3A4 in an attempt to rationalize the current in vitro findings and ultimately to characterize
the active site properties of CYP4F12.

Materials and Methods
Materials. Astemizole and d3-astemizole were obtained from United States Biological (Salem,
MA).
All other drug standards were purchased from Sigma-Aldrich (St. Louis, MO).
Recombinant cytochrome P450 3A4 and 4F12 were purchased from BD Biosciences (San Jose,
An Amplex® Red hydrogen
CA). NADPH was ordered from Calbiochem (San Diego, CA).
peroxide fluorometric detection kit and all HPLC-grade solvents (acetonitrile, methanol and
water) were from Thermo Fisher Scientific (Waltham, MA).
In Vitro Clearance and IC50 Determination. In vitro clearance values were determined for a
set of 53 compounds using a substrate depletion approach. Incubations consisted of 10 pmol
recombinant CYP4F12, 3 mM MgCl2 and 1 µM test compound in 100 mM potassium phosphate
buffer (pH 7.4). After a 3 minute pre-incubation period at 37 ºC, incubations were initiated with
1 mM NADPH (final concentration). Aliquots were removed at 0, 2.5, 5, 10, 20 and 30 minutes
and quenched with 2 volumes of ice cold acetonitrile containing 500 nM tolbutamide as an
internal standard. All in vitro clearance incubations were performed in duplicate. Samples were
subsequently centrifuged for 10 min at 1130 g in a Beckman Allegra 6R table top centrifuge
(Beckman Coulter, Fullerton, CA). The resulting supernatants were transferred to 96-well
polypropylene plates for LC-MS/MS analysis.
Substrate depletion rates were determined by first normalizing the analyte
concentrations to a percent remaining based on the T0 value and subsequently using linear
regression to determine the slope (-k) of the ln(percent remaining) values as a function of
incubation time (Obach et al., 1997). The rates (k) were converted to half-life values (T1/2
=

0.693 / k) and ultimately used to calculate an in vitro clearance with units of mL/min/nmol P450
value using Equation 1.
Incubation Volume
CYP Content
0.693
ClInVitro,app
=
∗
Equation 1
T1/ 2
Inhibition potency (IC50) was also determined for each of the 53 test compounds in a
recombinant CYP4F12 incubation. In brief, incubations consisted of 1 pmol recombinant
CYP4F12, 3 mM MgCl2, 0.25 µM terfenadine as the probe substrate and 0 – 25 µM test
compound. For incubations intended to determine the inhibition potency of terfenadine in
recombinant CYP4F12, astemizole (0.15 µM, final concentration) was used as the probe
substrate. Final organic solvent content was limited to less than 1% (v/v) and product formation
was linear with respect to time and protein concentration. Reactions were initiated with 1 mM
NADPH and were allowed to proceed for 5 minutes (terfenadine as probe substrate) or 1 minute
(astemizole as probe substrate) before being quenched with 2 volumes of ice cold acetonitrile
containing 500 nM tolbutamide as internal standard. IC50 determinations were performed in
duplicate. Samples were subsequently centrifuged for 10 min at 1130 g in a Beckman Allegra
6R table top centrifuge (Beckman Coulter, Fullerton, CA) and the resulting supernatants
transferred to 96-well polypropylene plates for LC-MS/MS analysis.
Inhibition parameters were estimated using Graphpad Prism (version 6.03; GraphPad
Software Inc., San Diego, CA). Data was expressed as a percent of control (no inhibitor) and fit
to a fixed-slope three parameter inhibition model in order to determine the IC50 (Equation 2),
where max and min refer to the metabolite formation rates of the probe substrate at the lowest

