Abstract
The cytochrome P450 enzymes (CYPs), a group of heme-containing enzymes, catalyze oxidative metabolism of a wide range of drugs and xenobiotics, as well as different endogenous molecules. Strong inhibition of human CYPs is the most common cause of clinically associated pharmacokinetic drug–drug/herb–drug interactions (DDIs/HDIs), which may result in serious adverse drug reactions, even toxicity. Accurate and rapid assessing of the inhibition potentials on CYP activities for therapeutic agents is crucial for the prediction of clinically relevant DDIs/HDIs. Over the past few decades, significant efforts have been invested into developing optical substrates for the human CYPs, generating a variety of powerful tools for high-throughput assays to detect CYP activities in biologic specimens and for screening of CYP inhibitors. This minireview focuses on recent advances in optical substrates developments for human CYPs, as well as their applications in screening CYP inhibitors and DDIs/HDIs studies. The examples for rational design and optimization of highly specific optical substrates for the target CYP enzyme, as well as applications in investigating CYP-mediated DDIs, are illustrated. Finally, the challenges and future perspectives in this field are proposed. Collectively, this review summarizes the reported optical-based biochemical assays for highly efficient CYP activities detection, which strongly facilitated the discovery of CYP inhibitors and the investigations on CYP-mediated DDIs.
SIGNIFICANCE STATEMENT Optical substrates for cytochrome P450 enzymes (CYPs) have emerged as powerful tools for the construction of high-throughput assays for screening of CYP inhibitors. This mini-review covers the advances and challenges in the development of highly specific optical substrates for sensing human CYP isoenzymes, as well as their applications in constructing fluorescence-based high-throughput assays for investigating CYP-mediated drug–drug interactions.
Introduction
The human cytochrome P450 (CYP) superfamily is a class of heme-containing monooxygenase enzymes capable of catalyzing the oxidation of extensive number of endogenous substances and exogenous chemicals (Manikandan and Nagini, 2018; Wright et al., 2019; Zhao et al., 2021). Human CYPs have exceptionally broad substrate spectra, their substrates include a wide range of physiologically, pharmacologically, and toxicologically related compounds, such as steroids, therapeutic drugs, environmental pollutants, and chemical carcinogens (Zhang et al., 2011; Nebert et al., 2013; Zanger and Schwab, 2013). In humans, 56 CYPs have been identified and divided into 18 families and 44 subfamilies according to their sequence similarity. Among them, the members of CYP1, CYP2, and CYP3 are mainly involved in xenobiotics metabolism, which metabolize ∼80% of marketed drugs and thus have been recognized as the key factors affecting clinical treatment outcomes (including therapeutic efficacy and toxicity) of therapeutic agents (Zhang et al., 2011; Zanger and Schwab, 2013; Tornio and Backman, 2018; Jin et al., 2022).
Both the expression and function of human CYPs could be influenced by intrinsic and extrinsic factors including sex, age, disease, hormone, and other factors (Congiu et al., 2009; Zhang et al., 2011; Harvey and Morgan, 2014). Meanwhile, a wide range of therapeutic agents may act as inhibitors or inducers of human CYPs, which can modulate the metabolic clearance of CYPs substrate drugs via regulating the activity/expression of CYPs. Coadministration with potent CYP inhibitors or inducers may trigger pharmacokinetic drug–drug/herb–drug interactions (DDIs/HDIs), resulting in drug accumulation or treatment failure, especially for the patients receiving some narrow therapeutic index medications (such as warfarin, digoxin, ciclosporin, etc.) (Kalra, 2007; Kumar et al., 2012; Wang et al., 2023). As a non-negligible cause of adverse drug reactions, DDI/HDI may bring many undesirable effects or even result in deaths (Kalra, 2007; Kumar et al., 2012). In some cases, to get improved clinical therapeutic effects, the combination drug therapies or the Western Medicines in combination with herbal products are frequently used in clinical settings, which could enhance the risks of clinically relevant DDIs/HDIs with potentially life-threatening consequences (Hohl et al., 2001; Smets et al., 2008). The regulatory agencies such as the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA) recommend evaluating the DDI potentials of drug candidates and their inhibitory effects on hepatic CYPs (like CYP1A2, CYP2C8, CYP2C9, CYP2C19, and CYP3A4) before drug approval, which emphasizes the importance of the validated CYP inhibition assays (Magro et al., 2021). Hence, there is an urgent need to develop more practical tools that can efficiently and accurately characterize CYPs activity/residual activity for CYP-related basic research and drug safety evaluation.
