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7-2017 Review of Ligand Specificity Factors for CYP1A Subfamily from Molecular Modeling Studies Reported to-Date Jayalakshmi Sridhar Xavier University of Louisiana, [email protected]

Navneet Goyal Xavier University of Louisiana, [email protected]

Jiawang Liu Xavier University of Louisiana, [email protected]

Maryam Foroozesh Xavier University of Louisiana, [email protected]

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Recommended Citation Molecules 2017, 22(7), 1143; https://doi.org/10.3390/molecules22071143

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Review Review of Ligand Specificity Factors for CYP1A Subfamily Enzymes from Molecular Modeling Studies Reported to-Date

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

Department of Chemistry, Xavier University of Louisiana, 1 Drexel Dr., New Orleans, LA 70125, USA; [email protected] (J.S.); [email protected] (N.G.); [email protected] (J.L.) * Correspondence: [email protected]; Tel.: +1-504-520-5078; Fax: +1-504-520-7942

Received: 5 June 2017; Accepted: 3 July 2017; Published: 8 July 2017

Abstract: The P450 (CYP) family 1A enzymes, CYP1A1 and CYP1A2, are two of the most important enzymes implicated in the metabolism of endogenous and exogenous compounds through oxidation. These enzymes are also known to metabolize environmental procarcinogens into carcinogenic species, leading to the advent of several types of cancer. The development of selective inhibitors for these P450 enzymes, mitigating procarcinogenic oxidative effects, has been the focus of many studies in recent years. CYP1A1 is mainly found in extrahepatic tissues while CYP1A2 is the major CYP in human liver. Many molecules have been found to be metabolized by both of these enzymes, with varying rates and/or positions of oxidation. A complete understanding of the factors that govern the specificity and potency for the two CYP 1A enzymes is critical to the development of effective inhibitors. Computational molecular modeling tools have been used by several research groups to decipher the specificity and potency factors of the CYP1A1 and CYP1A2 substrates. In this review, we perform a thorough analysis of the computational studies that are ligand-based and -ligand complex-based to catalog the various factors that govern the specificity/potency toward these two enzymes.

Keywords: cytochrome; P450 1A1; P450 1A2; docking; quantitative structure activity studies (QSAR); molecular modeling; dynamics;

1. Introduction Research on enzymes has evolved over the years from the initial in-vitro substrate metabolism studies to the studies on the metabolism of drugs and the role of these enzymes in various diseases including cancer. The Project revealed that there are 57 human P450 (CYP) and 58 pseudogenes [1]. CYP enzymes are found in all living organisms and catalyze the mono-oxygenation of many different substrates. Mammalian cytochrome P450 enzymes can oxidize both endogenous compounds and , thereby playing critical roles in cholesterol and hormone synthesis, metabolism of endogenous compounds such as vitamin D, drug deactivation, and xenobiotics detoxification. The CYP1 family of P450 enzymes comprises of three members 1A1, 1A2, and 1B1. CYP1A1 and CYP1A2 are closely related with the CYP1A cluster that is mapped to 15q24.1, with head-to-head orientation, sharing a 23 kb bi-directional promoter and a common 50-flanking region [2]. CYP1A1 gene spans 5.8 kb and CYP1A2 gene spans 7.8 kb with seven exons and six introns [3,4]. CYP1A2 is the highly expressed cytochrome enzyme in the human liver (~13–15%) [5], while CYP1A1 expression levels are low in the liver (<0.7%), and it is mostly found in extrahepatic tissues [6,7].

Molecules 2017, 22, 1143; doi:10.3390/molecules22071143 www.mdpi.com/journal/molecules Molecules 2017, 22, 1143 2 of 19

2. CYP1A Substrates The span of enzymatic activities of CYP1A1 and CYP1A2 vastly overlaps with hydroxylations and oxidations of aromatic compounds including polycyclic aromatic hydrocarbons. and eicosapentoic acid, involved in synthesis of inflammation factors and hormones such as melatonin and 17β-estradiol, are endogenous substrates for CYP1A1 [8–10]. CYP1A1-dependent metabolic activation of procarcinogens into via epoxides is well established in cancer initiation [11,12]. Other CYP1A1 exogenous substrates include heterocyclic aromatic amines of industrial origin or those that are found in burnt food such as meat, fuel combustion products, and tobacco products [12,13]. Some of the endogenous substrates that are metabolized by both CYP1A1 and CYP1A2 include melatonin, 6-hydroxylate melatonin, and arachidonic acid, etc. Several other endogenous substrates are metabolized by CYP1A2 including estradiol, estrone, bilirubin, and uroporphyrinogen, etc. CYP1A2 metabolizes numerous natural products that result in toxic products such as transformation of methyleugenol to 10-hydroxymethyleugenol, estragole to reactive metabolites, oxidation of nephrotoxins, and aristolochic acids [14–16]. CYP1A2 plays an important role in the metabolism of several clinical drugs including analgesics, antipyretics, antipsychotics, antidepressants, anti-inflammatory, and cardiovascular drugs [17–20].

