The metabolism of xenobiotics (foreign substances) in a living organism is vitally important to maintain homeostasis1. XDenobiotics are transformed in enzyme-catalyzed oxidafinition, reduction, hydrolysis, and conjugation reactions to more hydrophilic and excretable metabolites. Usually these reactions yield metabolites with diminished biological activity, but sometimes reactive and more toxic intermediates are formed.

In conjugation reactions, a cofactor (small endogenous molecule) is transferred to a functional group of a xenobiotic. The functional group is either already present or is created by reactions of oxidation, reduction or hydrolysis. Conjugations are highly important in overall metabolism because they usually inactivate xenobiotics or make reactive metabolites less harmful. In addition, conjugates are often substrates to transporters mediating excretion in the kidney and liver ^[1][2].

Cytochrome P450 (CYP) monooxygenaseluorescence-based assays are the most versatile xenobiotic-metabolizing enzymes (XMEs), which oxidize all kinds of lipophilic agents entering the body. CYP-mediated metabolism is an essential mechanism of elimination for both xenobiotics and lipophilic endogenous compounds (endobiotics) such as steroid hormones, bile acids, and lipid-soluble vitamins. CYP enzymes are supported by NADPH CYP reductase, which provides electrons to activate oxygen in the catalytic oxidation reactions of xenobiotics. In all nature, CYP enzymes comprise a very large superfamily, with 18 families in mammals and 57 individual forms in the human genome. Of these CYP forms, ~10 members in families CYP1, CYP2 and CYP3 catalyze the oxidation of xenobiotics to a significant degree. CYPs are most abundantly expressed in the liver, but they are also present in other barrier tissues such as the gastrointestinal tract, lung, skin, nose epithelium, and kidney ^[3][4][5].

It is estimated that >90% of enzymatic reactions of all organic compo the reaction produces a fluorescent produnds (general chemicals, natural and physiological compounds, and drugs) are catalyzed by CYP enzymes. About 75% of CYP reactions of clinically used drugs can be accounted for by a set of five hepatic CYP forms: CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 ^[6]. Cot from a njugation reactions, especially glucuronidation by uridine diphosphate glucuronosyltransferases (UGTs) and sulfonation by sulfotransferases (SULTs), also play a major role in the elimination of xenobiotics and harmful endobioticsfluorescent ^[7]. Rates of metabolic enzymes are modulated by numerous endogenous and exogenous factors, including genetic polymorphisms, sex, age, ethnicity, general health conditions, and induction and inhibition by xenobiotics ^[4][8].

When new chemical entities, especially dbstrate orugs, biocides, and food additives, are developed, it is essential to evaluate their metabolic pathways in target species and the major XMEs involved. Especially for drugs it is necessary to establish the kinetics and possible inhibition and induction of the XMEs involved.

After performing an in vitro envice versa. Fluorescence-based enzyme reaction, formation of metabolites or decrease of substrate can be detected by absorption of light (spectrophotometry), emitted fluorescence (fluorometry), mass/charge ratio (mass spectrometry, MS) or light pulses produced by a radioactive label. For the measurement of a substrate or its metabolites, the most versatile analytical approaches today are liquid chromatography (LC)-MS methods. The drawback of these methods is that the equipment is expensive, their use is labor intensive, and capacity may be saturated ^[9][10].

Fluorescessays are usually highly sence is a phenomenon where a molecule (fluorophore) absorbs and then emits lower-energy light. Fluorophores usually contain several conjugated aromatic groups or cyclic molecules with several π bonds or nonbonding electrons. The key parameters in this process are the absorption maxima (λ_max) (excitation) and the extitive and specinction coefficient at λ_max (ε, emission). Loss of energy occurs in excitation due to rapid relaxation to the first singlet excited state (S₁) and rellorganwization of solvent molecules around the altered dipole of the excited state. Fluorescence arises when this excited state is relaxed to the ground state (S₀) by eng mission of a photon. Each fluorophore has a characteristic pair of excitation (λ_max) and emission maximasum (λ_em). Chemiluminescence is similar to fluorescence but differs from it in that the electronic excited state is the product of a chemical reaction rather than of the absorption of a photon ^[11][12].

Fluorescence ments on smethods are widely used to evaluate rates and kinetic mechanisms of enzyme reactions. Fluorophore-based enzyme assays are usually highly sensitive and specific. This is particularly valuable when performed on small specimens ll specimens of tissues or other sources with low enzyme activities. Fluorescence assays are also amenable to miniaturization of the reaction setup and high throughput. Thus, further increases in sensitivity can be achieved with a decrease in demand for enzyme source and fluorescent substratemixtures and can thus ^[13][14][15]. An advantage of fluorophore-based enzyme assays is also that both fixed time and online continuous assays (milliseconds to hours) can be applied for multiple purposes.