and highest inhibitor concentrations, respectively, and [I] is the concentration of inhibitor in the
incubation.
(max− min)
Equation 2
%activity = min+
(
1+10(log[I ]−log IC50)
)
Metabolite Identification in Recombinant CYP4F12 and CYP3A4. Incubations were
performed in recombinant CYP3A4 and CYP4F12 to identify the metabolites of astemizole
formed by each enzyme. Reactions consisted of 20 pmol recombinant protein, 3 mM MgCl2,
and 10 µM astemizole or d3-astemizole in 100 mM potassium phosphate buffer (pH 7.4).
Following a 5 minute pre-incubation period at 37 ºC, the incubations were initiated with 1 mM
NADPH (final concentration) and allowed to proceed for 20 minutes. Metabolite identification
incubations were extracted into 3 volumes of ethyl acetate and vortexed for 5 minutes. The
organic layer was collected and evaporated to dryness under a gentle stream of N2. Samples
were reconstituted in water:acetonitrile (95:5) and placed into vials for LC-MS/MS analysis.
Kinetic Isotope Effects on Intrinsic Clearance and Metabolite Formation. O-demethylation
of the methoxyphenyl moiety of astemizole has previously been reported to be a major
metabolic pathway for astemizole by CYP4F12 (Matsumoto and Yamazoe, 2001). Substrate
depletion (intrinsic clearance) and enzyme kinetic experiments were conducted in recombinant
CYP3A4 or CYP4F12 to determine the effects of converting the methoxy group (-OCH3) of
astemizole to a d3-methoxy group (-OCD3) on the in vitro metabolism characteristics of

astemizole. Briefly, intrinsic clearance incubations consisted of 1 pmol CYP4F12 or CYP3A4, 3
mM MgCl2 and 0.15 µM astemizole or d3-astemizole in 100 mM potassium phosphate buffer (pH
7.4). Following a 3 minute pre-incubation at 37 ºC, incubations were initiated with 1 mM
NADPH (final concentration). Aliquots were removed at 0, 2.5, 5, 10, 20 and 30 minutes and
quenched with 2 volumes of ice-cold acetonitrile containing 500 nM tolbutamide as an internal
standard. In vitro half-lives were obtained using linear regression from 0 – 5 minutes for
astemizole and from 0 – 20 minutes for d3-astemizole and resulting in vitro clearance values
calculated using Equation 1. Enzyme kinetic experiments contained 1 pmol recombinant
CYP4F12 or CYP3A4, 3 mM MgCl2 and varying concentrations of astemizole or d3-astemizole
from 0 – 100 M depending on the metabolic pathway. Enzyme kinetics in recombinant
CYP4F12 and CYP3A4 incubations were terminated after 1 minute and 2.5 minutes,
respectively, in order to ensure Michaelis-Menten reaction conditions and product formation
linearity with respect to time and protein concentration. Reactions were quenched with 500 nM
tolbutamide in ice-cold acetonitrile, centrifuged (1160 g for 10 minutes) and transferred to 96-
well polypropylene plates for LC-MS/MS analysis. Enzyme kinetic parameters were determined
using nonlinear regression analysis fit to either a Michaelis-Menten model (Equation 3) or a
substrate inhibition model (Equation 4). For the enzyme kinetic equations, Km refers to half the
substrate concentration ([S]) at maximal reaction velocity (Vmax). Ki is the dissociation constant
for a single substrate molecule binding to the enzyme in such a way as to decrease the rate of
product formation. Kinetic isotope effects are expressed as the ratio of ((Vmax/Km),H
(Vmax/Km),D).
/
Vmax *[S]
V =
Equation 3
Km +[S]