3. Structure and Catalytic Cycle The overall fold structure of all of the cytochrome P450 enzymes is identical, even though the sequence identify across the entire superfamily is less than 20%. The three-dimensional structure consists of 12 α-helices, A-L, that form the bulk of the protein, and four β-sheets. The heme is housed within the L-helix and the highly conserved I-helix that is perpendicular to the F/G segment comprising of the F-helix, F/G-loop and the G-helix. The heme Fe atom is bound to the sulfur atom of the Cys in the adjacent loop that has the highly conserved sequence FxxGx(HRK)xCxG. Substrate access and specificity is governed by B-C and F-G helices. The sequence identity between the two CYP1A1 (512 aa) and CYP1A2 (515 aa) enzymes is 71%. The X-ray crystal structure of CYP1A2 (2HI4) was reported by Johnson’s group in 2006 [21] and the CYP1A1 X-ray structure (PDB code: 4I8V) was reported by Scott’s group in 2013 [22]. The two CYP1A enzymes exhibit an overall similarity in their three-dimensional structures (Figure1). However, there are some key structural differences as exemplified by their X-ray crystal structures (PDB codes 4I8V and 2HI4 for CYP1A1 and CYP1A2, respectively). Scott et al. reported the structural variations between the two enzymes. CYP1A1 and CYP1A2 differ from other cytochrome P450 enzymes in the rigidity of the H/I loop, the break in the F helix, and the secondary structure of the β4 region. The H/I loop of CYP1A2 contains two short β-strands made of two residues each, that are not seen in the crystal structure of CYP1A1. The most important structural difference is seen in the F helices of CYP1A1 and CYP1A2. There is a three-residue break in the middle of the F helix in the CYP1A2 structure and this break is extended to five residues in the CYP1A1 structure. The F helix plays an important role in substrate access/egress and is also involved in the regioselectivity and stereoselectivity preferences. The secondary structure adopted by the β4 system of two strands is absent in the structures of CYP1A1 and CYP1A2. The turn of the β4 loop that lines the binding cavity is known to exhibit ligand interactions and provide ligand access. In addition to these differences, the B’ helix exhibits minor differences in hydrogen bonding pattern between the CYP1A1 and CYP1A2. The notable differences in the features that define the substrate access/regress paths and the substrate-binding cavity indicate that the CYP1A enzymes may indeed allow for substrate specificity in binding, orientation and the point of oxidation. The catalytic cycle of the cytochrome P450 enzymes can be commonly defined by the protein heme iron—oxygen roles through the cycle. The resting state of the enzyme involves a low-spin ferric enzyme with a water molecule coordinated as the sixth ligand of the heme iron atom. The binding of a substrate to the enzyme sometimes changes it to a high-spin ferric state for the enzyme-substrate complex. The next step in the cycle is the oxygen binding to the heme iron to form an oxy-iron-P450 complex which is a stable intermediate. The next sequence of steps occurs rapidly to form unstable intermediates: Molecules 2017, 22, 1143 3 of 19 reduction of the oxy-iron-P450 complex to form the peroxo-ferric-P450 complex, protonation of the Molecules 2017, 22, 1143 3 of 18 distal oxygen to form the hydroperoxo-iron-P450 complex, and a second protonation of the same oxygenprotonation followed of by the heterolytic distal oxygen cleavage to form of the the bond hydroperoxo between-iron the- twoP450 oxygen complex, atoms and leading a second to the formationprotonation of a reactive of the same high-valent oxygen followed iron-oxo-P450 by heterolytic complex cleavage which of the is bond generally between called the two ‘Compound oxygen 1’. Compoundatoms leading1 then to abstracts the formation a hydrogen of a reactive from thehigh substrate-valent iron with-oxo subsequent-P450 complex radical which recombination is generally to called ‘Compound 1’. Compound 1 then abstracts a hydrogen from the substrate with subsequent producecalled the ‘Compound oxidized substrate 1’. Compound that is1 egressed.then abstracts The aresting hydrogen state from of the substrate P450 enzymes with subsequent is regenerated radical recombination to produce the oxidized substrate that is egressed. The resting state of the P450 by theradical binding recombination of a water to molecule produce tothe the oxidized heme substrate iron atom that (Figure is egressed.2). The resting state of the P450 enzymes is regenerated by the binding of a water molecule to the heme iron atom (Figure 2).

