DMD Fast Forward. Published on December 31, 2020 as DOI: 10.1124/dmd.120.000261
This article has not been copyedited and formatted. The final version may differ from this version.

DMD-AR-2020-000261

Functional characterization of 40 CYP3A4 variants by assessing midazolam
1′-hydroxylation and testosterone 6β-hydroxylation

Masaki KUMONDAI, Evelyn Marie GUTIÉRREZ RICO, Eiji HISHINUMA, Akiko
UEDA, Sakae SAITO, Daisuke SAIGUSA, Shu TADAKA, Kengo KINOSHITA,

MANO, Noriyasu HIRASAWA, Masahiro HIRATSUKA

Laboratory of Pharmacotherapy of Life-Style Related Diseases, Graduate School of
Pharmaceutical Sciences, Tohoku University, Sendai 980-8578, Japan (M.K., E.M.G.R.,
N.H., M.H.)
Tohoku Medical Megabank Organization, Tohoku University, Sendai 980-8575, Japan(E.H., S.S., S.T., D.S., K.K., M.H.)
Advanced Research Center for Innovations in Next-Generation Medicine, Tohoku
University, Sendai 980-8575, Japan (E. H., A. U., N.H., M.H.)
Faculty of Pharmacy, Meijo University, Nagoya 468-8503, Japan (T.N., A.O.)
Department of Pharmaceutical Sciences, Tohoku University Hospital, Sendai 980-8574,
Japan (M.M., N.M., N.H., M.H.)

1

Downloaded from dmd.aspetjournals.org at ASPET Journals on September 22, 2024

Tomoki NAKAYOSHI, Akifumi ODA, Ai ABE, Masamitsu MAEKAWA, Nariyasu

DMD Fast Forward. Published on December 31, 2020 as DOI: 10.1124/dmd.120.000261
This article has not been copyedited and formatted. The final version may differ from this version.

DMD-AR-2020-000261

Laboratory of Clinical Pharmacy, Faculty of Pharmaceutical Sciences, Tohoku
University, Sendai 980-8574, Japan (A.A., M.M., N.M.)

Downloaded from dmd.aspetjournals.org at ASPET Journals on September 22, 2024

2

DMD Fast Forward. Published on December 31, 2020 as DOI: 10.1124/dmd.120.000261
This article has not been copyedited and formatted. The final version may differ from this version.

DMD-AR-2020-000261

Running title: Functional characterization of 40 CYP3A4 variants

Corresponding author:
Dr. Masahiro Hiratsuka, Ph.D.
Laboratory of Pharmacotherapy of Life-Style Related Diseases, Graduate School of

980-8578, Japan
Tel. & Fax: +81-22-717-7049
E-mail: masahiro.hiratsuka.a8@tohoku.ac.jp

Number of text pages: 59
Number of tables: 2
Number of figures: 4
Number of references: 58
Number of words in Abstract: 181
Number of words in Introduction: 749
Number of words in Discussion: 1460

3

Downloaded from dmd.aspetjournals.org at ASPET Journals on September 22, 2024

Pharmaceutical Sciences, Tohoku University, 6-3 Aoba, Aramaki, Aoba-ku, Sendai

DMD Fast Forward. Published on December 31, 2020 as DOI: 10.1124/dmd.120.000261
This article has not been copyedited and formatted. The final version may differ from this version.

DMD-AR-2020-000261

Abbreviations: CO, carbon monoxide; CPR, cytochrome P450 oxidoreductase;
CYP3A4, cytochrome P450 3A4; DMDC, dimanganese decacarbonyl; HPLC,
high-performance liquid chromatography; HRP, horseradish peroxidase; kcat, turnover
number; Km, Michaelis constant; LOF, loss of function; MS, mass spectrometry; PCR,
polymerase chain reaction; SNV, single nucleotide variation; ToMMo, Tohoku Medical

4

Downloaded from dmd.aspetjournals.org at ASPET Journals on September 22, 2024

Megabank Organization; WGS, whole-genome sequencing.

DMD Fast Forward. Published on December 31, 2020 as DOI: 10.1124/dmd.120.000261
This article has not been copyedited and formatted. The final version may differ from this version.

DMD-AR-2020-000261

Abstract
Cytochrome P450 3A4 (CYP3A4) is among the most abundant liver andintestinal drug-metabolizing cytochrome P450 enzymes, contributing to the metabolism
of more than 30% of the clinically used drugs. Therefore, inter-individual variability in
CYP3A4 activity is a frequent cause of reduced drug efficacy and adverse effects. In

new variants, detected among 4,773 Japanese individuals by assessing CYP3A4
enzymatic activities for two representative substrates (midazolam and testosterone). The
reduced carbon monoxide-difference spectra of wild-type CYP3A4 and 31 CYP3A4
variants produced with our established mammalian cell expression system were
determined by measuring the increase in maximum absorption at 450 nm after carbon
monoxide treatment. The kinetic parameters of midazolam and testosterone
hydroxylation by wild-type CYP3A4 and 29 CYP3A4 variants (Km, kcat, and catalytic
efficiency) were determined, and the causes of their kinetic differences were evaluatedby three-dimensional structural modeling. Our findings offer insight into the mechanism
underlying inter-individual differences in CYP3A4-dependent drug metabolism.
Moreover, our results provide guidance for improving drug administration protocols byconsidering the information on CYP3A4 genetic polymorphisms.

5

Downloaded from dmd.aspetjournals.org at ASPET Journals on September 22, 2024

this study, we characterized wild-type CYP3A4 and 40 CYP3A4 variants, including 11

DMD Fast Forward. Published on December 31, 2020 as DOI: 10.1124/dmd.120.000261
This article has not been copyedited and formatted. The final version may differ from this version.

DMD-AR-2020-000261

Significance Statement
CYP3A4 metabolizes more than 30% of the clinically used drugs. Inter-individual
differences in drug efficacy and adverse effect rates have been linked toethnic-specific differences in CYP3A4 gene variants in Asian populations, including
Japanese individuals, indicating the presence of CYP3A4 polymorphisms resulting inin CYP3A4 activity due to amino acid substitutions by assessing the enzymatic
activities of coding variants for two representative CYP3A4 substrates.

6

Downloaded from dmd.aspetjournals.org at ASPET Journals on September 22, 2024

the increased expression of loss-of-function variants. This study detected alterations

DMD Fast Forward. Published on December 31, 2020 as DOI: 10.1124/dmd.120.000261
This article has not been copyedited and formatted. The final version may differ from this version.

DMD-AR-2020-000261

Introduction
Cytochrome P450 3A4 (CYP3A4) is the most abundant liver and intestinal drugmetabolizing cytochrome P450 enzyme, contributing to the metabolism of more than
30% of medications (Zanger and Schwab, 2013; Jackson et al., 2018; Niwa et al., 2020).
Inter-individual variability in CYP3A4 activity and expression influences drug

various factors that contribute to this variability, including CYP3A4 inducers andinhibitors and food-drug and drug-drug interactions, CYP3A4 genetic variations also
affect drug responses (Racha et al., 2003; Huang et al., 2008; Elens et al., 2013;
Hendriks et al., 2020). CYP3A5 genetic polymorphisms, including CYP3A5*3, havebeen widely investigated due to their high allele frequencies and clinical impact on thevariability of CYP3A activity. CYP3A4 gene variations may be overlooked due to thehigher impact of some CYP3A5 variants on activity. However, CYP3A4 gene variations
should also be investigated to elucidate their influence on the combinational effects of
CYP3A4andCYP3A5enzymatic

activity

and

CYP3A

in

vivo

activity(Fukushima-Uesaka et al., 2004; Lee et al., 2005; Zhou et al., 2017). For instance,
several CYP3A4 variant alleles, including CYP3A4*1B (a promoter variant) and *22 (anintronic variant), reduce mRNA levels or cause splicing defects, while CYP3A4*6, *17,

7

Downloaded from dmd.aspetjournals.org at ASPET Journals on September 22, 2024

responsiveness, efficacy, and adverse effect rates (Lamba et al., 2002a). Besides theDMD Fast Forward. Published on December 31, 2020 as DOI: 10.1124/dmd.120.000261
This article has not been copyedited and formatted. The final version may differ from this version.

DMD-AR-2020-000261*20, and *26 are associated with diminished enzymatic activities by loss-of-function(LOF) (Lamba et al., 2002a; Elens et al., 2013; Werk et al., 2014). The identification ofthese variants can improve patient safety and treatment efficacy; thus, increased
dose-adjusted tacrolimus concentration levels are achieved in transplant patients
carrying a LOF genotype (CYP3A4*20) (Gomez-Bravo et al., 2018). Hence, CYP3A4

The

Human

Cytochrome

P450

AlleleNomenclature

Database(https://www.pharmvar.org/gene/CYP3A4 [Accessed Sep 21th, 2020]) contains records
of CYP3A4 genetic polymorphisms (CYP3A4*2–*24, CYP3A4*26, and CYP3A4*28–*34). Moreover, several whole-genome sequencing (WGS) studies have identified
CYP3A4 genetic variations, including c.1165C>T (Pro389Ser) and c.1423C>G(Leu475Val) (Apellaniz-Ruiz et al., 2015). A WGS project of the Tohoku Medical
Megabank Organization (ToMMo), which targeted 4,773 Japanese individuals,
identified numerous genetic variations, including single nucleotide variations (SNVs),
as shown in Table 1 (https://jmorp.megabank.tohoku.ac.jp [Accessed Aug 30th, 2020]).
The identification of rare genetic variants, which may cause inter-individual differencesin drug efficacy and adverse reactions, facilitates the development of personalized
medicine (Fujikura et al., 2015; Schärfe et al., 2017). CYP3A4 rare genetic variants have8

Downloaded from dmd.aspetjournals.org at ASPET Journals on September 22, 2024

genotyping improves predictive outcomes.

DMD Fast Forward. Published on December 31, 2020 as DOI: 10.1124/dmd.120.000261
This article has not been copyedited and formatted. The final version may differ from this version.

DMD-AR-2020-000261

been implicated in high-dose simvastatin-related myopathy and affect serum
voriconazole concentrations linked to adverse drug reactions (Ingelman-Sundberg et al.,2018). Although the CYP3A4 SNVs analyzed in this study are relatively rare (Table 1),
their effects on CYP3A4 activity should be determined to improve treatment outcomesand manage adverse effects.

