Coumarin-Based Profluorescent and Fluorescent Substrates for Determining Xenobiotic-Metabolizing Enzyme Activities In Vitro

Activities of xenobiotic-metabolizing enzymes have been measured with various in vitro and in vivo methods, such as spectrophotometric, fluorometric, mass spectrometric, and radioactivity-based techniques. In fluorescence-based assays, the reaction produces a fluorescent product from a nonfluorescent substrate or vice versa. Fluorescence-based enzyme assays are usually highly sensitive and specific, allowing measurements on small specimens of tissues with low enzyme activities. Fluorescence assays are also amenable to miniaturization of the reaction mixtures and can thus be done in high throughput. 7-Hydroxycoumarin and its derivatives are widely used as fluorophores due to their desirable photophysical properties. They possess a large pi-pi conjugated system with electron-rich and charge transfer properties. This conjugated structure leads to applications of 7-hydroxycoumarins as fluorescent sensors for biological activities. We describe in this review historical highlights and current use of coumarins and their derivatives in evaluating activities of the major types of xenobiotic-metabolizing enzyme systems. Traditionally, coumarin substrates have been used to measure oxidative activities of cytochrome P450 (CYP) enzymes. For this purpose, profluorescent coumarins are very sensitive, but generally lack selectivity for individual CYP forms. With the aid of molecular modeling, we have recently described several new coumarin-based substrates for measuring activities of CYP and conjugating enzymes with improved selectivity

Molecular Docking-Based Design and Development of a Highly Selective Probe Substrate for UDP-glucuronosyltransferase 1A10

Intestinal and hepatic glucuronidation by the UDP-glucuronosyltransferases (UGTs) greatly affect the bioavailability of phenolic compounds. UGT1A10 catalyzes glucuronidation reactions in the intestine, but not in the liver. Here, our aim was to develop selective, fluorescent substrates to easily elucidate UGT1A10 function. To this end, homology models were constructed and used to design new substrates, and subsequently, six novel C3-substituted (4-fluorophenyl, 4-hydroxyphenyl, 4-methoxyphenyl, 4-(dimethylamino)phenyl, 4-methylphenyl, or triazole) 7-hydroxycoumarin derivatives were synthesized from inexpensive starting materials. All tested compounds could be glucuronidated to nonfluorescent glucuronides by UGT1A10, four of them highly selectively by this enzyme. A new UGT1A10 mutant, 1A10-H210M, was prepared on the basis of the newly constructed model. Glucuronidation kinetics of the new compounds, in both wild-type and mutant UGT1A10 enzymes, revealed variable effects of the mutation. All six new C3-substituted 7-hydroxycoumarins were glucuronidated faster by human intestine than by liver microsomes, supporting the results obtained with recombinant UGTs. The most selective 4(dimethylamino)phenyl and triazole C3-substituted 7-hydroxycoumarins could be very useful substrates in studying the function and expression of the human UGT1A10.Peer reviewe

Molecular docking and oxidation kinetics of 3-phenyl coumarin derivatives by human CYP2A13

CYP2A13 enzyme is expressed in human extrahepatic tissues, while CYP2A6 is a hepatic enzyme. Reactions catalysed by CYP2A13 activate tobacco-specific nitrosamines and some other toxic xenobiotics in lungs. To compare oxidation characteristics and substrate-enzyme active site interactions in CYP2A13 vs CYP2A6, we evaluated CYP2A13 mediated oxidation characteristics of 23 coumarin derivatives and modelled their interactions at the enzyme active site. CYP2A13 did not oxidise six coumarin derivatives to corresponding fluorescent 7-hydroxycoumarins. The K-m-values of the other coumarins varied 0.85-97 mu M, V-max-values of the oxidation reaction varied 0.25-60 min(-1), and intrinsic clearance varied 26-6190 kL/min*mol CYP2A13). K-m of 6-chloro-3-(3-hydroxyphenyl)-coumarin was 0.85 (0.55-1.15 95% confidence limit) mu M and V-max 0.25 (0.23-0.26) min(-1), whereas K-m of 6-hydroxy-3-(3-hydroxyphenyl)-coumarin was 10.9 (9.9-11.8) mu M and V-max 60 (58-63) min(-1). Docking analyses demonstrated that 6-chloro or 6-methoxy and 3-(3-hydroxyphenyl) or 3-(4-trifluoromethylphenyl) substituents of coumarin increased affinity to CYP2A13, whereas 3-triazole or 3-(3-acetate phenyl) or 3-(4-acetate phenyl) substituents decreased it. The active site of CYP2A13 accepts more diversified types of coumarin substrates than the hepatic CYP2A6 enzyme. New sensitive and convenient profluorescent CYP2A13 substrates were identified, such as 6-chloro-3-(3-hydroxyphenyl)-coumarin having high affinity and 6-hydroxy-3-(3-hydroxyphenyl)-coumarin with high intrinsic clearance

