Cytochrome P450 Family 1 Inhibitors and Structure-Activity Relationships

With the widespread use of O-alkoxyresorufin dealkylation assays since the 1990s, thousands of inhibitors of cytochrome P450 family 1 enzymes (P450s 1A1, 1A2, and 1B1) have been identified and studied. Generally, planar polycyclic molecules such as polycyclic aromatic hydrocarbons, stilbenoids, and flavonoids are considered to potentially be effective inhibitors of these enzymes, however, the details of the structure-activity relationships and selectivity of these inhibitors are still ambiguous. In this review, we thoroughly discuss the selectivity of many representative P450 family 1 inhibitors reported in the past 20 years through a meta-analysis.


Introduction
Cytochrome P450 enzymes are a large ubiquitous superfamily of enzymes, playing a significant physiological role in the detoxification of xenobiotics, and the biosynthesis of many endogenous compounds. P450 families 1, 2, and 3 contribute most extensively to the biotransformation of xenobiotics into more polar metabolites that are readily excreted. In humans and most mammals, P450 family 1 comprises three well-studied monooxygenases, 1A1, 1A2, and 1B1.
Cytochrome P450 1A1, a well-known aryl hydrocarbon hydroxylase, is implicated in the metabolic activation of environmental procarcinogens such as polycyclic aromatic hydrocarbons (PAHs)

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and polyhalogenated aromatic hydrocarbons (PHAHs). Human P450 1A1 is mainly expressed in extrahepatic tissues such as lung, gastrointestinal tract, placenta, and skin, and is present only at low levels in the liver [1,2]. P450 1A1 is one of the most important enzymes involved in tumorigenesis initiated by environmental pollutants [3]. A number of epidemiological studies have shown that genetic variants of human CYP1A1 gene are significantly associated with the susceptibilities to lung and breast cancers [4][5][6]. Because of the significant role of P450 1A1 enzyme in human carcinogenesis, modulation of P450 1A1 activity has been considered as a potential target for cancer chemoprevention.
Cytochrome P450 1B1 is found mainly in extrahepatic tissues as well as in a variety of human tumors. This has important implications for tumor development and progression. Metabolic activation of 17β-estradiol (E2) to 4-hydroxy-17β-estradiol by P450 1B1 has been postulated to be an important factor in mammary carcinogenesis [9][10][11]. Like P450 1A1, this enzyme also metabolically activates procarcinogens to carcinogenic forms, resulting in mutagenesis and tumorigenesis [9]. Thus, regulators of the expression and catalytic activity of P450 1B1 have been suggested to play an important role in cancer chemoprevention, especially at the early stage of tumorigenesis [12]. Recently, it has been observed that P450 1B1 could generate reactive oxygen species resulting in hypertension, which suggested that P450 1B1 inhibitors might be clinically useful in the treatment of hypertension and associated cardiovascular diseases [13].
To summarize, although P450 family 1 cytochromes are responsible for the metabolism of a wide variety of xenobiotics as well as endogenous compounds, the enzymatic reactions also produce reactive intermediates which can covalently bond to cellular macromolecules leading to toxicity, mutagenicity, and carcinogenicity. Hence, much emphasis has been on the inhibition of these enzymes to develop potential cancer preventative and therapeutic agents [2,3,9,14]. The potential anti-cancer strategies targeting P450 inhibition were postulated as: (i) preventing the conversion of environmental procarcinogens to active carcinogens; (ii) preventing the conversion of hormonal precursors to carcinogenic hormone derivatives; and (iii) preventing the metabolic inactivation of anti-cancer drugs [15]. Herein we will review multifarious inhibitors of cytochrome P450 family 1 enzymes, 1A1, 1A2, and 1B1.

Classified Inhibitors of P450s 1A1, 1A2, and 1B1
Because of their high structural and functional similarity, cytochrome P450s 1A1, 1A2, and 1B1 are known to have overlapping substrates, inducers, and inhibitors. Therefore, the development of selective inhibitors towards any of these enzymes is a great challenge. To date, thousands of compounds (including natural products, pharmaceuticals, and synthetic compounds) have been evaluated for their inhibitory activities toward P450 family 1 enzymes. In an early study, the inhibition effects of 12 compounds toward P450s 1A1 and 1A2 were investigated, including α-naphthoflavone, ellipticine, 7-ethoxyresorufin, 5-methoxypsoralen, 8-methoxypsoralen, 7-ethoxycoumarin, nifedipine, propranolol, caffeine, paraxanthine, theophylline, and theobromine [16]. The comparison between groups indicated that polycyclic aromatic hydrocarbons, flavones, and coumarins were highly potent P450 inhibitors with IC 50 values in the micromolar level. This review mainly focuses on the highly potent inhibitor groups, which are more likely to become chemopreventive agents.

