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4-24-2019

Coumarins and P450s, Studies Reported to-Date

Maryam Foroozesh Xavier University of Louisiana, [email protected]

Jayalakshmi Sridhar Xavier University of Louisiana, [email protected]

Navneet Goyal Xavier University of Louisiana, [email protected]

Jiawang Liu University of Tennessee Health Science Center, [email protected]

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Recommended Citation Foroozesh, Maryam; Sridhar, Jayalakshmi; Goyal, Navneet; and Liu, Jiawang, "Coumarins and P450s, Studies Reported to-Date" (2019). Faculty and Staff Publications. 42. https://digitalcommons.xula.edu/fac_pub/42

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Review Coumarins and P450s, Studies Reported to-Date

Maryam Foroozesh 1,*, Jayalakshmi Sridhar 1, Navneet Goyal 1 and Jiawang Liu 2

1 Department of Chemistry, Xavier University of Louisiana, 1 Drexel Dr., New Orleans, LA 70125, USA; [email protected] (J.S.); [email protected] (N.G.) 2 Department of Pharmaceutical Sciences, College of Pharmacy, University of Tennessee Health Science Center, 881 Madison Ave., Memphis, TN 38163, USA; [email protected] * Correspondence: [email protected]; Tel.: +1-504-520-5078; Fax: +1-504-520-7942



Academic Editor: Salvatore Genovese
 Received: 9 April 2019; Accepted: 22 April 2019; Published: 24 April 2019

Abstract: Cytochrome P450 enzymes (CYPs) are important phase I enzymes involved in the metabolism of endogenous and xenobiotic compounds mainly through mono-oxygenation reactions into more polar and easier to excrete species. In addition to their role in detoxification, they play important roles in the biosynthesis of endogenous compounds and the bioactivation of xenobiotics. Coumarins, phytochemicals abundant in food and commonly used in fragrances and cosmetics, have been shown to interact with P450 enzymes as substrates and/or inhibitors. In this review, these interactions and their significance in pharmacology and toxicology are discussed in detail.

Keywords: cytochrome P450; coumarins; docking; quantitative structure-activity relationship (QSAR); hologram quantitative structure-activity relationship (HQSAR); comparative molecular field analysis (CoMFA); comparative molecular similarity index analysis (CoMSIA); molecular modeling; active site; competitive inhibition; time-dependent inhibition

1. Introduction

1.1. Cytochrome P450s Enzymes Cytochrome P450 enzymes (CYPs), an important superfamily of Phase I enzymes, are involved in the metabolism of endogenous and exogenous (xenobiotic) compounds [1–4]. These versatile enzymes are found in all living organisms and catalyze the mono-oxygenation of a wide variety of substrates [1]. The primary purpose of P450s is detoxification by oxidizing non-polar molecules into more polar and excretable ones [5]. Mammalian P450s play important roles in cholesterol and hormone synthesis, metabolism of endogenous compounds such as vitamin D and bile acids, drug activation and deactivation, and xenobiotic detoxification [1–4]. About 75% of known enzymatic reactions on drugs are catalyzed by P450 enzymes [6]. In addition, these enzymes play an important role in carcinogenesis, as certain pro-carcinogenic compounds are metabolized by P450 enzymes into their ultimate carcinogenic forms, which are capable of binding DNA and initiating cancer [1,7,8]. P450s are estimated to catalyze about 66% of bioactivation reactions, many of which lead to cancer formation [9–11]. The Human Genome Project has shown that there are 57 human P450 genes and 58 pseudogenes [1,12]. As more substrates including environmental chemicals and drugs have been discovered for the various P450 enzymes, their versatility and importance have become more apparent, warranting additional studies on their mechanisms of action and roles in different diseases including cancer. The development of selective inhibitors for P450 enzymes involved in carcinogenesis has been the focus of many research groups, including ours and our collaborators [13–23].

Molecules 2019, 24, 1620; doi:10.3390/molecules24081620 www.mdpi.com/journal/molecules Molecules 2019, 24, 1620 2 of 17

Molecules 2019, 24, x FOR PEER REVIEW 2 of 17 1.1.1. Basic Enzyme Structure 1.1.1.P450 Basic Enzymes Enzyme are Structure a superfamily of hemoproteins [1]. Even though the amino acid sequence identityP450 across Enzymes the entire are P450a superfamily superfamily of hemoproteins is less than 20%, [1]. Even the enzymes though the have amino identical acid overallsequence fold structures.identity across The three-dimensional the entire P450 superfamily structure is of le P450sss than consists 20%, the of enzymes 12 α-helices have (A-L), identical forming overall the fold bulk ofstructures. the protein, The in three-dimensional addition to four βstructure-sheets. of The P450s heme consists is located of 12 α within-helices the (A-L), L-helix forming and the bulk highly conservedof the protein, I-helix in that addition is perpendicular to four β-sheets. to the The F/G heme segment is located made within from the the F-helix, L-helix Fand/G-loop the highly and the G-helixconserved [23]. I-helix The heme-Fe that is perpendicular atom is bound to to the the F/ sulfurG segment atom made of the from cysteine the F-helix, amino acidF/G-loop in the and adjacent the loop,G-helix which [23]. contains The heme-Fe the highly atom conserved is bound sequenceto the sulfur FxxGx(HRK)xCxG atom of the cysteine [23]. Substrateamino acid access in the and specificityadjacent forloop, the which various contains P450 active the highly sites arecons determinederved sequence by the FxxGx(HR B-C andK)xCxG F-G helices [23]. [ 23Substrate]. Crystal structuresaccess and of manyspecificity P450 for enzymes the various have P450 already active been sites published are determined [24–31]. by the B-C and F-G helices [23]. Crystal structures of many P450 enzymes have already been published [24–31]. 1.1.2. Catalytic Activity Cycle 1.1.2. Catalytic Activity Cycle The catalytic activity starts once a substrate is bound to the enzyme, and the low-spin ferric heme-ironThe (coordinatedcatalytic activity to a waterstarts moleculeonce a substrate as its sixth is bound ligand to in the its restingenzyme, state) and changes the low-spin to a high-spin ferric ferricheme-iron state in (coordinated the enzyme-substrate to a water complex.molecule Theas its heme-iron sixth ligand then in its binds resting an oxygenstate) changes molecule to a to high- form a spin ferric state in the enzyme-substrate complex. The heme-iron then binds an oxygen molecule to stable oxy-iron-enzyme complex. This oxy-iron-enzyme complex is then reduced to form an unstable form a stable oxy-iron-enzyme complex. This oxy-iron-enzyme complex is then reduced to form an peroxo-ferric-enzyme complex, followed by the protonation of the distal oxygen to form an unstable unstable peroxo-ferric-enzyme complex, followed by the protonation of the distal oxygen to form hydroperoxo-iron-enzyme complex. A second protonation of the same oxygen is then followed by an unstable hydroperoxo-iron-enzyme complex. A second protonation of the same oxygen is then heterolytic cleavage of the bond between the two oxygen atoms leading to the formation of a reactive followed by heterolytic cleavage of the bond between the two oxygen atoms leading to the high-valentformation iron-oxo-enzymeof a reactive high-valent complex iron-oxo-e and water.nzyme This complex complex and then water. abstracts This a complex hydrogen then atom fromabstracts the substrate a hydrogen with atom subsequent from the radical substrate recombination with subsequent to form radical the product recombination (oxidized to formform ofthe the substrate).product (oxidized After the releaseform of ofthe the substrate). product, After water the binds release to the of heme-iron,the product, regenerating water binds the to the resting heme- P450 enzymeiron, regenerating (Figure1)[1 ,the23]. resting P450 enzyme (Figure 1) [1,23].