CYP3A4 polymorphisms in vitro and complement in vivo analysis data. Performing invivo assays is challenging due to low allele frequencies and increased patient burden.
However, all CYP3A4 variant proteins should be evaluated using the same protocol andexperimental conditions because methodological differences between studies may leadto inconsistent kinetic parameters for some variants (Hiratsuka, 2012). To date,characterization studies have utilized cDNA-based heterologous expression systems,
including E. coli, baculoviruses, and mammalian cell systems (Eiselt et al., 2001;
Gutierrez Rico et al., 2020; Kumondai et al., 2018; Lee et al., 2005; Maekawa et al.,2010; Saito et al., 2018; Watanabe et al., 2018). However, accurate assessments arelimited by inter-species differences of post-translational modifications. Thus,
mammalian cell expression systems should be preferred to evaluate the function ofhuman CYP3A4 variants in drug metabolism (Clark and Pazdernik, 2016). Recently, an

9Downloaded from dmd.aspetjournals.org at ASPET Journals on September 22, 2024

Heterologous expression systems can be employed to evaluate the effects of

DMD Fast Forward. Published on December 31, 2020 as DOI: 10.1124/dmd.120.000261
This article has not been copyedited and formatted. The final version may differ from this version.

DMD-AR-2020-000261

intronic CYP2D6 mutation was evaluated using a mini-gene expression system withtransfected whole CYP2D6 sequences (<5 kb) (Zanger et al., 2020). Mini-genes may aid
the assessment of CYP3A4 levels. However, genomic DNA expression usingmini-genes, including the whole CYP3A4 gene (>20 kb), is challenging because
transfection efficiency decreases with increased DNA length (Hornstein et al., 2016).

co-expressing CYP3A4, cytochrome P450 oxidoreductase (CPR), and cytochrome b5(Kumondai et al., 2020).
Here, we functionally characterized wild-type CYP3A4 (CYP3A4.1) and 40
CYP3A4 allelic variants, including 11 novel variants identified in 4,773 Japaneseindividuals, by using two representative CYP3A4 substrates (midazolam andtestosterone). Notably, clinical and in vitro studies have assessed the functional
differences and drug-drug interactions related to CYP3A4, including CYP variants with
substrate-specific enzymatic activities (Foti et al., 2010; Hiratsuka et al., 2016; Xiao etal.,2019).

Moreover,carbonmonoxide(CO)-difference

spectroscopy

andthree-dimensional (3D) structure analysis were performed to gain insights intofunctional alterations caused by CYP3A4 variants.

10

Downloaded from dmd.aspetjournals.org at ASPET Journals on September 22, 2024

Therefore, we used our heterologous expression system consisting of 293FT cells

DMD Fast Forward. Published on December 31, 2020 as DOI: 10.1124/dmd.120.000261
This article has not been copyedited and formatted. The final version may differ from this version.

DMD-AR-2020-000261

Materials and Methods
Chemicals
The following reagents were purchased from commercial sources: midazolam andflunitrazepam (Wako Pure Chemical Industries, Osaka, Japan); 1′-hydroxymidazolam
and 6β-hydroxytestosterone-d7 (Corning Incorporated, New York, USA); testosterone

dinucleotide reduced form (NADH) and β-nicotinamide-adenine dinucleotide phosphate
reduced form (NADPH) (Oriental Yeast, Tokyo, Japan); polyclonal anti-human
CYP3A4 antibody (ab3572) (Abcam, Cambridge, UK); horseradish peroxidase(HRP)-conjugated goat anti-rabbit IgG (ProteinSimple, Tokyo, Japan); dimanganese
decacarbonyl (DMDC) (Sigma-Aldrich); and sodium cyanide and cytochrome c fromhorse heart (Nacalai Tesque, Kyoto, Japan). All other chemicals and reagents were of
the highest quality commercially available.

Detection of CYP3A4 sequence alterations by Sanger sequencing analysis
PCR amplification was conducted using peripheral blood leukocyte genome DNA
isolated from whole blood using the Gentra Puregene Blood Kit (Qiagen, Hilden,
Germany), as described previously (Nagasaki et al., 2015). Whole blood samples were11

Downloaded from dmd.aspetjournals.org at ASPET Journals on September 22, 2024

and 6β-testosterone (Sigma-Aldrich, Steinheim, Germany); β-nicotinamide-adenine

DMD Fast Forward. Published on December 31, 2020 as DOI: 10.1124/dmd.120.000261
This article has not been copyedited and formatted. The final version may differ from this version.

DMD-AR-2020-000261

obtained from Japanese subjects participating in the community-based cohort study
conducted by ToMMo; written informed consent was obtained from all subjects before
sample collection. Primers were used to amplify sequences containing each CYP3A4
exon (Supplementary Table 1). PCR amplification was performed with genomic DNA
samples (10 ng), 2×AmpliTaq Gold 360 Master Mix (Applied Biosystems, Foster City,

conditions included an initial denaturation step at 95ºC for 10 min, followed by 30
cycles of denaturation at 95ºC for 30 s, annealing at 60ºC for 30 s, extension at 72ºC for
30 s (for exons 2–12) or 1 min (for exons 1 and 13), and a final extension at 72ºC for 7
min. The PCR products were purified using columns and analyzed by Sanger
sequencing utilizing the same primers for each exon as in the PCR, except for theforward sequencing reaction of exon 1 (5´-CAGGCGTGGAAACACAATGGTGG-3´)
and reverse sequencing reaction of exon 11 (5´-TGTGGATGACTGTAGTTTTC-3´). To
determine the linkage between two SNVs identified in one subject, the respective PCR
fragments were ligated into the pcDNA3.4 vector (ThermoFisher Scientific), and theligation products were transformed into E. coli. Single colonies of the transformants
were collected, and the plasmids were isolated and analyzed by Sanger sequencing. This
analysis was performed in accordance with the Declaration of Helsinki, and prior

12

Downloaded from dmd.aspetjournals.org at ASPET Journals on September 22, 2024

CA, USA), and 0.5 µM of each primer in a total volume of 20 µL. Thermal cycling

DMD Fast Forward. Published on December 31, 2020 as DOI: 10.1124/dmd.120.000261
This article has not been copyedited and formatted. The final version may differ from this version.

DMD-AR-2020-000261

written informed consent was obtained from all participants.

Construction of CYP3A4 cDNA expression vectors
The wild-type CYP3A4 (CYP3A4*1) plasmid used in this study has been
described in a previous report (Sakurai et al., 2005). A plasmid containing CYP3A4*1(CYP3A4*2–CYP3A4*5,

CYP3A4*7–CYP3A4*21,

Pro389Ser,

CYP3A4*28,

Leu475Val,

CYP3A4*23,

CYP3A4*29,

CYP3A4*24,

CYP3A4*31–CYP3A4*34,

Thr138Ala, Met256Ile, Ile300Val, Thr323Ala, Met353Leu, Ile369Val, Ile369Asn,
Ala370Val, Arg372Thr, Tyr399Ser, and Ala448Ser) using primers sets described in
Supplementary Table 2 with the QuikChange Site-directed Mutagenesis Kit (Stratagene,
La Jolla, CA, USA) following the manufacturer’s instructions. All prepared constructs
were confirmed by Sanger sequencing. Wild-type and variant CYP3A4 cDNAs were
subcloned into the mammalian expression vector pcDNA3.4. A mock plasmid,
including the pcDNA3.4 vector, was prepared as previously reported (Kumondai et al.,2018; Saito et al., 2018).

Expression of CYP3A4 variants in 293FT cells

13

Downloaded from dmd.aspetjournals.org at ASPET Journals on September 22, 2024

cDNA was used as a template to generate CYP3A4 allelic variant constructs

DMD Fast Forward. Published on December 31, 2020 as DOI: 10.1124/dmd.120.000261
This article has not been copyedited and formatted. The final version may differ from this version.

DMD-AR-2020-000261

293FT cells (ThermoFisher Scientific) were cultured in Dulbecco’s modified
Eagle’s medium (Nacalai Tesque) containing 10% fetal bovine serum at 37ºC under 5%
CO2. Cells were plated at a density of 2.2 × 106 cells/100-mm dish; 24 h after plating,the cells were transfected with a plasmid carrying CYP3A4 (9.6 µg), CPR (0.2 µg), andcytochrome b5 (0.2 µg) cDNA using 30 μL of 1.0 mg/mL of Polyethylenimine “Max”(Kumondai et al., 2020). After incubation for 12 h at 37ºC, 0.25 mM of
5-aminolevulinic acid hydrochloride (Nacalai Tesque) and 0.25 mM iron (II) sulfate
heptahydrate (Wako) were added to the medium. After incubation for 48 h
post-transfection at 37ºC, the cells were scraped from the plates, and the microsomal
fractions were prepared as previously described (Kumondai et al., 2020). Protein
concentrations were determined using the BCA Protein Assay Kit (ThermoFisher
Scientific).

Western blotting
CYP3A4 levels were quantified with an immunoassay using the Wes system(ProteinSimple, Tokyo, Japan). Each well was loaded with 25 μg/mL microsomes.
CYP3A4 was detected using a polyclonal anti-human CYP3A4 antibody (diluted 1:100)

14

Downloaded from dmd.aspetjournals.org at ASPET Journals on September 22, 2024

(Polysciences, Inc., Warrington, PA, USA), according to previously described methods

DMD Fast Forward. Published on December 31, 2020 as DOI: 10.1124/dmd.120.000261
This article has not been copyedited and formatted. The final version may differ from this version.

DMD-AR-2020-000261

and HRP-conjugated goat anti-rabbit IgG. A total protein assay was performed tonormalize each signal using 25 μg/mL microsomes following the manufacturer’s
instructions. Recombinant human CYP3A4 supersomes (Corning Incorporated) wereused as the standard to quantify the CYP3A4 protein level.

The CYP holoprotein content was spectrophotometrically measured according toa previously reported method based on the established protocol by Omura and Sato
(Kumondai et al., 2020).

Measurement of NADPH-cytochrome c reduction activity and microsomal
cytochrome b5 content determinationThe

activity

of

CPR

and

the

contentofcytochrome

b5

were

spectrophotometrically evaluated as previously described (Kumondai et al., 2020).

Midazolam 1′-hydroxylation
CYP3A4 midazolam 1′-hydroxylation activity was determined using a previously
described method with several modifications (El Mahjoub and Staub, 2000). The

15

Downloaded from dmd.aspetjournals.org at ASPET Journals on September 22, 2024

CO-difference spectroscopy

DMD Fast Forward. Published on December 31, 2020 as DOI: 10.1124/dmd.120.000261
This article has not been copyedited and formatted. The final version may differ from this version.