Substrate Selectivity of Coumarin Derivatives by Human CYP1 Enzymes: In Vitro Enzyme Kinetics and In Silico Modeling

Of the three enzymes in the human cytochrome P450 family 1, CYP1A2 is an important enzyme mediating metabolism of xenobiotics including drugs in the liver, while CYP1A1 and CYP1B1 are expressed in extrahepatic tissues. Currently used CYP substrates, such as 7-ethoxycoumarin and 7-ethoxyresorufin, are oxidized by all individual CYP1 forms. The main aim of this study was to find profluorescent coumarin substrates that are more selective for the individual CYP1 forms. Eleven 3-phenylcoumarin derivatives were synthetized, their enzyme kinetic parameters were determined, and their interactions in the active sites of CYP1 enzymes were analyzed by docking and molecular dynamic simulations. All coumarin derivatives and 7-ethoxyresorufin and 7-pentoxyresorufin were oxidized by at least one CYP1 enzyme. 3-(3-Methoxyphenyl)-6-methoxycoumarin (19) was 7-O-demethylated by similar high efficiency [21-30 ML/(min.mol CYP)] by all CYP1 forms and displayed similar binding in the enzyme active sites. 3-(3-Fluoro-4-acetoxyphenyl)coumarin (14) was selectively 7-O-demethylated by CYP1A1, but with low efficiency [0.16 ML/(min mol)]. This was explained by better orientation and stronger H-bond interactions in the active site of CYP1A1 than that of CYP1A2 and CYP1B1. 3-(4-Acetoxyphenyl)-6-chlorocoumarin (20) was 7-O-demethylated most efficiently by CYP1B1 [53 ML/(min.mol CYP)], followed by CYP1A1 [16 ML/(min.mol CYP)] and CYP1A2 [0.6 ML/(min.mol CYP)]. Variations in stabilities of complexes between 20 and the individual CYP enzymes explained these differences. Compounds 14, 19, and 20 are candidates to replace traditional substrates in measuring activity of human CYP1 enzymes

Structure-Activity Relationship Analysis of 3-Phenylcoumarin-Based Monoamine Oxidase B Inhibitors

Monoamine oxidase B (MAO-B) catalyzes deamination of monoamines such as neurotransmitters dopamine and norepinephrine. Accordingly, small-molecule MAO-B inhibitors potentially alleviate the symptoms of dopamine-linked neuropathologies such as depression or Parkinson's disease. Coumarin with a functionalized 3-phenyl ring system is a promising scaffold for building potent MAO-B inhibitors. Here, a vast set of 3-phenylcoumarin derivatives was designed using virtual combinatorial chemistry or rationally de novo and synthesized using microwave chemistry. The derivatives inhibited the MAO-B at 100 nM-1 mu M. The IC50 value of the most potent derivative 1 was 56 nM. A docking-based structure-activity relationship analysis summarizes the atom-level determinants of the MAO-B inhibition by the derivatives. Finally, the cross-reactivity of the derivatives was tested against monoamine oxidase A and a specific subset of enzymes linked to estradiol metabolism, known to have coumarin-based inhibitors. Overall, the results indicate that the 3-phenylcoumarins, especially derivative 1, present unique pharmacological features worth considering in future drug development

Modeling of interactions between xenobiotics and cytochrome P450 (CYP) enzymes

The adverse effects to humans and environment of only few chemicals are well known. Absorption, distribution, metabolism, and excretion (ADME) are the steps of pharmaco/toxicokinetics that determine the internal dose of chemicals to which the organism is exposed. Of all the xenobiotic-metabolizing enzymes, the cytochrome P450 (CYP) enzymes are the most important due to their abundance and versatility. Reactions catalyzed by CYPs usually turn xenobiotics to harmless and excretable metabolites, but sometimes an innocuous xenobiotic is transformed into a toxic metabolite. Data on ADME and toxicity properties of compounds are increasingly generated using in vitro and modeling (in silico) tools. Both physics-based and empirical modeling approaches are used. Numerous ligand-based and target-based as well as combined modeling methods have been employed to evaluate determinants of CYP ligand binding as well as predicting sites of metabolism and inhibition characteristics of test molecules. In silico prediction of CYP–ligand interactions have made crucial contributions in understanding (1) determinants of CYP ligand binding recognition and affinity; (2) prediction of likely metabolites from substrates; (3) prediction of inhibitors and their inhibition potency. Truly predictive models of toxic outcomes cannot be created without incorporating metabolic characteristics; in silico methods help producing such information and filling gaps in experimentally derived data. Currently modeling methods are not mature enough to replace standard in vitro and in vivo approaches, but they are already used as an important component in risk assessment of drugs and other chemicals.peerReviewe

Development of New Coumarin-Based Profluorescent Substrates for Human Cytochrome P450 Enzymes

