Coordination Chemistry Reviews 427 (2021) 213600

Contents lists available at ScienceDirect

Coordination Chemistry Reviews

journal homepage: www.elsevier.com/locate/ccr

Molecular probes for human cytochrome P450 enzymes: Recent

progress and future perspectives
Jingjing Wu a,⇑, Xiaoqing Guan b, Ziru Dai c, Rongjing He b, Xinxin Ding d, Ling Yang b,⇑, Guangbo Ge b,⇑
a
Department of Clinical Pharmacology, College of Pharmacy, Dalian Medical University, Dalian 116044, China
b
Institute of Interdisciplinary Integrative Medicine Research, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
c
Key Laboratory of Bioactive Substances and Resources Utilization of Chinese Herbal Medicine, Ministry of Education, Institute of Medicinal Plant Development, Chinese Academy
of Medical Sciences & Peking Union Medical College, Beijing 100193, China
d
Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson, AZ 85721, USA

a r t i c l e i n f o a b s t r a c t

Article history: The cytochrome P450 enzymes (P450s or CYPs) are a class of heme-containing monooxygenases respon-
Received 22 July 2020 sible for the oxidative metabolism of a wide range of endogenous substances and foreign chemicals.
Accepted 7 September 2020 Deciphering the physiological functions of CYPs and their relevance to human diseases requires reliable
tools for sensing target enzyme in complex biological systems. Over the past few decades, great efforts
have been made to develop isoform-specific probe substrates for human CYP(s) and use them in both
Keywords: basic researches and translational applications. The growing database of the ligands and 3D-structures
Cytochrome P450 enzymes (CYPs)
of human CYPs strongly facilitates the design and development of CYP probe substrates with improved
Probe substrates
Design principles
specificity. Herein, we summarize the recent progress in the development and applications of molecular
Inhibitor screening probes for human CYPs. The structural and catalytic features of human CYPs, as well as the design prin-
Biomedical applications ciples and strategies used in the development of CYP probe substrates, have been well summarized. Two
comprehensive lists of molecular probes for various human CYP enzymes, including non-optical and opti-
cal probe substrates, along with their structural information and kinetic parameters, are presented.
Finally, the current challenges and future perspectives in this field are proposed and highlighted. The
information and knowledge presented here provides practical tools for sensing CYP activities in complex
biological systems, exploring the relevance of CYPs to human diseases, as well as screening and charac-
terizing of CYP modulators.
Ó 2020 Elsevier B.V. All rights reserved.

Abbreviations: ADR, adverse drug reactions; AHMC, 3-[2-(diethylamino)-ethyl]-7-hydroxy-4-methylcoumarin; AMMC, 3-[2-(N,N-diethyl-N-methylammonium)ethyl]-7-

methoxy-4-methylcoumarin; BFBFC, 2,5-bis(trifluoromethyl)-7-benzyloxy-4-trifluoromethyl-coumarin; BFC, 7-benzyloxy-4-(trifluoromethyl)-coumarin; BnXPI, (E)-2-(2-(6-
p-methoxybenzy
2,3-dihydro-1H-xanthen-4-yl)vinyl)-3,3-dimethyl-1-propyl-3H-indol-1-ium iodide; HXPI, (E)-2-(2-(6-hydroxy-2, 3-dihydro-1H-xanthen-4-yl)vinyl)-3,
3-dimethyl-1-propyl-3H-indol-1-ium iodide; BOMCC, 7-benzyloxy-methyloxy
3-cyano-coumarin; BOMF, benzyloxy-methyl-fluorescein; BOMFC, 7-benzyloxy-methyloxy-
4-(trifluoromethyl)-coumarin; BOMR, benzyloxy-methyl-resorufin; BQ, 7-benzyloxy-quinoline; CEC, 3-cyano-7-ethoxycoumarin; CHPO, chloroethyl derivative (7-(2-chlor
oethoxy)–3H-phenoxazin-3-one; CYP, cytochrome P450; rCYPs, recombinant cytochrome P450 enzymes; DBF, dibenzyl-fluorescein; DBOMF, di-(benzyl-O-methyl)-
fluorescein; DDI, drug-drug interactions; EMA, European Medicines Agency; ER, 7-ethoxy-resorufin; EOMCC, 7-ethoxy-methyloxy-3-cyano-coumarin; EFC, 7-ethoxy-4-
(trifluoromethyl)-coumarin; FDA, Food and Drug Administration; HAMC, 7-hydroxy-4-(aminomethyl)-coumarin; HDI, herb-drug interactions; HN, 4-hydroxy-1,
8-naphthalimide; HTS, high-throughput screening; Km, the substrate concentration that leads to half maximal velocity; MAMC, 7-methoxy-4-(aminomethyl)-coumarin;
MFC, 7-methoxy-4-(trifluoromethyl) -coumarin; MOBFC, 7-p-methoxy-benzyloxy-4-trifluoro-coumarin; NBCeN, N-(4-butyl)-4-chloroethoxy-1,8-naphthalimide; NBHN,
N-(4-butyl)-4-hydroxy-1,8-naphthalimide; iPrBN, 4-isopropoxy-7H-benzo[de]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one; HBN, 4-hydroxy-7H-benzo[de]benzo, [4,5]imi
dazo[2,1-a]isoquinolin-7-one; NCMN, N-(3-carboxy propyl)-4-methoxy-1, 8-naphthalimide; NCHN, N-(3-carboxy propyl)-4-hydroxy-1,8-naphthalimide; NEMN, N-((2-
hydroxyl ethoxy) ethyl)-4-methoxy-1,8-naphthalimide; NEHN, N-((2-hydroxyethoxy)ethyl)-4-hydroxy-1,8-naphthalimide; NEiPN, N-((2-hydroxyl ethoxy)ethyl)-4-isopro
poxy-1,8-naphthalimide; DPCI, 2-(3-(4-(2-chloroethoxy)styryl)-5,5-dimethylcyclohex-2-enylidene)malononitrile; DPOH, 2-(3-(4-hydroxystyryl)-5,5-dimethylcyclohex-2-e
nylidene) malononitrile; NEHN, N-(3-ethyl)-4-hydroxy-1,8-naphthalimide; NEN, N-(3-ethyl)-1,8-naphthalimide; NTI, narrow therapeutic index; OMF, 3-O-
methylfluorescein; PDB, Protein Data Bank; Vivid Cyan, 7-hydroxy-4-trifluoromethyl-coumarin; Vivid Blue, 3-cyano-7-hydroxy-coumarin; Vivid Red, resorufin; Vivid
Green, fluorescein; Vmax, the enzyme’s maximum velocity.
⇑ Corresponding authors.
E-mail addresses: wjj@dmu.edu.cn (J. Wu), yling@dicp.ac.cn (L. Yang), geguangbo@shutcm.edu.cn (G. Ge).

https://doi.org/10.1016/j.ccr.2020.213600
0010-8545/Ó 2020 Elsevier B.V. All rights reserved.
J. Wu et al. Coordination Chemistry Reviews 427 (2021) 213600

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2. Structural and catalytic features of human CYPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2.1. Structural features of human CYPs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2.1.1. General structural features of human CYPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2.1.2. Structural variability of human CYP enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
2.2. CYP-mediated reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
3. In silico prediction of CYP mediated metabolism. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
3.1. Prediction of ligand binding to CYP enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
3.2. Prediction of the site of metabolism (SOM) by CYP enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
4. Approaches for the development of CYP probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
4.1. Phenotype-based screening . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
4.2. Phenotype screening combined with computer-aided design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
5. Non-optical probes for human CYPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
6. Optical probes for human CYPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
6.1. Fluorogenic probes for human CYPs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
6.2. Bioluminogenic probes for human CYPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
7. Biomedical applications of the probes for human CYPs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
7.1. Sensing enzymatic activity in biological samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
7.2. Screening of CYP modulators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
7.3. CYP–ligand interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
8. Challenges and future perspectives. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Declaration of Competing Interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

1. Introduction variety of fatty acids, such as lauric acid, myristic acid, arachidonic
acid and eicosadienoic acid [7]. Generally, the xenobiotic-
The cytochrome P450 (P450s or CYPs) superfamily of metabolizing CYPs are mainly related to the metabolism and the
heme-containing monooxygenase enzymes are responsible for treatment outcomes (such as efficacy and toxicity) of the drugs
the oxidative metabolism of a wide range of endogenous sub- or toxins, while endobiotic-metabolizing CYPs may be associated
stances and foreign chemicals in almost all living organisms. Mam- with the etiology of some diseases [2].
malian CYPs have a broad substrate specificity, and they play The expression and function of mammalian CYPs, which can be
crucial roles in the biotransformation of numerous physiologically, affected by genetic, physiological, pathophysiological and environ-
pharmacologically, or toxicologically important molecules, includ- mental factors, have been recognized as critical determinants of
ing steroids, chemical carcinogens, environmental pollutants, and inter-individual variability in drug disposition and clinical out-
therapeutic drugs [1]. As one of the largest enzyme superfamily, come [8–10]. Significant changes in both expression and function
CYPs have been extensively investigated by many researchers in of human CYPs may bring various undesirable effects, including
various fields, such as biochemistry, pharmacology, toxicology, therapeutic failure, idiosyncratic toxicity [11], multiple-organ
genetics, environmental biology, chemical biology and molecular injury and drug-drug interactions (DDI) [12] (Fig. 1). By far, inhibi-
biology. In humans, 57 genes that encode CYPs have been identi- tion of CYP activities is the most common mechanism leading to
fied (Table 1), which are classified into 18 families and 44 subfam- DDI [13]. The incidence of serious adverse drug reactions (ADRs)
ilies based on sequence similarity [2]. Of the identified 18 human was found to be extremely high among hospitalized patients, mak-
CYP families, three (i.e., CYP1, CYP2, and CYP3) are primarily ing ADR as one of the leading causes of death [14]. Since DDI is an
responsible for the metabolism of xenobiotics [3]. By contrast, important cause of ADRs, various regulatory agencies including the
the majority of the other CYP families are involved in the metabo- United States Food and Drug Administration (USFDA) and the
lism of endobiotic substances. For instances, it has been reported European Medicines Agency (EMA), have recommended to assay
that CYP2S1 is involved in polyunsaturated fatty acid x-1 hydrox- the DDI potential of an investigational drug or drug candidate
ylation [4], while CYP2W1 selective catalyzes oxidation of toward the key hepatic CYPs involved in drug metabolism (such
lysophospholipids [5]. Meanwhile, CYP4F22 has been reported to as CYP1A2, CYP2C8, CYP2C9, CYP2C19 and CYP3A4) before
play an essential role in the transformation of ceramide to acylce- approval [15]. Therefore, the identification or development of pre-
ramide [6], and CYP2Z1 catalyzes the oxidative metabolism of a ferred substrates for individual CYPs, to be used as ‘‘probe sub-
strates” to accurately assess the activities of a target CYP enzyme
Table 1 in vivo, ex vivo, or in other biological systems such as microsomal
Classification of human CYPs based on their substrate spectra. preparations, and evaluate the potentials of DDI, have received
Substrate class Human CYP enzymes great attention from both basic science researchers and the phar-
maceutical industry over the past two decades.
Xenobiotics 1A1, 1A2, 2A6, 2A13, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6,
2E1, 2F1, 3A4, 3A5, 3A7, 3A43
Steroids 1B1, 7A1, 7B1, 8B1, 11A1, 11B1, 11B2, 17A1, 19A1, 21A2, 2. Structural and catalytic features of human CYPs
27A1, 39A1, 46A1, 51A1
Fatty Acids 2J2, 2S1, 2U1, 4A11, 4B1, 4F11, 4F12, 4V2, 4Z1
Lysophospholipids 2W1
2.1. Structural features of human CYPs
Eicosanoids 4F2, 4F3, 4F8, 5A1, 8A1
Vitamins 2R1, 24A1, 26A1, 26B1, 26C1, 27B1, 27C1 2.1.1. General structural features of human CYPs
Ceramides 4F22 The structural features of CYPs are invaluable for understanding
Unknown 2A7, 4A22, 4X1, 20A1
the structural basis of the selectivity of CYP ligands and gaining
2
J. Wu et al.
Fig. 1. Interindividual variability in CYPs and its clinical implications.
Fig. 2. The general structure of human CYPs. The heme iron and the cysteine
residue located on L helix are colored in red and blue, respectively.
deeper insight into the structure-function relationships. One of the
most conserved elements of CYPs is the cysteine residue located on
a loop region in front of the L helix, which provides the sulfur atom
to the heme iron; another one is the heme moiety surrounded by
helices I and L (Fig. 2) [16,17]. The catalytic domain is connected
to the NH2-terminal transmembrane helix of CYPs by a proline-
rich region, and the substrate-binding area is located on the distal
side of the heme [17,18]. Overall, the secondary structures of mam-
malian CYPs are conserved, and all human CYPs contain 12 a-
helices (A-L) and 4 b-sheets (1–4) [19]. The B0 helix and regions
between the F and G helices show the greatest degree of variation
and undergo remarkable conformational changes to allow the
ligands to enter [16]. Notably, several additional helices are pre-
sent, such as the F’ and G’ helices found in many human CYPs.
The F and G helices and the F–G loop, together with the F’ and G’
helices play a key role in substrate access to the active site, by
forming a ‘‘roof” for the active site opposite to the heme ‘‘floor”
[1,20]. The conserved I helix, the longest helix in human CYPs, is
adjacent to heme and runs through the catalytic domain [17].
Currently, the crystal structures of more than one hundred
human drug-metabolizing CYPs and 43 human steroid-
3
Coordination Chemistry Reviews 427 (2021) 213600
metabolizing CYPs have been revealed and found in the Protein
Data Bank (PDB). The representative protein crystals for major
human CYPs are summarized in Table 2. The three-dimensional
structures of CYP enzymes provide crucial insight into the active
site architectures, the key domains and amino acids responsible
for ligand binding [19]. With the help of the structures of CYP
enzymes, further investigations on the molecular basis of the
structure–activity relationships, and the altered enzyme activity
arising from genetic polymorphism, can be conducted more effi-
ciently. More importantly, the crystal structures of CYPs strongly
facilitate the rational design of CYP ligands (such as specific inhibi-
tors or probe substrates) that may confer potential therapeutic
benefits or serve as diagnostic tools [46].
2.1.2. Structural variability of human CYP enzymes
The variability in the amino acid sequence and structural fea-
tures of the active site among various CYP enzymes is substantial,
despite the overall conservation of three-dimensional structures in
all mammalian CYPs. The most structurally variable regions are
located in the B–C and F–G helices, and the L helix [22]. Some
examples of the structural diversity in these regions are high-
lighted below in the context of functional consequences. 1) CYP1A2
favors small planar aromatic or heterocyclic amine ligands, which
can be attributed to several polar residues (Thr118, Ser122, and
Thr124) on the B0 –C region that face toward the active site, as well
as Thr223 on F helix and Asp320 on I helix that line the roof of the
active cavity [22]. 2) CYP2A6 preferentially binds small aromatic
ligands [47], due to the fact that this enzyme has a correspondingly
small active site pocket (260 Å3), while Asn297 is considered as a
key residue for substrate orientation [23]. 3) CYP2C9 favors weakly
acidic ligands [27], owing to that its structure (PDB 1R9O) shows a
disordered conformation of the F–G loop and an extra turn at the
NH2-terminus of helix A, which is quite different from that of other
members from CYP2C subfamily [28]. The active site cavity of
CYP2C9 is smaller than that of CYP2C8 but larger than that of
CYP2A6 [48]. 4) CYP2E1 possesses one of the smallest active-site
pockets (190 Å3), and this enzyme favors neutral compounds with
low molecular weight and fatty acids [31]. CYP2E1 has a conserved
Asp295 in the active site cavity, and this amino acid plays a pivotal
role in substrate recognition and ligand binding. 5) CYP3A4 is a
highly flexible enzyme that possesses a large binding cavity
(1173 Å3 ~ 1332 Å3), leading to the binding of a wide range of
J. Wu et al. Coordination Chemistry Reviews 427 (2021) 213600

Table 2
Representative crystal structures of the major human CYP enzymes.

Enzyme PDB ID Resolution (Å) Ligand Refs.

