Current High-Throughput Approaches of Screening Modulatory Effects of Xenobiotics on Cytochrome P450 (CYP) Enzymes

Cytochrome P450 (CYP) is a critical drug-metabolizing enzyme superfamily. Modulation of CYP enzyme activities has the potential to cause drug–drug/herb interactions. Drug–drug/herb interactions can lead to serious adverse drug reactions (ADRs) or drug failures. Therefore, there is a need to examine the modulatory effects of new drug entities or herbal preparations on a wide range of CYP isoforms. The classic method of quantifying CYP enzyme activities is based on high-performance liquid chromatography (HPLC), which is time- and reagent-consuming. In the past two decades, high-throughput screening methods including fluorescence-based, luminescence-based, and mass-spectrometry-based assays have been developed and widely applied to estimate CYP enzyme activities. In general, these methods are faster and use lower volume of reagents than HPLC. However, each high-throughput method has its own limitations. Investigators may make a selection of these methods based on the available equipment in the laboratory, budget, and enzyme sources supplied. Furthermore, the current high-throughput systems should look into developing a reliable automation mechanism to accomplish ultra-high-throughput screening in the near future.


Introduction
Cytochrome P450 (CYP) is a superfamily of enzymes, and in this superfamily, enzymes belonging to the families 1, 2, and 3 play a major role in metabolizing a wide spectrum of xenobiotics [1]. Drugs such as one type of xenobiotics undergo processes of absorption, distribution, metabolism, and elimination throughout the human body [2]. The major CYP isoforms involved in drug metabolism include CYP3A4/5, CYP2D6, CYP2C9, CYP1A2, CYP2B6, CYP2C19, CYP2C8, CYP2A6, CYP2E1, and CYP2J2 [3]. The metabolism of xenobiotics including drugs is the key path of detoxification by adding hydrophilic groups to the molecular structures of parent compounds [4]. Drug-drug interactions may occur if one of the drugs inhibits CYP enzyme activity involved in metabolizing the coadministered drug. Alternatively, drug-drug interactions may also occur when coadministered drugs are metabolized by the same CYP enzyme but one drug has a lower specificity. These situations will lead to an elevation in plasma concentration of the coadministered drug (or drug with lower specificity) and potentially may result in adverse drug reactions (ADRs). ADRs are generally unwanted or harmful reactions following the administration of medications [5]. Various factors are responsible for ADRs, and drug-drug interactions contribute to up to 15% of hospitalized geriatric patients experiencing ADRs [6]. Since they are associated with morbidity and mortality, ADRs are one of the most serious clinical issues in relation to the use of drugs [7]. On the other hand, many  In general, fluorescence-based assays carried out in a 96-well plate are performed to screen the inhibitory potencies of a wide range of drugs and herbal constituents. Figure 1 illustrates the general work flow of a fluorescence-based assay performed in a single well. Human recombinant cDNA-expressed CYP3A4 with its nonfluorescent probe substrate 7-benzyloxy-4-(trifluoromethyl) coumarin (BFC) were incubated to investigate the simultaneous inhibition by 11 different compounds (erythromycin, verapamil, ethynilestradiol, miconazole, bromoergocriptine, nicardipine, clotrimazole, roxythromycin, cimetidine, nifedipine, and ketoconazole) [23]. Similarly, IC 50 values were obtained using recombinant microsomes from baculovirus-infected insect cells, liver CYPs (CYP1A1, CYP1A2, CYP2A6, and CYP3A4) and testing compounds (vorozole and letrozole) employing coumarin, 7-methoxy-4-(trifluoromethyl) coumarin (MFC), 3-cyano-7-ethoxycoumarin (CEC), and BFC as the substrates to screen the inhibitory