FigureFigure 1. The1. The catalytic catalytic oxidation oxidation cycle cycle of cytochrome of cytoch P450rome enzymes—TheP450 enzymes—The heme-Fe heme-Fe atom is atom covalently is boundcovalently to the bound sulfur to atom the ofsulfur a cysteine atom of amino a cysteine acid amino residue acid in theresidue enzyme in the active enzyme site active [23]. site [23]. Molecules 20192019,, 2424,, 1620x FOR PEER REVIEW 3 of 17

1.2. Coumarins 1.2. Coumarins Coumarins are natural products found in plants, fungi, and bacteria [32]. They are commonly foundCoumarins in food and are also natural widely products used in found cosmetics in plants, and fragrances fungi, and [33,34]. bacteria Coumarins [32]. They show are commonly a variety foundof biological in food activities, and also widelyincluding used anti-oxidant, in cosmetics an andti-cancer, fragrances anti-coagulant, [33,34]. Coumarins anti-inflammatory, show a variety anti- of biologicalfungal, anti-microbial, activities, including anti-viral, anti-oxidant, anti-neurodegenerative, anti-cancer, anti-coagulant, and anti-diabetic anti-inflammatory, activities anti-fungal, [32–34]. anti-microbial,Coumarin (2H-1-benzopyran-2-one) anti-viral, anti-neurodegenerative, backbone structure and anti-diabetic is made of activitiesfused benzene [32–34 and]. Coumarin α-pyrone (2H-1-benzopyran-2-one)rings [35,36]. This conjugated backbone structure structure also leads is made to coumarins’ of fused benzene applications and α as-pyrone fluorescent rings sensors [35,36]. Thisfor biological conjugated activities structure [32]. also There leads are tocoumarins’ six types of applications coumarins asbased fluorescent on their sensors extended for biologicalbackbone activitiesstructures: [32 1)]. ThereSimple are coumarins; six types of coumarins2) Dihydrofuranocoumarins; based on their extended 3) Furanocoumarins backbone structures: (linear 1) Simple and coumarins;angular); 4) 2) Pyranocoumarins Dihydrofuranocoumarins; (linear and 3) angular); Furanocoumarins 5) Phenylcoumarins; (linear and angular); and 6) Bicoumarins 4) Pyranocoumarins (Figure (linear2) [36]. and angular); 5) Phenylcoumarins; and 6) Bicoumarins (Figure2)[36].

OO O OO

1) Simple coumarin backbone 2) Dihydrofuranocoumarin backbone

O O OO OO

3) Furanocoumarin backbones (linear and angular)

O OO O OO

4) Pyranocoumarin backbones (linear and angular)

OO

O O O O

5) Phenylcoumarin backbone 6) Bicoumarin backbone Figure 2. Types of coumarin backbone structures.structures.

1.2.1. Coumarins and P450s 1.2.1. Coumarins and P450s Due to the importance of understanding the interactions between coumarins and cytochrome Due to the importance of understanding the interactions between coumarins and cytochrome P450 enzymes and their biological effects, especially on human health, this review is focused on studies P450 enzymes and their biological effects, especially on human health, this review is focused on of such interactions and lessons learned. studies of such interactions and lessons learned. The P450 enzymes have a wide range of natural product substrates and inhibitors including The P450 enzymes have a wide range of natural product substrates and inhibitors including coumarins [37–41]. For example, citrus fruits are rich in coumarins such as bergamottin, a linear coumarins [37–41]. For example, citrus fruits are rich in coumarins such as bergamottin, a linear furanocoumarin found in grapefruits, that potently inhibits several P450 enzymes including P450s 1A1, furanocoumarin found in grapefruits, that potently inhibits several P450 enzymes including P450s 1A2, 2A6, 2B6, 2D6, 3A4, and 3A5 [42,43]. These interactions are especially important in pharmacology 1A1, 1A2, 2A6, 2B6, 2D6, 3A4, and 3A5 [42,43]. These interactions are especially important in as many of these P450s are required for bioactivation and/or metabolism of drugs in vivo, and pharmacology as many of these P450s are required for bioactivation and/or metabolism of drugs in their inhibition can lead to lower bioavailability of the drugs’ active metabolites and/or higher vivo, and their inhibition can lead to lower bioavailability of the drugs’ active metabolites and/or concentrations of the drugs in the blood or tissues that are prone to drug toxicity. Furanocoumarins higher concentrations of the drugs in the blood or tissues that are prone to drug toxicity. are secondary metabolites in citrus plants and protect the plants against other organisms including Furanocoumarins are secondary metabolites in citrus plants and protect the plants against other insects [41]. Interestingly their biosynthesis in plants is also heavily P450 dependent [44]. A new organisms including insects [41]. Interestingly their biosynthesis in plants is also heavily P450 dependent [44]. A new X-ray crystal structure of P450 1A1 with bergamottin in its active site has Molecules 2019, 24, x FOR PEER REVIEW 4 of 17 been published recently, showing that the P450 1A1 active site is malleable and opens up as needed to accommodate large substrates [28]. This specific P450 enzyme is heavily involved in human carcinogenesis, thus its inhibition has been studied for cancer chemoprevention [28]. Bergamottin has been shown to have chemo-preventive effects in mice, inhibiting benzo[a]pyrene metabolism by P450 1A1 to its carcinogenic form, anti-benzo[a]pyrene-7,8-diol-9,10-epoxide, and the resulting production of DNA adducts and cancer formation [45]. Another naturally occurring linear furanocoumarin, methoxsalen, is a very potent mechanism-based, non-selective inhibitor of human P450s, and is used as a drug for the treatment of a number of skin diseases such as psoriasis [46,47]. Two other linear furanocoumarins, imperatorin and isopimpinellin, have been shown to have anti- cancer activities in a murine breast cancer model through the inhibition of P450 1A1/1B1 metabolism of 7,12-dimethylbenz[a]anthracene (DMBA) and blocking DMBA-DNA adduct Molecules 2019 24 formation [48]., , 1620 4 of 17 Figure 3 contains the structures of a number of natural coumarin P450 substrates and inhibitors X-ray[45]. Coumarins crystal structure exhibit ofsignificant P450 1A1 species with bergamottin differences in in metabolism its active site and has toxicity been published[37,40]. recently, showing that the P450 1A1 active site is malleable and opens up as needed to accommodate large substrates1.2.2. Coumarin [28]. Metabolism This specific by P450 P450s enzyme is heavily involved in human carcinogenesis, thus its inhibitionAs mentioned has been previously, studied for cancerP450 metabolism chemoprevention of coumarins [28]. Bergamottin is species dependent. has been shownCoumarin to have has chemo-preventivebeen found to be eaff ectsrat liver in mice, and inhibiting mouse lung benzo[a]pyrene toxicant due metabolism to the P450-de by P450pendant 1A1 to itsproduction carcinogenic of form,coumarin-3,4-epoxide anti-benzo[a]pyrene-7,8-diol-9,10-epoxide, by P450s 1A1, 1A2, and 2E1 and in liver the resulting and P450 production 2F2 in lung of (Figure DNA adducts4) [37,49]. and 3- cancerHydroxycoumarin formation [ 45has]. Anotherbeen found naturally as a minor occurring metabolite linear of furanocoumarin, coumarin metabolism methoxsalen, by recombinant is a very potentrat P450s mechanism-based, 1A1 and 1A2 [37]. non-selective inhibitor of human P450s, and is used as a drug for the treatmentIn humans, of a number only about of skin a dozen diseases of suchthe 57 as CYP psoriasis genes, [46 those,47]. belonging Two other to linear CYP furanocoumarins,families 1, 2, and imperatorin3, are involved and in isopimpinellin, the metabolism have of xenobiotics been shown including to have about anti-cancer 80% of activities current clinical in a murine drugs breast [50]. cancerExpression model of throughthese genes the inhibition to corresponding of P450 1A1 enzymes/1B1 metabolism (P450s 1A1, of 7,12-dimethylbenz[a]anthracene1A2, 1B1, 2A6, 2A13, 2B6, 2C8, (DMBA)2C9, 2C19, and 2D6, blocking 2E1, 2J2, DMBA-DNA 3A4, and 3A5) adduct is influenced formation by [48 combinations]. of different factors including geneticFigure polymorphisms,3 contains the structuressex, age, ethnicity, of a number general of natural health coumarin conditions, P450 and substrates induction and by inhibitors xenobiotics [ 45]. Coumarins[50]. exhibit significant species differences in metabolism and toxicity [37,40].

O OO O O OO

O

Bergamottin Methoxsalen

OOO OOO

O

Limettin Auraptene

O

O O O O O O O O O O

O

Imperatorin Isopimpinellin Angelicin Figure 3. Sample coumarin P450 substrates and inhibitors.