Figure 1. (A) The X-ray crystal structures of CYP1A1 (4I8V.pdb, pink) and CYP1A2 (2HI4.pdb, green) FigureFigure 1. (A 1). The(A) The X-ray X-ray crystal crystal structures structures of of CYP1A1CYP1A1 (4I8V.pdb, (4I8V.pdb, pink) pink) and and CYP1A2 CYP1A2 (2HI4.pdb, (2HI4.pdb, green) green) are superposed as ribbon model. Atoms are shown as ball and stick models. Heme is colored white are superposed as ribbon model. Atoms are shown as ball and stick models. Heme is colored white and the substrate flavone is colored yellow. The total rmsd for these structures is 1.4 Å ; (B) Native and the substrate flavone is colored yellow. The total rmsd for these structures is 1.4 Å; (B) Native state state of the heme bound to Cys sulfur atom as the fifth ligand and the water molecule as the sixth of theligand. heme bound to Cys sulfur atom as the fifth ligand and the water molecule as the sixth ligand.

Figure 2. The catalytic cycle of oxidation of substrates by CYP enzymes is shown. The heme iron atom Figure 2. The catalytic cycle of oxidation of substrates by CYP enzymes is shown. The heme iron atom is anchored to the enzyme through the entire cycle by its covalent linkage to the sulfur atom of the is anchored to the enzyme through the entire cycle by its covalent linkage to the sulfur atom of the Cys residue. Cys residue.

Molecules 2017, 22, 1143 4 of 19

4. Inhibition of CYP1A Enzymes Molecules 2017, 22, 1143 4 of 18 The role of CYP1A1 and CYP1A2 in metabolizing procarcinogens to carcinogens leading to mutagenesis4. Inhibition and of tumorigenesis CYP1A Enzymes has been well documented [13,23–27]. Targeting these enzymes for inhibitionThe can role help of in CYP1A1 cancer and prevention CYP1A2 by in inhibiting metabolizing the procarcinogens conversion of environmental to carcinogens leading procarcinogens to to theirmutagenesis carcinogenic and tumorigenesis forms, inhibiting has been the well formation documented of [13,23 carcinogenic–27]. Targeting hormone these enzymes derivatives for from hormonalinhibition precursors, can help and in inhibiting cancer prevention the metabolic by inactivation inhibiting the of several conversion therapeutic of enviro agentsnmental including anticancerprocarcinogens drugs [to28 ]. their Several carcinogenic small forms, natural inhibiting compounds the formation are also of inhibitors carcinogenic of hormone CYP1A1 and CYP1A2.derivatives The quinazolinecarboline from hormonal precursors, alkaloids- and evodiamine, inhibiting dehydroevodiaminethe metabolic inactivation and rutaecarpine of several from Evodiatherapeutic rutaecarpa, agentshave including been traditionally anticancer used drugs for [28] the. treatment Several small of hypertension,natural compounds and gastrointestinal are also disordersinhibitors in Chinese of CYP1A1 medicine and [29 ]. CYP1A2. Naturally The occurring quinazolinecarboline flavonoids are well alkaloids known- forevodiamine, their inhibition dehydroevodiamine and rutaecarpine from Evodia rutaecarpa, have been traditionally used for the of toxicological processes and drug disposition. These natural inhibitors of CYP1A1 and CYP1A2 treatment of hypertension, and gastrointestinal disorders in Chinese medicine [29]. Naturally couldoccurring have an important are role well in knowncancer preventionfor their inhibition by reducing of toxicological the metabolism processes of procarcinogens and drug by thesedisposition. enzymes. These Thus, natural they inhibitors have beenof CYP1A1 prescribed and CYP1A2 as essential could have dietary an important components role in by cancer regulating agenciesprevention worldwide. by reducing the metabolism of procarcinogens by these enzymes. Thus, they have been prescribedThe inhibitors as essential of P450 dietary enzymes components fall intoby regulating two main agencies categories- worldwide. direct competitive inhibitors and time-dependentThe inhibitors inhibitors.of P450 enzymes Competitive fall into two inhibitors main categories are capable- direct of competitive accessing inhibitors the active and site and reversiblytime-dependent binding to inhibitors. the active Competitive site. These inhibitors kinds of molecules are capable need of accessing to have a the much active higher site affinityand to the targetreversibly enzyme binding than to the active natural site. substrates. These kinds Time-dependent of molecules need inhibitors to have a are much also higher capable affinity of accessing to the activethe target site enzyme and binding than the to the natural active substrates. site [30 ,31 Time]. When-dependent these inhibitors inhibitors are are also initially capable incubated of accessing the active site and binding to the active site [30,31]. When these inhibitors are initially with the enzyme before the addition of the substrate, an increase in inhibition is observed, which incubated with the enzyme before the addition of the substrate, an increase in inhibition is observed, is awhich kinetic is phenomenon.a kinetic phenomenon. This category This catego canry be can further be further defined defined by by its its subset subset of of mechanism-based mechanism- inactivationbased inactivation wherein the wherein bound the inhibitor bound inhibitor is oxidized is oxidized by the enzyme by the enzyme to a highly to a reactive highly reactive intermediate thatintermediate subsequently that binds subsequently to a reactive binds amino to a acid reactive in its amino proximity. acid inThis its process proximity. permanently This process changes the enzymepermanently active chan site,ges the resulting enzyme in active the site, inactivation resulting in of the the inactivation enzyme. Thisof the processenzyme. isThis both process time- and cofactor-dependent.is both time- and cofactor Several-dependent. classes ofSeveral inhibitors classes haveof inhibitors been found have been that found act as that direct act as competitivedirect inhibitorscompetitive or time-dependent inhibitors or time inhibitors.-dependent inhibitors.