capability of inhibitors [24]. Moreover, baculovirus/insect cells, cDNA-expressed CYP3A4 using benzyloxyresorufin (BzRes), BFC, 7-benzyloxyquinoline (BQ), and dibenzylfluorescein (DBF) as the fluorometric probe substrates were used to test 27 compounds in inhibition assays [19]. Twenty-nine antiparasitic drugs and positive inhibitors (naphthoflavone, sulfaphenazole, ticlopidine, quinidine, and ketoconazole) were determined by their inhibition of recombinant human CYPs (1A2, 2C9, 2C19, 2D6, and 3A4) expressed from yeast employing CEC, MFC, 7-methoxy-4-(aminomethyl)-coumarin (MAMC), and BFC as substrates [25]. The mechanism-based inhibition of recombinant human c-DNA-expressed CYP2B6 by bergamottin was accessed by using 7-ethoxytrifluoromethyl coumarin (EFC) as the substrate [26]. Nowadays, commercial kits consisting of recombinant CYP, CYP reductase, cofactors, buffer, and fluorogenic substrates (such as BOMCC and EOMCC) have been developed to investigate the inhibitory potencies of compounds on CYP activities. Vivid ® CYP450 Screening Kits supplied by ThermoFisher Scientific (Waltham, MA, USA) were widely applied. Inhibitory potentials of standardized extracts of Tinospora cordifolia and its bioactive compound on CYP3A4, CYP2D6, CYP2C9, and CYP1A2 were determined using Vivid ® CYP450 Screening Kits [27]. Likewise, this kit was also employed to screen the inhibitory effects of Trigonella foenum-graecum (TFG) and trigonelline (TG) on several CYP isoforms [28]. Figure 2 demonstrates the scheme of the metabolism of the Vivid ® substrate to a fluorescent metabolite. It shows that the substrate is nonfluorescent as its fluorophores are blocked by R1 and R2. The fluorescence signal was triggered after R1 and/or R2 were removed by CYPs. (TG) on several CYP isoforms [28]. Figure 2 demonstrates the scheme of the metabolism of the Vivid ® substrate to a fluorescent metabolite. It shows that the substrate is nonfluorescent as its fluorophores are blocked by R1 and R2. The fluorescence signal was triggered after R1 and/or R2 were removed by CYPs.  This fluorescence-based approach has been attached to other systems to achieve ultra-highthroughput, which further accelerated the screening process. Human liver microsomes (CYP1A2, CYP2A6, CYP2B6, CYP2D6, CYP2E1, CYP2C8, CYP2C9, CYP2C19, CYP3A4, and CYP3A5) using DFB as a probe substrate and ketoconazole, miconazole, nicardipine, and nifedipine as the inhibitors for enzyme inhibition assays were carried out through an automated, fluorescent-based, 96-well assay. It was found that DFB was a selective substrate for CYP3A enzymes [21]. An automated, fluorometric, 384-well microplate assay was used to investigate the inhibitory effects of the test compound, leishmania, on human CYP3A4 and CYP2D6 from liver microsomes with BFC and 3-[2-(N,N-diethyl-N-methylammonium)ethyl]-7-methoxy-4-methylcoumarin (AMMC) as substrates [29]. On the other hand, BFC, MFC, 7-methoxy-4-(aminomethyl)coumarin (MAMC), and CEC were employed as substrates for CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 to determine the inhibitory effects of sulfaphenazole, ketoconazole, quinidine, troleandomycin, caffeine, and cimetidine on CYPs to reveal potential drug-herb interactions through a high-throughput, fluorescent, 96-well plate screening assay and a parallel artificial membrane permeability assay (PAMPA) [30]. Recently, CYP3A4 baculosomes were employed in inhibition assays to examine 49 herbal species through high-throughput, fluorometric screening together with Herbochip [31]. Vivid ® CYP450 enzyme screening kits and Vivid blue (EOMCC) and green substrates (DBOMF, BOMF) for CYP450 baculosomes (CYP3A4, CYP2C9 and CYP2D6) were used for mechanism-based inhibition by ketoconazole, sulfaphenazole, and quinidine through a fluorescent assay combining enzyme encapsulation techniques, the microarray method, and wide-field imaging [32].