DMD-AR-2020-000261

reaction mixture, a total volume of 100 μL, contained the following components: themicrosomal fraction (50 μg); midazolam (1, 2, 4, 6, 8, 10, and 15 μM); and 100 mM
potassium phosphate buffer (pH 7.4). Following pre-incubation at 37ºC for 3 min,reactions were initiated by adding 10 mM NADPH, and incubation continued at 37ºC
for 10 min. Reactions were terminated by adding 100 μL of acetonitrile containing 500

15,400 g for 10 min, the supernatant was injected into a liquid chromatography-tandem
mass spectrometry (LC-MS/MS) system, as previously described (Kumondai et al.,2020).

Testosterone 6β-hydroxylation assay
CYP3A4 testosterone 6β-hydroxylation activity was determined using apreviously described method with several modifications (Nguyen et al., 2020). The
reaction mixture, in a total volume of 100 μL, contained the following components: themicrosomal fraction (50 μg); testosterone (5, 10, 20, 50, 100, 200, or 500 μM); and 100
mM potassium phosphate buffer (pH 7.4). Following pre-incubation at 37ºC for 3 min,reactions were initiated by adding 10 mM NADPH, and the incubation was continued at
37ºC for 10 min. The reactions were terminated by adding 100 μL of acetonitrile

16

Downloaded from dmd.aspetjournals.org at ASPET Journals on September 22, 2024

nM flunitrazepam as an internal standard. After protein removal by centrifugation at

DMD Fast Forward. Published on December 31, 2020 as DOI: 10.1124/dmd.120.000261
This article has not been copyedited and formatted. The final version may differ from this version.

DMD-AR-2020-000261

containing 10 μM 6β-hydroxytestosterone-d7 as an internal standard. After protein
removal by centrifugation at 15,400 g for 10 min, the supernatant was injected into an
LC-MS/MS system.
The 6β-hydroxytestosterone content was measured using the LC-MS/MS systemin the positive ion detection mode at the electrospray ionization interface (QTRAP 6500

chromatography was conducted using the Nexera Ultra-High-Performance Liquid
Chromatography system (Shimadzu, Kyoto, Japan). Chromatographic separation was
performed with a Kinetex C18 column (2.1 × 50 mm, 5 µg particle size; Phenomenex,
Shimadzu) maintained at 40ºC. Mobile phases were prepared with deionized watercontaining 0.1% formic acid as eluent A and acetonitrile containing 0.1% formic acid aseluent B. The flow rate was 300 µL/min. The gradient program was as follows: elution
was initiated with 10% B, followed by a linear gradient to 60% B from 1 to 7 min, held
at 100% B for 2 min, and then immediately returned to the initial conditions, whichwere maintained for 3 min until the end of the run. Quantification analyses using
Analyst Software (Sciex) were performed in the selected reaction monitoring mode, inwhich ion transitions from the precursor into a product ion were monitored: m/z
289.1→97.0 for testosterone (collision energy, 14 V); m/z 305.0→269.1 for

17

Downloaded from dmd.aspetjournals.org at ASPET Journals on September 22, 2024

LC-MS/MS system; AB Sciex, MA USA). Separation by ultra-high-performance liquid

DMD Fast Forward. Published on December 31, 2020 as DOI: 10.1124/dmd.120.000261
This article has not been copyedited and formatted. The final version may differ from this version.

DMD-AR-2020-000261

6β-hydroxytestosterone (collision energy, 12 V); and m/z 312.1→276.1 for
6β-hydroxytestosterone-d6 (collision energy, 4 V). The optimized MS settings were as
follows: entrance potential, 2.0 V; curtain gas, 25.0 psi; ion transfer voltage, 5500.0 V;
temperature, 750.0ºC; gas 1, 70.0 psi; gas 2, 30.0 psi; and CAD gas, 12.0. Standard
curves for 6β-hydroxytestosterone were constructed within the range of 100–30000 nM

Data analysis
Kinetic data, including the Michaelis constant (Km), turnover number (kcat), andcatalytic efficiency (kcat/Km) values, were determined using the Enzyme Kinetics
Module of SigmaPlot 12.5 (Systat Software, Inc., Chicago, IL, USA), a curve-fitting
program based on nonlinear regression analysis. The normality of our datasets was
initially assessed using the Shapiro-Wilk test. Statistical analyses for multiple
comparisons were performed through variance analysis by Dunnett's T3 test or the
Kruskal-Wallis method (IBM SPSS Statistics Ver. 22, International Business Machines,
Armonk, NY, USA). All values are expressed as the mean ± SD of experiments
performed in triplicates. Differences with P < 0.05 were considered statisticallysignificant. All assays and samples were prepared in triplicates to permit statistical

18

Downloaded from dmd.aspetjournals.org at ASPET Journals on September 22, 2024

using metabolite standards, with a quantification limit of 100 nM.

DMD Fast Forward. Published on December 31, 2020 as DOI: 10.1124/dmd.120.000261
This article has not been copyedited and formatted. The final version may differ from this version.

DMD-AR-2020-000261

analysis.

3D structural modeling of CYP3A4
The 3D structural modeling of CYP3A4 was based on the CYP3A4 X-ray
structure of Sevrioukova and Poulos (2017) (Protein Data Bank code 5te8).

removing the substrate, midazolam or testosterone was docked with the CYP3A4 X-ray
structure according to the CDOCKER protocol of Discovery Studio 2.5 (BIOVIA, CA,
USA). Docking iterations were conducted, taking into consideration the bindingorientation and binding energies under the condition that the volume of the space wasdefined as 9, and the heme iron charge status was Fe3+. After replacing each substitution,
structural optimization was conducted as previously described (Oda et al., 2005).

19

Downloaded from dmd.aspetjournals.org at ASPET Journals on September 22, 2024

Swiss-model was used to complement a part of the lacking structure in CYP3A4. After

DMD Fast Forward. Published on December 31, 2020 as DOI: 10.1124/dmd.120.000261
This article has not been copyedited and formatted. The final version may differ from this version.

DMD-AR-2020-000261

Results
We performed resequencing for the subjects carrying novel SNVs, except for a
subject carrying c.1342G>T because this DNA sample was not available for further
sequencing. For SNVs located within exons, WGS identified 10 novel variants; thesefindings were identical with the results obtained using Sanger sequencing. Two SNVs

(Supplementary Fig. 1). Thus, 11 novel CYP3A4 variants, including the c.1342C>Tvariant, were identified among 4,773 Japanese individuals with low allele frequencies(0.01%–0.05%).
CYP3A4 protein levels normalized by total protein concentrations were assessedby western blotting with a polyclonal CYP3A4 antibody that recognized all CYP3A4
variants (Supplementary Figs. 2A and 2B). The protein levels of CYP3A4.1 and the
CYP3A4 variants did not differ significantly (Supplementary Fig. 2C). Moreover, theaspect ratio of CPR activity and cytochrome b5 content did not significantly vary among
the analyzed CYP3A4 variants (Supplementary Fig. 3). Thus, we used single
microsomes expressed per CYP3A4 variant for functional characterization.
In our study, CYP3A4 exhibited high activity with no significant solvent effects.
The holoprotein content of CYP3A4 variants expressed in 293FT cells was assessed by
CO-difference spectroscopy (Fig. 1). The reduced CO-difference spectra of microsomal
20

Downloaded from dmd.aspetjournals.org at ASPET Journals on September 22, 2024

(c.389G>A and c.412 A>G) carried by one subject were located in different alleles

DMD Fast Forward. Published on December 31, 2020 as DOI: 10.1124/dmd.120.000261
This article has not been copyedited and formatted. The final version may differ from this version.

DMD-AR-2020-000261

fractionsof(CYP3A4.2–.5,

CYP3A4.1
.7,

.9,

.10,

and
.14–.16,.18,

31

CYP3A4

.19,

.23,

.24,

variants
Pro389Ser,

Leu475Val, .28, .29, .31–.34, Thr138Ala, Met256Ile, Ile300Val, Thr323Ala,
Met353Leu, Ile369Val, Ile369Asn, Ala370Val, and Ala448Ser) were determined bymeasuring the increase in maximum absorption at 450 nm after CO treatment. The

Tyr399Ser) had no detectable holoprotein content based on the lack of a significant
increase in their maximum absorption at 450 nm.
We selected midazolam and testosterone as substrates because midazolam
1′-hydroxylation and testosterone 6β-hydroxylation have long been recognized as
CYP3A member-mediated reactions. First, we confirmed that the formation of
1′-hydroxymidazolam or 6β-hydroxytestosterone was linear for up to 10 min incubationby using 50 μg microsomal proteins and 15 µM midazolam or 500 µM testosterone,
respectively. Both metabolites were linear in the presence of 0–50 μg microsomal
proteins during 10 min incubation. The kinetic parameters were calculated byMichaelis-Menten

curvesofmidazolam

1′-hydroxylationandtestosterone

6β-hydroxylation for wild-type CYP3A4 and CYP3A4 variants (Fig. 2 and Table 2).
However, due to insufficient metabolite production, we could not determine the kinetic

21

Downloaded from dmd.aspetjournals.org at ASPET Journals on September 22, 2024

remaining nine variants (CYP3A4.8, .11–.13, .17, 488Frameshift, .21, Arg372Thr, andDMD Fast Forward. Published on December 31, 2020 as DOI: 10.1124/dmd.120.000261
This article has not been copyedited and formatted. The final version may differ from this version.

DMD-AR-2020-000261

parameters of midazolam 1′-hydroxylation for 11 variants (CYP3A4.8, .11–.13, .17,
488Frameshift, .21, Ile369Asn, Ala370Val, Arg372Thr, and Tyr399Ser). We found that
15 variants had significantly lower catalytic efficiency values (P < 0.05) than
CYP3A4.1, whereas there were no significant differences in Km and kcat values among
these CYP3A4 variants. Conversely, the Km values of testosterone 6β-hydroxylation< 0.05). Moreover, the kcat and catalytic efficiency values were significantly lower for
CYP3A4.12 and Leu475Val than for CYP3A4.1 (P < 0.05). Insufficient metabolite
production prevented us from determining the kinetic values for nine variants(CYP3A4.8, .11, .13, .17, 488Frameshift, .21, Ile369Asn, Ala370Val, and Arg372Thr).
Lastly, we calculated the correlation between the midazolam 1′-hydroxylation and thetestosterone 6β-hydroxylation catalytic efficiency values. As shown in Fig. 3, there wasa significant correlation between these values among the different variants (R2 = 0.724,
P < 0.001).
We used 3D structural modeling to assess the molecular properties of CYP3A4
variants and their interactions with midazolam. As shown in Figs. 4A and B, the residue
substitution in CYP3A4.8 and CYP3A4.12 abolished the interaction with heme. The
midazolam docking analysis indicated that the Thr138Ala substitution variant caused a

22

Downloaded from dmd.aspetjournals.org at ASPET Journals on September 22, 2024

were significantly higher for CYP3A4.7, .16, .33, and Thr138Ala than for CYP3A4.1 (PDMD Fast Forward. Published on December 31, 2020 as DOI: 10.1124/dmd.120.000261
This article has not been copyedited and formatted. The final version may differ from this version.