Polycyclic Aromatic Hydrocarbons
Since PAHs such as benzo[a]pyrene (B[a]P) are classical substrates of P450 family 1 enzymes, a number of PAHs were examined as inhibitors of the three enzymes through 7-ethoxyresorufin O-deethylation (EROD) assay by Shimada et al. The simplest PAHs, anthracene and phenanthrene ( Figure 1), showed stronger inhibition toward P450 1A2 over the other two enzymes. On the other hand, the four-ring pyrene structure showed extremely strong inhibition of P450 1B1 with an IC 50 of 2 nM, but not much selectivity relative to P450 1A2 [17]. The most potent inhibitor of P450 1A2 was benz[a]anthracene (IC 50 = 2 nM), exhibiting 8.9-fold and 7.0-fold more effective inhibition of this enzyme relative to P450s 1A1 and 1B1, respectively [17]. A follow-up study showed that the complex PAHs (Figure 1), such as chrysene, benzo[e]pyrene, 3-methylcholanthrene (3MC), fluoranthene (FA), and benzo[b]fluoranthene (B[b]FA), exhibited nanomolar level inhibition of P450 1B1-mediated EROD activity [18]. Mechanism investigation showed that all the studied non-substituted PAHs acted as competitive inhibitors [17,18]. Polycyclic aromatic hydrocarbons appear to be strong inhibitors of P450s 1A1, 1A2, and 1B1. However, PAHs (such as benzo[a]pyrene, 3-methylcholanthrene, and 7,12-dimethylbenz[a]anthracene (DMBA)) have also been linked to tumor formation and cancer in humans. Thus, even though PAHs have already become useful tools to investigate the structure and function of P450 family 1 enzymes, it is not logical to design therapeutic agents using them.
Since naphthoquinones and anthraquinones are natural products some of which have been used in Chinese traditional medicine for hundreds of years, their toxicity is much lower than that of PAHs. However, their efficacies for inhibiting P450 family 1 enzymes are also lower than those of PAHs and their structures need to be optimized to obtain highly potent and selective inhibitors of these enzymes.

Stilbenoids
trans-Stilbene, (E)-1,2-diphenylethene, is another main structural type of non-nitrogen P450 family 1 inhibitors. The first studied stilbenoid as a P450 inhibitor was resveratrol (3,5,4'-trihydroxystilbene), which is a natural product found in red grapes. Resveratrol showed chemopreventive effects in both cardiovascular disease and cancer [24]. Resveratrol exhibited mild inhibition of human P450 1A1 in a dose-dependent manner, with an IC 50 value of 23 µM for EROD and an IC 50 value of 11 µM for methoxyresorufin O-demethylation (MROD). However, the inhibition of human P450 1A2 by resveratrol was over 50-fold weaker than that of P450 1A1 (IC 50 1.2 mM for EROD and 580 µM for MROD) [24]. Mechanism investigation showed that resveratrol inhibited human P450 1A1 activity in a mixed-type inhibition (competitive-noncompetitive) with K i values of 9 µM (for competitive inhibition) and 89 µM (for non-competitive inhibition). Resveratrol is also a noncompetitive inhibitor of human P450 1B1 (with K i = 23 µM) [24] and a mechanism-based inactivator of cytochrome P450 3A4 (K I and k inact values of 20 µM and 0.20 min −1 , respectively) [25]. On the other hand, resveratrol was shown to be an arylhydrocarbon receptor (AhR) antagonist, inhibiting the induction of P450 1A1 [26]. Resveratrol inhibited TCDD-induced CYP1A1 expression in HepG 2 cells (in vitro) through blocking the induction of DRE (dioxin-responsive element)-driven transcription by AhR [27]. In an animal model, resveratrol was able to abrogate B[a]P-induced P450 1A1 activity and subsequent formation of B[a]P-DNA adducts [28].