CYP1A1 a
4I8V 2.60 Å a-naphthoflavone [21]
CYP1A2 a
2HI4 1.95 Å a-naphthoflavone [22]
a
CYP2A6 1Z10 1.90 Å Coumarin [23]
1Z11 2.05 Å Methoxsalen [23]
a
CYP2A13 4EJG 2.50 Å Nicotine [24]
CYP2B6 a 4I91 2.00 Å (+)-a-Pinene [25]
CYP2C8 a 2VN0 2.70 Å Troglitazone [26]
2NNJ 2.28 Å Felodipine [26]
2NNI 2.80 Å Montelukast [26]
a
CYP2C9 1OG5 2.55 Å S-wafarin [27]
1R9O 2.00 Å Flurbiprofen [28]
CYP2C19 a 4GQS 2.87 Å 0XV b [29]
CYP2D6 a 4WNU 2.26 Å Quinidine [30]
CYP2E1 a 3E6I 2.20 Å Indazole [31]
CYP3A4 a 5TE8 2.70 Å Midazolam [32]
3TJS 2.25 Å DTMCR c [33]
3NXU 2.00 Å Ritonavir [34]
CYP3A5 a 5VEU 2.91 Å Ritonavir [35]
CYP1B1 a 3PM0 2.70 Å a-naphthoflavone [36]
CYP7A1 3V8D 1.90 Å 7-ketocholesterol [37]
3SN5 2.75 Å Cholest-4-en-3-one [37]
CYP11A1 3N9Y 2.60 Å Cholesterol [38]
a
CYP17A1 3RUK 2.60 Å Abiraterone [39]
CYP19A1 3EQM 2.90 Å Androstenedione [40]
CYP21A2 4Y8W 2.64 Å Progesterone [41]
a
CYP46A1 2Q9F 1.90 Å Cholesterol-3-sulphate [42]
4ENH 2.50 Å Fluvoxamine [43]
3MDV 2.40 Å Clotrimazole [44]
CYP51A1 3LD6 2.80 Å Ketoconazole [45]
a
The recombinant CYPs are commercially available.
b
An inhibitor of CYP2C19.
c
Desthiazolylmethyloxycarbonyl ritonavir.

structurally diverse ligands [49]. CYP3A4 has several distinguish-
ing structural features, such as short F and G helices and several
large hydrophobic phenylalanine residues that form a cluster in
the ligand-free structure at the roof of the active site. The pheny-
lalanine cluster is distorted when the enzyme binds to ligands
[33,50,51]. 6) the structures of CYP3A5 and CYP3A4 are very simi-
lar due to their high homology (~83% identity) in amino acid
sequence, but the architectures of their active cavities are different.
Especially as the Phe210 located on the extended portion of helix F
in CYP3A5 is longer than Leu210 of CYP3A4, which expands the
size of the upper region of the substrate binding cavity in CYP3A5
by increasing the space between the helix F-F’ and helix G’-G con-
nectors, and this space can be further enlarged by replacing Phe108
in CYP3A4 using a shorter Leu108 in CYP3A5 [35].
A visual comparison of the substrate binding cavities of the
above mentioned CYPs is shown in Fig. 3. It is apparent that the
structural variability, primarily in the regions of F–G helices and
B0 helix among various CYPs, contributes to the distinct shapes
and sizes of the active cavities. Recent advances in deciphering
structural and mechanistic properties of human CYPs have signifi-
cantly facilitated the development of highly specific ligands for
various CYP enzymes.
via NADPH-CYP reductase, and generally involves a distal water
displacement from the heme iron [55]. Once the molecular oxygen
is bound to the heme, an oxy-iron-CYP complex is formed after the
transfer of a second electron and oxygen activation. The oxy-iron-
CYP complex is reduced, generating a peroxo-ferric-CYP complex,
which is further converted to a hydroperoxo-iron-P450 complex
via protonation of the terminal oxygen. Then, the latter complex
undergoes proton-assisted oxygen–oxygen bond scission, forming
a reactive, and high-valent oxo-iron-CYP complex. Finally, sub-
strate oxidation by this reactive complex produces the oxidized
metabolite, with CYP regenerated to its initial ferric state [56,57].
The schematic catalytic cycle of CYP enzymes is shown in Fig. 4.
CYP-catalyzed reactions are very diverse, including hydrocar-
bon hydroxylation; epoxidation; O-, S-, and N-dealkylation; dehy-
drogenation; dehalogenation, oxidative deamination,
decarboxylation; reductive dehalogenation; N-oxide, and epoxide
reduction; isomerization and ring formation. Among all known
CYP-catalyzed reactions, monooxygenation is the most common
reaction [55,58]. A clear understanding of the enzymology of vari-
ous types of CYP-catalyzed reactions is important for the rational
design and development of ligands for a CYP enzyme.

2.2. CYP-mediated reactions 3. In silico prediction of CYP mediated metabolism

As their name implies, the P450 hemoproteins have a character-
istic absorption band at 450 nm when the ferrous heme iron binds
to carbon monoxide [52]. During the catalytic process, CYPs bind
the molecular oxygen, split it into its component atoms and trans-
fer one oxygen atom to a substrate in the CYP active site [53]. The
binding of ligands to CYPs occur mainly when the heme iron is in
the ferric state, with water bound [54]. At most cases, the catalytic
cycle of CYPs is initiated by binding of a substrate to the ferric
enzyme, with the first electron provided to the CYP from NADPH
4
3.1. Prediction of ligand binding to CYP enzymes
The metabolism occurs when the compound has affinity for CYP
subtypes and is able to react with the activated heme group. The
existing approaches for predicting the specificity of CYP isoforms
can be divided into two major categories: ligand-based methods
and structure-based methods [59]. The ligand-based approach is
to establish mathematical models based on the biological activities
and descriptors of ligands, such as physicochemical and structural
J. Wu et al. Coordination Chemistry Reviews 427 (2021) 213600

Fig. 3. Comparisons of the substrate binding cavity of six major human hepatic CYPs: (a) CYP1A2 (2HI4)- a-naphthoflavone, (b) CYP2A6 (1Z10)-coumarin, (c) CYP2C9 (1OG5)-
flurbiprofen, (d) CYP2E1 (3E6I)- indazole, (e) CYP3A4 (3NXU)-ritonavir, (f) CYP3A5 (5VEU)-ritonavir. The greatest degree of variation includes the region between the F and G
helices (green) and the B0 helix (pink).

Fig. 4. The schematic catalytic cycle of CYPs.

5
J. Wu et al.
properties. Machine learning methods including neural networks,
support vector machines and random forests have been developed
as primary methods for predicting enzyme specificity on account
of the complex nonlinear relationships in enzyme-ligands interac-
tion data [60]. More detailed ligand-based methods and tools have
recently been reviewed [61]. In structure-based methods, protein
structure information is essential for understanding protein-
ligand interaction details. Structural characteristics of main CYP
subtypes have been described above. Both the key information of
substrate recognition and catalysis in target enzyme (ligand-
based methods) as well as protein structure information
(structure-based methods) would contribute to the computer-
aided design and development of the molecular probes for a target
CYP. Molecular docking and molecular dynamics (MD) simulations,
as well as quantum mechanics/molecular mechanics (QM/MM)
methods are the most commonly used structure-based methods
to predict binding affinity and binding mode [62]. Based on the
predicted binding affinity by scoring functions or more accurate
free energy values in MD simulations, binders and non-binders
can be classified. Notably, multiple binding poses are observed in
CYP2C9 and 2D6, which needs to be taken into account to interpret
the experimental data [63,64]. For CYP3A4, binding cooperativity
effect of more than one ligand is proposed [65]. Especially, due
to the active sites of CYP are deeply buried inside the enzyme,
the flexibility and adaptability of accessible channels have been
simulated and proved to be crucial for ligand passage and selectiv-
ity [66].
3.2. Prediction of the site of metabolism (SOM) by CYP enzymes
Because of the variety of CYP mediated reactions, it may exist a
few potential reactive sites of a substrate . Identification of SOMs is
critical for design of compounds with good ADMET properties. So
far, hybrid QM/MM calculation is considered as the most accurate
method which describes the active sites by accurate QM method,
while capturing the effects of protein/solvent environments by
MM method [54,67]. It is notable that the complete catalytic cycle
of CYP can now be studied by QM/MM methods. However, these
calculations are so time-consuming that it is not yet possible to
predict a large number of ligands [54,67]. Therefore, reactivity pre-
dictions of small ligands are always based on simplified models of
the heme interactions which ignore the protein environment
[59,61]. Studies have shown that for most CYP isoenzymes, such
as CYP3A4, 1A2, 2A6, 2B6, and 2E1, the prediction sites with low
activation energies are the major determinants of SOMs [50,68].
While in the case of CYP2D6 and 2C9, it is insufficient to predict
SOMs by assaying reactivity alone rather than combining with
binding poses [69,70]. The common SOM prediction tools have
been summarized by some previous literatures [61,62]. In addition
to the ligand reactivity prediction, the accessibility of ligands to the
vicinity of active site can be simulated by docking and MD
approaches. A simple metabolic criterion is based on the distance
(less than 6 Å) between the SOM of a substrate and the heme iron
ion, and more elaborate criteria including orientation are also pro-
posed for improvement [67].
4. Approaches for the development of CYP probes
4.1. Phenotype-based screening
The discovery and development of the first-generation CYP
probes was made primarily through phenotype-based screening.
Before the 1980 s, it was difficult to determine whether a com-
pound could act as a highly specific substrate (or ‘‘probe”) for a tar-
get human CYP, owing to the incomplete nature of the phenotype-
6
Coordination Chemistry Reviews 427 (2021) 213600
based screening systems and very limited access to purified human
CYP enzymes. During the 1980 s, in addition to the phenotype-
based assays using purified human CYPs, other approaches, includ-
ing correlation of enzyme activities toward individual substrates
and the application of selective chemical inhibitors, were devel-
oped and used in drug metabolism related fields [16]. After that,
application of recombinant DNA technology yielded a panel of
cDNAs for CYPs, and then heterologous expression of mammalian
CYPs was achieved in mammalian and insect cells, yeast and bac-
terial systems, in the early 1990 s [16,71].
The currently used specific probe substrates for human CYPs are
mainly derived from endogenous substance or therapeutic drugs.
For example, testosterone and cortisol can be selectively catalyzed
by CYP3A to form 6b-hydroxylated products [72,73]. Many drugs,
such as ethoxyresorufin (CYP1A), paclitaxel (CYP2C8), and nifedip-
ine (CYP3A), have been found with high specificity towards a par-
ticular CYP, which qualifies them as CYP probe substrates [74].
Over the past two decades, equipped with a deeper understanding
of the structure and catalytic behaviors of various CYPs, research-
ers have determined the relationships between substrate structure
and CYP selectivity, which are very helpful for discovery or rational
design of the probe substrate(s) for a target CYP enzyme.
In recent years, several groups have systematically investigated
the structure-metabolism relationships for a series of natural com-
pounds, one of which are the dibenzocyclooctadiene lignans iso-
lated from Fructus Schisandrae. These lignans exhibited obvious
metabolic preferences for CYP3A. For example, the dibenzocyclooc-
tadiene lignans with six methoxy groups on the biphenyl rings are
co-substrates of both CYP3A4 and CYP3A5, such as deoxyschizan-
drin [75]. By contrast, the C-7 hydroxylated dibenzocyclooctadiene
lignans are selectively metabolized by CYP3A4, but not CYP3A5,
such as gomisin A and schizandrin [76,77]. Conversely, schisan-
therin E, a natural dibenzocyclooctadiene lignan containing a ben-
zoyl group at C-6 and a hydroxyl group at C-12, can be selectively
metabolized to a 2-O-demethylated product by CYP3A5 [78]. The
above results suggest that the substituents on the octatomic and
biphenyl rings are crucial to the metabolic selectivity for CYP
enzymes. The structure-CYP metabolism selectivity relationships
of dibenzocyclooctadiene lignans are summarized in Fig. 5.
Another notable case is the naturally occurring bufadienolides,
the major constitutes in traditional Chinese medicine Chansu.
These compounds also showed evident metabolic preferences to
CYP3A enzymes. Several groups have reported that some bufa-
dienolides bearing an acetyl substituent at the C-16 site (such as
cinobufagin and bufotalin), can be metabolized by both CYP3A4
and CYP3A5 [79]. By contrast, resibufogenin and bufalin, two nat-
ural bufadienolides without an acetyl substituent at the C-16 site,
could be selectively metabolized by CYP3A4 to form the corre-
sponding 5b-hydroxylated product [80]. Further investigations
revealed that the hydroxyl group at the C-3 site and the epoxy
group at the C-14/15 site of these bufadienolides are crucial for
CYP3A4-catalyzed oxidation, while the acetyl substituent at the
C-16 site may contribute to metabolic increase of CYP3A5. The
structure-metabolic selectivity relationships of bufadienolide com-
pounds are summarized in Fig. 6.
4.2. Phenotype screening combined with computer-aided design
In view of the fact that identification of probe substrates for a
target CYP isoenzyme from endogenous substances or approved
drugs is always time-consuming, the researchers have begun to
rationally design CYP probes with the help of the structural and
catalytic features of target enzyme(s). Computer-aided design of
specific probes for a target enzyme is mainly based on the 3D
structure of the target enzyme and the key information of sub-
strate recognition and catalysis in target enzyme(s). Over the past
J. Wu et al.
Fig. 5. The substrate structure-CYP selectivity relationships of dibenzocyclooctadiene lignans.
Fig. 6. The substrate structure-CYP selectivity relationships of bufadienolides.
decade, in order to design a specific substrate for a particular
human CYP enzyme in a more efficient way, molecular docking
and molecular dynamic simulations are often utilized to explore
the key interactions between CYPs and their ligands
[46,78,81,82]. Commonly, the key interactions contributing to sub-
strate recognition and catalysis can guide the structural modifica-
tion of the ligands (such as substrate) to make their interactions
with target enzymes more specific and stronger [78,81]. With the
help of computer-aided design and virtual screening technology,
the biochemists can rationally design novel, man-made probe sub-
strates with improved specificity for target CYPs, as well as develop
enzyme-specific probe substrates via modification of a common
substrate for multiple CYPs [82,83].
In 2015, Dai et al. have developed several two-photon ratiomet-
ric fluorescent probes (i.e., NCMN and NBCeN) for CYP1A (including
CYP1A1 and CYP1A2) and CYP1A1 by virtual screening combined
with CYP phenotyping assays [81,83]. NCMN and NBCeN display
the best combination of selectivity, sensitivity, ideal kinetic behav-
iors and ratiometric fluorescence response following CYP1A or
CYP1A1-catalyzed O-dealkylation [81,83]. These probes are
7
Coordination Chemistry Reviews 427 (2021) 213600
designed on the basis of structural features (especially the amino
acids in the active site cavity) of CYP1A that play key roles in
ligand-enzyme interactions, while the substrate preferences of
CYP1A are also considered. In these studies, 4-hydroxy-1,8-
naphthalimide (HN) was selected as the basic fluorophore due to
its excellent optical properties, including high fluorescence quan-
tum yield, large stokes shifts (>120 nm), good photostability and
significant two-photon absorbability characteristics [84-86]. On
the baisi of the subtle differences in substrate preference and 3D
structure of CYP1A1 and CYP1A2, an isoform-specific probe
(NBCeN) for CYP1A1 was successfully developed by Dai et al, via
chemical modifications of the local structure at the metabolic site
on a known co-substrate for CYP1A isoenzymes [83]. To obtain a
highly sensitive and specific probe for the target enzyme, a panel
of O-alkylated HN derivatives were synthesized and used to
explore the structure-selectivity relationship with the help of
molecular docking and phenotyping-based screening (Fig. 7). The
results demonstrate that the introduction of a chloroethyl group
to HN can yield the best specificity towards CYP1A1 over other
CYPs including CYP1A2 [83]. This newly developed CYP1A1 probe
J. Wu et al.
Fig. 7. A practical strategy proposed for the design and development of a specific fluorogenic probe for a given CYP isozyme.
has been successfully used to real-time monitor the activities of
CYP1A1 in living systems with very high sensitivity and specificity.
These findings suggest that fluorescent probes hold great promise
for sensing the biological function of target CYPs in living systems;
their application will be very helpful for exploring the relevance of
CYPs to human diseases. Furthermore, the strategy used for the
development of NBCeN blazes a new trail for the development of
enzyme-specific probes for other CYPs.
Following the development of NBCeN as an isoform-specific flu-
orescent probe substrate for CYP1A1, Zhang et al. further investi-
gated a series of 1,8-naphthalimide-based two-photon
fluorescent chromophores and evaluated their potentials as
CYP1A1 substrate using computer-aided calculations [87]. The
docking results showed that NCMN-3, containing an electron-
withdrawing benzothiadiazole group, and NCMN-5, with an
electron-donating carbazole group, fit the active site cavity of
CYP1A1 well, with the carbon-iron distances of 3.31 Å and
4.18 Å, respectively, suggesting that NCMN-3 and NCMN-5 might
be specific substrates for CYP1A [87]. Nevertheless, it should be
noted that all virtual screening approaches require further experi-
mental validation by reaction phenotyping assays and enzymatic
kinetic analyses [88]. Furthermore, to ensure the isoform speci-
ficity, results from the biochemical assays for target enzyme activ-
ity using fluorogenic probes should be verified with other methods,
such as LC-MS/MS-based proteomics or western-blotting, or with
assay using known non-fluorogenic specific probe substrates for
the target enzyme [81,89,90].
More recently, Ning et al developed a near-infrared fluorescent
probe for CYP2J2 (BnXPI) by virtual screening combined with CYP
phenotyping assays. Based on the active cavity structure character-
istics, Ning et al. designed a series of self-immolative linker into
the HXPI derivatives with a long aromatic skeleton, in order to
adapt to the narrow substrate channel of CYP2J2. These modifica-
tions aim to shorten the spatial distance between the O-alkyl group
(metabolic moiety) and the catalytic center of CYP2J2. Further-
more, based on the CYP phenotyping assays, BnXPI possesses a
benzyl as a self-immolative linker, which was found to exhibit
the optimal combination of flexibility and specificity towards
CYP2J2 amongst all tested O-alkylated HXPI derivatives [91].
5. Non-optical probes for human CYPs
Probe substrates are defined as the compounds that are exclu-
sively metabolized in vitro by an individual CYP enzyme in a given
biological system [92]. Generally, an excellent probe targeting a
single enzyme is recommended to meet the following require-
ments: high selectivity, good sensitivity, and high conversion rate
8
Coordination Chemistry Reviews 427 (2021) 213600
(turnover) (Vmax or kcat), as well as commercial availability and
good chemical stability for both substrate and metabolite(s)
[93,94]. It is well-known that enzyme kinetic behavior is a key con-
cern for the quantitative applications of probe substrates, thus the
probe substrates that follow classic Michaelis-Menten kinetics are
highly desirable. The preferred and acceptable probe substrates for
the major hepatic human CYPs have been well documented and
recommended by FDA [95]. All these probes and their metabolites
are detected by LC combined with ultraviolet or tandem mass
spectrometry. However, most of these non-fluorogenic CYP probes
are derived from drugs, which commonly display low detection
sensitivity and inapplicable for high throughput screening of CYP
inhibitors [74,92]. Despite the aforementioned issues, non-
fluorogenic probes are still used by pharmaceutical researchers,
in consideration of their ‘‘drug-like” properties. The commonly
used non-optical substrates for human CYPs, along with the LC-
tandem mass spectrometry methods used for their analysis, are
listed in Table 3.
Besides of the CYPs in the CYP1-3 families, steroid-metabolizing
CYPs have also received increased attentions, as they are closely
related to certain pathological conditions. Multiple human CYP
enzymes are known to contribute to the production of steroid hor-
mones [117–120]. Among these enzymes, CYP17A1 is the sole
enzyme responsible for converting progestogens to androgens in
humans, and the latter drives prostate cancer proliferation. Thus,
CYP17A1 is recognized as an attractive therapeutic target for
blocking androgen biosynthesis [121], implying that inhibition of
this enzyme can be therapeutically useful [122]. One CYP17A1
inhibitor, abiraterone, progresses rapidly through clinical trials,
and has received FDA approval in 2012 for administration [123].
CYP46A1, which is a cholesterol 24-hydroxylase, is specifically
expressed in the brain. The metabolite 24S-hydroxycholesterol
shows elevated levels in the cerebrospinal fluid of Alzheimer
patients and could induce cell death, suggesting that CYP46A1 is
involved in the pathology of Alzheimer’s disease [124]. Therefore,
CYP46A1 inhibition therapy may be therapeutically beneficial for
the patients with Alzheimer’s disease [125]. Overall, either for
the human drug-metabolizing CYPs or for the steroid-
metabolizing CYPs, the highly specific probe substrates are crucial
for activity sensing and screening of selective modulators of the
target CYPs.
6. Optical probes for human CYPs
As mentioned above, quantification of the metabolite of non-
optical CYP substrates requires liquid chromatography combined
with UV or mass spectrometry detector, such assays are
J. Wu et al. Coordination Chemistry Reviews 427 (2021) 213600