1.2.2. Coumarin Metabolism by P450s As mentioned previously, P450 metabolism of coumarins is species dependent. Coumarin has been found to be a rat liver and mouse lung toxicant due to the P450-dependant production of coumarin-3,4-epoxide by P450s 1A1, 1A2, and 2E1 in liver and P450 2F2 in lung (Figure4)[ 37,49]. 3-Hydroxycoumarin has been found as a minor metabolite of coumarin metabolism by recombinant rat P450s 1A1 and 1A2 [37]. Molecules 2019, 24, 1620 5 of 17 Molecules 2019, 24, x FOR PEER REVIEW 5 of 17

Figure 4. Coumarin metabolism leading to toxicity in rodents.

TheIn humans, human onlyP450 about1 family a dozen has three of the enzymes 57 CYP genes,from two those subfamilies, belonging P450s to CYP 1A1families and 1A2 1, 2, from and subfamily3, are involved 1A and in the P450 metabolism 1B1 from of subfamily xenobiotics 1B. including P450s 1A1 about and 80% 1B1 of are current primarily clinical extrahepatic drugs [50]. enzymesExpression while of these P450 genes 1A2 is to mainly corresponding found in enzymes the liver. (P450s With 72% 1A1, amino 1A2, 1B1, acid 2A6, sequence 2A13, similarity, 2B6, 2C8, 2C9, the enzymatic2C19, 2D6, 2E1,activities 2J2, 3A4,of P450s and 3A5)1A1 isand influenced 1A2 greatly by combinations overlap and of mainly different include factors the including hydroxylation genetic polymorphisms,and oxidation of sex, aromatic age, ethnicity, compounds general healthincludin conditions,g polycyclic and inductionaromatic byhydrocarbons xenobiotics [50(PAHs).]. CoumarinThe human is metabolized P450 1 family by these has human three enzymes enzymes from into twocomarin-3,4-epoxide subfamilies, P450s at 1A1a much and lower 1A2 from rate thansubfamily observed 1A and in P450rodents, 1B1 fromand subfamilythus does 1B. not P450s cause 1A1 the and same 1B1 arehigh primarily toxicity extrahepatic [49]. P450 enzymes1B1 has whilerelatively P450 low 1A2 amino is mainly acid found sequence in the similarity liver. With with 72% both amino P450 acid 1A1 sequence and P450 similarity, 1A2, 38% the enzymatic and 37% respectively,activities of P450s however, 1A1 and it in 1A2 general greatly has overlap a similar and active mainly site include cavity the shape hydroxylation and size (441 and oxidationÅ3 for 1B1, of 469aromatic Å3 for compounds 1A2, and 524 including Å3 for polycyclic 1A1) leading aromatic to significant hydrocarbons substrate (PAHs). specificity Coumarin overlap is metabolized with these enzymesby these human(such as enzymes PAHs, heterocycle into comarin-3,4-epoxide aromatic amines, at aand much estradiol) lower rate[24]. thanP450s observed 1A1, 1A2, in and rodents, 1B1 doand not thus show does much not cause coumarin the same 7-hydroxylase high toxicity activi [49].ty. P450 P450 1B1 1B1 has also relatively does lownot aminoshow coumarin acid sequence 3,4- epoxidasesimilarity withactivity. both 3-Hydoxycoumarin P450 1A1 and P450 has 1A2, been 38% sh andown 37% to respectively,form as a minor however, metabolite it in general during has the a incubationsimilar active of sitecoumarin cavity shapewith recombinant and size (441 huma Å3 forn 1B1,P450 469 1A1 Å3 or for P450 1A2, 1A2 and [37]. 524 Å3All for three 1A1) enzymes leading showto significant 7-alkoxycoumarin substrate specificity dealkylation overlap activities with these [48]. enzymes The (such order as PAHs,of rates heterocycle of 7-ethoxy-4- aromatic trifluoromethylcoumarinamines, and estradiol) [24 deethylation]. P450s 1A1, by 1A2, these and three 1B1 enzymes do not show has muchbeen shown coumarin to be 7-hydroxylase P450 1A1 > P450activity. 1B1 P450 > P450 1B1 1A2 also [51]. does not show coumarin 3,4-epoxidase activity. 3-Hydoxycoumarin has been shownThere to form are 16 as CYP a minor2 genes metabolite in humans, during and themost incubation show high of levels coumarin of genetic with polymorphisms recombinant human [50]. FromP450 1A1the orCYP P4502A 1A2 subfamily, [37]. All only three CYPs enzymes 2A6 show and 7-alkoxycoumarin2A13 are functional dealkylation and both activitiesshow significant [48]. The geneticorder of polymorphisms. rates of 7-ethoxy-4-trifluoromethylcoumarin P450 2A6 is mainly hepatic, deethylationwhile P450 2A13 by these is mainly three enzymes expressed has in been the respiratoryshown to be tract. P450 1A1Most> P450substrates 1B1 > P450for these 1A2 [51enzymes,]. which have 94% amino acid sequence similarity,There are 16non-planar,CYP2 genes low in molecular humans, and weight most compounds show highlevels which of contain genetic two polymorphisms hydrogen bond [50]. acceptorsFrom the [50].CYP 2AThe subfamily, two only onlydifferCYPs in 32 2A6amino and acids, 2A13 ten are of functional which are and located both in show their significant relatively genetic polymorphisms. P450 2A6 is mainly hepatic, while P450 2A13 is mainly expressed in the small active sites (<300 Å3) [8,24]. P450 2A6 is responsible for the metabolism of about 3% of respiratory tract. Most substrates for these enzymes, which have 94% amino acid sequence similarity, clinically used drugs (such as disulfiram, halothane, and tegafur) in addition to the metabolism and are non-planar, low molecular weight compounds which contain two hydrogen bond acceptors [50]. bioactivation of tobacco nitrosamines (nicotine and NNK (4-methylnitrosamino-1-3-pyridyl-1- The two only differ in 32 amino acids, ten of which are located in their relatively small active sites butanone)) [50]. Polymorphisms in P450 2A6 are responsible for individual differences in the rate of (<300 Å3) [8,24]. P450 2A6 is responsible for the metabolism of about 3% of clinically used drugs nicotine metabolism, smoking behavior, and cancer risk associated with tobacco use [50]. P450 2A13 (such as disulfiram, halothane, and tegafur) in addition to the metabolism and bioactivation of tobacco is similar in substrate specificity in general. However, it is much more efficient in the bioactivation nitrosamines (nicotine and NNK (4-methylnitrosamino-1-3-pyridyl-1-butanone)) [50]. Polymorphisms of NNK [50]. Both enzymes are known to catalyze coumarin 7-hydroxylation and 7-alkoxycoumarin in P450 2A6 are responsible for individual differences in the rate of nicotine metabolism, smoking dealkylation [29,50]. The deethylation of 7-ethoxycoumarin and the demethylation of 7- behavior, and cancer risk associated with tobacco use [50]. P450 2A13 is similar in substrate specificity in methoxycomarin have been shown to produce both 7-hydroxycoumarin and 3-hydroxycoumarin as general. However, it is much more efficient in the bioactivation of NNK [50]. Both enzymes are known products, though the 3-hydroxylation was observed at a greater extent during the deethylation to catalyze coumarin 7-hydroxylation and 7-alkoxycoumarin dealkylation [29,50]. The deethylation of reaction [29,52]. Since position 7 has been shown to be the closest to the heme-iron, the production 7-ethoxycoumarin and the demethylation of 7-methoxycomarin have been shown to produce both of the 3-hydroxy product implies rotation of the substrate during the reaction to produce this 7-hydroxycoumarin and 3-hydroxycoumarin as products, though the 3-hydroxylation was observed at a product [53,54]. P450 2A6 is the major coumarin 7-hydroxylase in the human liver, and the X-ray greater extent during the deethylation reaction [29,52]. Since position 7 has been shown to be the closest crystal structure of the enzyme-substrate complex has been published showing a tight fit of the to the heme-iron, the production of the 3-hydroxy product implies rotation of the substrate during the coumarin molecule in the small P450 2A6 active site (260 Å3) [29,30]. Neither 2A6, nor 2A13, reaction to produce this product [53,54]. P450 2A6 is the major coumarin 7-hydroxylase in the human produce 3-hydroxycoumarin during oxidation of coumarin [29]. From the CYP2B subfamily, only liver, and the X-ray crystal structure of the enzyme-substrate complex has been published showing a CYP2B6 is functional in humans [55]. P450 2B6 has been shown to efficiently O-deethylate 7-ethoxy- tight fit of the coumarin molecule in the small P450 2A6 active site (260 Å3) [29,30]. Neither 2A6, nor 4-trifluoromethylcomarin [50]. Mammalian P450s 2B35 and 2B37 from the desert woodrat (Neotoma 2A13, produce 3-hydroxycoumarin during oxidation of coumarin [29]. From the CYP2B subfamily, lepida) have been studied for O-dealkylation activities with series of 7-alkoxycoumarins, 7-alkoxy-4- trifluoromethylcoumarins, and 7-alkoxy-4-methylcoumarins with a C1-C7 side chain, and have Molecules 2019, 24, 1620 6 of 17 only CYP2B6 is functional in humans [55]. P450 2B6 has been shown to efficiently O-deethylate 7-ethoxy-4-trifluoromethylcomarin [50]. Mammalian P450s 2B35 and 2B37 from the desert woodrat (Neotoma lepida) have been studied for O-dealkylation activities with series of 7-alkoxycoumarins, 7-alkoxy-4-trifluoromethylcoumarins, and 7-alkoxy-4-methylcoumarins with a C1-C7 side chain, and have been shown to display differences in their substrate selectivity based on the length of the O-alkyl group [31]. P450 2B35 shows higher activity in O-dealkylation of long-chain coumarin derivatives (7 and 6 carbon chains), while P450 2B37 shows preference for short-chain coumarin derivatives (such as 7-ethoxy-4-trifluoromethylcoumarin) [31]. The crystal structures of these two enzymes in complex with 4-(4-chlorophenyl)imidazole (4-CPI) have confirmed that P450 2B35 fits two 4-CPI molecules in its active site, while P450 2B37 only fits one in the active site, though it has an open conformation with two more 4-CPI molecules in its substrate access channel [31]. Rabbit P450 2B4 has a 78% amino acid sequence similarity with human P450 2B6. Crystal structures of P450s 2B4 and 2B6 have been shown to only contain one 4-CPI molecule in their active sites [56,57]. P450s 2B4 and 2B6 also show coumarin substrate selectivity based on the side chain length at position 7, as well as substitution at position 4 [55]. These observations confirm that P450 2B family enzymes show significant differences within their flexible active sites and substrate access channels across various species, and bind a wide range of ligand sizes and shapes [31]. The CYP2C subfamily members in humans are CYPs 2C8, 2C9, 2C18, and 2C19; though 2C18 mRNA is not efficiently translated to protein, and thus this enzyme is not expressed in high concentrations [50]. Polymorphisms in these genes significantly affect drug metabolism. P450 2C9 is the main enzyme from this subfamily involved in the metabolism of coumarins, and polymorphisms have been shown to lead to coumarin sensitivity and toxicity, especially for patients on coumarin anti-coagulants (such as warfarin, acenocoumarol, and phenprocoumon) [58]. Warfarin is used as a racemic mixture of R and S enantiomers, however, the S enantiomer is 2–5 times more potent. Both enantiomers are hydroxylated by P450 enzymes during metabolism leading to their excretion in urine (Figure5). S-warfarin is mainly metabolized by P450 2C9 to the 6-, and 7-hydroxy metabolites which are easily excreted and limit the drug’s toxicity. R-warfarin is metabolized by P450s 1A1, 1A2, and 3A4, so one enzyme containing a polymorphism does not have as much effect [59]. CYP2D6 is the only active gene from this subfamily in humans [50]. P450 2D6 is non-inducible, however, its hepatic concentration varies significantly between individuals due to polymorphisms [60]. This enzyme is involved in the metabolism of 15–25% of clinically used drugs, thus its genetic polymorphisms play important roles in the upregulation or downregulation of their pharmacological and toxicological effects [50]. P450 2D6 has been shown to perform 7-dealkylation reactions on 7-alkoxycoumarins such as 7-methoxy-4-aminomethylcoumarin [61]. Human P450 2E1 is an inducible enzyme and mainly metabolizes small polar molecules such as ethanol but also has some larger aromatic substrates including coumarins. P450 2E1 metabolism of ethanol leads to the formation of reactive oxygen species leading to liver cell damage in heavy drinkers [50]. Metabolism of coumarins by this enzyme leads to epoxidation at the 3,4 positions, dealkylation at the 7 position, and/or hydroxylation at different positions as previously discussed [37,49,62]. In addition, human P450 2F1 also efficiently catalyzes 7-ethoxycoumarin O-deethylation [63]. From the P450 family 3, the main human genes are CYP3A4, CYP3A5, CYP3A7, and CYP3A43. However, only P450s 3A4 and 3A5 are important in the metabolism of xenobiotics and clinical drugs, both of which show high levels of polymorphism. Tacrolimus, an immunosuppressant drug used in transplant patients, is metabolized by P450 3A5; thus, P450 3A5 genetic polymorphism testing is important to determine the correct dosage based on the patients’ rate of drug metabolism. P450s 3A4 and 3A5 have large active sites (~1400 Å3), over 85% amino acid sequence similarity, and are known to metabolize large lipophilic molecules [50]. These enzymes debenzylate 7-benzyloxy-4- trifluoromethylcoumarin, and their activities can be inhibited by some natural coumarins such as bergamottin leading to drug interactions [45–48,50,64]. Molecules 2019, 24, x FOR PEER REVIEW 7 of 17 Molecules 2019, 24, 1620 7 of 17 Molecules 2019, 24, x FOR PEER REVIEW 7 of 17