The fluorescence-based, high-throughput approach for the screening of the inhibitory effects of xenobiotic compounds on CYP activities demonstrates numerous advantages as compared to traditional, HPLC-based enzyme assays. It is faster as a shorter period of time is required for (TG) on several CYP isoforms [28]. Figure 2 demonstrates the scheme of the metabolism of the Vivid ® substrate to a fluorescent metabolite. It shows that the substrate is nonfluorescent as its fluorophores are blocked by R1 and R2. The fluorescence signal was triggered after R1 and/or R2 were removed by CYPs.  This fluorescence-based approach has been attached to other systems to achieve ultra-highthroughput, which further accelerated the screening process. Human liver microsomes (CYP1A2, CYP2A6, CYP2B6, CYP2D6, CYP2E1, CYP2C8, CYP2C9, CYP2C19, CYP3A4, and CYP3A5) using DFB as a probe substrate and ketoconazole, miconazole, nicardipine, and nifedipine as the inhibitors for enzyme inhibition assays were carried out through an automated, fluorescent-based, 96-well assay. It was found that DFB was a selective substrate for CYP3A enzymes [21]. An automated, fluorometric, 384-well microplate assay was used to investigate the inhibitory effects of the test compound, leishmania, on human CYP3A4 and CYP2D6 from liver microsomes with BFC and 3-[2-(N,N-diethyl-N-methylammonium)ethyl]-7-methoxy-4-methylcoumarin (AMMC) as substrates [29]. On the other hand, BFC, MFC, 7-methoxy-4-(aminomethyl)coumarin (MAMC), and CEC were employed as substrates for CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 to determine the inhibitory effects of sulfaphenazole, ketoconazole, quinidine, troleandomycin, caffeine, and cimetidine on CYPs to reveal potential drug-herb interactions through a high-throughput, fluorescent, 96-well plate screening assay and a parallel artificial membrane permeability assay (PAMPA) [30]. Recently, CYP3A4 baculosomes were employed in inhibition assays to examine 49 herbal species through high-throughput, fluorometric screening together with Herbochip [31]. Vivid ® CYP450 enzyme screening kits and Vivid blue (EOMCC) and green substrates (DBOMF, BOMF) for CYP450 baculosomes (CYP3A4, CYP2C9 and CYP2D6) were used for mechanism-based inhibition by ketoconazole, sulfaphenazole, and quinidine through a fluorescent assay combining enzyme encapsulation techniques, the microarray method, and wide-field imaging [32].
The fluorescence-based, high-throughput approach for the screening of the inhibitory effects of xenobiotic compounds on CYP activities demonstrates numerous advantages as compared to traditional, HPLC-based enzyme assays. It is faster as a shorter period of time is required for This fluorescence-based approach has been attached to other systems to achieve ultra-high-throughput, which further accelerated the screening process. Human liver microsomes (CYP1A2, CYP2A6, CYP2B6, CYP2D6, CYP2E1, CYP2C8, CYP2C9, CYP2C19, CYP3A4, and CYP3A5) using DFB as a probe substrate and ketoconazole, miconazole, nicardipine, and nifedipine as the inhibitors for enzyme inhibition assays were carried out through an automated, fluorescent-based, 96-well assay. It was found that DFB was a selective substrate for CYP3A enzymes [21]. An automated, fluorometric, 384-well microplate assay was used to investigate the inhibitory effects of the test compound, leishmania, on human CYP3A4 and CYP2D6 from liver microsomes with BFC and 3-[2-(N,N-diethyl-N-methylammonium)ethyl]-7-methoxy-4-methylcoumarin (AMMC) as substrates [29]. On the other hand, BFC, MFC, 7-methoxy-4-(aminomethyl)coumarin (MAMC), and CEC were employed as substrates for CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 to determine the inhibitory effects of sulfaphenazole, ketoconazole, quinidine, troleandomycin, caffeine, and cimetidine on CYPs to reveal potential drug-herb interactions through a high-throughput, fluorescent, 96-well plate screening assay and a parallel artificial membrane permeability assay (PAMPA) [30]. Recently, CYP3A4 baculosomes were employed in inhibition assays to examine 49 herbal species through high-throughput, fluorometric screening together with Herbochip [31]. Vivid ® CYP450 enzyme screening kits and Vivid blue (EOMCC) and green substrates (DBOMF, BOMF) for CYP450 baculosomes (CYP3A4, CYP2C9 and CYP2D6) were used for mechanism-based inhibition by ketoconazole, sulfaphenazole, and quinidine through a fluorescent assay combining enzyme encapsulation techniques, the microarray method, and wide-field imaging [32].