In the past few decades, a panel of endogenous substrates (testosterone, estradiol, cholesterol) and drug substrates (midazolam, S-warfarin, caffeine) (Kaminsky and Zhang, 1997; Elfaki et al., 2018; Guo et al., 2021; Kumondai et al., 2021) have been recommended by the FDA for screening and characterizing CYP inhibitors, and liver tissue preparations were used as the enzyme source. Such assays require liquid chromatography-tandem mass spectrometry, whereas the sample pretreatment and analysis is complex and time-consuming, which makes it very difficult to achieve high-throughput screening (HTS). Simultaneously, the high price of the instruments, as well as the stringent requirements of the tedious sample pretreatments and the skilled operators greatly increases the testing cost (Zhang et al., 2008; Yun et al., 2016). By contrast, optical substrate-based biochemical assays offer more practical tools for enzyme inhibition assays, owing to the inherent advantages of fluorescence signals (without sample pretreatment, simple detection, and operation processes, as well as capable of high-throughput and ultra–high-throughput detection) (Fig. 1). Over the past few decades, great efforts have been invested in developing highly specific optical substrates for targeting human CYPs, which generated a variety of powerful tools to construct high-throughput assays for detecting CYP activities in biologic specimens and for screening of CYP inhibitors. These practical optical substrates include fluorogenic substrates and bioluminogenic substrates, most of them have been used for accurately determination of the real/residual activities of the target CYP enzyme in complex biologic systems (such as liver preparations), thus significantly facilitating CYP-mediated DDIs/HDIs studies.
Two commonly used analytical techniques for screening inhibitors against human P450s.
Fluorogenic Substrates for Human P450s
By means of extensive studies on the substrate preferences of human CYPs and some practical computer-aided molecular design strategies, a panel of fluorogenic substrates for human CYPs has been developed by medicinal chemists. A summary of the available fluorogenic substrates for human CYPs is shown in Table 1. Although the specificity of most reported fluorogenic substrates for human CYPs is not very satisfactory, these substrates could still be applied for HTS of CYP inhibitors when using recombinant CYPs or specific cell preparations as enzyme sources. For example, dibenzyl-fluorescein (DBF), a multi-enzyme metabolizing substrate, was identified as a useful tool in HTS assay for CYP3A7 to realize the DDI/HDIs for neonatal population (Work et al., 2021). It is noteworthy that part of the fluorogenic compounds in Table 1 exhibit very high selectivity toward the target CYP enzyme, such as N-(4-butyl)-4-chloroethoxy-1,8-naphthalimide (NBCeN) (CYP1A1), 4-isopropoxy-7H-benzo[de]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one (iPrBN) (CYP1A1), chloroethyl derivative (7-(2-chloroethoxy)-3H-phenoxazin-3-one (CHPO) (CYP1A1), coumarin (CYP2A6), 1,3-dichloro-7-methoxy-9,9-dimethylacridin-2(9H)-one (DDAM) (CYP2C9), BnXPI (CYP2J2), 2-(3-(4-((4-methoxybenzyl)oxy)styryl)-5,5-dimethylcyclohex-2-en-1-ylidene)malononitrile (CYP2J2), (Z)-2-(2-(6-((4-methoxybenzyl)oxy)-2,3-dihydro-1H-xanthen-4-yl)vinyl)-3,3-dimethyl-1-(7-((2-((4-methylphenyl)sulfonamido)ethyl)amino)-6-oxoheptyl)-3H-indol-1-ium iodide (CYP2J2), (E)-2-(2-(6-methoxy-2,3-dihydro-1H-xanthen-4-yl)vinyl)-3-methylbenzo[d]thiazol-3-ium iodide (CYP2J2), 7-(benzyloxy)quinoline (CYP3A4), N-(3-ethyl)-1,8-naphthalimide (NEN) (CYP3A4), F8 (CYP3A4) and 2,5-bis(trifluoromethyl)-7-benzyloxy-4-trifluoromethyl-coumarin (CYP3A4) (Li et al., 1997; Renwick et al., 2001; Dai et al., 2017, 2021; Ning et al., 2018b, 2019a,c; Jin et al., 2020; Feng et al., 2022; Tian et al., 2022; He et al., 2023). 3-O-methylfluorescein, a reported fluorogenic substrate of CYP2C19, can also be metabolized by CYP2C9 and CYP1A1 (Sudsakorn et al., 2007). BFC, a CYP3A substrate, can be metabolized by CYP1A2 as well (Renwick et al., 2000).