OH O OH O OH O OH O

O O O O O O O O S-Warfarin R-Warfarin S-Warfarin Figure 5. Sites of warfarin hydroxylation R-Warfariby P450s. n Figure 5. SitesSites of of warfarin warfarin hydroxylation hydroxylation by by P450s. P450s. Recent explorations of controlled drug delivery have pointed to engineering of photo- responsiveRecent explorationsnanoparticlesexplorations of of controlledas controlledsite targeted drug drug delivery dr deliug very havedelivery pointedhave systemspointed to engineering [65–67].to engineering ofThese photo-responsive engineeredof photo- responsivenanoparticles nanoparticles should as site targeted have theas drug abilitysite deliverytargeted to hold systems ondr ugto the [delivery65 biologically–67]. Thesesystems active engineered [65–67]. molecule nanoparticles These (drug) engineered until should they nanoparticlesreachhave thethe abilitytargeted should to site, hold have where on the to uponability the biologically the to holdirradiatio on active ton the of molecule thebiologically photo-responsive (drug) active until molecule group they reach (“photo-cage”(drug) the until targeted they or reach“cagingsite, where the group”) targeted upon thesite, irradiationmolecule where upon in of its the active irradiatio photo-responsive staten ofcould the photo-responsive groupbe released (“photo-cage” [68,69]. group Due or (“photo-cage” “caging to their group”) water or “cagingsolubility,the molecule group”) versatility in its the active ofmolecule their state incorporation couldin its be active released into state varied [68 could,69 ].types Duebe ofreleased to nanoparticles, their water[68,69]. solubility, andDue their to versatilitytheir absorption water of solubility,intheir the incorporation visible versatility part of into theof their variedspectrum, incorporation types coumarins of nanoparticles, into have varied elicited andtypes theirextreme of nanoparticles, absorption interest inin theandthis visible area.their Theabsorption part photo- of the inresponsivespectrum, the visible coumarinsproperties part of the haveof spectrum, coumarins elicited coumarins extreme have made interest have th elicited inem this the area. extremephoto-cage The interest photo-responsive molecule in this of area. choice properties The forphoto- the of responsiverecentcoumarins studies haveproperties on made drug themof delivery. coumarins the photo-cage One have of the moleculemade main th limitationsem of choicethe photo-cage for of thesuch recent strategymolecule studies is ofthe on choice drugtoxicity/drug delivery.for the recentinteractionsOne of studies the main of on the limitations drug coumarins delivery. of such and One strategy the of theproducts is main the toxicity limitationsof metabolism/drug of interactions such of strategythe ofreleased the is coumarins the coumarins toxicity/drug and theby interactionsproductscytochrome of metabolismP450of the enzymes. coumarins of the Figure released and 6 theshows coumarins products the coumarin by of cytochrome metabolism structures P450 of that enzymes.the havereleased Figureshown coumarins6 promiseshows the byin cytochromephoto-cagingcoumarin structures P450 [68,70]. enzymes. that have Figure shown 6 promiseshows the in photo-cagingcoumarin structures [68,70]. that have shown promise in photo-caging [68,70].