Flavonoids
Flavonoids are widely present in fruits, nuts, vegetables, and medical herbs. These compounds have been described as health-promoting as well as disease-preventing dietary supplements [36]. The relative safety of flavonoids has allowed them to be used as therapeutic agents in traditional and modern medicine. Evidence indicates that certain flavonoids have high potency and selectivity for inhibition of P450 1A enzymes. This fact may have important implications for cancer prevention, as well as other pharmacological and toxicological effects [37].
As well-documented P450 inhibitors, the effects of flavonoids on AhR function and P450 family 1 enzyme expression were also investigated in many studies [36]. Ciolino et al. reported that galangin (3,5,7-trihydroxyflavone) caused an increase in the amount of CYP1A1 mRNA, but inhibited the induction of CYP1A1 mRNA by DMBA or by TCDD in MCF-7 human breast cancer cells. Galangin also inhibited the DMBA-or TCDD-induced transcription of a reporter vector containing the CYP1A1 promoter [43]. Quercetin and kaempferol are two of most abundant dietary flavonoids. Quercetin caused a time-and concentration-dependent increase in the level of CYP1A1 mRNA and P450 1A1 enzyme activity in MCF-7 cells. However, kaempferol inhibited the TCDD-induced CYP1A1 transcription instead of affecting normal CYP1A1 expression [44]. Baicalein (5,6,7-trihydroxyflavone, isolated from the plant Scutellariae baicalensis) reduce the CYP1A1/1B1 mRNA expression induced by DMBA, and the mRNA abundance for CYP1A1 appeared to be more responsive than that of CYP1B1 [45]. A flavone derivative, aminoflavone (Figure 4), also caused induction of CYP1A1 and CYP1B1 transcription through the AhR pathway [46]. Because of the two-way action of flavonoids on P450 enzyme inhibition and CYP gene expression, the true effects of a certain flavonoid in cell or in vivo are complex and need further investigation.

Alkaloids
Alkaloids are a large group of naturally occurring N-containing compounds. Since some of these compounds possess planar structural characteristics, they have the potential to be P450 inhibitors [16].
Ellipticine, a pyridocarbazole, was the first identified alkaloid inhibitor of P450 family 1 enzymes. It was isolated from Ochrosia elliptica Labill, well-known as an antineoplastic agent [56]. Although ellipticine showed a strong inhibition of EROD activity in rat liver microsomes (IC 50 = 0.11 µM), its inhibitory potential was non-specific and P450s from subfamilies 2B, 2D, and 3A were also inhibited by this compound [57].
A caffeine analog, furafylline, was the first selective P450 1A2 inhibitor identified. The first indication that it might inhibit P450 1A2 in vivo was that furafylline dramatically increased the half-life (7-to 10-fold) of caffeine in healthy volunteers [58]. Furafylline is a potent inhibitor of P450 1A2 phenacetin O-deethylase activity (IC 50 = 0.07 µM) in human liver microsomes. However, P450s 1A1, 2A6, 2C8, 2D6, and 3A3/4 were not inhibited by furafylline (IC 50 > 500 µM) [59]. In another study, furafylline was found to be a mechanism-based inhibitor of P450 1A2 [60]. The inactivation of P450 1A2 by this compound was characterized by a K i of 23 µM and a k inact of 0.87 min −1 .
Luotonin A, a pyrroloquinolinequinoline alkaloid originally isolated from Peganum nigellastrum, is a natural inhibitor of DNA topoisomerase I. Luotonin A inhibited P450 1A2-catalyzed phenacetin O-deethylation with an IC 50 of 6.3 μM in human liver microsomes, but did not inhibit it in a time-dependent manner. Luotonin A was found to decrease human recombinant cDNA-expressed P450 1A1-and 1A2-catalyzed phenacetin O-deethylase activities simultaneously, with IC 50 values of 4.05 and 7.35 μM, respectively ( Figure 6) [62].