Table 3
Commonly used non-optical CYP probes that are analyzed using LC/MS-based methods.
a
Enzyme Tissue Probe substrates Chemical structure Catalytic Applications Km Vmax MRM Refs.
sites monitor or Kcat or kmax
CYP1A2 Liver Phenacetin O-deethylation In vitro (rCYPs, 47.0 688b 151.9/ [96]
microsomes, living 110.0
cells)

CYP2A6 Liver, Coumarin 7-hydroxylation In vitro (rCYPs, 0.84 940b 161.0/ [96]
Lung microsomes, living 133.0 g
cells)

CYP2A13 Lung Phenacetin O-deethylation In vitro (rCYPs, 10.7 3.82f 151.9/ [97]
microsomes, living 110.0
cells)

CYP2B6 Liver, Bupropion Hydroxylation In vitro (rCYPs, 81.7 413b 256.2/ [96]
Lung microsomes, living 139.0
cells)

CYP2C8 Liver Paclitaxel 6a- In vitro (rCYPs, 18.3 250b 870.6/ [98]
hydroxylation microsomes, living 286.1
cells)In vivo (rat)

Amodiaquine N-dealkylation In vitro (rCYPs, 1.89 1480b 328.0/ [96]

microsomes, living 283.0
cells)

CYP2C9 Liver, Diclofenac 4-hydroxylation In vitro (rCYPs, 27.6 967b 312.0/ [99,100]
Intestine microsomes, living 231.1
cells)In vivo (rat)

d
(S)-Warfarin 7-hydroxylation In vitro (rCYPs, 4.60 2.33 323.0/ [101]
microsomes, living 177.0 g
cells)In vivo (human
and rat)

Tolbutamide 4-hydroxylation In vitro (rCYPs, 147 276b 287.0/ [96]

microsomes, living 171.0
cells)In vivo (human
and rat)
d
CYP2C19 Liver (R)-Omeprazole 5- hydroxylation In vitro (rCYPs, 3.99 12.9 362.2/ [102]
microsomes, living 214.1
cells)In vivo (human
and rat)

(S)-Mephenytoin 40 -hydroxylation In vitro (rCYPs, 57.2 58.3b 235.0/ [96,100]

microsomes, living 150.1
cells)

CYP2D6 Liver Bufuralol 10 -hydroxylation In vitro (rCYPs, 7.28 118.9b 278.1/ [100,103]
microsomes, living 186.1
cells)In vivo (human
and rat)

Dextromethorphan O-demethylation In vitro (rCYPs, 4.64 202b 258.2/ [96,100]

microsomes, living 157.0
cells)In vivo (human
and rat)

CYP2E1 Liver, Chlorzoxazone 6-hydroxylation In vitro (rCYPs, 73.9 2360b 184.2/ [96]
Lung microsomes, living 120.0 g
cells)In vivo (human
and rat)

(continued on next page)

9
J. Wu et al. Coordination Chemistry Reviews 427 (2021) 213600

Table 3 (continued)
a
Enzyme Tissue Probe substrates Chemical structure Catalytic Applications Km Vmax MRM Refs.
sites monitor or Kcat or kmax
CYP2J2 Heart, Astemizole O-demethylation In vitro (rCYPs, 1.80 152c 445.0/ [104]
Liver microsomes, living 204.0
cells)In vivo (human
and rat)

d
Terfenadine Hydroxylation In vitro (rCYPs, 1.60 29.4 488.3/ [105]
microsomes, living 452.2
cells)In vivo (human
and rat)

CYP3A4 Liver, Midazolam 10 -hydroxylation In vitro (rCYPs, 2.27 1220b 342.1/ [96]
Intestine microsomes, living 203.1
cells)In vivo (human
and rat)

Testosterone 6b- In vitro (rCYPs, 46.4 5260b 305.0/ [96]

hydroxylation microsomes, living 269.0
cells)

Nifedipine Dehydrogenation In vitro (rCYPs, 15.3 4461b 345.0/ [75,106]

microsomes, living 122.0
cells)

Bufalin 5b- In vitro (rCYPs, 8.00 2620b 403.3/ [94]

hydroxylation microsomes, living 349.3
cells)

Deoxyschizandrin 7-hydroxylation In vitro (rCYPs, 1.60 623b 250 nm [75]

microsomes, living
cells)

Gomisin A 8-hydroxylation In vitro (rCYPs, 6.30 47.5b 245 nm [76]

microsomes, living
cells)

d
CYP3A5 Liver, Schisantherin E 2-O- In vitro (rCYPs, 5.90 63.4 230 nm [78]
Lung, demethylation microsomes, living
Kidney cells)

10
J. Wu et al. Coordination Chemistry Reviews 427 (2021) 213600

Table 3 (continued)
a
Enzyme Tissue Probe substrates Chemical structure Catalytic Applications Km Vmax MRM Refs.
sites monitor or Kcat or kmax
d
T-5 N-oxidation In vitro (rCYPs, 2.60 13.9 584.2/ [107]
microsomes, living 476.2
cells)

d
Maraviroc Hydroxylation In vitro (rCYPs, 48.9 0.93 530.0/ [108]
microsomes, living 296.0
cells)

CYP4F2 Liver Leukotriene x-hydroxylation In vitro (rCYPs, 60.0 2.70 d

335.0/ [109,110]
microsomes, living 194.9 g
cells)

CYP7A1 Liver Cholesterol 7a- In vitro (rCYPs, 16.4 397b 385.1/ [111,112]
hydroxylation microsomes, living 159.1
cells)

CYP17A1 Adrenal Progesterone 17a- In vitro (rCYPs, 11.4 1.31f 331.2/ [39,113]
hydroxylation microsomes, living 97.00
cells)

e
CYP21A2 Adrenal Progesterone 21- In vitro (rCYPs, 1.05 3.73 331.2/ [114]
hydroxylation microsomes, living 97.00
cells)

CYP46A1 Brain 24(S)- 24,25- and In vitro (rCYPs, 3.90 0.92f 210 nm [42,115]
hydroxycholesterol 24,27- microsomes, living
dihydroxylation cells)

Cholesterol 24(S)- In vitro (rCYPs, 5.40 0.11f 420.3/ [42,116]

hydroxylation microsomes, living 385.5
cells)

a
Km values were lM; b Vmax values were in pmol/min/mg human liver microsome; c Vmax values were in pmol/min/mg human small intestinal microsome; d Vmax or kcat
values were in nmol/min/nmol P450 for CYP enzymes; e Vmax values were in nmol/min/mg transfected cells; f kcat values were in min
1; g: negative mode; MRM: multiple
reaction monitoring; kmax: wavelength of maximum UV absorption. Red arrows indicate the major site of metabolism by target CYP(s).

11
J. Wu et al.
time-consuming in both sample preparation and analysis, and
require skilled operators and expensive equipment. By contrast,
the fluorogenic or luminescent substrates based optical assays
are time- and- cost-saving, easy to made and can be adapted for
96- and 384-well microplate formats, representing a highly effi-
cient approach for high-throughput screening of inhibitors or stim-
ulators of a target enzyme. Hence, the optical probe substrates for
human CYPs are more and more favored by the researchers.
6.1. Fluorogenic probes for human CYPs
Compared to non-optical probes, fluorogenic probes have many
inherent advantages [81,117], including highly sensitive (the
detection limit of fluorogenic probes can be up to picomole level),
easy to manage, time- and- cost-saving (capable for high-
throughput detection) and can be adapted for 96- and 384-well
microplate formats, as well as in-situ imaging with high resolution
(commonly up to sub-nanometer spatial resolution and sub-
millisecond time resolution). These advantages allow most fluoro-
genic probes be used to real-time sensing CYP activities in living
cells or in vivo. Generally, CYP optical probes or sensors can be
divided into several categories, including substrate-based sensors
and electrochemical biosensors [118,119]. Among these,
substrate-based sensors are widely used for screening of CYP mod-
ulators, and in other drug metabolism and pharmacokinetics
(DMPK) related studies [119]. Substrate-based activity sensors
can readily record the alterations in CYP activities in the presence
of drug candidate(s), which strongly facilitate the development of
the new chemical entities, and decrease the rates of failure later
in the drug development process [120].
The reported fluorogenic probes for human CYPs have been
summarized in Table 4. It is should be noted that most of reported
fluorogenic probes for human CYPs displayed poor selectivity
[129–138], except for NBCeN (CYP1A1), iPrBN (CYP1A1), coumarin
(CYP2A6), BQ (CYP3A4), NEN (CYP3A4) and BFBFC (CYP3A4). The
non-specific fluorogenic probes for CYPs can only be used for
screening CYP inhibitors in vitro using individual recombinant
CYP enzyme as enzyme source. For example, 3-O-
methylfluorescein (OMF), a so-called fluorogenic probes for
CYP2C19, could also be O-demethylated by CYP2C9 and CYP1A1
[126]. Another example is 7-benzyloxy-4-(trifluoromethyl)-cou
marin (BFC), which can be metabolized by both CYP1A2 and
CYP3A4. Kinetic analyses demonstrated a lower Km with CYP1A2,
but a higher Vmax with CYP3A4 [127]. By contrast, 2,5-bis(trifluoro
methyl)-7-benzyloxy-4-trifluoromethyl-coumarin (BFBFC), which
has two additional trifluoromethyl groups compared to BFC, was
selectively metabolized by CYP3A4 [128]. Notably, in most cases,
the isoform-specificity of reported fluorogenic substrates for
CYP3A4 over CYP3A5 has not been well-studied, except for
dibenzyl-fluorescein (DBF).
A milestone fluorogenic probe to note is NBCeN, which is the
first reported isoform-specific fluorogenic probe for CYP1A1. From
the views of 3D structures of two CYP1A isoforms in humans
(CYP1A1 and CYP1A2), the volume of the catalytic cavity of CYP1A1
is larger than that of CYP1A2 (524 Å3 VS 375 Å3). Meanwhile, pre-
vious studies have found that CYP1A2 preferred to catalyze O-
demethylation of a set of substrates with O-methyl group(s), while
CYP1A1 preferred to catalyze O-dealkylation of relatively large
ethers, such as O-ethyl or O-chloroethyl [3,48]. These observations
inspired the researchers to develop a panel of isoform-specific flu-
orogenic probes for CYP1A1 via adjusting the volume of the leaving
ether group(s), such as CHPO and iPrBN [82,130]. Furthermore, the
strategies used in the design and development of NBCeN have
greatly promoted the development of fluorescent probes for other
target enzymes, such as human carboxylesterases and human
UDP-glucuronosyltransferases [84]. Inspired by the development
12
Coordination Chemistry Reviews 427 (2021) 213600
of NBCeN, Ning et al have reported a new two-photon fluorescent
probe for CYP1A1 (termed iPrBN) using 4-hydroxy-7H-benzo[de]
benzo[4,5]imidazo[2,1-a] isoquinolin-7-one (HBN) as a basic skele-
ton. This fluorogenic probe is designed on the basis of the substrate
preferences of CYP1A1 and the principle of intramolecular charge
transfer (ICT). Further investigations demonstrate iPrBN displays
excellent specificity and ultrahigh sensitivity towards CYP1A1,
with the detection limit as low as 0.036 nM [82]. More recently,
Jin et al have designed and developed a resorufin-based isoform-
specific fluorescent probe (CHPO) for highly selective and sensitive
sensing CYP1A1 activities in complex biological systems. CHPO is a
cell membrane permeable agent, which has been successfully used
for imaging of endogenous CYP1A1 in living cells and tissue slice
[130]. The reported fluorogenic probes for CYP1A1 have provided
promising tools for high-throughput screening of CYP1A1 inhibi-
tors and sensing CYP1A1 activities in living systems, which
strongly facilitate the investigations on the relevance of CYP1A1
to human diseases and the regulatory effects of CYP1A1 by xenobi-
otics. Besides of the development of a variety of CYP1A1 fluoro-
genic probes, another major breakthrough in this field is the
development of an isoform-specific fluorogenic probe for CYP3A4
(termed NEN), which has been reported recently by Ning et al.
[137]. NEN was developed by a novel two-dimensional strategy.
In the first dimension, docking based virtual screening was used
to select a suitable two-proton fluorophore as the substrate
candidate for CYP3A4. In the second dimensional design, chemical
modifications on the selected fluorophore (N-substituted
1,8-naphthalimide) were performed to optimize the catalytic
activity and isoform-selectivity. Notably, although great break-
through has been made by researchers in this field, the highly
specific fluorogenic probes for target human CYP isoforms are
rarely reported. Particularly, the highly specific fluorogenic probes
for target endobiotic-metabolizing CYPs have not been reported
yet. Hence, much more efforts should be made to develop more
practical and highly specific fluorogenic probes for target human
CYPs.
6.2. Bioluminogenic probes for human CYPs
Unlike fluorescent based assays, bioluminescent based CYP
activity assays always involves two reaction steps. First, the
luminogenic precusor produces D-luciferin, the natural substrate
for firefly luciferase. Second, the luciferase is added and catalyzes
luciferin to yield oxyluciferin that bring photons in the presence
of ATP, magnesium ion and oxygen (Fig. 8). Bioluminescence
occurs during the oxidation process, which can be detected as light
output in a luminometer [142]. The bioluminescent based assays
possess the following merits [142]: 1) obviates the need for pro-
duct extractions prior to detection by the method of chromatogra-
phy or mass spectrometry, 2) eliminates safety concerns associated
with radiometric assays, 3) obviates the interferences associated
with fluorescent analytes in fluorescent assays. Notably, biolumi-
nescent based assays also have some demerits. The major demerit
of bioluminescent assay is the limited tissue penetration ability of
emitted light resulting in quenching of the bioluminescence by tis-
sue components [143,144]. In recent years, many efforts has been
made to synthesize red-shifted D-luciferin analogues to improve
the tissue penetration [145]. Furthermore, oxyluciferin, the
luminescence product, could also regenerate into Luciferin. The
luciferin recycling process also follows two-step reaction: 1) trans-
formation of oxyluciferin to 2-cyano-6-hydroxybenzothiazole, and
2) condensation of 2-cyano-6-hydroxybenzothiazole with
D -cysteine to yield luciferin [146]. In addition, it has been reported
that some endogenous compounds can strongly inhibit the lucifer-
ase, which in turn, affect the readouts of the bioluminescent based
assays [147,148].
J. Wu et al. Coordination Chemistry Reviews 427 (2021) 213600

Table 4
Reported fluorogenic probes for human CYPs.