Figure 6. Structures of coumarin caging molecules [68,70]. [68,70].

1.2.3. P450 Inhibition MechanismFigure 6. Structures by Coumarins of coumarin caging molecules [68,70]. 1.2.3. P450 Inhibition Mechanism by Coumarins P450 inhibition follows two distinct mechanisms, direct competitive inhibition and time-dependent 1.2.3.P450 P450 Inhibitioninhibition Mechanismfollows two by distinct Coumarins mechanisms, direct competitive inhibition and time- inhibition. Direct competitive inhibitors are substrate-like molecules capable of entering and temporarily dependentP450 inhibition inhibition. follows Direct competitivetwo distinct inhibitors mechanisms, are substrate-like direct competitive molecules inhibition capable ofand entering time- binding to the enzyme active site while being metabolized; once the metabolites are released, the enzyme dependentand temporarily inhibition. binding Direct to thecompetitive enzyme activeinhibitors site arewhile substrate-like being metabolized; molecules once capable the metabolitesof entering returns back to its free state. For these compounds to be effective inhibitors, they have to show higher andare released,temporarily the binding enzyme to returnsthe enzyme back activeto its sitefree while state. being For metabolized;these compounds once theto bemetabolites effective affinity toward the enzyme than its natural substrates, thus keeping the enzyme busy and reducing areinhibitors, released, they the have enzyme to show returns higher back affinity to its to wardfree state.the enzyme For these than compounds its natural substrates,to be effective thus its substrate turn-around rate. Time-dependent inhibitors, also often resemble natural substrates in inhibitors,keeping the they enzyme have busyto show and higherreducing affinity its substr towardate turn-aroundthe enzyme rate.than Time-dependentits natural substrates, inhibitors, thus structure, giving them access to the enzyme active site; in addition, enzyme incubation in their presence keepingalso often the resemble enzyme natura busy andl substrates reducing in itsstructure, substrate giving turn-around them access rate. toTime-dependent the enzyme active inhibitors, site; in leads to increased inhibition. Mechanism-based (suicide) inhibitors are time-dependent inhibitors that alsoaddition, often enzymeresemble incubation natural substrates in their in presence structure, leads giving to themincreased access inhibition. to the enzyme Mechanism-based active site; in addition, enzyme incubation in their presence leads to increased inhibition. Mechanism-based Molecules 2019, 24, x FOR PEER REVIEW 8 of 17

(suicide) inhibitors are time-dependent inhibitors that are metabolized by the enzyme to reactive intermediates which covalently bond the active site residues and permanently inhibit the enzyme’s activity [71–73]. This process is both time- and cofactor-dependent. Coumarins can act as substrates, direct competitive inhibitors, or time-dependent inhibitors. Our group and collaborators have focused on the development of selective mechanism-based inhibitors for certain P450 enzymes involved in carcinogenesis including a number of coumarin derivatives [13–23,38,74]. P450s 1A1, 1A2, 1B1, 2A6, 2E1, and 3A4 are responsible for about 77% of all bioactivation reactions catalyzed by P450s, thus their inhibition has been studied as a potential means of cancer prevention [1,13,16,18,20,21,24,25,74,75]. However, P450 inhibitors have the potential for inducing the over-expression of P450 enzymes by activating the aryl hydrocarbon receptor (AhR) limiting their use as clinical cancer preventive agents. An ideal P450 inhibitor for use in cancer prevention should not cause AhR activation. Mechanism-based inhibitors could also beMolecules used 2019as probes, 24, 1620 of the enzyme active site structure and reaction mechanisms [1]. 8 of 17 P450 3A4 is involved in the bioactivation of environmental procarcinogens such as aflatoxin B1, aflatoxin G1, and dihydrodiol derivatives of PAHs [76]. 7-Coumarin propargyl ether and 7-(4- trifluoromethyl)coumarinare metabolized by the enzyme propargyl to reactive ether intermediates (structures which shown covalently in Figure bond 7) thewere active designed site residues and synthesizedand permanently by our inhibit group, the enzyme’scombining activity the structural [71–73]. This features process of isa both known time- substrate and cofactor-dependent. for P450s 3A4 andCoumarins 3A5, 7-benzyloxy-4-trifluoromethylcoumarin can act as substrates, direct competitive inhibitors,(BFC), with or time-dependent the propargyl inhibitors. functional group previouslyOur group shown and to collaboratorshave a potent haveial inhibitory focusedon effect the developmenton P450s [38]. of When selective correctly mechanism-based oriented, the terminalinhibitors triple for certain bond is P450 oxidized enzymes by the involved enzyme in into carcinogenesis a reactive ketene including intermediate a number through of coumarin a 1,2- hydrogenderivatives shift, [13– leading23,38,74 ].to P450sthe formation 1A1, 1A2, of 1B1,a covalent 2A6, 2E1, bond and with 3A4 a arenucleophilic responsible amino for about acid residue 77% of inall the bioactivation active site reactionsand loss of catalyzed enzymatic by P450s,activity thus [1,20]. their Both inhibition compounds has beenin this studied study asinhibited a potential the BFCmeans O-debenzylation of cancer prevention activity [1,13 of,16 P450,18,20 3A4,21, 24in, 25a ,74mechanism-based,75]. However, P450 manner inhibitors (time-, have concentration-, the potential andfor inducing NADPH-dependent) the over-expression [34]. Neither of P450 compound enzymes by showed activating mechanism-based the aryl hydrocarbon inhibition receptor of BFC (AhR) O- debenzylationlimiting their useby P450 as clinical 3A5, showing cancer preventive selectivity agents.for P450 An 3A4 ideal [38]. P450 This inhibitor observed for variation use in cancerin the catalyticprevention activity should of not the cause two AhRenzymes activation. with these Mechanism-based coumarin derivatives inhibitors has could been also attributed be used as to probes their differenceof the enzyme in 78 active amino site acids structure (out of and the reaction total 503 mechanisms in their sequences), [1]. of which 17 are located in their substrateP450 recognition 3A4 is involved sites in the(SRSs) bioactivation leading to of different environmental ligand-enzyme procarcinogens interactions such as aflatoxin [38]. B1, aflatoxin G1, andAs dihydrodiolpreviously discussed, derivatives P450s of PAHs 1A1 [76 and]. 7-Coumarin 1A2 are involved propargyl in ether the andbioactivation 7-(4-trifluoromethyl) of many environmentalcoumarin propargyl chemicals ether such (structures as PAHs shown[1,74,75]. in P450 Figure 1A17) prefers were designedlarger planar and molecules synthesized such by as benzo[a]pyreneour group, combining and DMBA, the structuralwhile P450 features 1A2 prefers of a known smaller substrate planar triangle-shaped for P450s 3A4 molecules and 3A5, including7-benzyloxy-4-trifluoromethylcoumarin aryl and heterocyclic amines (BFC), [74,75]. with A the series propargyl of 7-ethynylcoumarin functional group previouslyderivatives shown were designed,to have a potentialsynthesized, inhibitory and evaluated effect on P450sby us [ 38for]. po Whentential correctly inhibitory oriented, effects the and terminal selectivity triple toward bond is P450soxidized 1A1, by the1A2, enzyme 2A6, and into 2B1 a reactive [22]. keteneThese intermediatecompounds were through also a 1,2-hydrogendesigned by shift,combining leading the to coumarinthe formation backbone, of a covalent a known bond P450 with substrate, a nucleophilic with the amino ethynyl acid functional residue in group the active previously site and shown loss of toenzymatic cause mechanism-based activity [1,20]. Both inhibition compounds [1,38]. in All this of study the compounds inhibited the in BFC this O-debenzylationstudy, except 7-ethynyl-3- activity of methyl-4-phenylcoumarinP450 3A4 in a mechanism-based (7E3M4PC) manner showed (time-, concentration-,mechanism-based and inhibition NADPH-dependent) of P450s 1A1 [34 ].and Neither 1A2, andcompound no inhibition showed of P450s mechanism-based 2A6 and 2B1 inhibition[22]. The most of BFC potent O-debenzylation and selective compound by P450 3A5, in the showing study wasselectivity 7-ethynyl-3,4,8-trimethylcouma for P450 3A4 [38]. This observedrin (structure variation shown in the in catalytic Figure activity 8) [22]. of Methoxsalen the two enzymes has been with shownthese coumarin to inhibit derivatives P450 2A6 but has lacks been selectivity attributed as to theirit also di inhibitsfference other in 78 P450 amino enzymes acids (out such of as the P450s total 3A4503 inand their 2D6. sequences), 5-Methoxycoumarin of which 17 and are located6-methoxycoumarin in their substrate have recognition been shown sites to (SRSs)inhibit leadingP450 2A6 to selectivelydifferent ligand-enzyme (IC50 values of interactions 0.13 and 0.64 [38 mM,]. respectively) [77].