Vmax *[S]
Equation 4
V =
[S]
Km +[S]* 1+
Ki
Estimation of NADPH Consumption / H2O2 Formation. Incubations consisted of 1 pmol
recombinant CYP3A4 or CYP4F12 and 2 M astemizole or d3-astemizole in 100 mM potassium
phosphate buffer (pH 7.4). Reactions were initiated with 1 mM NADPH (final concentration). A
Cary 4000 UV-visible spectrophotometer (Agilent, Santa Clara, CA) was used to measure
NADPH consumption using previously reported methods by monitoring absorbance at 340 nm
(Xiao et al., 2011). A value of 6.22 mM-1 cm-1 for
was used in calculating the NADPH
340
consumption rate. Hydrogen peroxide formation was monitored using an Amplex® Red
hydrogen peroxide assay following guidelines provided by the manufacturer (Mishin et al.,
2010). A hydrogen peroxide detection cocktail of 100 M Amplex® Red reagent, 0.2 U/mL
horseradish peroxidase and 1x reaction buffer (final concentrations) was added to the
incubation after 2 minutes at 37 ºC and the formation of resorufin monitored at an excitation
wavelength of 545 nm and an emission wavelength of 590 nm.
In Vitro Sample Analysis.
Sample analysis for in vitro clearance and IC50 samples was
conducted using LC-MS/MS technology. Briefly, the LC-MS/MS platform consisted of an
Applied Biosystems 4000 Q-Trap fitted with an electrospray ionization source (Applied
Biosystems, Foster City, CA). Generic instrument parameters included curtain gas (40 arbitrary
units), CAD gas (medium), ion spray voltage (4700 V), source temperature (500 °C) and ion
source gas 1 and gas 2 (35 arbitrary units, each). Dwell time was 200 msec and interface

heaters were kept on for all analytes. Positive ionization MRM transitions for astemizole,
terfenadine and tolbutamide were 459.2 / 135.1, 471.9 / 436.3 and 271.2 / 91.1, respectively.
Specific instrument parameters for the 53 test compounds are listed in Supplemental Table 1.
The Q-Trap mass spectrometer was coupled to a liquid chromatography system
consisting of two LC-20AD pumps with an in-line CBM-20A controller and DGU-20A5 solvent
degasser (Shimadzu, Columbia, MD) and to a LEAP CTC HTS PAL autosampler equipped with
a dual-solvent self-washing system (CTC Analytics, Carrboro, NC). HPLC separation was
achieved using a Gemini C18 2.0 x 30 mm 5 µm column (Phenomenex, Torrance, CA). Rapid
gradient elution (flow rate = 400 µL/min) was carried out using a mobile phase system
consisting of (A) 5 mM ammonium formate with 0.1% formic acid and (B) acetonitrile with 0.1%
formic acid. HPLC flow was diverted to waste for the first minute to remove any non-volatile
salts. An injection volume of 10 L was used for all analytes.
For metabolite identification analyses, chromatographic separation was achieved using a
Kinetex C18 2.1 X 100 mm 2.7 m column (Phenomenex, Torrance, CA). Gradient elution (flow
rate = 200 µL/min) was carried out using a mobile phase system consisting of (A) water with
0.1% formic acid and (B) acetonitrile:methanol (1:1) with 0.1% formic acid. HPLC flow was
diverted to waste for the first two minutes to remove any non-volatile salts. An injection volume
of 15 L was used for all analyses. Mobile phase B was held at 2.5% for the first 2 minutes,
increased linearly from 2.5% to 33% from 2 to 6.5 minutes, from 33% to 45% from 6.5 to 16
minutes, increased to 95% at 17 minutes and held at 95% for 1 minute prior to re-equilibration at
2.5% B for 2 minutes. Metabolites were initially identified using a Q1 scan (100 – 800 amu).
Additional analysis was conducting using MRM transitions for astemizole and its metabolites.
The analytes monitored were astemizole (459.2 / 135.1), O-desmethylastemizole (445.3 /
121.4), N-desalkylastemizole (325.3 / 109.5) and hydroxylation at the 6-OH position of the