DMD-AR-2020-000261

hydrophobic interaction between Arg130 and Ser134 via the adjacently formedhydrophobic interaction between Ala138 and Ser134 (Fig 4C). In CYP3A4.17, thesubstitution Ser189 located at the F-G loop lacked a hydrophobic interaction with
Leu249 (Fig. 4D). CYP3A4.21 harbored its substitution in the I-helix, which formed ahydrophobic interaction between Cys319 and Val489 (Fig. 4E). Additionally, the4F). The substitution Tyr399Ser caused the formation of a hydrogen bond with Met371(Fig. 4G). We further analyzed Ile369 and Val370, as shown in Figs. 4H–M. The
Ile369Val substitution had an additional hydrophobic interaction with midazolam andheme, affecting the distance between heme and the active site (Fig. 4H). Furthermore,differences in the midazolam and heme interactions due to the amino acid substitutions(Figs. 4I, K, and L), as well as substrate-specific differences in the heme-surrounding
interactions (Figs. 4H, J, K, and M), were observed.

23

Downloaded from dmd.aspetjournals.org at ASPET Journals on September 22, 2024

substitution Ala448Ser abolished numerous hydrophobic interactions with heme (Fig.

DMD Fast Forward. Published on December 31, 2020 as DOI: 10.1124/dmd.120.000261
This article has not been copyedited and formatted. The final version may differ from this version.

DMD-AR-2020-000261

Discussion
Several CYP3A4 genetic variations may contribute to inter-individual differencesin drug efficacy and adverse effect rates (Lamba et al., 2002a). Several studies haveidentified CYP3A4 variants, including rare and region-specific variants, andcharacterized the associated in vitro enzyme kinetics, including previous studies of(Supplementary Table 3) (Fang et al., 2017; Xu et al., 2018; Li et al., 2019a; Li et al.,2019b; Lin et al., 2019a; Lin et al., 2019b; Yang et al., 2019; Zhou et al., 2019a; Chen etal., 2020). However, the underlying causes for functional differences produced by theamino acid alterations are not fully understood and require further research. This study
characterized 40 CYP3A4 variants, including 11 novel variants identified among 4,773
Japanese individuals, by assessing CO-difference spectra, enzymatic activities, and 3D
structural modeling analysis.
A single individual had two allelic variants (CYP3A4.8 and Thr138Ala,
Supplementary Fig. 1), each displaying a significantly decreased CYP3A4 activity for
midazolam and testosterone. These substitutions were located near the vital CPR-CYP
electron transfer site, where they could affect the enzymatic activities (Hasemann et al.,
1995; Bridges et al., 1998). Accordingly, an increase in the maximum absorption at 450

24

Downloaded from dmd.aspetjournals.org at ASPET Journals on September 22, 2024

CYP3A4 variants among the Han Chinese population using numerous substrates

DMD Fast Forward. Published on December 31, 2020 as DOI: 10.1124/dmd.120.000261
This article has not been copyedited and formatted. The final version may differ from this version.

DMD-AR-2020-000261

nm after CO treatment was observed for the Thr138Ala variant (Fig. 1) due tointeractions involving Ala138 that caused hydrogen bonds between Arg130 and Ser134,
which could affect electron transfer because Arg130 located in the C-D loop plays anessential role in electrostatic interactions (Fig. 4C). Interestingly, CYP3A4.8 did nothave an absorption peak at 450 nm after CO treatment (Fig. 1), reflecting a failed hemeon disrupted interactions with heme (Fig. 4A). Although it may be an infrequent case,carriers of two allelic variants that are predicted to be poor metabolizers may be at anincreased risk of adverse drug reactions when treated with drugs metabolized by
CYP3A4.
The midazolam and testosterone hydroxylation activities were slightly decreased
or absent in 11 variants (CYP3A4.8, .11–.13, .17, 488Frameshift, .21, Ile369Asn,
Ala370Val, Arg372Thr, and Tyr399Ser). Excluding Ile369Asn and Ala370Val, the nineremaining variants did not exhibit absorption peaks at 450 nm after CO treatment (Fig.
1), indicating failed heme incorporation. These findings suggest that the remaining nine
CYP3A4 variants lacked quantifiable enzymatic activity. Similarly, the zero holoprotein
content for variants CYP3A4.8 and CYP3A4.12 may be explained by disrupted
interactions with heme (Figs. 4A and B). The lack of activity exhibited by

25

Downloaded from dmd.aspetjournals.org at ASPET Journals on September 22, 2024

incorporation and resulting in zero CYP3A4.8 holoprotein content that could be based

DMD Fast Forward. Published on December 31, 2020 as DOI: 10.1124/dmd.120.000261
This article has not been copyedited and formatted. The final version may differ from this version.

DMD-AR-2020-000261

488Frameshift was due to extensive changes in its molecular conformation caused by
the frameshift mutation. The substitutions in CYP3A4.11 and .13, which were locatedin the conserved ERR triad and K region critical for maintaining the structure among
CYP isoforms, could affect holoprotein stability, as previously reported (Eiselt et al.,2001). The substitution in CYP3A4.17 located in the E-helix was associated with

Leu249 (Fig. 4D), resulting in conformational changes in the active site cavity aroundthe F-G loop, affecting the main active site channel as well as holoprotein stability(Benkaidali et al., 2019). CYP3A4.21 harbored a substitution in the I-helix, which may
affect the stability and flexibility of the β-sheet by forming a hydrophobic interaction
between Cys319 and Val489 (Fig. 4E). However, other substitutions, such as Ala448Ser,
which apparently affected the hydrophobic interactions with heme, did not result in acomplete lack of an absorption peak at 450 nm (Fig. 4G); however, the Ala448Ser
substitution decreased the enzymatic activities. Thus, it is important to evaluate
CO-difference spectroscopy results in combination with the findings of 3D structuralmodeling to improve our understanding of the consequential impact of amino acidsubstitutions.
In addition, a novel substrate recognition site (SRS) map for mammalian CYP

26

Downloaded from dmd.aspetjournals.org at ASPET Journals on September 22, 2024

slightly decreased enzymatic activity. Ser189 lacked a hydrophobic interaction with

DMD Fast Forward. Published on December 31, 2020 as DOI: 10.1124/dmd.120.000261
This article has not been copyedited and formatted. The final version may differ from this version.

DMD-AR-2020-000261

enzymes was developed based on the 3D crystal structures and docking of 868
substrates (Zawaira et al., 2011). Among 40 variants characterized in this study, thefollowing variants were classified as listed regions based on the SRS map: CYP3A4.4
and .29, SRS-1; CYP3A4.2 and .5, SRS-2; CYP3A4.18 and Ile300Val, SRS-4;
CYP3A4.12, .33, Ile369Val, Ile369Asn, Ala370Val, and Arg372Thr, SRS-5; andthe enzyme’s interactions with midazolam, resulting in an increased distance between
heme and the active site (Fig. 4H). Substitutions found in SRS-5, including Ile369 and
Ala370, had the most significant effect on CYP3A4 activity (Sevrioukova, 2017;
Sevrioukova and Poulos, 2017). Interestingly, interactions between the heme group andassayed substrates differed depending on amino acid substitutions, including Ile369Val,
Ile369Asn, CYP3A4.33 (Ala370Ser), and Ala370Val, which ultimately could cause theobserved differences in enzymatic activities (Figs. 4H, I, K, and L). Interestingly,
variants carrying amino acid substitutions around these sites showed a weakercorrelation with the decreasing catalytic efficiencies for midazolam and testosterone
than CYP3A4.1, as shown in Fig 3. The differences in the interactions between aminoacids (Ile369 and Ala370) and heme may play a critical role in the enzymatic activity(Figs. 4H, J, K, and M). The Tyr399Ser substitution also affected the SRS-5 region by

27

Downloaded from dmd.aspetjournals.org at ASPET Journals on September 22, 2024

Leu475Val, SRS-6. For instance, the substitution Ile369Val appeared to directly affectDMD Fast Forward. Published on December 31, 2020 as DOI: 10.1124/dmd.120.000261
This article has not been copyedited and formatted. The final version may differ from this version.

DMD-AR-2020-000261

forming a hydrogen bond between Ser399 and Met371 (Fig. 4G). Thus, amino acidsubstitutions in SRS regions interfered with CYP3A4 activities, but the extent of theresulting differences varied among substrates. Although our study detected a correlation
between midazolam and testosterone activities, in the context of previous data
(Supplementary Table 3), the degree of CYP3A4 activity reduction is apparently highly

The effects of CYP3A4*16 and CYP3A4*18 on several clinically used drugs havebeen evaluated due to their relatively high allele frequencies (1.4%–5.0% and 1.3%–
10%, respectively) (Lamba et al., 2002b; Yamamoto et al., 2003; Fukushima-Uesaka etal., 2004). However, their enzymatic activities also appear to be substrate-dependent(Supplementary Table 3). In addition to SNVs in the coding region, intron variants, such
as CYP3A4*16B and *18B, should also be evaluated in vivo because several promoter
and intronic variants, including CYP3A4*1B and *22, exhibit decreased activities due toreduced mRNA expression (Murayama et al., 2002; Elens et al., 2013). Notably,
CYP3A5*3 and other CYP3A5 allelic variants play important clinical roles because theeffects of CYP3A4 and CYP3A5 on the metabolism of numerous drugs overlap(Fukushima-Uesaka et al., 2004). Moreover, microRNA-27b was identified as apotential regulator of CYP3A4 mRNA level, underscoring the fact that other regulatory

28

Downloaded from dmd.aspetjournals.org at ASPET Journals on September 22, 2024

substrate-specific.

DMD Fast Forward. Published on December 31, 2020 as DOI: 10.1124/dmd.120.000261
This article has not been copyedited and formatted. The final version may differ from this version.