Other Natural Products
Ueng et al. isolated and identified a group of P450 inhibitors from danshen (the dried root of Salvia miltiorrhiza), which is used in the treatment of angina pectoris, hyperlipidemia, and acute ischemic stroke. Among the seven components of danshen extract, tanshinone I, tanshinone IIA, and cryptotanshinone ( Figure 7) were shown to be potent competitive inhibitors of P450 1A2 (IC 50 = 0.75, 1.3, and 0.75 µM, respectively) [67]. In Escherichia coli membranes expressing bicistronic human P450 1A enzymes, tanshinone IIA inhibited EROD activity of P450 1A1 with an IC 50 48 times higher than that for P450 1A2 [68]. In vitro assays showed that tanshinones caused a significant time-and concentration-dependent increase in the amount of CYP1A1 and CYP1A2 expression in HepG 2 cells through a transcriptional activation mechanism [69]. Oral treatment of tanshinone IIA caused a dose-dependent increase of liver microsomal MROD activity in the arylhydrocarbon (Ah)-responsive C57BL/6J (B6) mice but not in (Ah)-nonresponsive DBA/2J (D2) mice. These results demonstrated that induction of P450 1A2 by tanshinone IIA depended on the Ah-responsiveness and occurred at pre-translational level [70]. . The inhibitory effects of tested compounds were more pronounced in HaCaT cells than in HepG 2 cells, and dehydrosilybin was a much stronger inhibitor than silybin [71].
Inhibitory effects of cannabidiol (CBD) and cannabinol (CBN), the two major constituents in marijuana, on catalytic activities of human cytochrome P450 family 1 enzymes were investigated. These cannabinoids inhibited EROD activity of recombinant P450s 1A1, 1A2, and 1B1 in a competitive manner. CBD most potently inhibited P450 1A1; the apparent K i value (0.16 µM) was at least one-seventeenth of the values for the other P450 1 enzymes. On the other hand, CBN decreased the activity of P450s 1A2 and 1B1 (K i = 0.079 and 0.15 µM, respectively) more effectively than P450 1A1 (K i = 0.54 µM) [72]. ε-Viniferin, a dimer of resveratrol, has also been studies and showed higher inhibitory activities toward P450s 1A1 and 1A2 than resveratrol [73].

Drugs
Because of potential drug-drug interactions, the effects of drugs on P450 enzymes are of great importance in pre-clinical pharmacological studies as well as clinical trials. Since P450 1A2 is one of the major drug metabolizing enzymes, it has been the most investigated enzyme for the inhibitory effect of drugs.
The compound 343 ( Figure 9) described by Sienkiewicz et al. caused a concentration-dependent decrease in the EROD activity of P450 1A1 (IC 50 = 2 µM) and 1B1 (IC 50 = 6.5 µM). There was only a modest inhibition of P450 1A2 activity [93]. Compound 343 also inhibited the upregulation of cytochrome P450 enzyme activities and CYP mRNA levels in cells treated with the AhR ligands, DMBA or TCDD [93].

Mechanism-Based Inhibitors (MBIs)
As hemoprotein monooxygenases, cytochrome P450s are responsible for the oxidation of a wide variety of xenobiotics and endogenous compounds. Once the oxidation reaction with a potential inhibitor produces reactive intermediate(s) that can covalently bond to the P450 active site, irreversible inactivation occurs [96,97]. This is the basis of mechanism-based inactivation, and these potential inhibitors are known as mechanism-based or suicide inhibitors. Because of the complete irreversible inactivation of the P450 enzyme, a mechanism-based inhibitor is much stronger than a competitive inhibitor with the same affinity for the enzyme. Thus, mechanism-based inhibitors are used in the design of specific enzyme-targeted drugs since they only affect the enzyme which can metabolize them into oxygenated active intermediate(s). Mechanism-based inhibitors are also of great interest to researchers because of their utility in elucidating the characteristics of enzyme active site cavities and enzymatic reaction mechanisms.
Many mechanism-based inhibitors were intentionally designed and synthesized employing a basic reactive unit, acetylene. The triple bond of these terminal acetylenes is oxidized by the P450 enzyme to form a reactive ketene intermediate by a 1,2-hydrogen shift. The ketene intermediate can then react with the heme nitrogen resulting in a time-dependent destruction of the heme [98][99][100] or covalently bond with a nucleophilic residue of the active site protein, thereby inactivating the enzyme [101,102]. The other important component of these compounds is a planar conjugated system, fitting the narrow active site cavity of P450 family 1 enzymes. A large group of ethynyl and 1-propynyl pyrenes, phenanthrenes, naphthalenes, and biphenyls were investigated in a series of studies on P450s 1A1, 1A2, and 1B1 [17,18,103,104]. The results showed that inhibition mechanism varied for the different compounds and different enzymes. For example, 1EP (1-ethynylpyrene) is a mechanism-based inhibitor of P450 1A1, but a competitive inhibitor of P450s 1A2 and 1B1. While 1PP (1-(1-propynyl)pyrene) is a mechanism-based inhibitor of P450s 1A1 and 1A2, but a competitive inhibitor of P450 1B1 [18,103]. It was generally observed that replacing the terminal hydrogen of aryl acetylenes with a methyl group to convert ethynes into propynes enhanced the inhibition of P450 1A enzymes; in some instances, this modification converted a reversible inhibitor into a suicide inhibitor [103]. It was also observed that 2EPh (2-ethynylphenanthrene) and 2PPh (2-(1-propynyl)phenanthrene) more potently inhibited P450 1A1 than 1A2, whereas 9EPh (9-ethynylphenanthrene) and 9PPh (9-(1-propynyl)phenanthrene) selectively inhibited P450 1A2 over 1A1 [17,103]. This suggests that the molecular shape of an inhibitor is critical for the inhibitor's selectivity towards P450s 1A1 and 1A2, which will be further discussed in the following section. The studies on the 7-ethynylcoumarins and flavonyl propargyl ethers also provided mechanism-based inhibitors of P450 1As, and certain structural characteristics of P450 1A1 and 1A2's active site cavities [38,55].