The fluorescence-based, high-throughput approach for the screening of the inhibitory effects of xenobiotic compounds on CYP activities demonstrates numerous advantages as compared to traditional, HPLC-based enzyme assays. It is faster as a shorter period of time is required for sequential data acquisition; it also costs less as costs for reagents are minimized with little loss in data quality. Additionally, it is also particular useful for enzymes with low expression such as polymorphic variants and mechanism-based assays carried out through the dilution method. Nevertheless, caution need to be exercised when applying the fluorescence-based assays. At first, the test compounds should not exhibit fluorescence properties that interfere with the fluorometric measurement of metabolite. Excess NADPH may interfere with fluorometric detection of metabolites such as 3-hydroxy-5,5-dimethyl-4-(4-methylsulfonylphenyl)-(5H)-furan-2-one) (DFH), which requires further experimental steps to remove excess NADPH at the end of the incubation [21]. Some of these disadvantages have been resolved by structurally modifying common fluorogenic substrates supplied by Vivid ® CYP450 Screening Kits.

Luminescence-Based Assay
An alternative assay configured in multi-well plates is to measure luminescent readings to quantify CYP enzyme activities. Luminescence-based CYP assays employ derivatives of luciferin as CYP probe substrates, which are luminogenic [33]. In essence, the luminogenic probe substrates are metabolized by CYP to luciferin, which subsequently reacts with luciferase and produces luminescent light (See Figure 3). Most of the luminescence-based CYP assays available in literature utilized P450-Glo TM assay kits supplied by Promaga Corporation (Madison, WI, USA).
High-Throughput 2018, 7, x FOR PEER REVIEW 5 of 11 sequential data acquisition; it also costs less as costs for reagents are minimized with little loss in data quality. Additionally, it is also particular useful for enzymes with low expression such as polymorphic variants and mechanism-based assays carried out through the dilution method. Nevertheless, caution need to be exercised when applying the fluorescence-based assays. At first, the test compounds should not exhibit fluorescence properties that interfere with the fluorometric measurement of metabolite. Excess NADPH may interfere with fluorometric detection of metabolites such as 3-hydroxy-5,5-dimethyl-4-(4-methylsulfonylphenyl)-(5H)-furan-2-one) (DFH), which requires further experimental steps to remove excess NADPH at the end of the incubation [21]. Some of these disadvantages have been resolved by structurally modifying common fluorogenic substrates supplied by Vivid ® CYP450 Screening Kits.
Using a luminescence-based, high-throughput assay was an effective, cheap, and highly sensitive method for enzyme screening and was suitable for CYP screening during early drug discovery, especially for pharmacokinetics and toxicity studies. It is highly flexible in the types of tissue used, sample quantity, isozyme specificity, as well as the method of preparation. This method was rapid and safe as compared to Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) methods, and it was also able to reduce the interference between the optical properties of the test compound and CYP substrates [34]. However, the fluorescent assay tends to show readings with higher activity compared to a luminescent assay due to lower concentrations of luminescent substrate used in order to obtain a better luminescent signal-to-noise ratio reading, and this method alone should not be relied on.

Mass Spectrometry-Based Assay
Mass spectrometry (MS) analyzes gaseous ions via their mass-to-charge ratio (m/z) [39]. MS systems are usually attached to gas chromatography (GC) or liquid chromatography (LC). Once the individual components in a mixture are ionized, they are separated based on their m/z by GC or LC. Refer to Figure 4 for the general work flow of MS-based assay.
High-Throughput 2018, 7, x FOR PEER REVIEW 6 of 11 CYP2B6, and CYP1A2) employing luciferin pro-substrates (Promega) for CYP induction and inhibition by verapamil, ticlopidine, and α-napthoflavone [38]. Using a luminescence-based, high-throughput assay was an effective, cheap, and highly sensitive method for enzyme screening and was suitable for CYP screening during early drug discovery, especially for pharmacokinetics and toxicity studies. It is highly flexible in the types of tissue used, sample quantity, isozyme specificity, as well as the method of preparation. This method was rapid and safe as compared to Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) methods, and it was also able to reduce the interference between the optical properties of the test compound and CYP substrates [34]. However, the fluorescent assay tends to show readings with higher activity compared to a luminescent assay due to lower concentrations of luminescent substrate used in order to obtain a better luminescent signal-to-noise ratio reading, and this method alone should not be relied on.