1. Cytochrome P450 (CYP) enzymes constitute an essential xenobiotic metabolizing system that regulates the elimination of lipophilic compounds from the body. Convenient and affordable assays for CYP enzymes are important for assessing these metabolic pathways. 2. In this study, 10 novel profluorescent coumarin derivatives with various substitutions at carbons 3, 6 and 7 were developed. Molecular modeling indicated that 3- phenylcoumarin offers an excellent scaffold for the development of selective substrate compounds for various human CYP forms, as they could be metabolized to fluorescent 7-hydroxycoumarin derivatives. Oxidation of profluorescent coumarin derivatives to fluorescent metabolites by 13 important human liver xenobiotic-metabolizing CYP forms was determined by enzyme kinetic assays. 3. Four of the coumarin derivatives were converted to fluorescent metabolites by CYP1 family enzymes, with 6-methoxy-3-(4-trifluoromethylphenyl)coumarin being oxidized selectively by CYP1A2 in human liver microsomes. Another set of four compounds were metabolized by CYP2A6 and CYP1 enzymes. 7-Methoxy-3-(3- methoxyphenyl)coumarin was oxidized efficiently by CYP2C19 and CYP2D6 in a nonselective fashion. 4. The advantages of the novel substrates were 1) an excellent signal-to-background ratio, 2) selectivity for CYP1 forms, and 3) convenient multiwell plate measurement, allowing for precise determination of potential inhibitors of important human hepatic forms CYP1A2, CYP2C19 and CYP2D6.peerReviewe

Comparison of trapping profiles between d-peptides and glutathione in the identification of reactive metabolites

Qualitative trapping profile of reactive metabolites arising from six structurally different compounds was tested with three different d-peptide isomers (Peptide 1, gly–tyr–pro–cys–pro–his-pro; Peptide 2, gly–tyr–pro–ala–pro–his–pro; Peptide 3, gly–tyr–arg–pro–cys–pro–his–lys–pro) and glutathione (GSH) using mouse and human liver microsomes as the biocatalyst. The test compounds were classified either as clinically “safe” (amlodipine, caffeine, ibuprofen), or clinically as “risky” (clozapine, nimesulide, ticlopidine; i.e., associated with severe clinical toxicity outcomes). Our working hypothesis was as follows: could the use of short different amino acid sequence containing d-peptides in adduct detection confer any add-on value to that obtained with GSH? All “risky” agents’ resulted in the formation of several GSH adducts in the incubation mixture and with at least one peptide adduct with both microsomal preparations. Amlodipine did not form any adducts with any of the trapping agents. No GSH and peptide 2 and 3 adducts were found with caffeine, but with peptide 1 one adduct with human liver microsomes was detected. Ibuprofen produced one Peptide 1-adduct with human and mouse liver microsomes but not with GSH. In conclusion, GSH still remains the gold trapping standard for reactive metabolites. However, targeted d-peptides could provide additional information about protein binding potential of electrophilic agents, but their clinical significance needs to be clarified using a wider spectrum of chemicals together with other safety estimates

Acute changes in inflammatory biomarker levels in recreational runners participating in a marathon or half-marathon

Abstract Background: Strenuous physical activity activates the participant’s immune responses; however, few studies exist, observing exercise-induced simultaneous changes in mediators of inflammation. Methods: We examined individual responses in soluble urokinase-type plasminogen activator receptor (suPAR), a marker of immune activation, soluble endocytic receptor for haptoglobin-hemoglobin complexes (CD163), a marker of monocyte-macrophage activation, C-reactive protein (CRP), and pro- and anti-inflammatory cytokines from blood samples drawn at baseline, at 3- and 48-h post-races from recreational runners who successfully completed the marathon (199 ± 8 min, n = 4) or half-marathon (132 ± 4 min, n = 4) run. For comparisons, biomarkers reflecting muscle, heart, kidney, and liver functions were measured. Results: Significant 3-h post-race increases occurred in levels of suPAR (p < 0.01), CD163 (p < 0.05), white blood cells (p < 0.001), pro-inflammatory cytokines, interleukin-6 (IL-6) (p < 0.001), IL-8 (p < 0.05), and anti-inflammatory cytokine IL-10 (p < 0.05), whereas tumor necrosis factor-α (TNF-α) and transforming growth factor-β (TGF-β) remained relatively stable. Full-marathon running lead to more pronounced increases in suPAR, CD163, IL-8, and IL-10 than half-marathon running. In addition, 3-h post-race increases of all these parameters correlated significantly with changes in serum TNF-α and cortisol. The 48-h levels of serum suPAR and both pro- and anti-inflammatory cytokines had decreased to baseline levels, whereas CRP, a marker of acute phase response, increased in those with the most prominent IL-6 and IL-10 elevations in their preceding samples. The highest suPAR, CRP, IL-6, TNF-α, IL-10, and cortisol levels were noted in the individual with the most severe post-race fatigue. Conclusions: Prolonged running increases mediators of inflammation in an exercise-dose-dependent manner which should be considered in the assessment of health status of physically active individuals after recent acute bouts of strenuous exercise