DMD-AR-2020-000261

factors, and not only CYP3A4 allelic variants, affect CYP3A4 mRNA and protein levels(Pan et al., 2009; Ekstrom et al., 2015). Thus, future studies should establish a dosing
algorithm based on inter-individual differences in the in vivo activity of CYP3A.
CYP3A4 can contribute to the catalysis of several sites in the same compound.
For example, midazolam is metabolized by CYP3A4 to the main metabolite

et al., 2009). According to a previous report, the enzymatic activities of CYP3A4.16
and .18 for these two midazolam metabolites decreased to similar levels (Maekawa etal., 2010). Moreover, even with the amino acid sequence differences between CYP3A4
and CYP3A5 (83% homology), 4-hydroxymidazolam formation was several times
lower than 1′-hydroxymidazolam, suggesting that single amino acid substitutions in
CYP3A4 may not dramatically influence the catalysis of different primary products
dependent on changing mutation sites (Niwa et al., 2014). However, a detailed study
encompassing all midazolam metabolites is needed to fully understand the differencesin the metabolic profiles associated with CYP3A4 genetic polymorphisms.
CYP variant characterization by assessing the enzymatic activities for all
metabolites and using all possible substrates may prove essential to evaluate the impact
caused by amino acid substitutions and to improve treatment outcomes. However,29

Downloaded from dmd.aspetjournals.org at ASPET Journals on September 22, 2024

1′-hydroxymidazolam and the secondary metabolite 4-hydroxymidazolam (Christensen

DMD Fast Forward. Published on December 31, 2020 as DOI: 10.1124/dmd.120.000261
This article has not been copyedited and formatted. The final version may differ from this version.

DMD-AR-2020-000261

previously developed computational calculations have been successful in predicting
the activities of over 90% of representative CYP variants (Zhou et al., 2019b). Hence,using computational prediction to assess the effect of amino acid substitutions on
enzymatic CYP activities may be an acceptable surrogate approach, considering thechallenging nature of in vitro and in vivo analyses.

and 40 CYP3A4 variant proteins using our recently established heterologous
expression system. These data reveal the functional effects caused by CYP3A4 genetic
polymorphisms, including 11 novel variants identified among 4,773 Japaneseindividuals, and have been generated using a single microsomal protein preparation per
CYP3A4 variant to reveal their qualitative features. We recognize the limitations
associated with a single biological replicate, and additional biological replicates would
provide greater confidence in estimates of intrinsic clearance for use in futureapplications. Reduced activity or inactive variants caused by CYP3A4 allelic variations
located in the coding region may be the underlying cause of an altered therapeutic
response leading to poor treatment outcomes. However, our assay that uses a
cDNA-based expression system cannot evaluate the influence of splicing variants.
Therefore, further studies including additional biological replicates as well as the use

30

Downloaded from dmd.aspetjournals.org at ASPET Journals on September 22, 2024

In summary, we determined the in vitro metabolic activities of wild-type CYP3A4

DMD Fast Forward. Published on December 31, 2020 as DOI: 10.1124/dmd.120.000261
This article has not been copyedited and formatted. The final version may differ from this version.

DMD-AR-2020-000261

of mini-genes to assess the potential functional consequences of variants affecting
splice sites on catalytic activity are required prior to further downstream applications,such as in vitro-in vivo extrapolation. In the future, CYP3A4 genetic polymorphisms
should be used in combination with other factors affecting the in vivo activity of
CYP3A4, such as CYP3A4 inducers and inhibitors and CYP3A5 activity, to aid thepredictive risk panels. Our current findings should provide guidance in advancing these
efforts.

31

Downloaded from dmd.aspetjournals.org at ASPET Journals on September 22, 2024

implementation of personalized medicine, individualized dosing regimens, andDMD Fast Forward. Published on December 31, 2020 as DOI: 10.1124/dmd.120.000261
This article has not been copyedited and formatted. The final version may differ from this version.

DMD-AR-2020-000261

Acknowledgments
We thank the Biomedical Research Core at the Tohoku University Graduate
School of Medicine for their technical support. We would like to thank Prof. Mizuguchi(Graduate School of Pharmaceutical Sciences, Osaka University) for gifting the plasmid
vector. M.K. is grateful to the Nagai Memorial Research Scholarship from theto thank Editage (www.editage.com) for English language editing.

32

Downloaded from dmd.aspetjournals.org at ASPET Journals on September 22, 2024

Pharmaceutical Society of Japan for their research support (N-170603). We would like

DMD Fast Forward. Published on December 31, 2020 as DOI: 10.1124/dmd.120.000261
This article has not been copyedited and formatted. The final version may differ from this version.

DMD-AR-2020-000261

Authorship Contributions
Participated in research design: Kumondai and Hiratsuka
Conducted experiments: Kumondai, Gutiérrez Rico, Hishinuma, Ueda, Saito, Abe, andMaekawa
Contributed new reagents or analytic tools: Saigusa, Kinoshita, Maekawa, Mano,

Performed data analysis: Kumondai, Saito, Tadaka, Nakayoshi, Oda, and Hiratsuka
Wrote or contributed to the writing of the manuscript: Kumondai, Gutiérrez Rico, andHiratsuka

33

Downloaded from dmd.aspetjournals.org at ASPET Journals on September 22, 2024

Hirasawa, and Hiratsuka

DMD Fast Forward. Published on December 31, 2020 as DOI: 10.1124/dmd.120.000261
This article has not been copyedited and formatted. The final version may differ from this version.

DMD-AR-2020-000261

References
Apellaniz-Ruiz M, Lee MY, Sanchez-Barroso L, Gutierrez-Gutierrez G, Calvo I,
Garcia-Estevez L, Sereno M, Garcia-Donas J, Castelo B, Guerra E,
Leandro-Garcia LJ, Cascon A, Johansson I, Robledo M, Ingelman-Sundberg M,
and Rodriguez-Antona C (2015) Whole-exome sequencing reveals defective

Res 21:322-328.
Benkaidali L, Andre F, Moroy G, Tangour B, Maurel F, and Petitjean M (2019) Four
major channels detected in the cytochrome P450 3A4: A Step toward
understanding its multispecificity. Int J Mol Sci 20:987.
Bridges A, Gruenke L, Chang YT, Vakser IA, Loew G, and Waskell L (1998)
Identification of the binding site on cytochrome P450 2B4 for cytochrome b5
and cytochrome P450 reductase. J Biol Chem 273:17036-17049.
Chen B, Zhang XD, Wen J, Zhang B, Chen D, Wang S, Cai JP, and Hu GX (2020)
Effects of 26 recombinant CYP3A4 variants on brexpiprazole metabolism.
Chem Res Toxicol 33:172-180.
Christensen H, Mathiesen L, Postvoll LW, Winther B, and Molden E (2009) Different
enzyme kinetics of midazolam in recombinant CYP3A4 microsomes from

34

Downloaded from dmd.aspetjournals.org at ASPET Journals on September 22, 2024

CYP3A4 variants predictive of paclitaxel dose-limiting neuropathy. Clin Cancer

DMD Fast Forward. Published on December 31, 2020 as DOI: 10.1124/dmd.120.000261
This article has not been copyedited and formatted. The final version may differ from this version.

DMD-AR-2020-000261

human and insect sources. Drug Metab Dispos 24:261-268.
Clark DP and Pazdernik NJ (2016) Biotechnology. Elsevier/AP Cell, Academic Cell is
an imprint of Elsevier, Amsterdam; Boston.
Eiselt R, Domanski TL, Zibat A, Mueller R, Presecan-Siedel E, Hustert E, Zanger UM,
Brockmoller J, Klenk HP, Meyer UA, Khan KK, He YA, Halpert JR, andCYP3A4 protein variants. Pharmacogenetics 11:447-458.
Ekstrom L, Skilving I, Ovesjo ML, Aklillu E, Nylen H, Rane A, Diczfalusy U, andBjorkhem-Bergman L (2015) miRNA-27b levels are associated with CYP3A
activity in vitro and in vivo. Pharmacol Res Perspect 3:e00192.
El Mahjoub A and Staub C (2000) High-performance liquid chromatographic method
for the determination of benzodiazepines in plasma or serum using thecolumn-switching technique. J Chromatogr B Biomed Sci Appl 742:381-390.
Elens L, van Gelder T, Hesselink DA, Haufroid V, and van Schaik RH (2013)
CYP3A4*22: promising newly identified CYP3A4 variant allele for
personalizing pharmacotherapy. Pharmacogenomics 14:47-62.
Fang P, Tang PF, Xu RA, Zheng X, Wen J, Bao SS, Cai JP, and Hu GX (2017)
Functional assessment of CYP3A4 allelic variants on lidocaine metabolism in

35

Downloaded from dmd.aspetjournals.org at ASPET Journals on September 22, 2024

Wojnowski L (2001) Identification and functional characterization of eightDMD Fast Forward. Published on December 31, 2020 as DOI: 10.1124/dmd.120.000261
This article has not been copyedited and formatted. The final version may differ from this version.

DMD-AR-2020-000261

vitro. Drug Des Devel Ther 11:3503-3510.
Foti RS, Rock DA, Wienkers LC, and Wahlstrom JL (2010) Selection of alternative
CYP3A4 probe substrates for clinical drug interaction studies in vitro data andin vivo simulation. Drug Metab Dispos 38:981-987.
Fujikura K, Ingelman-Sundberg M, and Lauschke VM (2015) Genetic variation in the

25:584-594.
Fukushima-Uesaka H, Saito Y, Watanabe H, Shiseki K, Saeki M, Nakamura T, Kurose
K, Sai K, Komamura K, Ueno K, Kamakura S, Kitakaze M, Hanai S, Nakajima
T, Matsumoto K, Saito H, Goto Y, Kimura H, Katoh M, Sugai K, Minami N,
Shirao K, Tamura T, Yamamoto N, Minami H, Ohtsu A, Yoshida T, Saijo N,
Kitamura Y, Kamatani N, Ozawa S, and Sawada J (2004) Haplotypes of
CYP3A4 and their close linkage with CYP3A5 haplotypes in a Japanese
population. Hum Mutat 23:100.
Gomez-Bravo MA, Apellaniz-Ruiz M, Salcedo M, Fondevila C, Suarez F, Castellote J,
Rufian S, Pons JA, Bilbao I, Alamo JM, Millan O, Brunet M, andRodriguez-Antona

C(2018)

Influence

of

donor

liver

CYP3A4*20

loss-of-function genotype on tacrolimus pharmacokinetics in transplanted

36

Downloaded from dmd.aspetjournals.org at ASPET Journals on September 22, 2024

human cytochrome P450 supergene family. Pharmacogenet Genomics

DMD Fast Forward. Published on December 31, 2020 as DOI: 10.1124/dmd.120.000261
This article has not been copyedited and formatted. The final version may differ from this version.