Enzyme Substrate Substrate structure Metabolite Applications Km Vmax or kex kem Ref.
name (lM) Kcat (nm) (nm)
a
CYP1A1 NBCeN NBHN In vitro (rCYPs, microsomes, living 0.84 18.99 450 562 [83]
cells)

a
iPrBN HBN In vitro (rCYPs, microsomes, living 0.29 5.54 470 525 [82]
cells)

a
NCMN NCHN In vitro (rCYPs, microsomes, living 11.99 17.44 450 564 [81]
cells)

NEMN NEHN In vitro (rCYPs, microsomes, living N.A. N.A. 458 552 [129]
cells)

NEiPN NEHN In vitro (rCYPs, microsomes, living N.A. N.A. 446 550 [130]
cells)

DPCI DPOH In vitro (rCYPs, microsomes, living N.A. N.A. 555 673 [131]
cells)

a
ER Vivid Red In vitro (rCYPs, microsomes, living 2.80 18.2 530 590 [132]
cells)

a
CHPO Vivid Red In vitro (rCYPs, microsomes) 0.278 9.17 540 590 [133]

a
CEC Vivid Blue In vitro (rCYPs, microsomes) 1.2 24.2 409 460 [134]

a
CYP1A2 NCMN NCHN In vitro (rCYPs, microsomes, living 9.8 32.06 450 564 [81]
cells)

NEMN NEHN In vitro (rCYPs, microsomes, living N.A. N.A. 458 552 [129]
cells)

a
ER Vivid Red In vitro (rCYPs, microsomes, living 3.0 0.23 530 590 [132]
cells)

a
CEC Vivid Blue In vitro (rCYPs, microsomes) 1.14 7.13 409 460 [134]

EOMCC Vivid Blue In vitro (rCYPs, microsomes) 11 21.0c 415 460 [135]

a
BFC Vivid Cyan In vitro (rCYPs, microsomes) 4.1 11.1 410 510 [127]

a
CYP1B1 ER Vivid Red In vitro (rCYPs, microsomes, living 1.0 1.33 530 590 [132]
cells)

(continued on next page)

13
J. Wu et al. Coordination Chemistry Reviews 427 (2021) 213600

Table 4 (continued)

Enzyme Substrate Substrate structure Metabolite Applications Km Vmax or kex kem Ref.
name (lM) Kcat (nm) (nm)
CYP2A6 Coumarin 7-OH- In vitro (rCYPs, microsomes) 2.1 0.79b 390 460 [136]
coumarin

a
CYP2B6 EFC Vivid Cyan In vitro (rCYPs, microsomes) 8.5 14.0 409 530 [137]

a
MFC Vivid Cyan In vitro (rCYPs, microsomes) 6.79 6.48 409 530 [134]

BOMCC Vivid Blue In vitro (rCYPs, microsomes) 52 66.0c 415 460 [135]

BOMR Vivid Red In vitro (rCYPs, microsomes) 0.73 0.80c 550 590 [135]

CYP2C8 DBOMF Vivid In vitro (rCYPs, microsomes) 3.6 2.10c 490 520 [135]
Green

CYP2C9 BOMCC Vivid Blue In vitro (rCYPs, microsomes) 43 2.10c 425 460 [135]

BOMF Vivid In vitro (rCYPs, microsomes) 4.5 4.50c 490 520 [135]
Green

a
MFC Vivid Cyan In vitro (rCYPs, microsomes) 54.7 0.56 409 530 [134]

a
CYP2C19 CEC Vivid Blue In vitro (rCYPs, microsomes) 9.11 0.84 409 460 [134]

EOMCC Vivid Blue In vitro (rCYPs, microsomes) 16.6 5.70c 415 460 [135]

OMF Vivid In vitro (rCYPs, microsomes) 1.14 0.01b 485 530 [126]
Green

CYP2D6 MAMC HAMC In vitro (rCYPs, microsomes) 11.5 2.81c 390 460 [138]

a
AMMC AHMC In vitro (rCYPs, microsomes) 4.81 6.19 390 460 [134]

MOBFC Vivid Cyan In vitro (rCYPs, microsomes) 5.0 0.60c 405 490 [135]

14
J. Wu et al. Coordination Chemistry Reviews 427 (2021) 213600

Table 4 (continued)

Enzyme Substrate Substrate structure Metabolite Applications Km Vmax or kex kem Ref.
name (lM) Kcat (nm) (nm)
a
CYP2E1 EOMCC Vivid Blue In vitro (rCYPs, microsomes) 22 2.10 415 460 [139]

d
CYP2J2 BnXPI HXPI In vitro (rCYPs, microsomes, living 4.2 0.44 656 718 [91]
cells)

a
CYP3A4 NEN NEHN In vitro (rCYPs, microsomes, living 59.8 2.18 450 558 [140]
cells and tissues)

BQ Quinolinol In vitro (rCYPs, microsomes) 70 3.39b 420 530 [128]

a
DBF Vivid In vitro (rCYPs, microsomes) 1.03 1.55 485 538 [134]
Green

DBOMF Vivid In vitro (rCYPs, microsomes) 7.8 16.0c 490 520 [135]
Green

BOMR Vivid Red In vitro (rCYPs, microsomes) 1.4 1.10c 550 590 [135]

a
BFC Vivid Cyan In vitro (rCYPs, microsomes) 26 61.2 410 510 [127]

BFBFC Vivid Cyan In vitro (rCYPs, microsomes) 4.6 0.02b 410 510 [128]

BOMCC Vivid Blue In vitro (rCYPs, microsomes) 89 10.0c 415 460 [135]

BOMFC Vivid Cyan In vitro (rCYPs, microsomes) 22 4.00c 405 490 [135]

CYP3A5 BOMCC Vivid Blue In vitro (rCYPs, microsomes) N.A. N.A. 415 460 [141]

DBOMF Vivid In vitro (rCYPs, microsomes) N.A. N.A. 490 520 [141]
Green

(continued on next page)

15
J. Wu et al.
Table 4 (continued)
Enzyme Substrate Substrate structure Metabolite
name
DBF Vivid
Green
a
Vmax values were in nmol/min/nmol P450 for CYP enzymes; b Vmax values were in nmol/min/mg human liver microsome; c kcat or Vmax values were in min
1; d Vmax values
were in nmol/min/mg CYP2J2; kex: excitation wavelength; kem: emission wavelength. NA.: Not available or not assayed. Red arrows indicate the major site of metabolism by
CYP.
Fig. 8. Two reaction steps involved in bioluminescent CYP assays and luciferin recycling process.
Over the past few decades, multiple bioluminogenic CYP sub-
strates have been developed under the trade name P450-GloTM
from Promega Corp. [149]. Amongst these bioluminogenic sub-
strates, several have been identified as specific substrates for a tar-
get CYP isoenzyme, others have a broader enzyme profile. As listed
in Table 5, the relatively selective luminogenic substrates for
CYP1A1, 1A2, 2B6, 2D6, 2C9, 2C19, 2J2, 3A4, 3A7, 4A and CYP4F
have been reported. However, except Luciferin-H and Luciferin-
IPA have been validated as highly specific probe substrates for
CYP2A9, and CYP3A4, respectively, majority of luminogenic CYP
probes display poor selectivity, these luminogenic substrates are
limited to inhibitor screening using recombinant CYP enzymes as
enzyme sources. As far as we know, the specificity of a given
luminogenic substrate towards a target CYP was mainly affected
by the chemical structure of the luciferin derivatives and the most
labile sites of metabolism of CYPs. Two common CYP reactions, O-
dealkylation and aromatic hydroxylation, are usually involved in
the design and development of luminogenic probes [56,150]. In
addition, the modifications of these luminogenic substrates are
based on the structural features of target CYP (including catalytic
cavity and the key residues surrounding the catalytic site) and
the structures of their preferred ligands. For example, a previous
study haa found that the molecular weights and critical volumes
of Luciferin-H (a CYP2C9 luminogenic substrate) are similar to that
of flurbiprofen (a conventional CYP2C9 probe substrate), and
Luciferin-H was modeled in the CYP2C9 active site starting with
the carboxylic acid anchored by a hydrogen bond and salt bridge
to the same residues as observed with flurbiprofen [142]. In future,
more detailed in silico molecular modelling and docking simula-
tions should be carry out to find out the clarification of these highly
specific luminogenic substrates for CYPs.
Coordination Chemistry Reviews 427 (2021) 213600
Applications Km Vmax or kex kem Ref.
(lM) Kcat (nm) (nm)
a
In vitro (rCYPs, microsomes) 2.17 1.28 490 520 [134]
7. Biomedical applications of the probes for human CYPs
7.1. Sensing enzymatic activity in biological samples
As the most important xenobiotic-metabolizing enzymes in
mammals, CYPs play a crucial role in detoxification and metabolic
clearance of a wide range of xenobiotics. Dysfunction or inhibition
of CYPs may have great impact on drug safety, efficacy and dose
requirement. Increasing evidence has indicated that the large vari-
ability in the function of some key CYPs may result in significant
interindividual variations in drug disposition and treatment out-
comes, especially those drugs with narrow therapeutic index
(NTI), such as warfarin, digoxin and paclitaxel [157,158]. The
highly specific molecular probes for target human CYPs have pro-
vided powerful tools for reliable sensing the real activities of target
CYPs in various biological systems. In fact, some molecular probes
for human CYPs have been utilized to investigate the interindivid-
ual variability in CYP activities. For example, Court’s group has
investigated the interindividual variations in the activities of sev-
eral key drug metabolizing enzymes in human liver microsomes
(n = 55) from individual donors, using several specific non-
fluorogenic probes [10]. The results reveal that CYP3A4 (midazo-
lam 10 -hydroxylation) shows the highest interindividual variability
(111% CV), while other hepatic CYPs, including CYP2E1 (chlorzox-
azone 6-hydroxylation), display moderate interindividual variabil-
ity (35% CV) [10]. Furthermore, Qiao’s group has determined the
activities of nine CYP enzymes in 78 individual human liver micro-
somes using non-fluorogenic CYP probes [159]. The CYP activities
show large individual variations, with the largest interindividual
variations occurred in the microsomal activities (VM) of CYP2C19
and CYP2A6, reaching 232 and 109-fold, respectively, followed by
16
J. Wu et al. Coordination Chemistry Reviews 427 (2021) 213600

Table 5
The bioluminogenic substrates for human CYPs.
c
Enzyme Substrate Substrate structure Metabolite Applications Km k (nm) Refs.
name (lM)
CYP1A1 Luciferin- D- In vitro (rCYPs, 3.0 560 [142,151]
a
1A1 Luciferin microsomes)

CYP1A2 Luciferin- D- In vitro (rCYPs, 6.0 560 [142,149]

a
1A2 Luciferin microsomes)

CYP2B6 Luciferin- D- In vitro (rCYPs, 3.0 560 [149]

a
2B6 Luciferin microsomes)

CYP2C9 Luciferin-H D- In vitro (rCYPs, 100 560 [142,149]

Luciferin microsomes)

CYP2C19 Luciferin-H D- In vitro (rCYPs, 10 560 [149]

EGE Luciferin microsomes)

CYP2D6 Luciferin- D- In vitro (rCYPs, 30 560 [149]

ME EGE Luciferin microsomes)

CYP2J2 Luciferin- D- In vitro (rCYPs, 1.0 560 [142,152]

2 J2/4F12 * Luciferin microsomes)

CYP3A4 Luciferin- D- In vitro (rCYPs, 3.0 560 [142,149]

IPA Luciferin microsomes, living
cells)

CYP3A7 Luciferin- D- In vitro (rCYPs, 33b 560 [142,153]

3A7 Luciferin microsomes)

CYP4A11 Luciferin- D- In vitro (rCYPs, 80 560 [142,154]

4A* Luciferin microsomes)

CYP4F3B Luciferin- D- In vitro (rCYPs, ~10b 560 [142,155]

4F2/3* Luciferin microsomes)

(continued on next page)