Reported fluorogenic substrates for human CYPs
Inspired by the substrate preference of CYPs, some hydrophobic fluorophores bearing extended polyaromatic rings (such as resorufin, coumarin, and naphthalimide) were used as substrate candidates with various modifications (Fig. 2). One of the first identified isoform-specific fluorogenic substrate for CYP1A1 was NBCeN, a ratiometric substrate, which was successfully employed for monitoring the CYP1A1 activity in situ and efficiently screening CYP1A1 modulators in living system (Dai et al., 2017). CYP1A1 and CYP1A2 share 72.55% sequence homology, and their catalytic cavity volumes are 524 and 375 Å, respectively. These two dealkylating enzymes have subtle metabolic bias; CYP1A1 prefers relatively large groups, O-ethyl or O-chloroethyl, for instance, whereas CYP1A2 preferably catalyzes demethylation reaction. Given this, more CYP1A1-specific substrates have emerged. On the basis of the preference for CYP1A1 substrate characteristics and the intramolecular charge transfer principle, Ning et al. (2018) have offered a novel two-photon fluorogenic substrate iPrBN. It displayed remarkable specificity and high sensitivity, the detection limit of which for CYP1A1 was 0.036 nM. Recently, Jin et al. (2020) provided a CYP1A1 specific substrate CHPO via adjusting the volume of O-alkylation. CHPO has been applied to imaging endogenous CYP1A1 in living cells and tissues and also for high-throughput screening of CYP1A1 inhibitors, which inspired the research on human diseases in which CYP1A1 plays a role, as well as on biomedical applications of CYP1A1 modulators.
The commonly used strategies for constructing optical substrates for target drug metabolizing enzymes. (A) The strategy of substrate preference inspired optical substrate discovery. (B) The strategy of structure-guided substrate design and discovery. The schematics are modified from that previous published literature with copyright permission by American Chemical Society (He et al., 2023).
Although the structural preference-based substrate discovery strategy is practical, the exploration of structure–metabolism relationship is time-consuming, and another strategy has been applied to the efficient discovery of fluorogenic substrates (Fig. 2). Based on structure-based substrate design, another great innovation was a practical, isoform-specific, and sensitive two-photon fluorogenic substrate for CYP3A4 (F8) (He et al., 2023). Of note is that a two-dimension design strategy was proposed, which is functional for designing and developing isoform-specific substrates for target enzymes. F8 was applied for the real-time visualization in living cells and tissues.
Bioluminogenic Substrates for Human P450s
In addition to the progress in the fluorescence-based CYP activity assays, bioluminescence-based assays were also developed prosperously in the past few decades. The polycyclic structure of D-luciferin renders bioluminogenic substrates biocompatible, and corporate-led researchers have developed numerous bioluminogenic substrates (Table 2). Unlike fluorescence-based assays, bioluminescence-based assays don’t need an excitation source. The bioluminogenic signal is generated by the addition of ATP and luciferase, thus the bioluminescence has almost no background interferences, but the poor tissue penetration property causes the quench of emission light by tissue composition (Syed and Anderson, 2021). Of note, it has been reported that the luciferase used in HTS assays may suffer from unexpected inhibition, which could then disturb the outputs of such assays (Leitão and Esteves da Silva, 2010; Poutiainen et al., 2013; Zhang et al., 2017).
Reported bioluminogenic substrates for human CYPs
Although a series of bioluminogenic substrates have been designed and developed, highly selective bioluminogenic substrates for sensing a specific human CYP isoenzyme are not yet well established, except for luciferin-H (Table 2). Luciferin-H shows high specificity toward CYP2C9, which can be applied to screen CYP2C9 inhibitors using liver S9 fraction or microsomes as enzyme sources (Cali et al., 2012).