Of done in high the various fluorophore-based approaches, we focus here on those that measure fluorescence change of coumarin substrates in reactions catalyzed by several types of XMEs, with main emphasis on our recent experience. In these reactions, a nonfluorescent coumarin derivative is metabolized to corresponding fluorescent 7-houghput. 7-Hydroxycoumarin metabolite(s) or vice versa (Figure 1). Other fluorophores commonly used for determining activities of XMEs include alkoxyresorufins ^[16], quinolines ^[17], furanones ^[14][17] and some proprietary ones such as Viand its derivid® fluorogenic substrates ^[18].

The coumarin (2H-1-benzopyran-2-one) scafas fold is made of fused benzene and α-pyrone rings. Coumarins can be grouped in six types based on their extended backbone structures (Figure 2): (1) simple coumarins, (2) furanocoumarins, (3) pyranocoumarins, (4) sesquiterpenyl coumarins, (5) oligomeric coumarins and (6) miscellaneous coumarins ^[19]. Thuorophores due coumarin scaffold occurs in constituents of food and numerous natural and synthetic products such as drugs, perfumes, rodenticides, and laser dyes ^[19][20].

Coumarin is widely used as a fluorophore due to its desirable photophysical properties. CoumarinThey possesses a large π-π conjugated system with electron-rich and charge transfer properties. This conjugated structure leads to their applications as fluorescent sensors for biological activities. Substitution at the 7-position with electron-donating groups yields highly fluorescent molecules, 7-hydroof 7-hydroxycoumarin (umbelliferone) being the core structure of these molecules. Many suitable combinations of substitutions at the 7-hydroxycoumarin scaffold do not destroy the intense fls as fluorescence ^[11][21]. However, we have noticed that 4, 5-dialkyl substituents suppress the fluorescence intensity to a minimum (unpublished data), and fluorescence of 7-hydroxywarfarin is low. Coumarin and many of its derivatives are converted to 7-hydroxycoumarin metabolites in the typical oxidation reaction of CYP enzymes (Figure 1).

The fsensors for bioluorescent properties of 7-hydroxycoumarin were already used in the 1950s to evaluate its metabolism in the laboratory of R.T. Williams. It was observed that, although fluorescence of 7-hydroxycoumarin was strong in ultraviolet light at pH ~10, its glucuronide and sulfate conjugates showed little or no fluorescence. Based on this, a sensitive fluorescence method was developed for the determination of β-glucuronidase activity, in which nonfluorescent glucuronide conjugate was hydrolyzed to fluorescent 7-hydroxycoumarin ^[22]gical activities. An in vitro method for measuring coumarin 7-hydroxylation activity using fluorescence detection was subsequently developed for CYP enzymes in the same laboratory

Numerous new 7-substituted coumarin derivatives for measuring activities of CYP enzymes have been introduced since the 1970s. These include 7-ethoxycoumarin, 3-cyano-7-ethoxycoumarin (CEC), 7-ethoxy-4-trifluoromethylcoumarin (EFC), 7-methoxy-4-trifluoromethylcoumarin (MFC), 7-methoxy-4-aminomethylcoumarin (MAMC), 3-[2-(N,N-diethyl-N-methylammonium)ethyl]- 7-methoxy-4-methylcoumarin (AMMC), and 7-benzyloxy-4-trifluoromethylcoumarin (BFC) (Figure 3). These alkylated derivatives of 7-hydroxycoumarin are ‘blocked’ substrates, whose fluorescence signal is minimal compared to the 7-O-dealkylated oxidation product. Some of these have been commercialized for use in CYP-mediated drug interaction studies, pioneered by the Gentest Corporation (now Corning) ^[13][17][25]. A major shortcoming of these coumarin derivatives is that, with few exceptions, they are not selective but oxidized by several human CYP forms ^{[13][17][25][26][27]}. Coumarin itself is an example of a highly selective CYP substrate, as it is oxidized practically exclusively by human CYP2A6 to fluorescent 7-hydroxycoumarin ^[28][29]. In addition to being excellent substrates, numerous coumarins are potent CYP inhibitors, particularly furanocoumarins such as methoxsalen ^[30][31][32].

Ideally, sensitive and selective fluorophore substrates for different forms of XMEs would provide effective and low-cost tools for studying form-specific metabolism in simple assays using all kinds of enzyme sources, including whole tissue or subcellular fractions. However, although many fluorophore substrates are available, new substrates are needed as most human XMEs are still missing the optimal substrate, especially regarding selectivity for different enzyme forms. The flexible chemistry of the coumarin core offers diverse options for functionalization to design novel fluorescent substrates.