benzimidazole moiety (475.3 / 135.2), fluorophenyl moiety (475.3 / 135.2) or methoxyphenyl
moiety (475.2 / 151.4).
CYP4F12 Homology Modeling. A three-dimensional homology model of CYP4F12 was
developed using Prime (Schrodinger, Portland, OR). The CYP4F12 amino acid sequence was
obtained from GenBank (AAQ89336.1) with the crystal structure of P450 BM3 (PDB 1JPZ)
serving as the template for the homology model (30% sequence identity). While BLAST
searches identified CYP3A4 as another plausible template based on sequence identity and total
sequence alignment score, BM3 was chosen as the template as to not bias the subsequent
comparison to the CYP3A4 crystal structure. The structural plausibility of the CYP4F12
homology model was determined through evaluation of the Ramachandran plot and through
visualization of bond angles and lengths. Local flexibility/rigidity was assessed by visual
inspection of helices as compared to loop regions and through secondary structure predictions
performed in PSIPRED (University College London, UK) and SSPro (Schrodinger, Portland,
OR). The active site of the CYP4F12 homology model was defined using SiteMap (Schrodinger,
Portland, OR) and a docking grid generated using a 14 x 14 x 14 Å3 box that was used to
constrain the center of mass of the docked ligand. The astemizole structure was prepared prior
to docking using LigPrep 2.0 (Schrodinger, Portland, OR) which converts two-dimensional .mol
files to three-dimensional molecular structures with defined stereocenters.
Following
conversion, the astemizole structure underwent a minimization step using a distance dependent
dielectric model and the OPLS_2005 force field. Astemizole was then docked into the
homology model of CYP4F12 or the crystal structure of CYP3A4 (PDB 1W0G) using Glide
(Schrodinger, Portland, OR) and evaluated by GlideScore and eModel.
Induced-fit docking
algorithms (Sherman et al., 2006a; Sherman et al., 2006b) which allow for reduced van der

Waals radii, removal and reorientation of non-rigid protein side chains and a second energy
minimization step upon ligand docking were used to dock astemizole into the homology model
of CYP4F12 and for two of the three orientations within the active site of the CYP3A4 crystal
structure model (O-demethylation and N-dealkylation orientations).
CAVER 3.0.1 (CaverSoft, Brno, Czech Republic) was used to estimate the active site
volumes of the CYP4F12 homology model and the CYP3A4 crystal structure as previously
described (Chovancova et al., 2012; Petrek et al., 2006; VandenBrink et al., 2012). Protein
structures were prepared in Maestro (Schrodinger, Portland OR) prior to CAVER analysis.
Initial optimization steps included correction for missing hydrogen atoms, removal of water
molecules and definition of the formal charge on the heme iron at Fe3+. The Maestro script
automatically assigned protonation states for all arginine, glutamine and histidine residues.
Following optimization of the hydrogen bonding network, the entire protein structure was subject
to a restrained energy minimization after which each protein structure was imported into
CAVER. The active site of each enzyme was defined with respect to a water ligand that was
positioned axial to the heme prosthetic group. Active site volume estimates were obtained
using embedded molecular dynamics trajectory files within CAVER and graphically visualized
through PyMOL (Schrodinger, Portland, OR).