Screening of CYP Inhibitors Using Optical Substrates
DDIs/HDIs have been highlighted as a major clinical issue of pharmacotherapy. As the most critical phase I xenobiotic-metabolizing enzymes, human CYPs have been regarded as the most crucial targets/mediators to trigger DDIs/HDIs. Although the drug substrates for CYPs are routinely used for assessing of the inhibition potentials of new chemical entities against CYPs over the past few decades, the optical substrates offer a more efficient method to do it and to discover potential CYP inhibitors. Currently, the optical tools, including non-specific and specific optical substrates for CYPs, are widely used in both academia and industry. For instances, Trubetskoy et al. (2005) used Vivid fluorogenic substrates (7-ethoxy-methyloxy-3-cyano-coumarin [EOMCC] for CYP1A2, benzyloxymethylfluorescein (BOMF) and n-octyloxymethyl-resorufin for 2C9, EOMCC for 2C19, EOMCC for 2D6, di-(benzyl-O-methyl)-fluorescein [DBOMF], and benzyloxy-methyl-resorufin [BOMR] for 3A4) to coincubate with each recombinant human CYP enzyme, for evaluating the CYP inhibition profiles of the tested compounds in 1536-well plates. Sekiguchi et al. (2009) performed time-dependent inhibition profiles of 46 drugs using BFC as the fluorogenic substrate, and identified 26 of which as time-dependent inhibition. Awortwe et al. (2014) tested the IC50 values of seven herbal medicines and products against five important hepatic CYPs, including CYP1A2, 2C9, 2C19, 2D6, 3A4, using 3-cyano-7-ethoxycoumarin (CEC), 7-methoxy-4-(trifluoromethyl)-coumarin (MFC), MFC, 7-methoxy-4-(aminomethyl)-coumarin, BFC as the fluorogenic substrates, and found that Hypoxis hemerocallidea and Echinacea purpurea extracts could potently inhibit CYP1A2 and 3A4, while Leucophyllum frutescens and H. hemerocallidea extract could time-dependently inhibit CYP3A4.
In recent years, some isoform-specific fluorogenic substrates have been developed that offer practical tools to test CYP-mediated drug interactions in complex biologic systems. CYP2C9 was recognized as one of the most pivotal CYP enzymes, accounting for approximately 15% of therapeutic medications. In clinical, inhibition of CYP2C9 activity has been considered as a primary cause for life-threatening DDI, particularly when coadministration of warfarin, thus considerably impairing the drug’s pharmacokinetic profiles and increasing the risk of bleeding particularly in surgical patients. DDAM, has been used as a specific fluorogenic substrate for CYP2C9 applied for HTS, the response mechanism of which is demonstrated in Fig. 3A (Feng et al., 2022). Five other potent inhibitors (raloxifene, sulconazole, miconazole, fluvastatin, and sulfaphenazole) and eight activators (omeprazole, mebendazole, loratadine, pantoprazole, estradiol, dapsone, dopamine, and lansoprazole) were rapidly screened out from 93 clinical drugs using HLM as enzyme source (Fig. 3B). Coadministration of miconazole with warfarin in vivo could remarkably elevate the plasma exposure to warfarin, prolong prothrombin times of animals, and fatal subcutaneous abdominal wall hemorrhaging was also observed (Fig. 3, C and D). Consequently, such efficient and sensitive fluorescence-based screening assay could be applied to rapidly discover modulators of CYP2C9 from complex systems. In addition to the identification of inhibitors from drugs, DDAM was applied to the discovery the effective constitutes from herbal medicines using chromatographic-activity analysis. As shown in Fig. 4, the inhibitory potentials of herbal medicines and the involved specific inhibitory components were investigated. CYP2J2 plays a significant role in the metabolism of polyunsaturated fatty acids. Elevated levels of CYP2J2 have been related to various cancer types, and therefore it is considered to be a potential drug target. Tian et al. (2021) used a fluorogenic substrate BnXPI to perform a high-throughput screening assay including 108 common herbal medicines and identified a powerful inhibitor, piperine, with an IC50 value of 0.44 μM. Then, a series of derivatives were designed and synthesized, two of which exhibited a 10-fold increase in their inhibitory potentials, namely IC50 values of 40 and 50 nM.