5. Substrate5. Substrate Binding Binding Site Site Characteristics Characteristics TheThe substrate substrate binding binding cavity cavity is is defined defined by by the the I, I, F, F, G, G, C C and and B’B’ helices,helices, thethe loop between the the K K helix helix and β1–4 sheets and the residues at the turn of the β4 region. The X-ray crystal structures of the and β sheets and the residues at the turn of the β region. The X-ray crystal structures of the CYP1A1 CYP1A11–4 and CYP1A2 demonstrate several similarities4 between the two enzymes’ active sites (Figure and CYP1A2 demonstrate several similarities between the two enzymes’ active sites (Figure3). 3).

(A) (B)

FigureFigure 3. The3. The molecular molecular surface surface representationrepresentation of of the the active active site site pocket pocket of the of ( theA) CYP1A1 (A) CYP1A1 and (B and) (B) CYP1A2 enzymes colored by lipophilicity where the pink region depicts hydrophilic region of the CYP1A2 enzymes colored by lipophilicity where the pink region depicts hydrophilic region of the pocket and the green region depicts the lipophilic region of the pocket. The heme residue is pocket and the green region depicts the lipophilic region of the pocket. The heme residue is represented represented as white stick model, the ligand (α-naphthoflavone) is shown as yellow stick model, and α as whitethe enzyme stickmodel, residues the are ligand shown (as-naphthoflavone) cyan stick models. is shown as yellow stick model, and the enzyme residues are shown as cyan stick models.