Results
The in vitro inhibition potency and in vitro clearance of 53 compounds selected to
sample a diverse cross-section of the chemical space of ‘drug-like’ compounds was investigated
using recombinant CYP4F12. The resulting IC50 values of the molecules ranged from 78 nM
(amiodarone) to > 100 M (multiple compounds). Only astemizole and terfenadine exhibited
measurable turnover in recombinant CYP4F12 incubations with in vitro clearance values of 70.1
mL/min/nmol P450 and 60.8 mL/min/nmol P450, respectively (Supplemental Table 1).
Examination of the structural and physicochemical properties of the 53 test compounds
indicated that the lipophilic nature of the compounds (cLogP) had the greatest impact on
inhibition potency (Figure 1). Furthermore, there were no significant trends observed between
inhibition potency and molecular weight, polar surface area, pKa, the number of acidic or basic
groups, the number of hydrogen bond donors or acceptors or the number of aromatic rings was
observed (Pearson coefficient < 0.25; data not shown).
Experiments carried out to identify the sites of astemizole metabolism in recombinant
CYP4F12 confirmed previously published reports that O-demethylation is the primary metabolic
clearance pathway for astemizole by CYP4F12 (fm = 0.99). The fraction metabolized was
calculated as a ratio of the peak area : internal standard area for each respective metabolite to
the sum of the peak area : internal standard areas for all observed metabolites. Trace amounts
of 6-hydroxyastemizole, fluorophenyl-OH astemizole and N-desalkylastemizole were also
observed (Figure 2). Incubations with recombinant CYP3A4 identified multiple oxidative
metabolites. In addition to O-demethylation (fm = 0.39), 6-hydroxyastemizole (fm = 0.38),
hydroxylation on the methoxyphenyl moiety (fm = 0.16), fluorophenyl hydroxylation (fm = 0.06)
and N-desalkylastemizole (fm = 0.01) were also identified in recombinant CYP3A4 incubations.

When deuterium was incorporated at the site of O-demethylation, the in vitro clearance
of astemizole in recombinant CYP4F12 incubations decreased by 8.3-fold (Figure 3).
Conversely, the effect of deuteration on the in vitro clearance of astemizole was negligible for
incubations conducted with recombinant CYP3A4. An isotope effect ((Vmax/Km),H / (Vmax/Km),D) of
7.1 and 3.8 was observed for the formation of O-desmethyl astemizole by CYP4F12 and
CYP3A4, respectively (Figures 4 and 5; Table 1).
For CYP3A4, isotopically sensitive
branching resulted in an increased formation of 6-hydroxyastemizole ((Vmax/Km),H / (Vmax/Km),D =
0.80), fluorophenyl-OH astemizole ((Vmax/Km),H / (Vmax/Km),D = 0.47) and methoxyphenyl-OH
astemizole ((Vmax/Km),H / (Vmax/Km),D = 0.45). No effect was observed on the formation of 6-
hydroxyastemizole or fluorophenyl-OH astemizole by CYP4F12 or for N-dealkylation by either
isoform. Recombinant CYP4F12 incubations with deuterated astemizole also resulted in a
NADPH consumption rate that was approximately 1.5 – 2.2-fold higher than the NADPH
consumption rate that was observed for astemizole (Figure 6). No significant increase in the
formation of H2O2 was observed for CYP4F12 incubations with deuterated astemizole (data not
shown). Similar to the effects on in vitro clearance, deuteration of the methoxy moiety of
astemizole had negligible effects on the NADPH consumption rate and formation of H2O2 when
incubations were conducted with recombinant CYP3A4.
A homology model was designed for CYP4F12 using P450 BM3 (PDB 1JPZ) as a
template and compared to CYP3A4 using crystal structure data (PDB 1W0G). Induced fit
docking was used to orient astemizole in the active site of each model. For CYP4F12, only a
single substrate orientation was achieved with the homology model, with the protons of the
methoxy carbon located approximately 2.25 Å from the heme iron (Figure 7a). Key amino acid
residues included Phe397 and Leu421 which were located within 2 Å of the fluorophenyl moiety,
and Asp121 and Pro396 which were within 2 Å of the benzimidazole and piperidine moieties,