(A) Sensing mechanism of DDAM toward CYP2C9. (B) Fluorescence-based screening of the inhibitory effects of therapeutic agents on CYP2C9-catalyzed DDAM demethylation in 96-well microplates (A7, raloxifene; B4, sulconazole; C5, miconazole; G1, fluvastatin; G5, sulfaphenazole). The influence of miconazole (MCN) on the pharmacokinetics (C) and pharmacodynamics (D) of warfarin (WAR). PT, prothrombin time. The figures are modified from that previous published literature with copyright permission by Elsevier (Feng et al., 2022).
Inhibitory effects of herbal medicines toward human CYP2C9. (A) Fluorescent response to the inhibitory effect of extracts of herbal medicines against CYP2C9 measured by fluorescent image analyzer (λex = 600 nm, λem = 670 ± 15 nm; A1, B1, and C1 are control samples; D1, E1, and F1 are positive control samples treated with 0.1, 1, 10 μM miconazole, respectively). (B) Corresponding inhibitory effects of herbal medicines on CYP2C9 were measured using the present fluorescent assay (red) by the fluorescent image analyzer (λex = 635 nm, λem = 670 nm ± 15 nm) and relative fluorescence intensity (blue) based on microplate reader (λex = 600 nm, λem = 658 nm), respectively. (C) HPLC chromatogram of chloroform extracts of Evodia rutaecarpa (Juss.) Benth. and prepared fraction profiles. The activity remaining of CYP2C9 in presence of individual extracts was inserted. (D) Inhibitory effects of the pre-HPLC fractions from the extracts of Evodia rutaecarpa (Juss.) Benth. toward CYP2C9. (E) The IC50 and inhibition kinetics of CYP2C9 by fraction 11 (Fr. 11) of Evodia rutaecarpa (Juss.) Benth. The figures are modified from that previous published literature with copyright permission by Elsevier (Feng et al., 2022).
In addition, parts of the molecules are metabolized by human CYPs to generate chemically reactive intermediate(s) that may bind to a range of biologic macromolecules covalently, resulting in protein dysfunction and/or other unexpected effects, such as idiosyncratic toxicity (Hollenberg et al., 2008; He and Feng, 2015; Gu et al., 2022). Therefore, the accurate identification of compounds that are mechanism-based CYP inactivators is urgent and necessary, because it can promote early warning of drug toxicity or reevaluation of therapeutic candidates that have the potential to cause unforeseen toxicity. To discover potent CYP3A inactivators, Tu et al. (2022) exploited more than one hundred natural products and assessed the inactivation effects against hCYP3A by using NEN as a CYP3A4 fluorogenic substrate (Fig. 5A). As shown in Fig. 5, B and C, bufalin (BF) could time-dependently inactivate hCYP3A via complex CYP-catalyzed oxidative cascade. In addition, 3-keto-bufalin, the unique nonpolar oxidative metabolite of BF, was further metabolized by hCYPs to produce two monohydroxylated metabolites that were easily dehydrated and then covalently bound to glutathione or hCYP3A4 (Fig. 5, D–F). In summary, that study revealed a unique and complicated bioactivation pathway of BF by using a novel developed high-throughput biochemical assay, which is very helpful for expanding the scopes of the suspects alert groups for hCYP3A4 inactivators, as well as for developing new steroid-type drug candidates without adverse effects via inactivating hCYP3A4. Recently, Ning et al. (2022) employed BN-1 as a CYP3A4 and CYP3A5 fluorogenic substrate to provide a HTS platform, enabling characterization of mechanism-based inactivation-related hepatotoxicity by CYP3A from clinical drugs and herbal medicines.