O OO O OO

CF3 7-Coumarin propargyl ether 7-(4-Trifluoromethyl)coumarin propargyl ether Figure 7. StructuresStructures of of two two coumarin cytochrome P450 3A4 mechanism-based inhibitors [[38].38].

As previously discussed, P450s 1A1 and 1A2 are involved in the bioactivation of many environmental chemicals such as PAHs [1,74,75]. P450 1A1 prefers larger planar molecules such as benzo[a]pyrene and DMBA, while P450 1A2 prefers smaller planar triangle-shaped molecules including aryl and heterocyclic amines [74,75]. A series of 7-ethynylcoumarin derivatives were designed, synthesized, and evaluated by us for potential inhibitory effects and selectivity toward P450s 1A1, 1A2, 2A6, and 2B1 [22]. These compounds were also designed by combining the coumarin backbone, a known P450 substrate, with the ethynyl functional group previously shown to cause mechanism-based inhibition [1,38]. All of the compounds in this study, except 7-ethynyl-3-methyl-4- phenylcoumarin (7E3M4PC) showed mechanism-based inhibition of P450s 1A1 and 1A2, and no inhibition of P450s 2A6 and 2B1 [22]. The most potent and selective compound in the study was 7-ethynyl-3,4,8-trimethylcoumarin (structure shown in Figure8)[ 22]. Methoxsalen has been shown to inhibit P450 2A6 but lacks selectivity as it also inhibits other P450 enzymes such as P450s 3A4 and 2D6. Molecules 2019, 24, 1620 9 of 17

Molecules5-Methoxycoumarin 2019, 24, x FOR PEER and REVIEW 6-methoxycoumarin have been shown to inhibit P450 2A6 selectively9 (IC of 1750 valuesMolecules of 2019 0.13, 24 and, x FOR 0.64 PEER mM, REVIEW respectively) [77]. 9 of 17

Figure 8. Structure of coumarin mechanism-based inhibitors with selectivity toward P450s 1A1 and 1A2Figure compared 8. Structure to P450s of coumarin 2A6 and mechanism-based2B1 [22]. inhibitors with selectivity toward P450s 1A1 and 1A2 compared toto P450sP450s 2A62A6 andand 2B12B1 [[22].22]. In a more recent study, our group designed and studied a number of furano-, pyrano-, pyridino-andIn aa more more dioxolo-coumarin recent recent study, study, our groupour derivatives group designed designed as and potential studied and inhibitors astudied number ofafor furano-,number P450 1A2 pyrano-,of furano-,[63]. pyridino-and One pyrano-, of the compoundsdioxolo-coumarinpyridino-and in dioxolo-coumarin this derivatives study, 4-trifluoromethyl-7,8-pyranocoumarin, as potentialderivatives inhibitors as potential for P450 inhibitors 1A2 [63 was ].for One foundP450 of the1A2 to compoundsbe [63]. a competitiveOne inof thisthe inhibitor,study,compounds 4-trifluoromethyl-7,8-pyranocoumarin, showing in this high study, selectivity 4-trifluoromethyl-7,8-pyranocoumarin, for the inhibition was found of this to beenzyme, a competitive was with found selectivity inhibitor, to be indicesa showing competitive ofhigh 155 andselectivityinhibitor, 52 for showing forP450 the 1A2 inhibition high over selectivity P450s of this 1A1 enzyme,for and the 1B1, inhibition with respectively selectivity of this (structure indicesenzyme, of withshown 155 andselectivity in 52 Figure for P450indices 9) [63]. 1A2 of This over 155 selectivityP450sand 52 1A1 for is andP450 of 1B1,importance 1A2 respectively over P450s since (structure1A1most and known 1B1, shown P450respectively in 1A2 Figure inhibitors 9(structure)[ 63]. Thisalso shown inhibit selectivity in P450sFigure is of1A1 9) importance [63]. and This1B1 (80%sinceselectivity and most 40%is known of aminoimportance P450 acid 1A2 sincesequence inhibitors most similarity, known also inhibit P450 respectively). P450s1A2 inhibitors 1A1 In and yeast also 1B1 inhibit (80%AhR andactivation P450s 40% 1A1 amino assays, and acid1B1 at concentrationssequence(80% andsimilarity, 40% loweramino respectively). than acid 1 μsequenceM, this In yeastpyranocoumarin similarity, AhR activation respectively). did assays, not activate In at yeast concentrations the AhR receptor activation lower suggesting thanassays, 1 thatµ M,at itthisconcentrations would pyranocoumarin not up-regulate lower did than not AhR-caused1 activateμM, this the pyranocoumarin receptorP450 enzyme suggesting expressiondid not that activate it would[63]. theThese not receptor up-regulate results suggesting show AhR-caused that that 4- trifluoromethyl-7,8-pyranocoumarinP450it would enzyme notexpression up-regulate [63 ].AhR-caused These results is selective P450 show enzyme that for 4-trifluoromethyl-7,8-pyranocoumarinthe expressioninhibition of[63]. P450 These 1A2 andresults has showthe is potential selective that 4- forfortrifluoromethyl-7,8-pyranocoumarin use the inhibitionin cancer-prevention of P450 1A2 [63]. and has is the selective potential for for the use inhibition in cancer-prevention of P450 1A2 and [63 ].has the potential for use in cancer-prevention [63].

O OO O OO

F F F F F FigureFigure 9. 9. StructureStructure of of 4-trifluorom 4-trifluoromethyl-7,8-pyranocoumarin.ethyl-7,8-pyranocoumarin.F