respectively.
Phe124, Leu137, Phe327 and Thr332 were located within 3 Å of the
methoxyphenyl ring. Ile67, Leu74, Ser77 and Ile239 appeared to delineate the boundaries of
the CYP4F12 active site beyond the fluorophenyl ring of astemizole. A CAVER analysis
predicted the volume of the CYP4F12 active site to be approximately 947 Å3. When astemizole
was docked in the CYP3A4 in an orientation consistent with O-demethylation (methoxy protons
approximately 2.66 Å from heme iron), Phe57 and Phe215 were located within 2 Å of the
benzimidazole and fluorophenyl moieties, respectively. Arg105, Ser119, Ala305, Met371 and
Arg372 were also located within 2 – 3 Å of astemizole (Figure 7b). When the orientation of
astemizole was reversed to allow for hydroxylation on either the benzimidazole or fluorophenyl
moieties (benzimidazole protons approximately 3.23 Å from the heme iron), Arg105 and Arg106
were located within 2 Å of the piperidine backbone of astemizole with Ser119 located within 2 Å
of one of the benzimidazole nitrogens (Figure 7c). Phe215 was within 3 Å of the methoxyphenyl
ring and Ile369 and Ala370 were within 3 Å of the fluorophenyl moiety. Tyr53, Phe57 and
Glu374 were also located within 3 – 4 Å of astemizole. Finally, when astemizole was positioned
in the active site of the CYP3A4 homology model in a pose conducive with N-dealkylation ( -
carbon protons approximately 5.87 Å from the heme iron), Phe241 and Phe304 were within 3 Å
of the benzimidazole ring with Thr309 located within 3 Å of the methoxyphenyl moiety (Figure
7d). Arg106, Pro107, Ser119, Arg212 and Ala370 were also located within 3 – 4 Å of
astemizole. The active site volume from the crystal structure of CYP3A4 was estimated to be
approximately 1239 Å3, similar to previous reports (Ekroos and Sjogren, 2006; Ohkura et al.,
2009).

Discussion
CYP4F12 catalyzes the -1 through -3 hydroxylations of compounds such as
arachidonic acid, 9,11-diazo-prostaglandin H2 and 9,11-methanoepoxy-prostaglandin H2
(Bylund et al., 2001). The enzyme can also metabolize xenobiotics such as astemizole,
terfenadine and ebastine (Lee et al., 2010; Parikh et al., 2003). Though no mechanistic
explanation was available at the time, studies to compare the substrate profiles of ten P450
isoforms concluded that while CYP4F12 is susceptible to inhibition by many of the same ligands
as other P450 enzymes, the ability of the enzyme to metabolize the same compounds is
relatively restricted (Foti et al., 2011). The objective of this manuscript was to characterize the
active site properties of CYP4F12 as a means to rationalize the decreased catalytic promiscuity
through kinetic analyses and homology modeling.
Efforts to characterize the inhibitory properties of 53 compounds against CYP4F12
resulted in IC50 values from 78 nM for amiodarone to > 100 M for 13 of the compounds.
Lipophilicity is a well characterized property of P450 ligands that can influence inhibition potency
(Lewis and Dickins, 2003; Lewis et al., 2004). This was true for CYP4F12 as the molecular
descriptors associated with lipophilicity (cLogP, ALogP, LogD) trended with inhibitor potency
and the most potent inhibitor, amiodarone, was also the most lipophilic (cLogP = 8.95). Of the
53 compounds, 47 had IC50 values for CYP4F12 that were either more potent or similar to the
IC50 observed for CYP3A4, indicating that the active site of CYP4F12 is capable of
accommodating much of the chemical space that is able to fit in the active site of CYP3A4.
Conversely, only two of the compounds (astemizole and terfenadine) exhibited measurable
clearance by CYP4F12, suggesting that while the many of these ligands can act as an inhibitor
of CYP4F12, the likelihood of achieving a catalytically favorable orientation in the active site is a
less common phenomena.