DMD-AR-2020-000261

patients. Pharmacogenet Genomics 28:41-48.
Gutierrez Rico EM, Kikuchi A, Saito T, Kumondai M, Hishinuma E, Kaneko A, Chan
CW, Gitaka J, Nakayoshi T, Oda A, Saito S, Hirasawa N, and Hiratsuka M(2020) CYP2D6 genotyping analysis and functional characterization of novel
allelic variants in a Ni-Vanuatu and Kenyan population by assessing

35:89-101.
Hasemann CA, Kurumbail RG, Boddupalli SS, Peterson JA, and Deisenhofer J (1995)
Structure and function of cytochromes P450: a comparative analysis of three
crystal structures. Structure 3:41-62.
Hendriks DFG, Vorrink SU, Smutny T, Sim SC, Nordling A, Ullah S, Kumondai M,
Jones BC, Johansson I, Andersson TB, Lauschke VM, and Ingelman-Sundberg
M (2020) Clinically relevant cytochrome P450 3A4 induction mechanisms anddrug screening in three-dimensional spheroid cultures of primary humanhepatocytes. Clin Pharmacol Ther 108:844-855.
Hiratsuka M (2012) In vitro assessment of the allelic variants of cytochrome P450.
Drug Metab Pharmacokinet 27:68-84.
Hornstein BD, Roman D, Arevalo-Soliz LM, Engevik MA, and Zechiedrich L (2016)37

Downloaded from dmd.aspetjournals.org at ASPET Journals on September 22, 2024

dextromethorphan O-demethylation activity. Drug Metab Pharmacokinet

DMD Fast Forward. Published on December 31, 2020 as DOI: 10.1124/dmd.120.000261
This article has not been copyedited and formatted. The final version may differ from this version.

DMD-AR-2020-000261

Effects of circular DNA length on transfection efficiency by electroporation into
HeLa cells. PLoS One 11:e0167537.
Huang SM, Strong JM, Zhang L, Reynolds KS, Nallani S, Temple R, Abraham S, Habet
SA, Baweja RK, Burckart GJ, Chung S, Colangelo P, Frucht D, Green MD,
Hepp P, Karnaukhova E, Ko HS, Lee JI, Marroum PJ, Norden JM, Qiu W,

H, and Lesko LJ (2008) New era in drug interaction evaluation: US Food andDrug Administration update on CYP enzymes, transporters, and the guidance
process. J Clin Pharmacol 48:662-670.
Ingelman-Sundberg M, Mkrtchian S, Zhou Y, and Lauschke VM (2018) Integrating rare
genetic variants into pharmacogenetic drug response predictions. Hum Genomics
12:26
Jackson KD, Durandis R, and Vergne MJ (2018) Role of Cytochrome P450 Enzymes in
the metabolic activation of tyrosine kinase inhibitors. Int J Mol Sci 19:2367.
Kumondai M, Hosono H, Maekawa M, Yamaguchi H, Mano N, Oda A, Hirasawa N,
and Hiratsuka M (2018) Functional characterization of 9 CYP2A13 allelicvariants by assessment of nicotine C-oxidation and coumarin 7-hydroxylation.
Drug Metab Pharmacokinet 33:82-89.

38

Downloaded from dmd.aspetjournals.org at ASPET Journals on September 22, 2024

Rahman A, Sobel S, Stifano T, Thummel K, Wei XX, Yasuda S, Zheng JH, Zhao

DMD Fast Forward. Published on December 31, 2020 as DOI: 10.1124/dmd.120.000261
This article has not been copyedited and formatted. The final version may differ from this version.

DMD-AR-2020-000261

Kumondai M, Hishinuma E, Gutierrez Rico EM, Ito A, Nakanishi Y, Saigusa D,
Hirasawa N, and Hiratsuka M (2020) Heterologous expression of high-activity
cytochrome P450 in mammalian cells. Sci Rep 10:14193.
Lamba JK, Lin YS, Schuetz EG, and Thummel KE (2002a) Genetic contribution tovariablehuman

CYP3A-mediatedmetabolism.

Adv

Drug

Deliv

Rev

Lamba JK, Lin YS, Thummel K, Daly A, Watkins PB, Strom S, Zhang J, and Schuetz
EG (2002b) Common allelic variants of cytochrome P4503A4 and theirprevalence in different populations. Pharmacogenetics 12:121-132.
Lee SJ, Bell DA, Coulter SJ, Ghanayem B, and Goldstein JA (2005) Recombinant
CYP3A4*17 is defective in metabolizing the hypertensive drug nifedipine, andthe CYP3A4*17 allele may occur on the same chromosome as CYP3A5*3,
representing a new putative defective CYP3A haplotype. J Pharmacol Exp Ther
313:302-309.
Li YH, Lin QM, Pang NH, Zhang XD, Huang HL, Cai JP, and Hu GX (2019a)
Functional characterization of 27 CYP3A4 protein variants to metabolize
regorafenib in vitro. Basic Clin Pharmacol Toxicol 125:337-344.
Li YH, Lu XR, Lin QM, Huang HL, Liang XL, Cai JP, Cui J, and Hu GX (2019b)

39

Downloaded from dmd.aspetjournals.org at ASPET Journals on September 22, 2024

54:1271-1294.

DMD Fast Forward. Published on December 31, 2020 as DOI: 10.1124/dmd.120.000261
This article has not been copyedited and formatted. The final version may differ from this version.

DMD-AR-2020-000261

Functional characterization of 27 CYP3A4 variants on macitentan metabolism invitro. J Pharm Pharmacol 71:1677-1683.
Lin QM, Li YH, Liu Q, Pang NH, Xu RA, Cai JP, and Hu GX (2019a) Functional
characteristics of CYP3A4 allelic variants on the metabolism of loperamide invitro. Infect Drug Resist 12:2809-2817.

Characterization of genetic variation in CYP3A4 on the metabolism ofcabozantinib in vitro. Chem Res Toxicol 32:1583-1590.
Maekawa K, Harakawa N, Yoshimura T, Kim SR, Fujimura Y, Aohara F, Sai K, Katori
N, Tohkin M, Naito M, Hasegawa R, Okuda H, Sawada J, Niwa T, and Saito Y(2010) CYP3A4*16 and CYP3A4*18 alleles found in East Asians exhibit
differential catalytic activities for seven CYP3A4 substrate drugs. Drug Metab
Dispos 38:2100-2104.
Murayama N, Nakamura T, Saeki M, Soyama A, Saito Y, Sai K, Ishida S, Nakajima O,
Itoda M, Ohno Y, Ozawa S, and Sawada J (2002) CYP3A4 gene polymorphisms
influence testosterone 6beta-hydroxylation. Drug Metab Pharmacokinet
17:150-156.
Nagasaki M, Yasuda J, Katsuoka F, Nariai N, Kojima K, Kawai Y, Yamaguchi-Kabata Y,

40

Downloaded from dmd.aspetjournals.org at ASPET Journals on September 22, 2024

Lin QM, Li YH, Lu XR, Wang R, Pang NH, Xu RA, Cai JP, and Hu GX (2019b)

DMD Fast Forward. Published on December 31, 2020 as DOI: 10.1124/dmd.120.000261
This article has not been copyedited and formatted. The final version may differ from this version.

DMD-AR-2020-000261

Yokozawa J, Danjoh I, Saito S, Sato Y, Mimori T, Tsuda K, Saito R, Pan X,
Nishikawa S, Ito S, Kuroki Y, Tanabe O, Fuse N, Kuriyama S, Kiyomoto H,
Hozawa A, Minegishi N, Douglas Engel J, Kinoshita K, Kure S, Yaegashi N, To
MJRPP, and Yamamoto M (2015) Rare variant discovery by deep whole-genome
sequencing of 1,070 Japanese individuals. Nat Commun 6:8018.

and specific LC-MS/MS cocktail assay for CYP450 enzymes: Application to
study the effect of catechin on rat hepatic CYP activity. Biomed Chromatogr
34:e4789.
Niwa T, Okamoto A, Narita K, Toyota M, Kato K, Kobayashi K, and Sasaki S (2020)
Comparison of steroid hormone hydroxylation mediated by cytochrome P450
3A subfamilies. Arch Biochem Biophys 682:108283.
Niwa T, Yasumura M, Murayama N, and Yamazaki H (2014) Comparison of catalytic
properties of cytochrome P450 3A4 and 3A5 by molecular docking simulation.
Drug Metab Lett 8:43-50.
Oda A, Yamaotsu N, and Hirono S (2005) New AMBER force field parameters of heme
iron for cytochrome P450s determined by quantum chemical calculations ofsimplified models. J Comput Chem 26:818-826.

41

Downloaded from dmd.aspetjournals.org at ASPET Journals on September 22, 2024

Nguyen V, Espiritu M, and Elbarbry F (2020) Development and validation of a sensitive

DMD Fast Forward. Published on December 31, 2020 as DOI: 10.1124/dmd.120.000261
This article has not been copyedited and formatted. The final version may differ from this version.

DMD-AR-2020-000261

Pan YZ, Gao W, and Yu AM (2009) MicroRNAs regulate CYP3A4 expression via direct
and indirect targeting. Drug Metab Dispos 37:2112-2117.
Racha JK, Zhao ZS, Olejnik N, Warner N, Chan R, Moore D, and Satoh H (2003)
Substrate dependent inhibition profiles of fourteen drugs on CYP3A4 activity
measured by a high throughput LCMS/MS method with four probe drugs,
testosterone,

nifedipine

andterfenadine.

Drug

Metab

Pharmacokinet 18:128-138.
Saito T, Gutierrez Rico EM, Kikuchi A, Kaneko A, Kumondai M, Akai F, Saigusa D,
Oda A, Hirasawa N, and Hiratsuka M (2018) Functional characterization of 50
CYP2D6 allelic variants by assessing primaquine 5-hydroxylation. Drug Metab
Pharmacokinet 33:250-257.
Sakurai F, Kawabata K, Yamaguchi T, Hayakawa T, and Mizuguchi H (2005)
Optimization of adenovirus serotype 35 vectors for efficient transduction inhuman hematopoietic progenitors: comparison of promoter activities. Gene Ther
12:1424-1433.
Schärfe CPT, Tremmel R, Schwab M, Kohlbacher O, and Marks DS (2017) Genetic
variation in human drug-related genes. Genome Med 9:117.
Sevrioukova IF (2017) High-level production and properties of the cysteine-depleted

42

Downloaded from dmd.aspetjournals.org at ASPET Journals on September 22, 2024

midazolam,

DMD Fast Forward. Published on December 31, 2020 as DOI: 10.1124/dmd.120.000261
This article has not been copyedited and formatted. The final version may differ from this version.