1.2.4. Computational MolecularFigure 9. Structure Modeling of 4-trifluorom Studies ethyl-7,8-pyranocoumarin. 1.2.4. Computational Molecular Modeling Studies Coumarin metabolism studies have shown that sites of oxidation are species dependent and 1.2.4.Coumarin Computational metabolism Molecular studies Modeling have shown Studies that sites of oxidation are species dependent and human P450 enzyme oxidation of coumarins mainly produces 7-hydroxycoumarin, with four other humanCoumarin P450 enzyme metabolism oxidation studies of coumarins have shown mainly that produces sites of oxidation 7-hydroxycoumarin, are species withdependent four other and products formed only in minor amounts due to 3,4-epoxidation, and hydroxylation at the 3-, 6-, or productshuman P450 formed enzyme only oxidation in minor ofamounts coumarins due mainlyto 3,4-epoxidation, produces 7-hydroxycoumarin, and hydroxylation withat the four 3-, 6-,other or 8- positions (Figure 10) [78–87]. Kinetic studies have been used to show that P450s 1A1, 1A2, 2B6 8-products positions formed (Figure only 10) in [78–87]. minor amountsKinetic studies due to have3,4-epoxidation, been used toand show hydroxylation that P450s at1A1, the 1A2, 3-, 6-, 2B6 or and8- positions 2E1 catalyze (Figure metabolism 10) [78–87]. of coumarin Kinetic studies to its 3,4-epoxide have been whichused to then show leads that to P450s the formation 1A1, 1A2, of 2B6the orthoand 2E1-hydroxyphenylacetaldehyde catalyze metabolism of coumarin as the metabolite to its 3,4-epoxide [37,82]. which P450 2A6then oxidizesleads to thecoumarin formation at theof the 7- positionortho-hydroxyphenylacetaldehyde leading to the formation as theof themetabolite 7-hydroxycoumarin [37,82]. P450 2A6metabolite oxidizes [37,82].coumarin P450 at the3A4 7- position leading to the formation of the 7-hydroxycoumarin metabolite [37,82]. P450 3A4 Molecules 2019, 24, x FOR PEER REVIEW 10 of 17 metabolism leads to the formation of two metabolites, namely, 3-hydroxycoumarin and ortho- hydroxyphenylacetaldehyde, through the coumarin 3,4-epoxidation pathway [82]. Computational molecular modeling tools have been used by several research groups to understand the interactions of coumarin molecules with the P450 enzymes. Studies have included docking studies of coumarins in the active sites of the P450 enzymes. Quantitative structure-activity relationship (QSAR) studies have provided important information on structural features of coumarins as P450 substrates [19,22,39,61,83,84]. The binding modes of coumarins vary depending on the nature and position of substituents and the P450 enzyme binding cavity topography [13,23]. Docking studies have been performed by Lewis et al. in order to understand the role of P450 binding cavity residues in the oxidative pathways of coumarin [39]. One of the key interactions shown by coumarins is the π-π stacking interactions with the phenylalanine residues in the binding pocket [39,84]. Additionally, interactions with a hydrogen-bonding residue determine the binding orientation of coumarin in the binding pocket and its site of oxidation [39,84]. The differences in spatial arrangements of the hydrogen-bonding residues and the phenylalanine residues have been shown to result in differences in the binding mode and product formation. For example, in P450 2A6, the Asn290 forms a hydrogen bond with the coumarin carbonyl group, resulting in the orientation of the 7- positionMolecules 2019 towards, 24, 1620 the heme-Fe, resulting in the formation of 7-hydroxycoumarin [39]. In P45010 3A4, of 17 the hydrogen bonding of the coumarin carbonyl with a Ser119 in the binding pocket leads to the 3- position facing the heme-Fe, and the formation of the 3-hydroxycoumarin [39]. and 2E1Coumarin catalyze is metabolismthe marker ofsubstrate coumarin for tothe its cytoch 3,4-epoxiderome P450 which 2A then family, leads consisting to the formation of P450s of 2A6 the andortho 2A13.-hydroxyphenylacetaldehyde As previously mentioned, as the these metabolite enzymes [37, 82have]. P450 94% 2A6 amino oxidizes acid coumarin sequence at theidentity. 7-position The catalyticleading to efficiency the formation of coumarin of the 7-hydroxycoumarin 7-hydroxylation was metabolite found to [37 be,82 higher]. P450 for 3A4 P450 metabolism 2A6 than leads P450 to 2A13the formation (10 fold) ofby two He metabolites,et al. [85], while namely, Weymarn 3-hydroxycoumarin and Murphy andfoundortho that-hydroxyphenylacetaldehyde, both enzymes had similar efficiencythrough the [86]. coumarin DeVore 3,4-epoxidationet al. measured pathwayspectral binding [82]. Computational affinities (KD) molecular for coumarin modeling and found tools equal have bindingbeen used affinities by several to both research P450s groups2A6 and to 2A13 understand [87]. The the planar interactions active site of and coumarin Asn297 molecules are conserved with inthe both P450 enzymes. enzymes. DeVore Studies et have al. performed included docking receptor-based studies ofQSAR coumarins studies in using the activethe comparative sites of the bindingP450 enzymes. energy Quantitative(COMBINE) structure-activityapproach and identified relationship individual (QSAR) protein/ligand studies have providedinteractions. important These studiesinformation revealed on structural that the binding features orientation of coumarins was assimilar P450 for substrates both proteins [19,22, 39with,61 ,coumarin83,84]. The forming binding a hydrogenmodes of coumarinsbond to Asn297. vary depending This was onconsistent the nature with and both position enzymes of substituents generating and 7-hydroxycoumarin the P450 enzyme withbinding identical cavity K topographyD values. While [13,23 P450]. Docking 2A6 exclusively studies have formed been performed7-hydroxycoumarin, by Lewis et von al. inWeymarn order to andunderstand Murphy the [86] role found of P450 that binding P450 cavity2A13 formed residues significant in the oxidative amounts pathways of 3,4-epoxide of coumarin and [39 smaller]. One amountsof the key of interactions 6-hydroxycoumarin shown by coumarinsand 8-hydroxycou is the π-πmarinstacking in interactionsaddition to with 7-hydroxycoumarin. the phenylalanine DeVoreresidues et in al. the found binding that pocket the approach [39,84]. Additionally,path of coumarin interactions substrate with to athe hydrogen-bonding activated oxygen residueon the hemedetermine was theless binding constrained orientation in P450 of coumarin2A13 than in in the P450 binding 2A6, pocket leading and to its hydroxylation site of oxidation at [the39, 846-]. or The 8- positions,differences and in spatial also arrangementsthe inverted oforientation the hydrogen-bonding of coumarin residues that andresulted the phenylalanine in 3,4-eopxide residues [87]. Comparisonhave been shown of the to active result site in differences residues between in the binding P450s mode2A6 and and 2A13 product revealed formation. that For10 out example, of 32 residuesin P450 2A6, differ the between Asn290 formsthe two a hydrogenenzymes, bondpotentia withlly the correlating coumarin to carbonyl the functional group, resulting differences in the in substrateorientation metabolism. of the 7-position The COMBINE towards the QSAR heme-Fe, model resulting generated in the by formation DeVore ofet 7-hydroxycoumarinal. suggested that steric [39]. rolesIn P450 played 3A4, theby the hydrogen active bondingsite residues of the were coumarin primarily carbonyl responsible with a Ser119for the in differing the binding ligand pocket affinities leads [87].to the 3-position facing the heme-Fe, and the formation of the 3-hydroxycoumarin [39].