17
J. Wu et al.
Table 5 (continued)
Enzyme Substrate Substrate structure
name
CYP4F12 Luciferin-
4F12*
a
The product of the Luciferin-1A1, Luciferin-1A2 and Luciferin-2B6 is a luciferin precursor, which can further generate D-luciferin by addition of luciferin detection reagent
supplemented with d-Cysteine (Fig. 8). b The reactions showed atypical kinetics. c k: the bioluminescence wavelength. Red arrows indicate the major site of metabolism by
CYP. * Currently, Luciferin-2J2/4F12, Luciferin-4A, Luciferin-4F2/3, and Luciferin-4F12, are not commercially available.
that of CYP3A4/5, CYP2D6, CYP2C8, CYP2B6, CYP1A2 and CYP2E1
(Fig. 9). Notably, interindividual variations of CYP activities based
on liver tissues (VL) were even more pronounced, except for
CYP2C19 [159]. More recently, Vos’s group has measured multiple
drug metabolizing enzyme activities in a set of 20 individual liver
homogenates, among which, the strongest inter-individual vari-
ability in enzyme-specific activities were observed for CYP2C19
and CYP2B6 (CV ~ 78%) by employing mephenytoin and bupropion
as the probe substrates, respectively [8].
7.2. Screening of CYP modulators
Numerous clinical surveys have highlighted continuing issues
related to drug/herb-drug interactions (DDI/HDI) and the resulting
serious adverse events in clinical settings [160,161]. Assessing the
potential risks of DDI/HDI is an important task in the development
of new drugs. Inhibition of human CYPs responsible for metabolic
clearance of many therapeutic drugs has been considered as one of
the key causes of DDI/HDI. Therefore, assessing the inhibition
potency of human CYPs by a new chemical entity has become a
routine practice during the drug discovery stage [162]. The large
changes in exposure triggered by modulation (inhibition, induction
or stimulation) of CYP activities can alter the efficacy and safety
profile of a drug. This is most obvious for NTI drugs, but is also pos-
Fig. 9. The individual fold-change of CYP activity in human livers (n = 78). The fold-
change of CYP activity is expressed as the ratio between the maximal (Max) and
minimal (Min) values of CYP metabolic rate. Phenacetin (CYP1A2), coumarin
(CYP2A6), bupropion (CYP2B6), paclitaxel (CYP2C8), tolbutamide (CYP2C9),
omeprazole (CYP2C19), dextromethorphan (CYP2D6), chlorzoxazone (CYP2E1),
and midazolam (CYP3A4/5) were used as the isoform-specific probes, respectively.
VM: metabolic rate of each CYP isozymes per mg microsomal protein; VL: metabolic
rate of each CYP isozymes per gram liver tissue, which derived from the VM values
multiplied by the individual microsomal protein per gram of liver. See ref. [159].
18
Coordination Chemistry Reviews 427 (2021) 213600
c
Metabolite Applications Km k (nm) Refs.
(lM)
D- In vitro (rCYPs, 10 560 [142,156]
Luciferin microsomes)
sible for non-NTI drugs, such as statins. Regulators, including FDA,
have therefore issued guidance for in vitro and in vivo drug interac-
tion studies that are recommended to be conducted during devel-
opment [11]. Predicting the DDI potential for a new drug usually
relies on the evaluation of the effect on the rate of a probe reaction
that represents the activity of a specific CYP enzyme [74]. Over the
past few decades, various non-fluorogenic CYP probes have been
utilized for screening and characterization of CYP modulators,
and subsequently to assess their potential risks to trigger CYP-
mediated DDI in humans [11]. Meanwhile, fluorogenic probes have
gotten more and more attention in the assessment of DDI, due to
their advantage of throughput capabilities [163,164]. For example,
7-ethoxy-resorufin (ER) has been used as a fluorogenic probe for
CYP1A, which allows high-throughput screening (HTS). The inhibi-
tory potentials of rutaecarpine, evodiamine, and dehydroevodi-
amine, the alkaloids isolated from a traditional Chinese medicine
towards CYP1A1 and CYP1A2, were evaluated using ER, and the
results indicated that rutaecarpine is a potent inhibitor of CYP1A2
in human liver microsomes [165]. Furthermore, the fluorogenic
probe of CYP is highly favoured in evaluating the induction poten-
tial of CYP inducers in living cells, due to its high sensitivity. For
example, NBCeN, a CYP1A1 probe, has been successfully used to
evaluate the activity of CYP1A1 in hepG2 cells pre-treated with
2,3,7,8-tetrachlorodibenzo-p-dioxin, a well-known CYP1A inducer
[81], and the results showed that CYP1A1 activity agreed well with
its protein levels (Fig. 10). Taken together, chemical probes have
become promising tools for screening and characterization of CYP
modulators, and more practical probe substrates with high speci-
ficity and high sensitivity are highly desirable.
Fig. 10. Comparison of the CYP1A1 activity and protein levels in HepG2 cells pre-
treated with CYP1A inducer by using NBCeN as a CYP1A1 probe and western blot
analysis, respectively. See ref. [83].
J. Wu et al.
7.3. CYP–ligand interactions
Understanding the key structural elements and the key moiety
of ligands that dictate their orientation in the active site of the tar-
get CYP is very helpful for deep understanding of CYP–ligand inter-
actions, which may aid the design and development of highly
specific ligands (such as highly specific inhibitors and substrates)
[49]. For some key human CYPs (such as CYP3A4), there are multi-
ple ligand-binding sites in the catalytic cavity. In these cases, the
researches should know whether the optical substrates for the tar-
get enzyme could replace its physiological substrates or its
substrate-drugs. [75,84,166]. An inappropriate selection of a probe
substrate may lead to false positive or negative results to predict
the DDI risks [74]. For example, testosterone, midazolam, and
nifedipine are frequently used as three substrate subgroups for
CYP3A4, and multiple molecular probes are recommended to be
used for CYP3A4-mediated DDI assays [166]. Wu et al. have
reported that deoxyschizandrin is a good substrate to replace
midazolam and testosterone, but not nifedipine (Fig. 11) [75]. This
was concluded from mutual inhibition assays. Testosterone and
midazolam were shown to compete with deoxyschizandrin for
the mutual binding site. However, deoxyschizandrin showed only
slight inhibitory effects on nifedipine oxidation even though
nifedipine was able to inhibit deoxyschizandrin hydroxylation via
a noncompetitive manner [75]. Taken together, site-specific probes
for CYPs are in high demand for the studies on interactions
between the small-molecule ligands and CYPs, especially the CYPs
with multiple ligand-binding sites.
8. Challenges and future perspectives
Molecular probes are practical tools for deciphering the physio-
logical functions of target enzyme, as well as for exploring the
interactions between target enzyme and its ligands [167–170].
Over the past few decades, many strategies and approaches have
been used to develop specific molecular probes for sensing the
activities of CYPs in complex biological systems, living cells or
in vivo [81–83]. At present, CYP probes have been widely used in
biological and biomedical fields, including sensing CYP activity in
various biological samples, high-throughput screening of CYP inhi-
bitors or inducers, as well as biological imaging of intracellular
Fig. 11. The proposed binding site distribution of MDZ, TST, NIF and DS within
CYP3A4 enzyme. DS: deoxyschizandrin, MDZ: midazolam, TST: testosterone, NIF:
nifedipine.
19
Coordination Chemistry Reviews 427 (2021) 213600
CYPs in living cells or tissues. Although a wide range of specific
probes for human CYPs have been reported, most of known CYP
probe substrates are hardly used in living systems, due to low sen-
sitivity, poor chemical stability, high cytotoxicity or other safety
concerns. Thus, more practical molecular probes for highly specific
and highly sensitive sensing of target CYP enzymes in living sys-
tems are always desirable.
The major challenge to develop practical CYP probes is the lim-
ited ability to enhance their specificity and sensitivity. With the
help of structural biology techniques that make the crystal struc-
tures of more CYP enzymes available, the structure-based probe
development using computer-aided design and virtual screening
will likely improve the isoform-specificity of CYP probes to a large
extent. To improve the sensitivity of candidate probe substrates,
several feasible strategies, such as increasing turnover rate,
enhancing the fluorescence quantum yield of fluorogenic sub-
strates, or dwindling the reaction group size to reduce the energy
consumption of CYP reactions, could be used for future design
and development of chemical probes for CYPs.
In light of the fact that fluorescence-based assays have multiple
inherent advantages, such as highly sensitive, easy to manage, and
applicable to HTS, there is a great deal of interest in the design and
development of highly specific and practical fluorescent probes for
target CYPs. The design strategies and experiences in the develop-
ment of isoform-specific fluorogenic substrates for CYP1A1 and
other CYP enzymes may assist others to develop more practical flu-
orescent substrates for highly specific and sensitive sensing of tar-
get CYP enzyme(s) in complex biological systems. These optical
probes can be used to develop a set of fluorescence-based CYP
detection microarrays, which in turn, at least partially, take place
of the physiological substrates or those CYP substrates with rela-
tively poor enzyme-specificity and low conversion rate [171]. Of
particular note that the optical probes has been tried ~15 years
ago in the pharmaceutical industry, but there are a number of
issues & many companies still use the physiological substrates
(such as testosterone) or the drug substrates (such as diclofenac)
for CYP inhibition assays, owing to the man-made substrates
may bind on the target enzyme (such as CYP3A4) at a distinct
ligand-binding site [166]. Therefore, it is highly recommended that
the difference in ligand-binding site and the binding mode of the
man-made optical substrates and the physiological or drug sub-
strates on a target enzyme should be tested, prior to inhibition
assays [84].
Although significant breakthroughs have been achieved in the
design and development of optical probes for human CYPs, the
emission wavelengths of most reported molecular probes for
human CYPs and their corresponding metabolites are less than
560 nm, which may be interfered by the background fluorescence
signals in living systems. To get more practical CYP probes, more
fluorogens or luminogens with diverse chemical structures (such
as green fluorescent protein (GFP)-like and red fluorescent protein
(GFP)-like fluorophores, as well as aggregation-induced emission
luminogens) should be used to construct the fluorogenic probe
substrates for a target human CYP [172–174]. Meanwhile, exten-
sive investigations on the design and development of CYP probes
with high specificity, excellent optical properties (e.g., near-
infrared wavelength probes), improved safety profiles and good
biological compatibility are highly desirable, which will strongly
facilitate real-time monitoring and in situ imaging of the activities
of target CYPs in vivo, and exploring the relevance of CYP enzymes
to human diseases. Furthermore, most previous studies are focused
on the development of molecular probes for xenobiotic-endobio
tic-metabolizing CYPs. Until now, no optical probes have been
developed for in vivo or in situ sensing or imaging of endobiotic-
metabolizing CYPs, owing to the lack of 3D structural information
of the target endobiotic-metabolizing CYPs and the limited
J. Wu et al.
knowledge about their substrate preferences. In future, more
investigations still need to be done using isoform-specific sub-
strates for target human CYP(s), to gain better understanding of
the biological functions, substrate preferences and the mechanisms
of catalysis of these key oxidative enzymes, as well as to explain
how regulation of these enzymes is beneficial for improving or
maintaining of human health.
Declaration of Competing Interest
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared
to influence the work reported in this paper.
Acknowledgements
This work was supported by the National Natural Science Foun-
dation of China (81922070, 81973286, 81773687), the National
Key Research and Development Program of China
(2020YFC0845400, 2017YFC1700200, 2017YFC1702000), the
Three-year Action Plan of Shanghai TCM Development (ZY-(2018-
2020)-CCCX-5001), Liaoning Revitalization Talents Program
(XLYC1907103), Program of Shanghai Academic/Technology
Research Leader (18XD1403600), the 111 Project (Grant B16047),
and Shuguang Program (No. 18SG40) supported by Shanghai Edu-
cation Development Foundation and Shanghai Municipal Educa-
tion Commission.
References
[1] A.W. Munro, H.M. Girvan, A.E. Mason, A.J. Dunford, K.J. McLean, What makes a
P450 tick?, Trends Biochem. Sci. 38 (2013) 140–150, https://doi.org/10.1016/
j.tibs.2012.11.006.
[2] F.P. Guengerich, Human cytochrome P450 enzymes, in: P.R. Ortiz de
Montellano (Ed.), Cytochrome P450, Springer, Cham, New York, 2015, pp.
523–785.
[3] U.M. Zanger, M. Schwab, Cytochrome P450 enzymes in drug metabolism:
regulation of gene expression, enzyme activities, and impact of genetic
variation, Pharmacol. Ther. 138 (2013) 103–141, https://doi.org/10.1016/j.
pharmthera.2012.12.007.
[4] M.I. Fekry, Y. Xiao, J.Z. Berg, F.P. Guengerich, A role for the orphan human
cytochrome P450 2S1 in polyunsaturated fatty acid x-1 hydroxylation using
an untargeted metabolomic approach, Drug Metab. Dispos. 47 (2019) 1325–
1332, https://doi.org/10.1124/dmd.119.089086.
[5] Y. Xiao, F.P. Guengerich, Metabolomic analysis and identification of a role for
the orphan human cytochrome P450 2W1 in selective oxidation of
lysophospholipids, J. Lipid Res. 53 (2012) 1610–1617, https://doi.org/
10.1194/jlr.M027185.
[6] Y. Ohno, S. Nakamichi, A. Ohkuni, N. Kamiyama, A. Naoe, H. Tsujimura, U.
Yokose, K. Sugiura, J. Ishikawa, M. Akiyama, Essential role of the cytochrome
P450 CYP4F22 in the production of acylceramide, the key lipid for skin
permeability barrier formation, Proc. Natl. Acad. Sci. 112 (2015) 7707–7712,
https://doi.org/10.1073/pnas.1503491112.
[7] Q. Yan, D. Machalz, A. Zöllner, E.J. Sorensen, G. Wolber, M. Bureik, Efficient
substrate screening and inhibitor testing of human CYP4Z1 using
permeabilized recombinant fission yeast, Biochem. Pharmacol. 146 (2017)
174–187, https://doi.org/10.1016/j.bcp.2017.09.011.
[8] S.P. Den Braversewradj, M.W. Den Braver, M. Van Dijk, Y. Zhang, S.J. Dekker, L.
Wijaya, N.P.E. Vermeulen, L. Richert, J.N.M. Commandeur, J.C. Vos, Inter-
individual variability in activity of the major drug metabolizing enzymes in
liver homogenates of 20 Individuals, Curr. Drug Metab. 19 (2018) 370–381,
https://doi.org/10.2174/1389200219666180108160046.
[9] B. Achour, M.R. Russell, J. Barber, A. Rostami-Hodjegan, Simultaneous
quantification of the abundance of several cytochrome P450 and uridine 5
’-diphospho-glucuronosyltransferase enzymes in human liver microsomes
using multiplexed targeted proteomics, Drug Metab. Dispos. 42 (2014) 500–
510, https://doi.org/10.1124/dmd.113.055632.
[10] M.H. Court, Interindividual variability in hepatic drug glucuronidation:
studies into the role of age, sex, enzyme inducers, and genetic
polymorphism using the human liver bank as a model system, Drug Metab.
Rev. 42 (2010) 209–224, https://doi.org/10.3109/03602530903209288.
[11] T. D. Bjornsson, J. T. Callaghan, H. J. Einolf, V. Fischer, L. Gan, S. Grimm, J. Kao,
S. P. King, G. Miwa, L. Ni, G. Kumar, J. McLeod, R. S. Obach, S. Roberts, A. Roe, A.
Shah, F. Snikeris, J. T. Sullivan, D. Tweedie, J. M. Vega, J. Walsh, S. A. Wrighton,
R. Pharmaceutical, G. Manufacturers of America Drug Metabolism/Clinical
Pharmacology Technical Working, F. D. A. C. f. D. Evaluation,Research, The
20
Coordination Chemistry Reviews 427 (2021) 213600
conduct of in vitro and in vivo drug-drug interaction studies: a
Pharmaceutical Research and Manufacturers of America (PhRMA)
perspective, Drug Metab Dispos, 31 (2003) 815-832. https://doi.org/
10.1124/dmd.31.7.815.
[12] A. Guglielmi, A. Ruzzenente, S. Conci, A. Valdegamberi, C. Iacono, How much
remnant is enough in liver resection?, Dig Surg 29 (2012) 6–17, https://
doiorg/10.1159/000335713.
[13] A.S. Kalgutkar, R.S. Obach, T.S. Maurer, Mechanism-based inactivation of
cytochrome P450 enzymes: chemical mechanisms, structure-activity
relationships and relationship to clinical drug-drug interactions and
idiosyncratic adverse drug reactions, Curr. Drug Metab. 8 (2007) 407–447,
https://doi.org/10.2174/138920007780866807.
[14] J. Lazarou, B.H. Pomeranz, P.N. Corey, Incidence of adverse drug reactions in
hospitalized patients: a meta-analysis of prospective studies, JAMA 279
(1998) 1200–1205, https://doi.org/10.1001/jama.279.15.1200.
[15] R. Leone, L. Magro, U. Moretti, P. Cutroneo, M. Moschini, D. Motola, M.
Tuccori, A. Conforti, Identifying adverse drug reactions associated with drug-
drug interactions, Drug Saf. 33 (2010) 667–675, https://doi.org/10.2165/
11534400-000000000-00000.
[16] T.L. Poulos, E.F. Johnson, Structures of Cytochrome P450 Enzymes, in: P.R.
Ortiz de Montellano (Ed.), Cytochrome P450: Structure, Mechanism, and
Biochemistry, Springer, Cham, New York, 2015, pp. 3–32.
[17] M. Otyepka, J. Skopalik, E. Anzenbacherova, P. Anzenbacher, What common
structural features and variations of mammalian P450s are known to date?,
Bba-Gen Subjects 1770 (2007) 376–389, https://doi.org/10.1016/j.
bbagen.2006.09.013.
[18] E.F. Johnson, C.D. Stout, Structural diversity of human xenobiotic-
metabolizing cytochrome P450 monooxygenases, Biochem. Biophys. Res.
Commun. 338 (2005) 331–336, https://doi.org/10.1016/j.bbrc.2005.08.190.
[19] E.F. Johnson, C.D. Stout, Structural diversity of eukaryotic membrane
cytochrome P450s, J. Biol. Chem. 288 (2013) 17082–17090, https://doi.org/
10.1074/jbc.R113.452805.
[20] P.A. Williams, J. Cosme, D.M. Vinkovic, A. Ward, H.C. Angove, P.J. Day, C.
Vonrhein, I.J. Tickle, H. Jhoti, Crystal structures of human cytochrome P450
3A4 bound to metyrapone and progesterone, Science 305 (2004) 683–686,
https://doi.org/10.1126/science.1099736.
[21] A.A. Walsh, G.D. Szklarz, E.E. Scott, Human Cytochrome P450 1A1 Structure
and Utility in Understanding Drug and Xenobiotic Metabolism, J. Biol. Chem.
288 (2013) 12932–12943, https://doi.org/10.1074/jbc.M113.452953.
[22] S. Sansen, J.K. Yano, R.L. Reynald, G.A. Schoch, K.J. Griffin, C.D. Stout, E.F.
Johnson, Adaptations for the oxidation of polycyclic aromatic hydrocarbons