(A) Sensing mechanism of NEN toward CYP3A4. (B) Time-dependent inhibition assays of 103 natural compounds against hCYP3A4. The remaining activities of hCYP3A with different incubation times (RA0 minute) or (RA30 minute) were determined in the NADPH-generating system. (C) Time- and NADPH-dependent inhibition of BF against CYP3A4-catalyzed midazolam 1′-hydroxylation in HLM. (D) Proposed pathways for the formation of two reactive intermediates derived from 3-KBF by hCYPs. (E) Tandem mass spectrometry spectrum for the modified peptide from hCYP3A4 by the reactive intermediate deriving from 3-KBF. (F) The possible covalent binding poses of the reactive metabolites of 3-KBF bearing Michael acceptor I and Michael acceptor II on hCYP3A4 at C442. The figures were adapted with copyright permission by American Chemical Society (Tu et al., 2022).
The bioluminogenic signal output depends on firefly luciferase luminescence that enables fast bioluminescence signal collection. The colleagues from Promega have constructed a panel of bioluminescence-based HTS assays for the rapid discovery of CYP inhibitors (Cali et al., 2006). With the help of these bioluminescence-based biochemical assays, Veith et al. (2009) determined the in vitro inhibitory potentials of 17,143 molecules against five hCYPs (CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4). Kabir et al. (2022) used a CYP3A7 bioluminogenic substrate, 2-(6-(benzyloxy)benzo[d]thiazol-2-yl)-4,5-dihydrothiazole-4-carboxylic acid (luciferin-BE), to screen a library of ∼5000 compounds, including FDA-approved drugs and drug-like molecules, to find potential CYP3A7-specific modulators. After the primary screening, 782 potential CYP3A7-selective compounds were identified (Kabir et al., 2022). Recently, a report on pancreatic ductal adenocarcinoma demonstrated that CYP3A5 mediated the chemoresistance in different cancer subtypes, and RNA interference that resulted in CYP3A5 knockdown desensitized the drug-resistant pancreatic ductal adenocarcinoma cells, thereby clarifying the probable involvement of CYP3A5 in drug resistance. To find potent CYP3A5 inhibitors, Wright et al. (2020) have efficiently screened a St. Jude bioactive library against CYP3A5 by using luciferin-IPA (a CYP3A selective bioluminogenic substrate) (Fig. 6A) as the probe substrate and recombinant human CYP3A5 as the enzyme source. The assays were performed in 384-well plates, while clobetasol propionate was identified as a potent and selective CYP3A5 inhibitor (Fig. 6B). By general comparison of all the IC50 values, clobetasol propionate (clobetasol) was determined as a powerful and selective CYP3A5 inhibitor (Fig. 6, C and D). The IC50 values of clobetasol were 0.206 μM for CYP3A5 and 15.6 μM for CYP3A4, which was the strongest selective inhibitor from the screening pool. Such rapid screening assays may facilitate the research on the reversion of CYP3A5-mediated drug resistance and decode the characteristic of CYP3A5 in catalytic patterns and disease-associated contexts.
(A) The sensing mechanism of luciferin-IPA toward CYP3A. (B) CYP3A5 inhibition profile of the St. Jude bioactive library when screened at a concentration of 5 μM. (C) Dose-inhibition curves of ritonavir (Rit) or clobetasol (Clob) against CYP3A4 and CYP3A5. IC50 values for each curve are shown in parentheses. The IC90 value of clobetasol against CYP3A5 is shown in red text. (D) Chemical structure of clobetasol. The figures were adapted with copyright permission by American Chemical Society (Wright et al., 2020).
Challenges and Future Perspectives
Although substantial advancements in the development and application of optical substrates for CYPs have already been achieved, more efforts should be invested in designing and constructing more practical optical substrates and related high-throughput biochemical assays for investigating CYP-mediated DDIs/HDIs. Mammalian CYPs are intracellular enzymes, and most of them are located in endoplasmic reticulum. Strong inhibition of intracellular CYPs will affect CYP-mediated oxidative metabolism and then trigger DDIs/HDIs (Hakkola et al., 2020). Thus, ideal high-throughput assays for investigating CYP-mediated DDIs/HDIs should be constructed in living cells (Riede et al., 2021). In these cases, the ideal optical CYP substrates for constructing high-throughput biochemical assays should fulfill the following four requirements, namely high specificity, rapid response, good cell-membrane permeability, and excellent optical properties. However, to the best of our knowledge, most of the reported optical substrates for CYPs cannot fulfill the above requirements at the same time. For instance, coumarin derivatives have been frequently used as CYP substrates for screening CYP inhibitors, but the fluorescence response and isoform-specificity of most reported coumarin-based CYP substrates toward target CYP are not good enough, whereas the emission wavelengths of the reported coumarin-based CYP substrates and their fluorogenic metabolites are lower than 560 nm, which strongly limit their applications in living cells. 1,8-Naphthalimide derivatives have also been constructed as specific substrates for several important human CYPs, but the poor cell-membrane permeability and short emission wavelengths (∼550 nm) also limit their applicability in living cells or in living organs.