O-demethylation has been reported to be a metabolic pathway for astemizole by multiple
P450s (Hashizume et al., 2002; Lee et al., 2010; Matsumoto and Yamazoe, 2001; Parikh et al.,
2003). Consistent with these results, O-demethylation accounted for over 99% of the
metabolites formed by CYP4F12. Trace amounts of 6-hydroxyastemizole, fluorophenyl-OH
astemizole and N-desalkylastemizole were also observed suggesting that while it is plausible for
astemizole to achieve multiple binding orientations in the active site of CYP4F12, the additional
orientations are less catalytically favorable. While O-demethylation was also catalyzed by
CYP3A4, it accounted for only 39% of the metabolites formed, with 6-hydroxyastemizole,
fluorophenyl-OH and hydroxylation on the methoxyphenyl ring all accounting for significant
fractions of the overall metabolism. Unlike CYP4F12, the relative similarity of the fm values for
CYP3A4 suggests that conditions exist within the active site of CYP3A4 for astemizole to
achieve multiple catalytically favorable orientations.
To further compare the active sites of CYP4F12 and CYP3A4, incubations were
performed using d3-astemizole. The use of isotope effects to evaluate a ligand’s ability to freely
reorient within an active site has been described previously (Henne et al., 2001; Iyer et al.,
1997). The basis for these experiments begins with the symmetry of the transition state for the
bond-cleaving reaction step (C-H or C-D) and results in an alteration to the rate constant for that
step (Kim et al., 2006; Lu et al., 1984; Manchester et al., 1997; Miwa and Lu, 1987; Miwa et al.,
1984; Nelson and Trager, 2003; Westheimer, 1961). When d3-astemizole was incubated with
recombinant CYP4F12, an isotope effect of 7.1 ((Vmax/Km),H / (Vmax/Km),D) was observed for the
formation of O-desmethylastemizole with an 8.3-fold decrease in the clearance of the substrate.
No alternative metabolites were observed with CYP4F12 suggesting that the active site
architecture of CYP4F12 is such that the substrate is unable to rapidly reorient itself. When
kinetic isotope effect experiments were conducted with recombinant CYP3A4, an isotope effect

was also observed for the formation of O-desmethylastemizole, however it was reduced to 3.8
((Vmax/Km),H / (Vmax/Km),D) with no effect on the clearance of astemizole. Inverse isotope effects
were observed for the formation of 6-hydroxyastemizole (0.8), fluorophenyl-OH astemizole
(0.47) and methoxyphenyl-OH astemizole (0.45) with CYP3A4. One explanation may be that
isotopically sensitive branching to the three hydroxylated metabolites is masking the kinetic
isotope effect at the site of O-demethylation and that the lack of an effect on the clearance of
astemizole is a combination of the metabolic branching and the relatively low fm value for this
metabolic pathway (Atkins and Sligar, 1987; Harada et al., 1984; Jones et al., 1986; Korzekwa
et al., 1989; Miwa and Lu, 1987; Nelson and Trager, 2003). Furthermore, the P450 catalytic
cycle is known to include three nonproductive (uncoupling) pathways of which only the
formation of water from Compound I results in an NADPH to O2 ratio of 2:1. The approximate 2-
fold increase in the NADPH consumption rate which was observed for CYP4F12 and d3-
astemizole with no increase in the formation of H2O2 suggests that the enzymatic reaction may
be branching to this uncoupling pathway and resulting in the formation of an additional water
molecule from Compound I (Atkins and Sligar, 1988; Grinkova et al., 2013).
Finally, to rationalize the previously discussed results using the active site geometries of
CYP4F12 and CYP3A4, a homology model was designed for CYP4F12 and compared to an x-
ray crystal structure model of CYP3A4. Upon docking, only a single orientation was achieved
with CYP4F12 while three orientations were obtained with CYP3A4. Binding of astemizole in
the CYP4F12 model required an induced-fit docking approach, indicating that the active site of
the enzyme is relatively constrained. The majority of the active site residues that were located
within 2 – 3 Å of astemizole were hydrophobic in nature, consistent with hydrophobic
interactions influencing the binding of ligands within the CYP4F12 active site. The active site
volume of the CYP4F12 homology model was approximately 76% of the total volume of the