exhibited by the structure of human P450 1A2, J. Biol. Chem. 282 (2007)
14348–14355, https://doi.org/10.1074/jbc.M611692200.
[23] J.K. Yano, M.H. Hsu, K.J. Griffin, C.D. Stout, E.F. Johnson, Structures of human
microsomal cytochrome P450 2A6 complexed with coumarin and
methoxsalen, Nat. Struct. Mol. Biol. 12 (2005) 822–823, https://doi.org/
10.1038/nsmb971.
[24] N.M. DeVore, E.E. Scott, Nicotine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-
butanone binding and access channel in human cytochrome P450 2A6 and
2A13 enzymes, J. Biol. Chem. 287 (2012) 26576–26585, https://doi.org/
10.1074/jbc.M112.372813.
[25] P.R. Wilderman, M.B. Shah, H.H. Jang, C.D. Stout, J.R. Halpert, Structural and
thermodynamic basis of (+)-alpha-pinene binding to human cytochrome
P450 2B6, J. Am. Chem. Soc. 135 (2013) 10433–10440, https://doi.org/
10.1021/ja403042k.
[26] G.A. Schoch, J.K. Yano, S. Sansen, P.M. Dansette, C.D. Stout, E.F. Johnson,
Determinants of cytochrome P450 2C8 substrate binding: structures of
complexes with montelukast, troglitazone, felodipine, and 9-cis-retinoic acid,
J. Biol. Chem. 283 (2008) 17227–17237, https://doi.org/10.1074/jbc.
M802180200.
[27] P.A. Williams, J. Cosme, A. Ward, H.C. Angove, D. Matak, Vinkovic H. Jhoti,
Crystal structure of human cytochrome P450 2C9 with bound warfarin,
Nature 424 (2003) 464–468, https://doi.org/10.1038/nature01862.
[28] M.R. Wester, J.K. Yano, G.A. Schoch, C. Yang, K.J. Griffin, C.D. Stout, E.F.
Johnson, The structure of human cytochrome P450 2C9 complexed with
flurbiprofen at 2.0-A resolution, J. Biol. Chem. 279 (2004) 35630–35637,
https://doi.org/10.1074/jbc.M405427200.
[29] R.L. Reynald, S. Sansen, C.D. Stout, E.F. Johnson, Structural characterization of
human cytochrome P450 2C19: active site differences between P450s 2C8,
2C9, and 2C19, J. Biol. Chem. 287 (2012) 44581–44591, https://doi.org/
10.1074/jbc.M112.424895.
[30] A. Wang, C.D. Stout, Q.H. Zhang, E.F. Johnson, Contributions of ionic
interactions and protein dynamics to cytochrome P450 2D6 (CYP2D6)
substrate and inhibitor binding, J. Biol. Chem. 290 (2015) 5092–5104,
https://doi.org/10.1074/jbc.M114.627661.
[31] P.R. Porubsky, K.M. Meneely, E.E. Scott, Structures of human cytochrome P-
450 2E1. Insights into the binding of inhibitors and both small molecular
weight and fatty acid substrates, J Biol Chem 283 (2008) 33698–33707,
https://doi.org/10.1074/jbc.M805999200.
[32] I.F. Sevrioukova, T.L. Poulos, Structural basis for regiospecific midazolam
oxidation by human cytochrome P450 3A4, Proc. Natl. Acad. Sci. U.S.A. 114
(2017) 486–491, https://doi.org/10.1073/pnas.1616198114.
[33] I.F. Sevrioukova, T.L. Poulos, Interaction of human cytochrome P4503A4 with
ritonavir analogs, Arch. Biochem. Biophys. 520 (2012) 108–116, https://doi.
org/10.1016/j.abb.2012.02.018.
J. Wu et al.
[34] I.F. Sevrioukova, T.L. Poulos, Structure and mechanism of the complex
between cytochrome P4503A4 and ritonavir, Proc. Natl. Acad. Sci. U.S.A. 107
(2010) 18422–18427, https://doi.org/10.1073/pnas.1010693107.
[35] M.H. Hsu, U. Savas, E.F. Johnson, The X-ray crystal structure of the human
mono-oxygenase cytochrome P450 3A5-ritonavir complex reveals active site
differences between P450s 3A4 and 3A5, Mol. Pharmacol. 93 (2018) 14–24,
https://doi.org/10.1124/mol.117.109744.
[36] A. Wang, U. Savas, C.D. Stout, E.F. Johnson, Structural characterization of the
complex between alpha-naphthoflavone and human cytochrome P450 1B1, J.
Biol. Chem. 286 (2011) 5736–5743, https://doi.org/10.1074/jbc.
M110.204420.
[37] W. Tempel, I. Grabovec, F. MacKenzie, Y.V. Dichenko, S.A. Usanov, A.A. Gilep,
H.W. Park, N. Strushkevich, Structural characterization of human cholesterol
7alpha-hydroxylase, J. Lipid Res. 55 (2014) 1925–1932, https://doi.org/
10.1194/jlr.M050765.
[38] N. Strushkevich, F. MacKenzie, T. Cherkesova, I. Grabovec, S. Usanov, H.W.
Park, Structural basis for pregnenolone biosynthesis by the mitochondrial
monooxygenase system, Proc. Natl. Acad. Sci. U.S.A. 108 (2011) 10139–
10143, https://doi.org/10.1073/pnas.1019441108.
[39] N.M. DeVore, E.E. Scott, Structures of cytochrome P450 17A1 with prostate
cancer drugs abiraterone and TOK-001, Nature 482 (2012) 116–119, https://
doi.org/10.1038/nature10743.
[40] D. Ghosh, J. Griswold, M. Erman, W. Pangborn, Structural basis for androgen
specificity and oestrogen synthesis in human aromatase, Nature 457 (2009)
219–223, https://doi.org/10.1038/nature07614.
[41] P. S. Pallan, C. Wang, L. Lei, F. K. Yoshimoto, R. J. Auchus, M. R. Waterman, F. P.
Guengerich,M. Egli, Human Cytochrome P450 21A2, the Major Steroid 21-
Hydroxylase: STRUCTURE OF THE ENZYME.PROGESTERONE SUBSTRATE
COMPLEX AND RATE-LIMITING C-H BOND CLEAVAGE, J Biol Chem, 290
(2015) 13128-13143. https://doi.org/10.1074/jbc.M115.646307.
[42] N. Mast, M.A. White, I. Bjorkhem, E.F. Johnson, C.D. Stout, I.A. Pikuleva, Crystal
structures of substrate-bound and substrate-free cytochrome P450 46A1, the
principal cholesterol hydroxylase in the brain, Proc. Natl. Acad. Sci. U.S.A. 105
(2008) 9546–9551, https://doi.org/10.1073/pnas.0803717105.
[43] N. Mast, M. Linger, M. Clark, J. Wiseman, C.D. Stout, I.A. Pikuleva, In silico and
intuitive predictions of CYP46A1 inhibition by marketed drugs with
subsequent enzyme crystallization in complex with fluvoxamine, Mol.
Pharmacol. 82 (2012) 824–834, https://doi.org/10.1124/mol.112.080424.
[44] N. Mast, C. Charvet, I.A. Pikuleva, C.D. Stout, Structural basis of drug binding
to CYP46A1, an enzyme that controls cholesterol turnover in the brain, J. Biol.
Chem. 285 (2010) 31783–31795, https://doi.org/10.1074/jbc.M110.143313.
[45] N. Strushkevich, S.A. Usanov, H.W. Park, Structural basis of human CYP51
inhibition by antifungal azoles, J. Mol. Biol. 397 (2010) 1067–1078, https://
doi.org/10.1016/j.jmb.2010.01.075.
[46] P.C. Nair, R.A. Mckinnon, J.O. Miners, Cytochrome P450 structure–function:
insights from molecular dynamics simulations, Drug Metab. Rev. 48 (2016)
434–452, https://doi.org/10.1080/03602532.2016.1178771.
[47] K.M. Huttunen, N. Mahonen, H. Raunio, J. Rautio, Cytochrome P450-activated
prodrugs: targeted drug delivery, Curr. Med. Chem. 15 (2008) 2346–2365,
https://doi.org/10.2174/092986708785909120.
[48] D. Dong, B. Wu, D. Chow, M. Hu, Substrate selectivity of drug-metabolizing
cytochrome P450s predicted from crystal structures and in silico modeling,
Drug Metab. Rev. 44 (2012) 192–208, https://doi.org/10.3109/
03602532.2011.645580.
[49] S.C. Gay, A.G. Roberts, J.R. Halpert, Structural features of cytochromes P450
and ligands that affect drug metabolism as revealed by X-ray crystallography
and NMR, Future Med. Chem. 2 (2010) 1451–1468, https://doi.org/10.4155/
fmc.10.229.
[50] M. Ekroos, T. Sjogren, Structural basis for ligand promiscuity in cytochrome
P450 3A4, Proc. Natl. Acad. Sci. U.S.A. 103 (2006) 13682–13687, https://doi.
org/10.1073/pnas.0603236103.
[51] J.K. Yano, M.R. Wester, G.A. Schoch, K.J. Griffin, C.D. Stout, E.F. Johnson, The
structure of human microsomal cytochrome P450 3A4 determined by X-ray
crystallography to 2.05-A resolution, J. Biol. Chem. 279 (2004) 38091–38094,
https://doi.org/10.1074/jbc.C400293200.
[52] T. Omura, R. Sato, The Carbon Monoxide-Binding Pigment of Liver
Microsomes. I. Evidence for Its Hemoprotein Nature, J. Biol. Chem. 239
(1964) 2370–2378.
[53] A.W. Munro, H.M. Girvan, K.J. McLean, Variations on a (t)heme–novel
mechanisms, redox partners and catalytic functions in the cytochrome
P450 superfamily, Nat. Prod. Rep. 24 (2007) 585–609, https://doi.org/
10.1039/b604190f.
[54] S. Shaik, S. Cohen, Y. Wang, H. Chen, D. Kumar, W. Thiel, P450 enzymes: their
structure, reactivity, and selectivitys modeled by QM/MM calculations, Chem.
Rev. 110 (2010) 949–1017, https://doi.org/10.1021/cr030722j.
[55] E.M. Isin, F.P. Guengerich, Complex reactions catalyzed by cytochrome P450
enzymes, Bba-Gen Subjects 1770 (2007) 314–329, https://doi.org/10.1016/j.
bbagen.2006.07.003.
[56] F.P. Guengerich, Common and uncommon cytochrome P450 reactions related
to metabolism and chemical toxicity, Chem. Res. Toxicol. 14 (2001) 611–650,
https://doi.org/10.1021/tx0002583.
[57] I. Schlichting, J. Berendzen, K. Chu, A.M. Stock, S.A. Maves, D.E. Benson, B.M.
Sweet, D. Ringe, G.A. Petsko, S.G. Sligar, The catalytic pathway of cytochrome
P450cam at atomic resolution, Science 287 (2000) 1615–1622, https://doi.
org/10.1126/science.287.5458.1615.
21
Coordination Chemistry Reviews 427 (2021) 213600
[58] R. Bernhardt, Cytochromes P450 as versatile biocatalysts, J. Biotechnol. 124
(2006) 128–145, https://doi.org/10.1016/j.jbiotec.2006.01.026.
[59] L. Olsen, C. Oostenbrink, F.S. Jorgensen, Prediction of cytochrome P450
mediated metabolism, Adv. Drug Deliv. Rev. 86 (2015) 61–71, https://doi.org/
10.1016/j.addr.2015.04.020.
[60] Y. Xiong, Y. Qiao, D. Kihara, H. Zhang, X. Zhu, D. Wei, Survey of machine
learning techniques for prediction of the isoform specificity of cytochrome
P450 substrates, Curr. Drug Metab. 20 (2019) 229–235, https://doi.org/
10.2174/1389200219666181019094526.
[61] J.D. Tyzack, J. Kirchmair, Computational methods and tools to predict
cytochrome P450 metabolism for drug discovery, Chem. Biol. Drug Des. 93
(2019) 377–386, https://doi.org/10.1111/cbdd.13445.
[62] J. Kirchmair, M.J. Williamson, J.D. Tyzack, L. Tan, P.J. Bond, A. Bender, R.C.
Glen, Computational prediction of metabolism: sites, products, SAR, P450
enzyme dynamics, and mechanisms, J. Chem. Inf. Model. 52 (2012) 617–648,
https://doi.org/10.1021/ci200542m.
[63] E. Stjernschantz, C. Oostenbrink, Improved ligand-protein binding affinity
predictions using multiple binding modes, Biophys. J. 98 (2010) 2682–2691,
https://doi.org/10.1016/j.bpj.2010.02.034.
[64] L. Perichassler, E. Stjernschantz, C. Oostenbrink, D.P. Geerke, CYP 2D6 binding
affinity predictions using multiple ligand and protein conformations, Int. J.
Mol. Sci. 14 (2013) 24514–24530, https://doi.org/10.3390/ijms141224514.
[65] U. Bren, C. Oostenbrink, Cytochrome P450 3A4 inhibition by ketoconazole:
tackling the problem of ligand cooperativity using molecular dynamics
simulations and free-energy calculations, J. Chem. Inf. Model. 52 (2012)
1573–1582, https://doi.org/10.1021/ci300118x.
[66] M. Paloncýova, V. Navratilova, K. Berka, A. Laio, M. Otyepka, Role of enzyme
flexibility in ligand access and egress to active site: bias-exchange
metadynamics study of 1,3,7-trimethyluric acid in cytochrome P450 3A4, J.
Chem. Theory Comput. 12 (2016) 2101–2109, https://doi.org/10.1021/acs.
jctc.6b00075.
[67] M.A. Olah, J.N. Harvey, Understanding the determinants of selectivity in drug
metabolism through modeling of dextromethorphan oxidation by
cytochrome P450, Proc. Natl. Acad. Sci. U.S.A. 108 (2011) (2011) 6050–
6055, https://doi.org/10.1073/pnas.1010194108.
[68] P. Rydberg, M. Rostkowski, D.E. Gloriam, L. Olsen, The contribution of atom
accessibility to site of metabolism models for cytochromes P450, Mol. Pharm.
10 (2013) 1216–1223, https://doi.org/10.1021/mp3005116.
[69] P. Rydberg, L. Olsen, Predicting drug metabolism by cytochrome P450 2C9:
comparison with the 2D6 and 3A4 isoforms, Chem. Med. Chem. 7 (2012)
1202–1209, https://doi.org/10.1002/cmdc.201200160.
[70] R. Liu, J. Liu, G. Tawa, A. Wallqvist, 2D SMARTCyp reactivity-based site of
metabolism prediction for major drug-metabolizing cytochrome P450
enzymes, J. Chem. Inf. Model. 52 (2012) 1698–1712, https://doi.org/
10.1021/ci3001524.
[71] H.J. Barnes, M.P. Arlotto, M.R. Waterman, Expression and enzymatic-activity
of recombinant cytochrome-P450 17-alpha-hydroxylase in Escherichia-Coli,
Proc. Natl. Acad. Sci. U.S.A. 88 (1991) 5597–5601, https://doi.org/10.1073/
pnas.88.13.5597.
[72] M.M. Galteau, F. Shamsa, Urinary 6b-hydroxycortisol: a validated test for
evaluating drug induction or drug inhibition mediated through CYP3A in
humans and in animals, Eur. J. Clin. Pharmacol. 59 (2003) 713–733, https://
doi.org/10.1007/s00228-003-0690-3.
[73] T. Miura, M. Komori, M. Iwasaki, K. Kurozumi, K. Ohta, S. Ohmori, M. Kitada, T.
Kamataki, Sex-related difference in oxidative metabolism of testosterone and
erythromycin by hamster liver microsomes, FEBS Lett. 231 (1988) 183–186,
https://doi.org/10.1016/0014-5793(88)80727-0.
[74] R. Yuan, S. Madani, X.X. Wei, K. Reynolds, Evaluation of cytochrome P450
probe substrates commonly used by the pharmaceutical industry to study
in vitro drug interactions, Drug Metab. Dispos. 30 (2002) 1311–1319, https://
doi.org/10.1124/dmd.30.12.1311.
[75] J.J. Wu, Y.F. Cao, Y.Y. Zhang, Y. Liu, J.Y. Hong, L.L. Zhu, G.B. Ge, L. Yang,
Deoxyschizandrin, a naturally occurring lignan, is a specific probe substrate
of human cytochrome P450 3A, Drug Metab. Dispos. 42 (2014) 94–104,
https://doi.org/10.1124/dmd.113.053884.
[76] J.J. Wu, G.B. Ge, Y.Q. He, P. Wang, Z.R. Dai, J. Ning, L.H. Hu, L. Yang, Gomisin A
is a novel isoform-specific probe for the selective sensing of human
cytochrome P450 3A4 in liver microsomes and living cells, AAPS J. 18
(2016) 134–145, https://doi.org/10.1208/s12248-015-9827-4.
[77] Y.F. Cao, Y.Y. Zhang, J. Li, G.B. Ge, D. Hu, H.X. Liu, T. Huang, Y.C. Wang, Z.Z.
Fang, D.X. Sun, H. Huo, J. Yin, L. Yang, CYP3A catalyses schizandrin
biotransformation in human, minipig and rat liver microsomes, Xenobiotica
40 (2010) 38–47, https://doi.org/10.3109/00498250903366052.
[78] J.J. Wu, Y.F. Cao, L. Feng, Y.Q. He, J.Y. Hong, T.Y. Dou, P. Wang, D.C. Hao, G.B.
Ge, L. Yang, A naturally occurring isoform-specific probe for highly selective
and sensitive detection of human cytochrome P450 3A5, J. Med. Chem. 60
(2017) 3804–3813, https://doi.org/10.1021/acs.jmedchem.7b00001.
[79] Z.R. Dai, J. Ning, G.B. Sun, P. Wang, F. Zhang, H.Y. Ma, L.W. Zou, J. Hou, J.J. Wu, G.B.
Ge, X.B. Sun, L. Yang, Cytochrome P450 3A enzymes are key contributors for
hepatic metabolism of bufotalin, a natural constitute in chinese medicine chansu,
Front. Pharmacol. 10 (2019) 52, https://doi.org/10.3389/fphar.2019.00052.
[80] J. Ning, Z. Yu, L. Hu, C. Wang, X. Huo, S. Deng, J. Hou, J. Wu, G. Ge, X. Ma,
Characterization of phase I metabolism of resibufogenin and evaluation of the
metabolic effects on its antitumor activity and toxicity, Drug Metab. Dispos.
43 (2015) 299–308, https://doi.org/10.1124/dmd.114.060996.
J. Wu et al.
[81] Z.R. Dai, G.B. Ge, L. Feng, J. Ning, L.H. Hu, Q. Jin, D.D. Wang, X. Lv, T.Y. Dou, J.N.
Cui, L. Yang, A highly selective ratiometric two-photon fluorescent probe for
human cytochrome P450 1A, J. Am. Chem. Soc. 137 (2015) 14488–14495,
https://doi.org/10.1021/jacs.5b09854.
[82] J. Ning, Z.H. Tian, B. Wang, G.B. Ge, Y. An, J. Hou, C. Wang, X.Y. Zhao, Y.N. Li, X.
G. Tian, Z.L. Yu, X.K. Huo, C.P. Sun, L. Feng, J.N. Cui, X.C. Ma, A highly sensitive
and selective two-photon fluorescent probe for real-time sensing of
cytochrome P450 1A1 in living systems, Mater. Chem. Front. 2 (2018)
2013–2020, https://doi.org/10.1039/c8qm00372f.
[83] Z.R. Dai, L. Feng, Q. Jin, H. Cheng, Y. Li, J. Ning, Y. Yu, G.B. Ge, J.N. Cui, L. Yang, A
practical strategy to design and develop an isoform-specific fluorescent probe
for a target enzyme: CYP1A1 as a case study, Chem. Sci. 8 (2017) 2795–2803,
https://doi.org/10.1039/c6sc03970g.
[84] X. Lv, L. Feng, C.Z. Ai, J. Hou, P. Wang, L.W. Zou, J. Cheng, G.B. Ge, J.N. Cui, L.
Yang, A Practical and High-Affinity fluorescent probe for uridine diphosphate
glucuronosyltransferase 1A1: a good surrogate for bilirubin, J. Med. Chem. 60
(2017) 9664–9675, https://doi.org/10.1021/acs.jmedchem.7b01097.
[85] H. Yu, Y. Xiao, L. Jin, A lysosome-targetable and two-photon fluorescent probe
for monitoring endogenous and exogenous nitric oxide in living cells, J. Am.
Chem. Soc. 134 (2012) 17486–17489, https://doi.org/10.1021/ja308967u.
[86] X.L. Liu, X.J. Du, C.G. Dai, Q.H. Song, Ratiometric two-photon fluorescent
probes for mitochondrial hydrogen sulfide in living cells, J. Org. Chem. 79
(2014) 9481–9489, https://doi.org/10.1021/jo5014838.
[87] C. Zhang, A.M. Ren, J.F. Guo, D. Wang, L.Y. Yu, Theoretical design and
investigation of 1,8-naphthalimide-based two-photon fluorescent probes for
detecting cytochrome P450 1A with separated fluorescence signal, PCCP 20
(2018) 13290–13305, https://doi.org/10.1039/c8cp01754a.
[88] P.H. Keizers, C. de Graaf, F.J. de Kanter, C. Oostenbrink, K.A. Feenstra, J.N.
Commandeur, N.P. Vermeulen, Metabolic regio- and stereoselectivity of
cytochrome P450 2D6 towards 3,4-methylenedioxy-N-alkylamphetamines:
in silico predictions and experimental validation, J. Med. Chem. 48 (2005)
6117–6127, https://doi.org/10.1021/jm050338+.
[89] X. Liu, L. Hu, G. Ge, B. Yang, J. Ning, S. Sun, L. Yang, K. Pors, J. Gu, Quantitative
analysis of cytochrome P450 isoforms in human liver microsomes by the
combination of proteomics and chemical probe-based assay, Proteomics 14
(2014) 1943–1951, https://doi.org/10.1002/pmic.201400025.
[90] F. Weis, H. Hammer, K. Klein, H. Planatscher, U.M. Zanger, A. Noren, C. Wegler,
P. Artursson, T.O. Joos, O. Poetz, Direct quantification of cytochromes P450
and drug transporters—a rapid, targeted mass spectrometry-based
immunoassay panel for tissues and cell culture lysates, Drug Metab. Dispos.
46 (2018) 387–396, https://doi.org/10.1124/dmd.117.078626.
[91] J. Ning, T. Liu, P. Dong, W. Wang, G.B. Ge, B. Wang, Z.L. Yu, L. Shi, X. Tian, X.K.
Huo, L. Feng, C. Wang, C.P. Sun, J.N. Cui, T.D. James, X. Ma, Molecular design
strategy to construct the near-infrared fluorescent probe for selectively
sensing human cytochrome P450 2J2, J. Am. Chem. Soc. 141 (2019) 1126–
1134, https://doi.org/10.1021/jacs.8b12136.
[92] D.S. Streetman, J.S. Bertino Jr., A.N. Nafziger, Phenotyping of drug-
metabolizing enzymes in adults: a review of in-vivo cytochrome P450
phenotyping probes, Pharmacogenetics 10 (2000) 187–216, https://doi.org/
10.1097/00008571-200004000-00001.
[93] R.S. Foti, D.A. Rock, L.C. Wienkers, J.L. Wahlstrom, Selection of alternative
CYP3A4 probe substrates for clinical drug interaction studies using in vitro
data and in vivo simulation, Drug Metab. Dispos. 38 (2010) 981–987, https://
doi.org/10.1124/dmd.110.032094.
[94] G.B. Ge, J. Ning, L.H. Hu, Z.R. Dai, J. Hou, Y.F. Cao, Z.W. Yu, C.Z. Ai, J.K. Gu, X.C.
Ma, L. Yang, A highly selective probe for human cytochrome P450 3A4:
isoform selectivity, kinetic characterization and its applications, Chem.
Commun. (Camb.) 49 (2013) 9779–9781, https://doi.org/10.1039/
c3cc45250f.
[95] FDA, Clinical Drug Interaction Studies — Cytochrome P450 Enzyme- and
Transporter-Mediated Drug Interactions Final Guidance. https://www.
fda.gov/drugs/guidances-drugs/clinical-drug-interaction-studies-
cytochrome-p450-enzyme-and-transporter-mediated-drug-interactions,