Molecules 2017, 22, 1143 5 of 19

Molecules 2017, 22, 1143 5 of 18 A comparative protein structural analysis between the X-ray crystal structures of CYP1A1 and A comparative protein structural analysis between the X-ray crystal structures of CYP1A1 and CYP1A2 has been performed by Kesharwani et al. [32,33]. They describe several differences in the CYP1A2 has been performed by Kesharwani et al. [32,33]. They describe several differences in the six six identified substrate recognition sites between the two CYP1A enzymes. They have also identified the identified substrate recognition sites between the two CYP1A enzymes. They have also identified the residuesresidues in in CYP1A1 CYP1A1 and and CYP1A2 CYP1A2 showingshowing higher higher B B-factor-factor values values than than the the average average B-factor. B-factor. They They are- are- Asn221,Asn221, Leu254, Leu254, Asp320 Asp320 and and Lys499 Lys499 in thein the F, G, F, IG, and I and L helices L helices of CYP1A1, of CYP1A1, and Thr118,and Thr118, Asp320, Asp320, Thr321, β Leu382Thr321, and Leu382 Ile386 andin the Ile386 B’ and in I the helices B’ and and I the helices loop and connecting the loop K connectinghelix to 2 sheet K helix of CYP1A2. to β2 sheet Several of identicalCYP1A2. residues Several are identical aligned residues in identical are aligned orientations in identical in the orientations active site spacesin the active such assite the spaces Ile-115/117, such Phe-123/125,as the Ile-115/117, Phe-224/226, Phe-123/125, Thr-321, Phe-224/226, Asp-320, Thr Ile-386,-321, Asp Leu-496/497,-320, Ile-386, Leu Asn-255/257,-496/497, Asn and-255/257, Thr-497/498 and inThr CYP1A1/CYP1A2.-497/498 in CYP1A1/CYP1A2. The two non-conserved The two non residues-conserved with residues similar with properties similar in properties the active in sites the of CYP1A1/CYP1A2active sites of CYP1A1/CYP1A2 are the Ser116/Thr118 are the and Ser116/Thr118 the Ser122/Thr124. and the The Ser three122/Thr124. non-conserved The three residues non with- differentconserved properties residues in the with active different sites of properties CYP1A1/CYP1A2 in the active are the sites Asn222/Thr223, of CYP1A1/CYP1A2 the Leu312/Asn312, are the andAsn222/Thr223, the Val382/Leu382. the Leu312/Asn312, The B-factor analysis and the indicatedVal382/Leu382. that the The non-conserved B-factor analysis residues indicated and the that residues the withnon higher-conserved B-factors residues demonstrated and the residues greater wi mobilityth higher and B- flexibility.factors demonstrated greater mobility and flexibility. 6. Ligand-Based Studies on Isoform Selectivity 6. Ligand-Based Studies on Isoform Selectivity While the X-ray crystal structures provide a detailed map of the substrate recognition sites and the activeWhile sites, the the X-ray wide crystal range structures of the substrates provide anda detailed inhibitors map for of the substrate two enzymes recognition with varied sites shapesand andthe sizes active indicate sites, the the wide plasticity range of of the the activesubstrates sites and defined inhibitors by the for flexibility the two enzymes and movement with varied of the helicesshapes surrounding and sizes indicate the active the plasticity sites. The of shapesthe active of thesites active defined sites by arethe definedflexibility by and the movement F and I helices of inthe the helices two enzymes surrounding CYP1A1 the active and CYP1A2,sites. The shapes forming of athe flat active surface sites between are defined these by the helices, F and that I helices clearly in the two enzymes CYP1A1 and CYP1A2, forming a flat surface between these helices, that clearly indicate the preference for planar molecules. The selective inhibition of either CYP1A1 or CYP1A2 has indicate the preference for planar molecules. The selective inhibition of either CYP1A1 or CYP1A2 been a challenge due to the large number of substrates with varied structures that are metabolized by has been a challenge due to the large number of substrates with varied structures that are metabolized both enzymes. by both enzymes. To study the nature of the active site cavities in these enzymes, a series of planar molecules, To study the nature of the active site cavities in these enzymes, a series of planar molecules, naphthoflavonesnaphthoflavones and and pyranoflavones, pyranoflavones, were synthesized by by our our group. group. A A meta meta-analysis-analysis of of thes thesee moleculesmolecules was was performed performed toto studystudy the inhibitor selectivity selectivity toward toward CYP1A1 CYP1A1 and and CYP1A2 CYP1A2 (Figure (Figure 4). 4). InIn light light of of the the variations variations in in thethe inhibitoryinhibitory data from from different different methods methods of of determination, determination, a selectivity a selectivity ratioratio which which is is a a measure measure of of the the ratioratio ofof thethe IC50 valvaluesues of of the the inhibitors inhibitors for for CYP1A1 CYP1A1 and and CYP1A2 CYP1A2 was was usedused to to define define two two groups groups of of inhibitors inhibitors (IC (IC5050 forfor CYP1A1/ICCYP1A1/IC5050 forfor CYP1A2 CYP1A2 ratios ratios <0.2 <0.2—group1—group1 and and >5—group>5—group 2) 2) for for the the comparative comparative meta-analysismeta-analysis (Figure 55).).

Figure 4. Cont.

Molecules 2017, 22, 1143 6 of 18 Molecules 2017, 22, 1143 6 of 18 Molecules 2017, 22, 1143 6 of 19 Figure 4. Cont. Figure 4. Cont.