CYP3A4 active site and was constrained in a long, cylindrical nature. The region of the
CYP4F12 active site with the greatest structural divergence from CYP3A4 appeared to be in the
region of the F and G helices, where CYP4F12 appears to have a rigid helical motif similar to
P450 BM3 as opposed to the more flexible helix-loop-helix motifs of the CYP3A4 F and G helix
structures (Ekroos and Sjogren, 2006; Joyce et al., 2004; Ohkura et al., 2009). The overall
mobility of the F / G loop in P450 isoforms has been proposed as a determinant of a P450 to
bind diverse ligands and one could argue that rigidity in this region of CYP4F12 contributes to
the narrow substrate profile of the enzyme (Poulos, 2003; Scott et al., 2003). The estimated
volume of the active site of CYP4F12 suggests it is similar in size to the larger P450 active sites
such as CYP3A4 and CYPC8, though the overall flexibility and volume changes upon substrate
binding remain to be determined.
Consistent with its metabolic profile, multiple binding orientations were obtained with the
CYP3A4 model. Multiple crystal structures and evaluations of the ligand promiscuity of CYP3A4
have been reported, resulting in a prominent role for the enzyme in the biotransformation of
xenobiotics and endogenous compounds (Ekroos and Sjogren, 2006; Foti et al., 2011; Foti et
al., 2010; Gibbs and Hosea, 2003; Kenworthy et al., 1999; Wienkers and Heath, 2005; Williams
et al., 2004; Yano et al., 2004). Binding within the active site of CYP3A4 has also been shown
to be related to lipophilicity and conformational changes to the active site of CYP3A4 upon
ligand binding have been reported (Ishigami et al., 2001; Kenworthy et al., 2001; Khan et al.,
2002; Korzekwa et al., 1998; Lu et al., 2001; Riley et al., 2001; Shou et al., 1994). Interactions
between astemizole and the phenylalanine cluster of CYP3A4 were observed, with Phe215,
Phe241 and Phe304 generally involved in - interactions with the aromatic ring of astemizole
most distal from the site of metabolism. Ser119 and Ala305, previously shown to be involved in
the substrate recognition of testosterone, progesterone and 7-hexoxycoumarin were also

located within 3 – 4 Å of astemizole for all three orientations and may influence binding
interactions with the benzimidazole or piperidine ring, depending on the orientation of
astemizole (Domanski et al., 1998; He et al., 1997; Khan and Halpert, 2000; Tanaka et al.,
2004). The three binding models for astemizole with CYP3A4 also predict significant
interactions with a polar region of the active site which includes Arg106 and Glu374, a region
known to be involved in the binding of ketoconazole as well as with Arg212, which is known to
undergo significant conformational changes between the ligand-free and ligand-bound
structures of CYP3A4 (Ekroos and Sjogren, 2006; Sevrioukova and Poulos, 2012; Yano et al.,
2004).
In conclusion, previous studies have identified CYP4F12 as being catalytically restricted
as compared to other P450 isoforms and the results presented herein provide a plausible
mechanistic explanation for the narrow ligand profile of the enzyme. Metabolite identification
studies identified astemizole O-demethylation as the single route of metabolism by CYP4F12
while CYP3A4 was capable of catalyzing the formation of multiple metabolic products with
relatively low fm values. Kinetic isotope effects demonstrate that deuteration of the primary site
of metabolism results in a direct effect on the clearance of astemizole by CYP4F12 while
CYP3A4 can account for the isotope effect through metabolic branching. Overall, it appears
that while the CYP4F12 active site may be capable of accommodating and binding similar
ligands as the other P450 isoforms, the ability to achieve a catalytically favorable orientation
may be more difficult which ultimately results in a decrease in catalytic promiscuity.

Author Contribution
Participated in research design: J. Eksterowicz, D. Rock, B. Rock, L. Wienkers, R. Foti
Conducted experiments: J. Eksterowicz, B. Rock, R. Foti
Contributed new reagents or analytic tools: None
Performed data analysis: J. Eksterowicz, B. Rock, R. Foti
Wrote or contributed to the writing of the manuscript: J. Eksterowicz, R. Foti

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