DMD-AR-2020-000261

cytochrome P450 3A4. Biochemistry 56:3058-3067.
Sevrioukova IF and Poulos TL (2017) Structural basis for regiospecific midazolam
oxidation by human cytochrome P450 3A4. Proc Natl Acad Sci U S A
114:486-491.
Watanabe T, Saito T, Gutierrez Rico EM, Hishinuma E, Kumondai M, Maekawa M,

Yamaguchi H, Mano N, Hirasawa N, and Hitatsuka M (2018) Functional
characterization of 40 CYP2B6 allelic variants by assessing efavirenz
8-hydrozylation. Biochem Pharmacol 156:420-430.
Werk AN, Lefeldt S, Bruckmueller H, Hemmrich-Stanisak G, Franke A, Roos M,
Kuchle C, Steubl D, Schmaderer C, Brasen JH, Heemann U, Cascorbi I, andRenders L (2014) Identification and characterization of a defective CYP3A4
genotype in a kidney transplant patient with severely diminished tacrolimus
clearance. Clin Pharmacol Ther 95:416-422.
Xiao K, Gao Jie, Weng SJ, Fang Y, Gao N, Wen Qiang, Jin H, and Qiao HL (2019)
CYP3A4/5 activity probed with testosterone and midazolam: correlation
between two substrates at the microsomal and enzyme levels. Mol
Pharmaceutics 16:382-392.

43

Downloaded from dmd.aspetjournals.org at ASPET Journals on September 22, 2024

Oda A, Saigusa D, Saito S, Yasuda J, Nagasaki M, Minegishi N, Yamamoto M,

DMD Fast Forward. Published on December 31, 2020 as DOI: 10.1124/dmd.120.000261
This article has not been copyedited and formatted. The final version may differ from this version.

DMD-AR-2020-000261

Xu RA, Wen J, Tang P, Wang C, Xie S, Zhang BW, Zhou Q, Cai JP, and Hu GX (2018)
Functional characterization of 22 CYP3A4 protein variants to metabolize
ibrutinib in vitro. Basic Clin Pharmacol Toxicol 122:383-387.
Yamamoto T, Nagafuchi N, Ozeki T, Kubota T, Ishikawa H, Ogawa S, Yamada Y, Hirai
H, and Iga T (2003) CYP3A4*18: it is not rare allele in Japanese population.

Yang CC, Zheng X, Liu TH, Wang CC, Tang PF, Chen Z, Zhang BW, Fang P, Hu GX,
and Cai JP (2019) Functional characterization of 21 CYP3A4 variants on
amiodarone metabolism in vitro. Xenobiotica 49:120-126.
Zanger UM, Momoi K, Hofmann U, Schwab M, and Klein K (2020) Tri-allelichaplotypes determine and differentiate functionally normal allele CYP2D6*2
and impaired allele CYP2D6*41. Clin Pharmacol Ther Oct 12 Online ahead ofprint.
Zanger UM and Schwab M (2013) Cytochrome P450 enzymes in drug metabolism:
regulation of gene expression, enzyme activities, and impact of genetic variation.
Pharmacol Ther 138:103-141.
Zawaira A, Ching LY, Coulson L, Blackburn J, and Wei YC (2011) An expanded,unified substrate recognition site map for mammalian cytochrome P450s:

44

Downloaded from dmd.aspetjournals.org at ASPET Journals on September 22, 2024

Drug Metab Pharmacokinet 18:267-268.

DMD Fast Forward. Published on December 31, 2020 as DOI: 10.1124/dmd.120.000261
This article has not been copyedited and formatted. The final version may differ from this version.

DMD-AR-2020-000261

analysis of molecular interactions between 15 mammalian CYP450 isoforms and868 substrates. Curr Drug Metab 12:684-700.
Zhou XY, Hu XX, Wang CC, Lu XR, Chen Z, Liu Q, Hu GX, and Cai JP (2019a)
Enzymatic activities of CYP3A4 allelic variants on quinine 3-hydroxylation In
Vitro. Front Pharmacol 10:591.

cytochrome P450 alleles: a meta-analysis of population-scale sequencing
projects. Clin Pharmacol Ther 102:688-700.
Zhou Y, Mkrtchian S, Kumondai M, Hiratsuka M, and Lauschke VM (2019b) An
optimized prediction framework to assess the functional impact ofpharmacogenetic variants. Pharmacogenomics J 19:115-126.

45

Downloaded from dmd.aspetjournals.org at ASPET Journals on September 22, 2024

Zhou Y, Ingelman-Sundberg M, and Lauschke VM (2017) Worldwide distribution of

DMD Fast Forward. Published on December 31, 2020 as DOI: 10.1124/dmd.120.000261
This article has not been copyedited and formatted. The final version may differ from this version.

DMD-AR-2020-000261

Footnotes:
M.H. was supported by grants from AMED (Grant Number 20kk0305009), Takahashi
Industrial and Economic Research Foundation, and Smoking Research Foundation.
M.K. was supported by the Japan Society for the Promotion of Science (Grant Number
19J10744). S.S, K.K, S.T., and D.S. were supported by grants from AMED (Grant

Megabank Project (MEXT), Promoting Public Utilization of Advanced Research
Infrastructure (MEXT), and the Sharing and Administrative Network for Research
Equipment (MEXT).

Competing interests
The authors declare no conflict of interest.

46

Downloaded from dmd.aspetjournals.org at ASPET Journals on September 22, 2024

Number JP20km0105001 and JP20km0105002), in part from the Tohoku Medical

DMD Fast Forward. Published on December 31, 2020 as DOI: 10.1124/dmd.120.000261
This article has not been copyedited and formatted. The final version may differ from this version.

DMD-AR-2020-000261

Figure legends
Fig. 1. Representative CO-difference spectra of CYP3A4 variant proteins expressed in
293FT cells. All assays and measurements were performed in triplicates using a single
microsomal preparation. Mock 1 indicates transfection with 10 μg mock plasmids.
Mock 2 indicates transfection with 9.6 μg mock plasmids, 0.2 μg CPR plasmids, and 0.2

Fig. 2. Michaelis-Menten curves for CYP3A4 variants. Determined kinetic parameters(Km, kcat, and catalytic efficiency) of midazolam 1′-hydroxylation and testosterone
6β-hydroxylation. All assays and measurements were performed in triplicates using asingle microsomal preparation.

Fig. 3. Correlation between the

catalytic efficiency ratios for midazolam

1′-hydroxylation and testosterone 6β-hydroxylation among CYP3A4 variants.
Midazolam 1′-hydroxylation catalytic efficiency ratios are plotted on the horizontal axis,
and testosterone 6β-hydroxylation catalytic efficiency ratios are plotted on the verticalaxis. The numbers correspond to known CYP3A4 variants, and the amino acidsubstitutions indicate the newly identified variants.

47

Downloaded from dmd.aspetjournals.org at ASPET Journals on September 22, 2024

μg cytochrome b5 plasmids.

DMD Fast Forward. Published on December 31, 2020 as DOI: 10.1124/dmd.120.000261
This article has not been copyedited and formatted. The final version may differ from this version.

DMD-AR-2020-000261

Fig. 4. Diagram pairs showing the partial crystal structure of CYP3A4.1 (left image)and CYP3A4 variants (right image). Pink line; hydrophobic interactions. Green line;
conventional hydrogen bonds. Gray line; carbon-hydrogen bonds. MDZ; midazolam.
TST; testosterone.
Downloaded from dmd.aspetjournals.org at ASPET Journals on September 22, 2024

48

Variants

Protein

CYP3A4*1

CYP3A4.1

CYP3A4*2

CYP3A4.2

664T>CCYP3A4*3

CYP3A4.3

1334T>CCYP3A4*4

CYP3A4.4

352A>GCYP3A4*5

CYP3A4.5

653C>G

Downloaded from dmd.aspetjournals.org at ASPET Journals on September 22, 2024

CYP3A4*7

CYP3A4.7

167G>A

Gly56Asp

CYP3A4*8

CYP3A4.8

389G>A

Arg130Gln

CYP3A4*9

CYP3A4.9

508G>A

Val170Ile

CYP3A4*10

CYP3A4.10

520G>C

Asp174His

Table 1. CYP3A4 allelic variants characterized in this study.
Nucleotide mutations

49

DMD-AR-2020-000261

Amino acid substitutions

Ser222Pro
Met445Thr
Ile118Val
Pro218Arg

CYP3A4*11

CYP3A4.11

1088C>TCYP3A4*12

CYP3A4.12

1117C>TCYP3A4*13

CYP3A4.13

1247C>TCYP3A4*14

CYP3A4.14

44T>C

CYP3A4*15

CYP3A4.15

485G>ACYP3A4*16

CYP3A4.16

554C>GCYP3A4*17

CYP3A4.17

566T>C

Downloaded from dmd.aspetjournals.org at ASPET Journals on September 22, 2024

CYP3A4*18

CYP3A4.18

878T>C

Leu293Pro

CYP3A4*19

CYP3A4.19

1399C>T

Pro467Ser

1461_1462insA

488Frameshift956A>G

Tyr319Cys

CYP3A4*20
CYP3A4*21

CYP3A4.21

50

DMD-AR-2020-000261

Thr363Met
Leu373Phe
Pro416Leu
Leu15Pro
Arg162Gln
Thr185Ser
Phe189Ser

CYP3A4*28

CYP3A4.28

64C>G

CYP3A4*29

CYP3A4.29

337T>ACYP3A4*31

CYP3A4.31

972C>A

Downloaded from dmd.aspetjournals.org at ASPET Journals on September 22, 2024

CYP3A4*32

CYP3A4.32

1004T>CIle335Thr

CYP3A4*33

CYP3A4.33

1108G>TAla370Ser

CYP3A4*34

CYP3A4.34

1279A>GIle427Val

Novel variant 1

412A>G

Thr138Ala

CYP3A4*23

CYP3A4.23

484C>T

CYP3A4*24

CYP3A4.24

600A>T
1165C>T
1423C>G51

DMD-AR-2020-000261

Arg162Trp
Gln200His
Pro389Ser
Leu475Val
Leu22Phe
Phe113Ile
His324GlnNovel variant 2

768G>C

Novel variant 3

898A>G

Novel variant 4

967A>G

Novel variant 51057A>TNovel variant 61105A>G

Novel variant 7

1106T>A

Novel variant 81109C>T

Downloaded from dmd.aspetjournals.org at ASPET Journals on September 22, 2024

Novel variant 91115G>CArg372Thr

Novel variant 10

1196A>C

Tyr399Ser

Novel variant 111342G>TAla448Ser

52

DMD-AR-2020-000261

Met256Ile
Ile300Val
Thr323Ala
Met353Leu
Ile369Val
Ile369Asn
Ala370Val

Downloaded from dmd.aspetjournals.org at ASPET Journals on September 22, 2024

DMD-AR-2020-000261

Table 2. Kinetic parameters of midazolam 1′-hydroxylation and testosterone 6β-hydroxylation by microsomes from 293FT cells
expressing wild-type CYP3A4 and variant CYP3A4 proteins.