2020 (accessed 02/28 2020).
[96] R. Walsky, R. Obach, Validated assays for human cytochrome P450 activities,
Drug Metab. Dispos. 32 (2004) 647–660, https://doi.org/10.1124/
dmd.32.6.647.
[97] N.M. DeVore, B.D. Smith, M.J. Urban, E.E. Scott, Key residues controlling
phenacetin metabolism by human cytochrome P450 2A enzymes, Drug
Metab. Dispos. 36 (2008) 2582–2590, https://doi.org/10.1124/
dmd.108.023770.
[98] Y.Y. Zhang, Y. Liu, J.W. Zhang, G.B. Ge, L.M. Wang, J. Sun, L. Yang,
Characterization of human cytochrome P450 isoforms involved in the
metabolism of 7-epi-paclitaxel, Xenobiotica 39 (2009) 283–292, https://doi.
org/10.1080/00498250802714907.
[99] U. Yasar, E. Eliasson, C. Forslund-Bergengren, G. Tybring, M. Gadd, F. Sjöqvist,
M. Dahl, The role of CYP2C9 genotype in the metabolism of diclofenac in vivo
and in vitro, Eur. J. Clin. Pharmacol. 57 (2001) 729–735, https://doi.org/
10.1007/s00228-001-0376-7.
[100] M.G. Shou, R. Norcross, G. Sandig, P. Lu, Y.H. Li, Y. Lin, Q. Mei, A.D. Rodrigues,
T.H. Rushmore, Substrate specificity and kinetic properties of seven
heterologously expressed dog cytochromes P450, Drug Metab. Dispos. 31
(2003) 1161–1169, https://doi.org/10.1124/dmd.31.9.1161.
[101] V. Kumar, D.A. Rock, C.J. Warren, T.S. Tracy, J.L. Wahlstrom, Enzyme source
effects on CYP2C9 kinetics and inhibition, Drug Metab. Dispos. 34 (2006)
1903–1908, https://doi.org/10.1124/dmd.106.010249.
22
Coordination Chemistry Reviews 427 (2021) 213600
[102] A. Abelo, T.B. Andersson, M. Antonsson, A.K. Naudot, I. Skanberg, L. Weidolf,
Stereoselective metabolism of omeprazole by human cytochrome P450
enzymes, Drug Metab. Dispos. 28 (2000) 966–972.
[103] R.S. Foti, M.B. Fisher, Impact of incubation conditions on bufuralol human
clearance predictions: enzyme lability and nonspecific binding, Drug Metab.
Dispos. 32 (2004) 295–304, https://doi.org/10.1124/dmd.32.3.295.
[104] S. Matsumoto, T. Hirama, T. Matsubara, K. Nagata, Y. Yamazoe, Involvement
of CYP2J2 on the intestinal first-pass metabolism of antihistamine drug,
astemizole, Drug Metab. Dispos. 30 (2002) 1240–1245, https://doi.org/
10.1124/dmd.30.11.1240.
[105] E.A. Evangelista, R. Kaspera, N.A. Mokadam, J.P. Jones 3rd, R.A. Totah, Activity,
inhibition, and induction of cytochrome P450 2J2 in adult human primary
cardiomyocytes, Drug Metab. Dispos. 41 (2013) 2087–2094, https://doi.org/
10.1124/dmd.113.053389.
[106] J. Zhang, Y. Chen, Y. Sun, R. Wang, J. Zhang, Z. Jia, Plateau hypoxia attenuates
the metabolic activity of intestinal flora to enhance the bioavailability of
nifedipine, Drug Deliv. 25 (2018) 1175–1181, https://doi.org/10.1080/
10717544.2018.1469687.
[107] X. Li, V. Jeso, S. Heyward, G.S. Walker, R. Sharma, G.C. Micalizio, M.D.
Cameron, Characterization of T-5 N-oxide formation as the first highly
selective measure of CYP3A5 activity, Drug Metab. Dispos. 42 (2014) 334–
342, https://doi.org/10.1124/dmd.113.054726.
[108] Y.H. Lu, C.W. Hendrix, N.N. Bumpus, Cytochrome P450 3A5 Plays a Prominent
Role in the Oxidative Metabolism of the Anti-Human Immunodeficiency
Virus Drug Maraviroc, Drug Metab. Dispos. 40 (2012) 2221–2230, https://doi.
org/10.1124/dmd.112.048298.
[109] Y. Kikuta, E. Kusunose, M. Kusunose, Characterization of human liver
leukotriene B(4) omega-hydroxylase P450 (CYP4F2), J. Biochem. 127 (2000)
1047–1052, https://doi.org/10.1093/oxfordjournals.jbchem.a022696.
[110] W. Lin, M.Q. Huang, X. Xue, K. Bertelsen, G. Chen, H. Zhao, Z.J. Lin, A. Fourie, J.
de Jong, N. Weng, A highly sensitive and selective method for the
determination of leukotriene B4 (LTB4) in ex vivo stimulated human
plasma by ultra fast liquid chromatography-tandem mass spectrometry, J
Chromatogr B Analyt Technol Biomed Life Sci 925 (2013) 54–62, https://doi.
org/10.1016/j.jchromb.2013.02.038.
[111] K.O. Martin, K. Budai, N.B. Javitt, Cholesterol and 27-hydroxycholesterol 7
alpha-hydroxylation: evidence for two different enzymes, J. Lipid Res. 34
(1993) 581–588.
[112] C.H. Yun, T.J. Yin, K. Shatzer, D.G. Burrin, L.W. Cui, Y.F. Tu, M. Hu,
Determination of 7 alpha-OH cholesterol by LC-MS/MS: application in
assessing the activity of CYP7A1 in cholestatic minipigs, J. Chromatogr. B
1025 (2016) 76–82, https://doi.org/10.1016/j.jchromb.2016.05.005.
[113] S. Zhang, S.R. Mada, S. Sharma, M. Torch, D. Mattison, S. Caritis, R.
Venkataramanan, Simultaneous quantitation of 17alpha-
hydroxyprogesterone caproate, 17alpha-hydroxyprogesterone and
progesterone in human plasma using high-performance liquid
chromatography-mass spectrometry (HPLC-MS/MS), J. Pharmaceut. Biomed.
48 (2008) 1174–1180, https://doi.org/10.1016/j.jpba.2008.08.024.
[114] P. Concolino, E. Mello, M.C. Patrosso, S. Penco, C. Zuppi, E. Capoluongop.,
H282N and p.Y191H: 2 novel CYP21A2 mutations in Italian congenital
adrenal hyperplasia patients, Metabolism 61 (2012) 519–524, https://doi.
org/10.1016/j.metabol.2011.08.008.
[115] Z. Zhang, D.S. Li, D.E. Blanchard, S.R. Lear, S.K. Erickson, T.A. Spencer, Key
regulatory oxysterols in liver: analysis as Delta(4)-3-ketone derivatives by
HPLC and response to physiological perturbations, J. Lipid Res. 42 (2001)
649–658.
[116] V.V. Bandaru, N.J. Haughey, Quantitative detection of free 24S-
hydroxycholesterol, and 27-hydroxycholesterol from human serum, BMC
Neurosci. 15 (2014) 137, https://doi.org/10.1186/s12868-014-0137-z.
[117] V. Miller, D. Stresser, A. Blanchard, S. Turner, C. Crespi, Fluorometric high-
throughput screening for inhibitors of cytochrome P450, Ann. N. Y. Acad. Sci.
919 (2000) 26–32, https://doi.org/10.1111/j.1749-6632.2000.tb06864.x.
[118] E. Schneider, D.S. Clark, Cytochrome P450 (CYP) enzymes and the
development of CYP biosensors, Biosens. Bioelectron. 39 (2013) 1–13,
https://doi.org/10.1016/j.bios.2012.05.043.
[119] A.T. Wright, J.D. Song, B.F. Cravatt, A suite of activity-based probes for human
cytochrome P450 enzymes, J. Am. Chem. Soc. 131 (2009) 10692–10700,
https://doi.org/10.1021/ja9037609.
[120] A.T. Wright, B.F. Cravatt, Chemical proteomic probes for profiling cytochrome
p450 activities and drug interactions in vivo, Chem. Biol. 14 (2007) 1043–
1051, https://doi.org/10.1016/j.chembiol.2007.08.008.
[121] C. Fehl, C.D. Vogt, R. Yadav, K.L. Li, E.E. Scott, J. Aube, Structure-based design
of inhibitors with improved selectivity for steroidogenic cytochrome P450
17A1 over cytochrome P450 21A2, J. Med. Chem. 61 (2018) 4946–4960,
https://doi.org/10.1021/acs.jmedchem.8b00419.
[122] G.A. Potter, S.E. Barrie, M. Jarman, M.G. Rowlands, Novel steroidal inhibitors
of human cytochrome P45017 alpha (17 alpha-hydroxylase-C17,20-lyase):
potential agents for the treatment of prostatic cancer, J. Med. Chem. 38
(1995) 2463–2471, https://doi.org/10.1021/jm00013a022.
[123] K. Fizazi, H.I. Scher, A. Molina, C.J. Logothetis, K.N. Chi, R.J. Jones, J.N. Staffurth,
S. North, N.J. Vogelzang, F. Saad, P. Mainwaring, S. Harland, O.B. Goodman, C.
N. Sternberg, J.H. Li, T. Kheoh, C.M. Haqq, J.S. de Bono, Abiraterone acetate for
treatment of metastatic castration-resistant prostate cancer: final overall
survival analysis of the COU-AA-301 randomised, double-blind, placebo-
controlled phase 3 study, Lancet Oncol. 13 (2012) 983–992, https://doi.org/
10.1016/s1470-2045(12)70379-0.
J. Wu et al.
[124] P. Schonknecht, D. Lutjohann, J. Pantel, H. Bardenheuer, T. Hartmann, K. von
Bergmann, K. Beyreuther, J. Schroder, Cerebrospinal fluid 24S-
hydroxycholesterol is increased in patients with Alzheimer’s disease
compared to healthy controls, Neurosci. Lett. 324 (2002) 83–85, https://doi.
org/10.1016/s0304-3940(02)00164-7.
[125] F. Djelti, J. Braudeau, E. Hudry, M. Dhenain, J. Varin, I. Bieche, C. Marquer, F.
Chali, S. Ayciriex, N. Auzeil, S. Alves, D. Langui, M.C. Potier, O. Laprevote, M.
Vidaud, C. Duyckaerts, R. Miles, P. Aubourg, N. Cartier, CYP46A1 inhibition,
brain cholesterol accumulation and neurodegeneration pave the way for
Alzheimer’s disease, Brain 138 (2015) 2383–2398, https://doi.org/10.1093/
brain/awv166.
[126] S. Sudsakorn, J. Skell, D.A. Williams, T.J. O’Shea, H.L. Liu, Evaluation of 3-O-
methylfluorescein as a selective fluorometric substrate for CYP2C19 in
human liver microsomes, Drug Metab. Dispos. 35 (2007) 841–847, https://
doi.org/10.1124/dmd.106014472.
[127] A.B. Renwick, D. Surry, R.J. Price, B.G. Lake, D.C. Evans, Metabolism of 7-
benzyloxy-4-trifluoromethyl-coumarin by human hepatic cytochrome P450
isoforms, Xenobiotica 30 (2000) 955–969, https://doi.org/10.1080/
00498250050200113.
[128] A.B. Renwick, D.F.V. Lewis, S. Fulford, D. Surry, B. Williams, P.D. Worboys, X.
Cai, R.W. Wang, R.J. Price, B.G. Lake, D.C. Evans, Metabolism of 2,5-bis
(trifluoromethyl)-7-benzyloxy-4-trifluoromethylcoumarin by human hepatic
CYP isoforms: evidence for selectivity towards CYP3A4, Xenobiotica 31
(2001) 187–204, https://doi.org/10.1080/00498250110043526.
[129] X. Zhang, Y. Zhou, X. Gu, Y. Cheng, M. Hong, L. Yan, F. Ma, Z. Qi, Synthesis of a
selective ratiometric fluorescent probe based on Naphthalimide and its
application in human cytochrome P450 1A, Talanta 186 (2018) 413–420,
https://doi.org/10.1016/j.talanta.2018.04.079.
[130] H. Ji, X. Zhang, Y. Dai, T. Xue, S. Misal, Z. Qi, A highly selective ratiometric
fluorescent probe based on naphthalimide for detection and imaging of
CYP1A1 in living cells and zebrafish, Analyst 144 (2019) 7390–7397, https://
doi.org/10.1039/c9an01767d.
[131] T. Xue, Y. Dai, X. Zhang, Y. Cheng, X. Gu, H. Ji, S. Misal, Z. Qi, Ultrasensitive
near-infrared fluorescent probe with large stokes shift for real-time tracing of
CYP1A1 in living cells and zebrafish model, Sens. Actuat. B-Chem. 293 (2019)
265–272, https://doi.org/10.1016/j.snb.2019.04.147.
[132] T. Shimada, E.M. Gillam, T.R. Sutter, P.T. Strickland, F.P. Guengerich, H.
Yamazaki, Oxidation of xenobiotics by recombinant human cytochrome P450
1B1, Drug Metab. Dispos. 25 (1997) 617–622.
[133] Q. Jin, Y. Ma, L. Feng, P. Wang, R. He, L. Yang, G.B. Ge, Sensing cytochrome
P450 1A1 activity by a resorufin-based isoform-specific fluorescent probe,
Chin. Chem. Lett. (2020), https://doi.org/10.1016/j.cclet.2020.05.038.
[134] A. Ghosal, N. Hapangama, Y. Yuan, X. Lu, D. Horne, J.E. Patrick, S. Zbaida, Rapid
determination of enzyme activities of recombinant human cytochromes
P450, human liver microsomes and hepatocytes, Biopharm. Drug Dispos. 24
(2003) 375–384, https://doi.org/10.1002/bdd.374.
[135] L. Makings, G. Zlokarnik, Optical molecular sensors for cytochrome P450
activity. https://patents.google.com/patent/US6514687B1/en, 2003 (accessed
19 Junly 2020).
[136] Y. Li, N.Y. Li, E.M. Sellers, Comparison of CYP2A6 catalytic activity on
coumarin 7-hydroxylation in human and monkey liver microsomes, Eur. J.
Drug Metab. 22 (1997) 295–304, https://doi.org/10.1007/BF03190960.
[137] M. Spatzenegger, H. Liu, Q. Wang, A. Debarber, D. Koop, J. Halpert, Analysis of
differential substrate selectivities of CYP2B6 and CYP2E1 by site-directed
mutagenesis and molecular modeling, J. Pharmacol. Exp. Ther. 304 (2002)
477–487, https://doi.org/10.1124/jpet.102.043323.
[138] J. Venhorst, A. ter Laak, J. Commandeur, Y. Funae, T. Hiroi, N. Vermeulen,
Homology modeling of rat and human cytochrome P450 2D (CYP2D)
isoforms and computational rationalization of experimental ligand-binding
specificities, J. Med. Chem. 46 (2003) 74–86, https://doi.org/10.1021/
jm0209578.
[139] B.D. Marks, R.W. Smith, H.A. Braun, T.A. Goossens, M. Christenson, M.S. Ozers,
C.S. Lebakken, O.V. Trubetskoy, A high throughput screening assay to screen
for CYP2E1 metabolism and inhibition using a fluorogenic vivid p450
substrate, Assay Drug Dev. Technol. 1 (2002) 73–81, https://doi.org/
10.1089/154065802761001329.
[140] N. Ning, W. Wang, G.B. Ge, P. Chu, F. Long, Y. Yang, Y. Peng, L. Feng, X. Ma, T.D.
James, Targeted enzyme activated two-photon fluorescent probes: a case
study of CYP3A4 using a two-dimensional design strategy, Angew. Chem. Int.
Ed. Engl. 58 (2019) 9959–9963, https://doi.org/10.1002/anie.201903683.
[141] R. Foti, L. Wienkers, J. Wahlstrom, Application of cytochrome P450 drug
interaction screening in drug discovery, Comb. Chem. High T Scr. 13 (2010)
145–158, https://doi.org/10.2174/138620710790596718.
[142] J.J. Cali, D. Ma, M.G. Wood, P.L. Meisenheimer, D.H. Klaubert, Bioluminescent
assays for ADME evaluation: dialing in CYP selectivity with luminogenic
substrates, Expert Opin. Drug Metab. Toxicol. 8 (2012) 1115–1130, https://
doi.org/10.1517/17425255.2012.695345.
[143] J. Li, L. Chen, L. Du, M. Li, Cage the firefly luciferin!–a strategy for developing
bioluminescent probes, Chem. Soc. Rev. 42 (2013) 662–676, https://doi.org/
10.1039/C2CS35249D.
[144] T.A. Su, K.J. Bruemmer, C.J. Chang, Caged luciferins for bioluminescent
activity-based sensing, Curr. Opin. Biotechnol. 60 (2019) 198–204, https://
doi.org/10.1016/j.copbio.2019.05.002.
[145] Z.M. Kaskova, A.S. Tsarkova, I.V. Yampolsky, 1001 lights: luciferins,
luciferases, their mechanisms of action and applications in chemical
23
Coordination Chemistry Reviews 427 (2021) 213600
analysis, biology and medicine, Chem. Soc. Rev. 45 (2016) 6048–6077,
https://doi.org/10.1039/C6CS00296J.
[146] K. Gomi, N. Kajiyama, Oxyluciferin, a luminescence product of firefly
luciferase, is enzymatically regenerated into luciferin, J. Biol. Chem. 276
(2001) 36508–36513, https://doi.org/10.1074/jbc.M105528200.
[147] E.V. Vetrova, N.S. Kudryasheva, K.H. Cheng, Effect of quinone on the
fluorescence decay dynamics of endogenous flavin bound to bacterial
luciferase, Biophys. Chem. 141 (2009) 59–65, https://doi.org/10.1016/j.
bpc.2008.12.012.
[148] E. Vetrova, N. Kudryasheva, V. Kratasyuk, Redox compounds influence on the
NAD (P) H: FMN–oxidoreductase–luciferase bioluminescent system,
Photochem. Photobiol. Sci. 6 (2007) 35–40, https://doi.org/10.1039/
B608152E.
[149] Promega, P450-Glo assay technical bulletin. . http://www.promega.com/
~/media/Files/Resources/Protocols/Technical%20Bulletins/101/P450%20Glo%
20Assays%20Protocol.pdf, 2012 (accessed 19 Junly 2020).
[150] J.J. Cali, D. Ma, M. Sobol, D.J. Simpson, S. Frackman, T.D. Good, W.J. Daily, D.
Liu, Luminogenic cytochrome P450 assays, Expert Opin. Drug Metab. Toxicol.
2 (2006) 629–645, https://doi.org/10.1517/17425255.2.4.629.
[151] J. J. Cali,D. Ma, A Rapid Cell Based Bioluminescent CYP1A1 Aryl Hydrocarbon
Receptor Activation Assay. https://www.promega.com.cn/
resources/scientific_posters/posters/a-rapid-cell-based-bioluminscent-
cyp1a1-aryl-hydrocarbon-receptor-assay-poster/, 2012 (accessed 19 Junly
2020).
[152] D. Ma, M. Sobol, C. C. Woodroofe,J. J. Cali, Cytochrome P450 2J2 enzyme assay
using a novel bioluminescent probe substrate. . https://www.promega.com.
cn/resources/pubhub/enotes/cytochrome-p450-2j2-enzyme-assay-using-a-
novel-bioluminescent-probe-substrate/, 2008 (accessed 19 Junly 2020).
[153] M. Sobol, D. Ma,J. J. Cali, Selective cytochrome p450 3A7 enzyme assay using
a novel bioluminescent probe substrate. https://www.promega.com.cn/
resources/pubhub/enotes/selective-cytochrome-p450-3a7-enzyme-assay-
using-a-novel-bioluminescent-probe-substrate/, 2007 (accessed 19 Junly
2020).
[154] M. Sobol, D. Ma,J. J. Cali, Selective Cytochrome P450 4A11 Enzyme Assay
Using a Novel Bioluminescent Probe Substrate. http://www.promega.com/
resources/articles/pubhub/enotes/selective-cytochrome-p450-4a11-
enzyme-assay-using-a-novel-bioluminescent-probe-substrate/, 2012
(accessed 16 January 2012).
[155] M. Sobol, D. Ma, C. C. Woodroofe,J. J. Cali, Cytochrome P450 4F2 and 4F3B
Enzyme Assays Using a Novel Bioluminescent Probe Substrate. https://www.
promega.com.cn/Resources/PubHub/eNotes/Cytochrome%20P450%204F2%
20and%204F3B%20Enzyme%20Assays%20Using%20a%20Novel%
20Bioluminescent%20Probe%20Substrate/, 2008 (accessed 19 Junly 2020).
[156] D. Ma, M. Sobol, C. C. Woodroofe,J. J. Cali, Cytochrome P450 4F12 Enzyme
Assay Using a Novel Bioluminescent Probe Substrate. https://www.
promega.com.cn/Resources/PubHub/eNotes/Cytochrome%20P450%204F12%
20Enzyme%20Assay%20Using%20a%20Novel%20Bioluminescent%20Probe%
20Substrate/, 2007 (accessed 27 August 2020).
[157] H.S. Blix, K.K. Viktil, T.A. Moger, A. Reikvam, Drugs with narrow therapeutic
index as indicators in the risk management of hospitalised patients, Pharm.
Pract. 8 (2010) 50–55, https://doi.org/10.4321/s1886-36552010000100006.
[158] M.E. de Jonge, A.D. Huitema, J.H. Schellens, S. Rodenhuis, J.H. Beijnen,
Population pharmacokinetics of orally administered paclitaxel formulated in
Cremophor EL, Br. J. Clin. Pharmacol. 59 (2005) 325–334, https://doi.org/
10.1111/j.1365-2125.2004.02325.x.
[159] H. Zhang, N. Gao, X. Tian, T. Liu, Y. Fang, J. Zhou, Q. Wen, B. Xu, B. Qi, J. Gao, H.
Li, L. Jia, H. Qiao, Content and activity of human liver microsomal protein and
prediction of individual hepatic clearance in vivo, Sci. Rep. 5 (2015) 17671,
https://doi.org/10.1038/srep17671.
[160] D.N. Juurlink, Drug-drug interactions among elderly patients hospitalized for
drug toxicity, JAMA 289 (2003) 1652–1658, https://doi.org/
10.1001/jama.289.13.1652.
[161] C. Zhan, R. Correa-de-Araujo, A.S. Bierman, J. Sangl, M.R. Miller, S.W. Wickizer,
D. Stryer, Suboptimal prescribing in elderly outpatients: potentially harmful
drug-drug and drug-disease combinations, J. Am. Geriatr. Soc. 53 (2005) 262–
267, https://doi.org/10.1111/j.1532-5415.2005.53112.x.
[162] S. Fowler, H. Zhang, In vitro evaluation of reversible and irreversible
cytochrome P450 inhibition: current status on methodologies and their
utility for predicting drug-drug interactions, AAPS J. 10 (2008) 410–424,
https://doi.org/10.1208/s12248-008-9042-7.
[163] L.H. Cohen, M.J. Remley, D. Raunig, A.D. Vaz, In vitro drug interactions of
cytochrome p450: an evaluation of fluorogenic to conventional substrates,
Drug Metab. Dispos. 31 (2003) 1005–1015, https://doi.org/10.1124/
dmd.31.8.1005.
[164] L. Bell, S. Bickford, P.H. Nguyen, J. Wang, T. He, B. Zhang, Y. Friche, A. Zimmerlin,
L. Urban, D. Bojanic, Evaluation of fluorescence- and mass spectrometry-based
CYP inhibition assays for use in drug discovery, J. Biomol. Screen. 13 (2008)
343–353, https://doi.org/10.1177/1087057108317480.
[165] Y.F. Ueng, W.C. Jan, L.C. Lin, T.L. Chen, F.P. Guengerich, C.F. Chen, The alkaloid
rutaecarpine is a selective inhibitor of cytochrome P450 1A in mouse and
human liver microsomes, Drug Metab. Dispos. 30 (2002) 349–353, https://
doi.org/10.1124/dmd.30.3.349.
[166] A. Galetin, K. Ito, D. Hallifax, J.B. Houston, CYP3A4 substrate selection and
substitution in the prediction of potential drug-drug interactions, J.
Pharmacol. Exp. Ther. 314 (2005) 180–190, https://doi.org/10.1124/
jpet.104.082826.
J. Wu et al. Coordination Chemistry Reviews 427 (2021) 213600