Mass Spectrometry-Based Assay
Mass spectrometry (MS) analyzes gaseous ions via their mass-to-charge ratio (m/z) [39]. MS systems are usually attached to gas chromatography (GC) or liquid chromatography (LC). Once the individual components in a mixture are ionized, they are separated based on their m/z by GC or LC. Refer to Figure 4 for the general work flow of MS-based assay.
Nevertheless, the analyte derivatization process prior to GC-MS is laborious, thus limiting its utility for metabolite identification. On the other hand, LC-MS or LC-MS/MS (liquid chromatography tandem mass spectrometry) has been evidenced to be a more powerful, analytical approach to characterize structures and to quantify drug metabolites in CYP reactions.
Nevertheless, the analyte derivatization process prior to GC-MS is laborious, thus limiting its utility for metabolite identification. On the other hand, LC-MS or LC-MS/MS (liquid chromatography tandem mass spectrometry) has been evidenced to be a more powerful, analytical approach to characterize structures and to quantify drug metabolites in CYP reactions.

LC-MS or LC-MS/MS-Based Assay
CYP1A2 and CYP3A4 from human C3A and HepaRG cells, together with phenacetin and testosterone as the substrates were screened for their phenotypic and metabolic parameters through LC-MS/MS by prototypical inducers (omeprazole and rifampicin) and CYP isoform-specific inhibitors (fluvoxamine and ketoconazole) [42]. Additionally, LC-MS/MS was used to perform metabolic studies using human liver microsomes with a range of substrates (cefotaxime, gemifloxacin, ciprofloxacin, fluconazole, gentamicin, clindamycin, linezolid, and metronidazole) [43]. Similarly, pharmacokinetics analysis targeting CYP1A2, CYP2A, CYP2C19, CYP2D6, CYP2E1, and CYP3A from rat liver microsomes involved the use of the substrates phenacetin, coumarin, omeprazole, dextromethorphan, and chlorzoxazone with the test compound, chrysosplenetin, through the LC-MS/MS method [44].
LC-MS/MS system has been further equipped with 96-well microplates to strengthen high-throughput.
LC-MS/MS is considered to be a fast and highly sensitive analytical approach for the quantification of metabolites. It is able to produce large sets of experimental data and also has the ability to produce data with high accuracy. Furthermore, LC-MS/MS can be coupled with the cocktail method, which allows the determination of multiple CYP activities simultaneously. LC-MS/MS with a cocktail assay is adaptable for automation and small volumes. On the contrary, LC-MS/MS requires electrospray ionization prior to screening, and the equipment is relatively expensive. Additionally, some metabolites are not sensitive enough to be detected by this method. Stable-isotope compounds were required for CYP-specific probe substrate metabolites as an internal standard to avoid interference by ion suppression in LC-MS/MS quantification, and this method was not suitable for initial drug discovery as compared to fluorescent and luminescent assays, which provided the highest throughput [50]. A cocktail assay should be used with concern as interactions might occur among the probe substrates, and the use of high microsomal protein concentration levels might complicate the data interpretation due to the nonspecific binding of inhibitors or substrates to the microsomal protein.

Conclusions
CYP family of enzymes are important phase I enzymes responsible for drug clearance or the bioactivation of prodrugs. At the early stage of drug discovery and development, in vitro investigations of modulatory effects of new drug entities or herbal preparations on CYP activities play key roles in selecting suitable therapeutic candidates for subsequent in vivo and clinical trials. A high-throughput screening approach optimizes the chance of identifying lead compounds from a large number of agents. Currently, several high-throughput methods including fluorescence-based, luminescence-based, and MS-based assays have been developed and widely applied to quantify CYP activities. Despite fast screening processes, each approach is has certain advantages and disadvantages (see Table 2). It is advised to consider multiple factors such as available equipment in the laboratory, budget, and enzyme sources supplied before making a choice from these methods. It would be ideal to apply more than one approach for the screening of modulatory effects of compounds on CYP activities if possible. In the future, ultra-high-throughput screening methods with an automated system should be further developed and validated to meet the requirement of drug discovery and development in the new era. Funding: This research received no external funding.

Conflicts of Interest:
The authors declare no conflict of interest.