2A13

2A6, 2A13

OO 8 1 7 2 6 3 3A4 5 4

2A13 1A1, 1A2, 2A13, 2B6, 2E1, 3A4 Figure 10. Oxidation ofof coumarincoumarin atat various various positions positions by by di ffdifferenterent cytochrome cytochrome P450 P450 enzymes enzymes [78 –[78–87]. 87]. Coumarin is the marker substrate for the cytochrome P450 2A family, consisting of P450s 2A6 and 2A13. As previously mentioned, these enzymes have 94% amino acid sequence identity. The catalytic efficiency of coumarin 7-hydroxylation was found to be higher for P450 2A6 than P450 2A13 (10 fold) by He et al. [85], while Weymarn and Murphy found that both enzymes had similar efficiency [86]. DeVore et al. measured spectral binding affinities (KD) for coumarin and found equal binding affinities to both P450s 2A6 and 2A13 [87]. The planar active site and Asn297 are conserved in both enzymes. DeVore et al. performed receptor-based QSAR studies using the comparative binding energy (COMBINE) approach and identified individual protein/ligand interactions. These studies revealed that the binding orientation was similar for both proteins with coumarin forming a hydrogen bond to Asn297. This was consistent with both enzymes generating 7-hydroxycoumarin with identical KD values. While P450 2A6 exclusively formed 7-hydroxycoumarin, von Weymarn and Murphy [86] found that P450 2A13 formed significant amounts of 3,4-epoxide and smaller amounts of 6-hydroxycoumarin and 8-hydroxycoumarin in addition to 7-hydroxycoumarin. DeVore et al. found that the approach path of coumarin substrate to the activated oxygen on the heme was less constrained in P450 2A13 than in P450 2A6, leading to hydroxylation at the 6- or 8- positions, and also the inverted orientation of coumarin that resulted in 3,4-eopxide [87]. Comparison of the active site residues between P450s 2A6 and 2A13 revealed that 10 out of 32 residues differ between the two enzymes, potentially correlating to Molecules 2019, 24, 1620 11 of 17 the functional differences in substrate metabolism. The COMBINE QSAR model generated by DeVore et al. suggested that steric roles played by the active site residues were primarily responsible for the differing ligand affinities [87]. Homology models of rat and human P450 2D isozymes (2D1, 2D2, 2D3, 2D4, and 2D6) have been built and studied [61]. Docking followed by molecular dynamic simulations were carried on 7-methoxy-4-aminomethylcoumarin [61]. The models showed that the Asp301 residue of these enzymes form hydrogen bonds with the basic nitrogen of 7-methoxy-4-aminomethylcoumarin, resulting in its O-demethylation [61]. Docking studies were performed in our laboratory for a series of ethynylcoumarins with P450 enzymes 1A1 and 1A2 using the LigandFit in the Accelrys DS studio® (Figure 11)[22]. These studies showed that π-π interactions of the coumarins with the phenylalanine residues of the active site are critical determinants of the binding mode of the inhibitors. Changes in orientations guided by the position of the phenyl substituents dictate the nature of inhibition as a competitive or time-dependent inhibitor of the P450 1A2 enzyme. In the absence of a phenyl substituent, the ethynyl groups were the closest to the heme-Fe, enabling the generation of a highly reactive oxidation intermediate that could then react with the receptor residues to form covalent interactions leading to a time-dependent inhibition and inactivation of the enzyme. The presence of a 3-phenyl substituent on the coumarin ring flipped the molecule’s orientation in the active site to facilitate the phenyl group’s facing the heme-Fe, and the ethynyl group facing away from the heme. This leads to these molecules acting as competitive inhibitors of P450 1A2. P450 1A1 has a larger active site cavity in comparison to P450 1A2. This results in multiple binding modes for the ethynyl coumarin series studied leading to some of the binding modes orienting the ethynyl group in close proximity to the heme-Fe causing time-dependent inhibition by all of these compounds. QSAR studies were performed on a series of 7-coumarin propargyl ethers using comparative molecular field analysis (CoMFA), comparative molecular similarity index analysis (CoMSIA) and hologram quantitative structure-activity relationship (HQSAR) modules in Tripos-Sybyl [19]. These analyses showed that there are equal contributions from steric, electrostatic and ClogP descriptors towards the inhibition potency of these compounds toward P450 1A2, indicating that lipophilicity is an important factor. The contour maps of steric and electrostatic plots were generated for the binding pocket of P450 1A2 [19]. Lewis et al. performed QSAR analysis on a series of 7-alkoxycoumarins that are used as diagnostic probes for the P450 2B enzymes [84]. The logP data which is a lipophilicity index was obtained experimentally and by calculations via the ClogP. They found a quadratic relationship between the logP of 7-alkoxycoumarins and the experimentally determined logarithm of the dealkylation activity in phenobarbital-induced animals. Hydrophobicity was found to be a key factor in the P450 2B and substrate interactions. Additional interactions such a hydrogen bonding and pi-pi interactions were also found to be relevant. Leong et al. used a hierarchical support vector regression (HVSR) approach to predict the interactions of several drugs with P450 2B6 [88]. Cytochrome P450 2B6 is a hepatic enzyme constituting 1% of the total hepatic P450 complement [89], but metabolizes 3% of the clinical drugs [90]. Five coumarins, 7-ethoxycoumarin, 7-ethoxy-4-trifluoromethylcoumarin, 7-methoxy-4-trifluoromethylcoumarin, 3-cyano -7-ethoxycoumarin, and 4-chloromethyl-7-ethoxycoumarin were used in this study [88]. The generated HVSR model showed a relation between the experimental and predicted values, which were statistically true (correlation coefficient q2 of 0.93 and r2 values of 0.97 and 0.82 for the training set and test set, respectively for the 10-cross validation of the model) [88]. Out of a total of 10 descriptors, logP’s were shown to be of critical importance, and the descriptors relating to the molecular size and shape were also important contributors to the model [88]. The docking and QSAR studies by various research groups have provided a deeper understanding of the nature and types of interactions of coumarins with the active site residues of P450 1A, 1B, 2B, 2C, 2D and 3A enzymes. Molecules 2019, 24, 1620 12 of 17 Molecules 2019, 24, x FOR PEER REVIEW 12 of 17

Figure 11. ReprintedReprinted with with permission permission from from reference 22. ( (AA)) Diagram Diagram of of the the active active site site cavity of P450 1A2: 1A2: a anarrow, narrow, but but extended extended channel channel (part (part A),A and), and a spherical a spherical hydrophobic hydrophobic pocket pocket (part (part B). (B).) The(B) Thebinding binding patterns patterns of compound of compound 7ETMC 7ETMC with with P450 P450 1A2, 1A2, which which suggests suggests that that the themidsize midsize 7- ethynylcoumarins7-ethynylcoumarins could could freely freely rota rotatete in in the the narrow narrow cavity cavity (part (part AA) )if if only only it it is is parallel parallel with with Phe-226 and the peptide bond in the α-helix I in order toto formform π––ππ interactions.interactions. ( C) The possible binding patterns of compound 7E3PC with P450 1A2. Phenyl Phenyl-facing-heme-facing-heme orientation (left) takes an obvious priority overover thethe acetylene-facing-hemeacetylene-facing-heme orientation orientation (right). (right). (D ()D The) The possible possible binding binding pattern pattern (left) (left) and andimpossible impossible binding binding pattern pattern (right) (right) of compound of compound 7E3M4PC 7E3M4PC with with P450 P450 1A2, 1A2, suggesting suggesting that therethat there is no isreactive no reactive pose forpose the for docking the docking of 7E3M4PC of 7E3M4PC with thiswith enzyme. this enzyme.

2. Conclusion Conclusions Structure-activity relationshiprelationship studies studies through through bioassays bioassays and computational and computational molecular molecular modeling modelinghave provided have importantprovided important insights into insights the interaction into the interaction patterns andpatterns mechanisms and mechanisms of the action of the of actioncytochrome of cytochrome P450 enzymes P450 withenzymes their with substrates their subs andtrates inhibitors. and inhibitors. These studies These have studies led to have important led to importantfindings about findings the structuralabout the structural features required features forrequired substrate for substrate metabolism metabolism and/or inhibition and/or inhibition of these ofenzymes. these enzymes. Docking Docking studies and studies molecular and molecular dynamic simulationsdynamic simulations of protein-ligand of protein-ligand docked complexes docked π π complexeshave been have performed. been performed. Docking studiesDocking have studies shown have the shown importance the importance of - interactions of π-π interactions between betweencoumarins coumarins and the phenylalanine and the phenylalanine residues in the residues active site,in alongthe active with importantsite, along hydrogen-bonding with important hydrogen-bondingresidues that can change residues the that binding can modechange of th thee coumarins.binding mode The of optimum the coumarins. distances The between optimum the distancesheme-iron between and the the site heme-iron of oxidation and on the the site substrates of oxidation for the on various the substrates P450 enzymes for the have various also P450 been enzymesdetermined have through also been docking determined studies. through Ligand-based docking studiesstudies. such Ligand-based as QSAR studies andsuch structuralas QSAR studiesmeta-analysis and structural studies havemeta-analysis shown that studies three have descriptors- shown steric,that three electrostatic, descriptors- and steric, ClogP electrostatic, play critical androles ClogP in determining play critical the roles inhibition in determining potency of the coumarinsinhibition potency toward P450of the 1A2. coumarins toward P450 1A2.Funding: This research was funded by the NIH NIGMS BUILD grants (TL4GM118968, RL5GM118966, and Funding:UL1GM118967), This research the NIH-RCMI was funded grant by 2G12MD007595,the NIH NIGMS and BUILD the Louisianagrants (TL4GM118968, Cancer Research RL5GM118966, Center (LCRC), and including the LCRC Tobacco-Free Living Pilot Program. UL1GM118967), the NIH-RCMI grant 2G12MD007595, and the Louisiana Cancer Research Center (LCRC), includingConflicts ofthe Interest: LCRC Tobacco-FreeThe authors declareLiving Pilot no conflict Program. of interest.

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

Molecules 2019, 24, 1620 13 of 17

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