Structural Characteristics of P450s 1A1 and 1A2 Inhibitors
The crystal structures of P450s 1A1, 1A2, and 1B1 have been elucidated in the recent years, and the apparent active site sizes ranked as 1A1 (524 Å 3 ) > 1B1 (469 Å 3 ) > 1A2 (441 Å 3 ) [1,105,106]. These crystal structures are very useful for exploring active sites, enzyme-ligand binding, and related interactions. P450s 1A1 and 1A2 are most similar, sharing 72% amino acid sequence identity, while P450 1B1 has relatively low amino acid sequence identity with both P450s 1A1 (38%) and 1A2 (37%) [1]. However, in terms of the 3D structures and the function of enzymes, P450 1A1 shows much more similarity to P450 1B1 than P450 1A2. In fact, P450 1A2 is largely responsible for the metabolism of aromatic amines, whereas P450s 1A1 and 1B1 metabolize polycyclic aromatic hydrocarbons and polyhalogenated aromatic hydrocarbons [2,[107][108][109]. Furthermore, observations of crystal structures also show that similarity of active site cavities of P450s 1A1 and 1B1 is considerably more than that of P450s 1A1 and 1A2, indicating that selectively inhibiting P450s 1A1 and 1B1 is much more difficult than P450s 1A1 and 1A2 [1]. On the other hand, the P450 1A1 and 1A2 encoding genes (CYP1A1 and CYP1A2) are located head-to-head on human chromosome 15q24.1, suggesting a close correlation of CYP1A1 and CYP1A2 expression, while CYP1B1 gene is located on human chromosome 2p22.2. Thus, a certain gene expression regulator, such as an AhR ligand, that leads to the same effects on CYP1A1 and 1A2 transcription, probably leads to less or reverse effect on transcription of CYP1B1. Since some P450 inhibitors also regulate CYP gene expression, the effects of a single chemical entity on the level and activity of a P450 enzyme are complex and hard to predict.
The inhibitor structures were sketched in ChemDraw 13.0 (PerkinElmer, Inc., Waltham, MA, USA), and alignment was performed manually. The aligned images ( Figure 11) obviously show that the skeleton of selective P450 1A1 inhibitors is a long strip shape with 12.3 Å in length and 4.6 Å in width, reflecting the narrow and long cavity of P450 1A1. While the skeleton of selective P450 1A2 inhibitors is a triangle with side lengths 9.3 Å, 8.7 Å, and 7.2 Å, respectively, suggesting a narrow compact cavity for P450 1A2. These results demonstrate the structural characteristics of P450s 1A1 and 1A2 inhibitors as well as the differences of P450s 1A1 and 1A2's active site cavities, which could not be determined through the analysis of X-ray crystal structures of the two enzymes.

Conclusions
In this paper, we have reviewed a large number of P450 family 1 enzyme inhibitors and determined the differences between P450s 1A1 and 1A2's active site cavities by a meta-analysis, which will benefit the future inhibitor development. Since some P450 enzyme inhibitors are inducers of CYPs expression, the evaluation of selective P450 family 1 inhibitors should also consider their effects on the expression of CYP1s and other related genes. Thus, an integrated in solution, in cell, and in vivo bioassay system should be established to comprehensively evaluate the effects of a P450 inhibitor on the enzymes, on the other drugs, and on the related diseases. In addition, because of the notable relationship between P450 1B1 and carcinogenesis, the development of highly selective P450 1B1 inhibitors should be an important focus of research on the family 1 P450 enzymes. Due to the limited data about the selectivity toward P450 1B1, more comparative studies on P450 family 1 inhibitors are needed.