[167] L. Yu, Y.M. Qiao, L.X. Miao, Y.Q. He, Y. Zhou, Recent progress in fluorescent
and colorimetric sensors for the detection of ions and biomolecules, Chin.
Chem. Lett. 29 (2018) 1545–1559, https://doi.org/10.1016/j.
cclet.2018.09.005.
[168] J. Zhang, X. Chai, X.P. He, H.J. Kim, J. Yoon, H. Tian, Fluorogenic probes for
disease-relevant enzymes, Chem. Soc. Rev. 48 (2019) 683–722, https://doi.
org/10.1039/c7cs00907k.
[169] H.W. Liu, L. Chen, C. Xu, Z. Li, H. Zhang, X.B. Zhang, W. Tan, Recent progresses
in small-molecule enzymatic fluorescent probes for cancer imaging, Chem.
Soc. Rev. 47 (2018) 7140–7180, https://doi.org/10.1039/c7cs00862g.
[170] W. Chyan, R.T. Raines, Enzyme-activated fluorogenic probes for live-cell and
in vivo imaging, ACS Chem. Biol. 13 (2018) 1810–1823, https://doi.org/
10.1021/acschembio.8b00371.
[171] S.M. Sukumaran, B. Potsaid, M.Y. Lee, D.S. Clark, J.S. Dordick, Development of
a fluorescence-based, ultra high-throughput screening platform for
nanoliter-scale cytochrome p450 microarrays, J. Biomol. Screen. 14 (2009)
668–678, https://doi.org/10.1177/1087057109336592.
[172] J. Hao, J. Long, B. Li, X. Li, S. Zhang, F. Yang, X. Zeng, Z. Yang, W.K. Pang, Z. Guo,
Toward high-performance hybrid zn-based batteries via deeply
understanding their mechanism and using electrolyte additive, Adv. Funct.
Mater. 29 (2019) 1903605, https://doi.org/10.1002/adfm.201903605.
[173] M. Gao, B.Z. Tang, AIE-based cancer theranostics, Coord. Chem. Rev. 402
(2020), https://doi.org/10.1016/j.ccr.2019.213076 213076.
[174] A.D. Halleran, A. Swaminathan, R.M. Murray, Single day construction of
multigene circuits with 3G assembly, ACS Synth. Biol. 7 (2018) 1477–1480,
https://doi.org/10.1021/acssynbio.8b00060.

24