As a class of NADPH-dependent oxidases, P450 enzymes catalyze the oxidative metabolism of the substrates via an extremely complicated catalytic cycle, which includes several rate-limiting steps (Manikandan and Nagini, 2018; Dubey and Shaik, 2019). In most cases, the turnover rates of human CYPs are much lower than that of hydrolases, such as carboxylesterases, and transferases, such as UDP-glucuronosyltransferase enzymes and sulfotransferases in humans (Cederbaum, 2015). Thus, the first challenge for developing practical CYP optical probes is to find good substrates with high turnover rates in the target P450 enzyme(s) from fluorogenic compounds. To highly efficient discovery of good substrates for a target P450 enzyme, several molecular design strategies (such as substrate preference-based ligand design and computer-aided ligand design) have been proposed for constructing CYP optical substrates in the past decade. In the future, more feasible molecular design strategies or their combinations with experimental validations should be used to find more fluorogenic compounds as basic scaffolds or substrate candidates to develop ideal optical substrates for each target CYP enzyme.
The second challenge in this field is to improve the specificity of CYP optical substrates in real biologic samples. Several strategies for improving the specificity of candidate substrates could be applied, such as enhancing binding affinity or increasing the turnover rate toward target enzyme, designing the most suitable ligand based on the 3D structure of the catalytic cavity of the target enzyme, and reducing the catalytic efficiency of other P450 enzymes or non-P450 enzymes in the tested cell lines. For CYP enzymes with large catalytic cavities, such as CYP3A4 and CYP2J2, the specificity of the substrates could be improved by expanding the molecular volume, to block the formation of catalytic conformations in those P450 enzymes with small catalytic pockets.
The third challenge in this field is to improve the practicality of CYP optical substrates in living systems, such as living cells and tissues, even combined with newer technologies like 3D cell spheres, microfluidics chips, and other tissue-engineered models. HTS in living cells minimizes the enzyme damage, leaving the endoplasmic reticulum intact, while the exogenetic cofactors or NADPH-regeneration systems are not required, and the transport processes are maintained. With the aim of preliminary screening of inhibitors in intact P450-overexpressed cells, Donato et al. (2004) re-evaluated the catalytic properties of fluorogenic substrates. The one showing highest metabolic rate and lowest background was selected: CEC for CYP1A2 and CYP2C19, coumarin for CYP2A6, 7-ethoxy-4-(trifluoromethyl)-coumarin (EFC) for CYP2B6, DBF for CYP2C8, MFC for CYP2C9 and CYP2E1, 3-[2-(N,N-diethyl-N-methylammonium)ethyl]-7-methoxy-4-methylcoumarin (AMMC) for CYP2D6, and BFC for CYP3A4. Several strategies, such as improving water-solubility, enhancing cell-membrane permeability, reducing cytotoxicity, improving 7-ethoxy-resorufin (ER)-targeting ability, could be employed to improve the practicality of CYP optical substrates in living systems. Furthermore, excellent optical properties of the fluorogenic substrates, such as long emission wavelength and high and stable quantum yield in physiologic environment, are key factors affecting their detection sensitivity and practicality in living systems. Notably, most of the reported CYP fluorogenic substrates emit their fluorescent signals in the low wavelength region (<560 nm), which may be affected by the endogenous substances or biologic matrix with fluorescence emission, as well as the tested agents, such as therapeutic agents or herbal constituents in DDI/HDI studies.