As part of an effort to find novel coumarin-based, pharmacologically active molecules, we have synthesized multiple derivatives of the coumarin scaffold. The synthesis protocols were kept simple and robust, and inexpensive starting materials were used. The detailed protocols are published ^[33][34][35]. In a typical procedure, a mixture of a salicylaldehyde derivative, a phenylacetic acid derivative, acetic acid anhydride and trimethylamine is heated at 100°C. After cooling, NaHCO

₃

¹H-NMR the purity of compounds is typically >95%.

When setting up fluorescence assays to quantify enzyme activities, the first step is to establish incubation conditions supporting the reaction. Phosphate or Tris-HCl buffers are commonly used to gain the optimal ion concentration and pH of 7.4. All enzymes require their cofactors: NADPH for CYP oxidations, uridine 5’-diphosphoglucuronic acid (UDPGA) for UGTs, 3’-phosphoadenylyl sulfate (PAPS) for SULTs and S-adenosylmethionine (SAM) for COMT. The fluorescent substrates must have sufficient aqueous solubility (at least µM range), efficient metabolite formation, low background fluorescence, high signal-to-noise ratio, and appropriate excitation and emission wavelengths in the UV range ^[12][36].

During incubation a metabolite is formed, and the amount of substrate is decreased. Fluorescent 7-hydroxycoumarins are excellent for measuring substrate decrease, because several conjugating enzymes transfer a substituent to the 7-hydroxyl group making the product nonfluorescent (Figure 1B). A practical advantage of measuring conjugating reactions is that 7-hydroxycoumarins act as standards, allowing for reliable and accurate quantification of the reaction, whereas CYP oxidation reactions require the availability of a selective reaction standard for exact quantitation. However, in the latter case surrogate standards such as 7-hydroxycoumarin can be applied.

The solubility of coumarin in water is very high (20 mM). Lipophilic substituents decrease solubility, causing much variability to solubility among coumarin derivatives. The solubility of 7-hydroxycoumarins is higher than that of the corresponding derivatives. In our experience, even the poorest soluble coumarin derivatives have been soluble at µM concentrations. We routinely use 0.05–10 µM concentrations in the assays. Enzyme sources (especially tissues) and cofactors can produce background fluorescence. Contamination of tissue samples with blood causes problems because hemoglobin absorbs light efficiently at 370–450 nm. Too high concentrations of enzyme sample reduce fluorescence due to absorption of excitation or emission light and produce insufficient signal-to-noise ratio in the reaction. In the continuous (kinetic) assays up to 5% tissue in the reaction mixture does not excessively suppress fluorescence. Recombinant or purified enzymes added in small volumes in the incubation mixture yield no increase in the background. Tissue samples can be removed in the fixed time measurements by precipitation, but this adds extra steps. When very low activities are measured in fixed time assays, small differences in composition between sample and negative control may cause background fluorescence even after precipitation. In continuous assays background fluorescence is usually not a major issue, as fluorescence change is monitored against background during the entire measurement period.

Although both the excitation and emission peaks are broad for fluorescent compounds, it is important that these are optimized in the assays. Excitation wavelengths typically range from 300 to 420 nm and emission wavelengths from 350 to 500 nm for 7-hydroxycoumarin derivatives. Coumarins are also fluorescent; however, their excitation wavelengths are lower than 7-hydroxycoumarins. NADPH is strongly fluorescent <390 nm if excitation ~350 nm is used, but it is nonfluorescent at excitation >400 nm. The cofactors UDPGA and PAPS do not cause background fluorescence. Using excitation >400 nm minimizes background fluorescence and gives high enough emission fluorescence at 450–500 nm for coumarin-based assays. Negative control incubations lacking the enzyme, substrate or cofactor are needed to monitor background fluorescence.

When selecting the enzyme source, one must keep in mind that tissues and their cellular subfractions contain several forms of XMEs, most notably the liver. Since almost all fluorescent probes are not selective or lose selectivity at high substrate concentrations, heterologously expressed individual XMEs should be used when enzyme-specific reactions are studied. Nowadays, there is a good commercial supply of expressed human CYP, UGT and SULT enzymes.

To understand an enzyme reaction and its physiological role, it is essential to determine the effect of substrate concentration on the reaction rate (K

_m

_max—the rate at saturating substrate concentration). Experiments can then be rationally planned for evaluating potential inhibitors and characteristics of inhibition, including inhibitor potency and whether the inhibition mechanism is reversible, irreversible, competitive, un-competitive (catalytic) or mixed type of inhibition ^[12][36]. When interpreting the results, it should be considered that especially CYP3 enzymes may deviate from Michaelis–Menten kinetics with several probes, including fluorescent substrates ^[37][38]. In summary, sensitivity of fluorescence-based assays consists of the fluorescence properties of the substrates, products, cofactors, amount and purity of samples, and incubation time. All can be optimized as described here.