Testosterone 6β-hydroxylation

Midazolam 1′-hydroxylation
Km (μM)
Variants

Km (μM)

kcat

Catalytic(pmol/min/pmol

efficiency(fmol/min/pmol

efficiency

CYP3A4)(kcat/Km)CYP3A4)(kcat/Km)(% of wild-type)

kcat

Catalytic(% of wild-type)

CYP3A4.1

2.96 ± 0.19

4.79 ± 0.13

1.63 ± 0.11

67.5 ± 7.15

26.6 ± 1.77

0.40 ± 0.02

CYP3A4.2

3.49 ± 1.12

1.43 ± 0.14

0.45 ± 0.13*

55.7 ± 6.88

6.75 ± 0.32*

0.12 ± 0.01***(27.4%)CYP3A4.3

3.15 ± 0.15

3.00 ± 0.21

0.95 ± 0.04

53

(30.9%)
63.8 ± 6.23

8.41 ± 1.12*

0.13 ± 0.01***CYP3A4.4

3.56 ± 0.14

2.78 ± 0.09

0.78 ± 0.03

98.9 ± 7.36

(47.9%)CYP3A4.5

7.64 ± 2.99

0.75 ± 0.09

0.11 ± 0.03*

256 ± 23.3

(6.7%)CYP3A4.7

4.20 ± 0.36

1.37 ± 0.09

0.33 ± 0.04*

240 ± 11.1***(20.2%)

Downloaded from dmd.aspetjournals.org at ASPET Journals on September 22, 2024

(58.4%)DMD-AR-2020-000261(33.2%)

27.3 ± 1.49

0.28 ± 0.01*(69.7%)

6.37 ± 0.55*

0.02 ± 0.00**(6.3%)

3.63 ± 0.03*

0.02 ± 0.00**(3.8%)CYP3A4.8

N.D.

N.D.

N.D.

N.D.

N.D.

N.D.

CYP3A4.9

3.36 ± 0.11

4.23 ± 0.08

1.26 ± 0.05

68.2 ± 1.43

20.2 ± 0.34

0.30 ± 0.01

(77.5%)CYP3A4.10

4.11 ± 0.34

2.50 ± 0.01

0.61 ± 0.05*

54(74.8%)
103 ± 8.67

12.1 ± 0.22

0.12 ± 0.01***CYP3A4.11

N.D.

N.D.

N.D.

N.D.

CYP3A4.12

N.D.

N.D.

N.D.

152 ± 13.4

CYP3A4.13

N.D.

N.D.

N.D.

N.D.

CYP3A4.14

3.97 ± 0.43

4.31 ± 0.28

1.10 ± 0.11

92.2 ± 17.3

(67.2%)CYP3A4.15

3.46 ± 0.21

3.29 ± 0.15

0.95 ± 0.02

65.5 ± 2.48

Downloaded from dmd.aspetjournals.org at ASPET Journals on September 22, 2024

(37.6%)DMD-AR-2020-000261(29.9%)
N.D.

N.D.

1.69 ± 0.10*

0.01 ± 0.00**(2.8%)

N.D.

N.D.

17.4 ± 0.98

0.19 ± 0.03*

15.9 ± 0.09

(58.4%)CYP3A4.16

5.51 ± 0.37

1.60 ± 0.07

0.29 ± 0.01*(17.8%)

55(48.9%)
0.25 ± 0.01*(63.3%)

128 ± 4.74*

15.9 ± 0.31

0.12 ± 0.01**(31.5%)

N.D.

N.D.

N.D.

N.D.

CYP3A4.18

3.69 ± 0.27

2.80 ± 0.03

0.76 ± 0.07*

73.8 ± 3.85

(46.9%)CYP3A4.19

3.97 ± 0.06

4.05 ± 0.14

1.02 ± 0.02

99.5 ± 20.4

(62.6%)
488Frameshift

N.D.

N.D.

N.D.

N.D.

CYP3A4.21

N.D.

N.D.

N.D.

N.D.

CYP3A4.23

3.28 ± 0.37

2.48 ± 0.11

0.76 ± 0.05

69.8 ± 1.81

Downloaded from dmd.aspetjournals.org at ASPET Journals on September 22, 2024

CYP3A4.17

DMD-AR-2020-000261

N.D.

N.D.

9.07 ± 0.40*

0.12 ± 0.00*(31.1%)

16.3 ± 0.48

(43.1%)
N.D.

N.D.

N.D.

N.D.

7.66 ± 0.22*

0.11 ± 0.00*(46.7%)CYP3A4.24

3.27 ± 0.38

3.84 ± 0.08

1.19 ± 0.12
(73.0%)

56

0.17 ± 0.03

(27.7%)
63.1 ± 10.7

14.5 ± 0.52

0.23 ± 0.03**(59.2%)

4.44 ± 0.37

1.31 ± 0.02

0.30 ± 0.03*

73.5 ± 4.84

(18.2%)
Leu475Val

3.83 ± 0.17

1.77 ± 0.04

0.46 ± 0.01*

68.8 ± 3.80

(28.4%)CYP3A4.28

3.15 ± 0.25

3.32 ± 0.19

1.06 ± 0.09

96.1 ± 8.18

(65.0%)CYP3A4.29

3.56 ± 0.33

1.91 ± 0.04

0.54 ± 0.06*

98.3 ± 8.96

Downloaded from dmd.aspetjournals.org at ASPET Journals on September 22, 2024

Pro389Ser

5.16 ± 0.08*

5.47 ± 0.01*

15.7 ± 0.58

11.0 ± 0.36

(33.2%)CYP3A4.31

3.25 ± 0.42

4.72 ± 0.19

1.47 ± 0.18

5.33 ± 0.47

3.23 ± 0.13

0.61 ± 0.06*

57

0.07 ± 0.00**(17.8%)
0.08 ± 0.00**(20.1%)
0.16 ± 0.01***(41.5%)
0.11 ± 0.01**(28.4%)

72.6 ± 8.08

22.7 ± 1.27

(90.4%)CYP3A4.32

DMD-AR-2020-000261

0.31 ± 0.02
(79.4%)

73.7 ± 3.36

15.9 ± 0.40

0.22 ± 0.01*

CYP3A4.33

3.02 ± 0.16

2.30 ± 0.12

0.76 ± 0.08*

214 ± 3.35***(46.9%)CYP3A4.34

3.35 ± 0.50

4.86 ± 0.12

1.48 ± 0.21

75.3 ± 3.87

(91.0%)
Thr138Ala

10.2 ± 0.97

1.10 ± 0.06

0.11 ± 0.01*

134 ± 6.81*(6.7%)
Met256Ile

6.87 ± 0.87

2.66 ± 0.13

0.39 ± 0.06*

105 ± 4.88

Downloaded from dmd.aspetjournals.org at ASPET Journals on September 22, 2024

(37.5%)

12.3 ± 0.33

19.9 ± 0.13

1.39 ± 0.00*

17.0 ± 0.91

(24.2%)
Ile300Val

3.33 ± 0.49

2.82 ± 0.15

0.86 ± 0.08
(52.8%)58

DMD-AR-2020-000261(54.6%)
0.06 ± 0.00*(14.5%)
0.26 ± 0.01*(66.8%)
0.01 ± 0.00**(2.6%)
0.16 ± 0.00*(40.7%)

105 ± 5.62

18.0 ± 0.48

0.17 ± 0.00*(43.4%)

4.45 ± 0.44

2.79 ± 0.21

0.63 ± 0.02

83.8 ± 7.36

(38.6%)
Met353Leu

3.33 ± 0.13

4.00 ± 0.10

1.20 ± 0.03

83.9 ± 5.34

(73.7%)
Ile369Val

5.26 ± 0.34

1.34 ± 0.05

0.26 ± 0.01*

83.7 ± 5.78

(15.7%)
Ile369Asn

N.D.

N.D.

N.D.

N.D.

Ala370Val

N.D.

N.D.

N.D.

Arg372Thr

N.D.

N.D.

Tyr399Ser

N.D.

N.D.

Downloaded from dmd.aspetjournals.org at ASPET Journals on September 22, 2024

Thr323Ala

DMD-AR-2020-000261

17.3 ± 0.79

0.21 ± 0.01**(52.5%)

15.2 ± 0.44

0.18 ± 0.01**(45.9%)

20.1 ± 0.79

0.24 ± 0.01*(60.7%)

N.D.

N.D.

N.D.

N.D.

N.D.

N.D.

N.D.

N.D.

N.D.

N.D.

131 ± 15.7

3.29 ± 0.28*

0.03 ± 0.00**(6.4%)

59

3.58 ± 0.35

2.45 ± 0.10

0.69 ± 0.05*

85.6 ± 10.9

(42.4%)
Mock 1

N.D.

N.D.

N.D.

N.D.

Mock 2

N.D.

N.D.

N.D.

N.D.

The data represent the mean ± SD of the three independently performed catalytic assays.
*P < 0.05, **P < 0.01, and ***P < 0.005 compared to CYP3A4.1 by Dunnett T3 test.

Downloaded from dmd.aspetjournals.org at ASPET Journals on September 22, 2024

Ala448Ser

DMD-AR-2020-000261

10.6 ± 1.32*(31.2%)
N.D.

N.D.

N.D.

N.D.

N.D. represents not determined.
All assays and measurements were performed in triplicates using a single microsomal preparation.
Mock 1 indicates transfection with 10 μg mock plasmids.
Mock 2 indicates transfection with 9.6 μg mock plasmids, 0.2 μg CPR plasmids, and 0.2 μg cytochrome b5 plasmids.

60

0.12 ± 0.00*

Fig. 1

Downloaded from dmd.aspetjournals.org at ASPET Journals on September 22, 2024

Fig. 2

Downloaded from dmd.aspetjournals.org at ASPET Journals on September 22, 2024

Testosterone 6β-hydroxylation
Midazolam 1ʹ-hydroxylation

Fig. 3

Downloaded from dmd.aspetjournals.org at ASPET Journals on September 22, 2024

Fig. 4

Downloaded from dmd.aspetjournals.org at ASPET Journals on September 22, 2024