In the future, much more efforts should be undertaken to develop more practical and versatile fluorescence-based assays for HTS of CYP inhibitors or inducers in living systems. Extensive investigations are still needed to gain deeper insight into the substrate preferences of the target P450 enzymes and the detailed binding conformations of the good substrates in the target P450 enzymes, which will significantly facilitate the design and development of the highly specific optical substrates for human CYPs as powerful molecular tools to develop the fluorescence-based high-throughput assays for investigating CYP-mediated DDIs. Although fluorescence-based CYP inhibition assays realize high-throughput screening and are easy to perform, in vitro CYP inhibition data should be verified by in vivo studies or clinical investigations. CYP-substrate drugs, such as midazolam and felodipine for CYP3A are often used in in vivo or clinical DDI studies for assessment of potential DDI risks (Ito et al., 2004). The risks of clinically relevant CYP-mediated DDIs could also be explored by using physiologically based pharmacokinetic modeling and in vitro–in vivo extrapolation approaches, on the basis of in vitro inhibition data, such as fluorescence-based CYP inhibition data (Dong et al., 2022). In fact, several previous studies have already extrapolated the in vitro data deriving from drug/physiologic substrates and partial fluorescent substrates to predict clinically potential DDIs (Ito et al., 2004; Riley and Grime, 2004; Zlokarnik et al., 2005; Obach et al., 2006, 2007; Bell et al., 2008; Fang et al., 2010; Goey et al., 2013; Bhatnagar et al., 2021; Steinbronn et al., 2021; Ramsden et al., 2022) and suggested that fluorogenic substrates for the target CYP enzymes could be used to replace the corresponding drug/physiologic substrates. In light of the complex setting for assessment of the DDI risks in vivo, more attention should be paid to the in vitro data from fluorescence-based biochemical assays and their relevance for clinical data.
Authorship Contributions
Wrote or contributed to the writing of the manuscript: He, Dai, Finel, Zhang, Tu, Yang, Ge.
Footnotes
- Received August 14, 2022.
- Accepted May 25, 2023.
This work was supported by the National Natural Science Foundation of China [Grants 82273897, 81922070, and 81973286]; Shanghai Science and Technology Innovation Action Plans [Grant 21S21900600] supported by Shanghai Science and Technology Committee; the Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences [Grants 2021-I2M-1-071, 2022-I2M-2-001]; Innovation Team and Talents Cultivation Program of National Administration of Traditional Chinese Medicine [Grant ZYYCXTDD-202004]; Shanghai Municipal Health Commission’s Traditional Chinese Medicine research project [Grant 2022CX005]; the State Key Laboratory of Fine Chemicals, Dalian University of Technology [Grant KF2022], and Three-year Action Plan for Shanghai Traditional Chinese Medicine Development and Inheritance Program [Grant ZY(2021-2023)-0401].
↵1R.H. and Z.D. contributed equally to this work.
The authors are not aware of any affiliations, memberships, funding, or financial holdings that might be perceived as affecting the objectivity of this review.
Abbreviations
- BF
- bufalin
- 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
- BOMR
- benzyloxy-methyl-resorufin
- CEC
- 3-cyano-7-ethoxycoumarin
- CHPO
- chloroethyl derivative (7-(2-chloroethoxy)-3H-phenoxazin-3-one
- CYP
- cytochrome P450 enzymes
- DBF
- dibenzyl-fluorescein
- DBOMF
- di-(benzyl-O-methyl)-fluorescein
- DDAM
- 1, 3-dichloro-7-methoxy-9, 9-dimethylacridin-2(9H)-one
- DDI
- drug–drug interaction
- EMA
- the European Medicines Agency
- EOMCC
- 7-ethoxy-methyloxy-3-cyano-coumarin
- FDA
- Food and Drug Administration
- HDI
- herb-drug interaction
- HTS
- high-throughput screening
- iPrBN
- 4-isopropoxy-7H-benzo[de]benzo[4, 5]imidazo[2, 1-a]isoquinolin-7-one
- luciferin-BE
- 2-(6-(benzyloxy)benzo[d]thiazol-2-yl)-4, 5-dihydrothiazole-4-carboxylic acid
- MFC
- 7-methoxy-4-(trifluoromethyl)-coumarin
- NBCeN
- N-(4-butyl)-4-chloroethoxy-1, 8-naphthalimide
- NEN
- N-(3-ethyl)-1, 8-naphthalimide
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