Figure 4. Structures and CYP1A1 & CYP1A2 inhibition data for (A) & (C) linear planar molecules and FigureFigure 4. 4Structures. Structures and and CYP1A1 CYP1A1 && CYP1A2CYP1A2 inhibition data data for for (A (A) &)&( (CC) linear) linear planar planar molecules molecules and and (B) & (D) triangular planar molecules [(A) and (B) reprinted from reference 34 with permission ]. (B)&((B) &D ()D triangular) triangular planar planar molecules molecules [([(AA)) andand (B) reprinted from from reference reference 34 34 with with permission permission ]. ].

FigureFigure 5. Alignment 5. Alignment images images of group of group 1 and 1 andgroup group 2 inhibitors. 2 inhibitors. (A) and (A) (C) and Group (C) Group 1 represents 1 represents selective Figure 5. Alignment images of group 1 and group 2 inhibitors. (A) and (C) Group 1 represents P450selective 1A1 inhibitors. P450 1A1 (Binhibitors.) and (D) ( GroupB) and 2 (D) represents Group 2 selective represents P450 selective 1A2 inhibitors. P450 1A2 [Figure inhibitors. was [ reprintedFigure selective P450 1A1 inhibitors. (B) and (D) Group 2 represents selective P450 1A2 inhibitors. [Figure fromwas reference reprinted 34 from with reference permission]. 34 with permission]. was reprinted from reference 34 with permission].

Structural alignments between the two groups of inhibitors showed that selective CYP1A1 StructuralStructural alignments alignments between between the two groups of of inhibitors inhibitors showed showed that that selective selective CYP1A1 CYP1A1 inhibitors have a long strip shape with an average length of 12.3 Å , and average width of 4.6 Å , inhibitorsinhibitors have have a a long long stripstrip shapeshape withwith an average average length length of of 12.3 12.3 Å Å, , and and average average width width of 4.6 of 4.6Å , Å, showing a narrow and long cavity for CYP1A1. The alignment of the groups of selective CYP1A2 showingshowing a a narrow narrow and and long long cavitycavity forfor CYP1A1.CYP1A1. The The alignment alignment of of the the group groupss of ofselective selective CYP1A2 CYP1A2 inhibitors form a triangular shape with side lengths of 9.3 Å , 8.7 Å , and 7.2 Å , that are indicative of a inhibitorsinhibitors form form a a triangular triangular shapeshape with with side side lengths lengths of of 9.3 9.3 Å ,Å, 8.7 8.7 Å , Å,and and 7.2 7.2Å , that Å, that are areindicative indicative of a of narrow compact triangular cavity for CYP1A2 [34]. The impact of the ligand shape on the enzyme a narrownarrow compact triangular cavity cavity for for CYP1A2 CYP1A2 [34] [34. ].The The impact impact of ofthe the ligand ligand shape shape on onthe the enzyme enzyme specificity for CYP1A1 and CYP1A2 was confirmed by the study of the derivatives of long planar specificity for CYP1A1 and CYP1A2 was confirmed by the study of the derivatives of long planar specificitymolecules for such CYP1A1 as anthracene, and CYP1A2 phenazines, was confirmedphenothiazines, by the acridines, study of and the anthracene derivatives-9,10 of-diones, long planar all molecules such as anthracene, phenazines, phenothiazines, acridines, and anthracene-9,10-diones, all moleculesshowing suchgreater as inhibition anthracene, potency phenazines, for CYP1A1 phenothiazines, than CYP1A2acridines, [34–37] (Figure and anthracene-9,10-diones,4). On the other hand, showing greater inhibition potency for CYP1A1 than CYP1A2 [34–37] (Figure 4). On the other hand, alltriangular showinggreater shaped inhibition molecules potency such as forphenanthrenes CYP1A1 than and CYP1A2 carbazoles [34– 37clearly] (Figure show4). greatly On the increased other hand, triangular shaped molecules such as phenanthrenes and carbazoles clearly show greatly increased triangularpotency toward shaped CYP1A2 molecules compared such as to phenanthrenes CYP1A1 [34,36,37] and (Figure carbazoles 4). clearly show greatly increased potency toward CYP1A2 compared to CYP1A1 [34,36,37] (Figure 4). potencySeveral toward structure CYP1A2-activity compared relationship to CYP1A1 studies [34,36 such,37] (Figure as CoMFA,4). SAR, 3D-QSAR have been Several structure-activity relationship studies such as CoMFA, SAR, 3D-QSAR have been undertakenSeveral structure-activity by researchers to relationshipunderstand the studies ligand such interactions as CoMFA, with t SAR,he residues 3D-QSAR in the have protein been undertaken by researchers to understand the ligand interactions with the residues in the protein undertakenactive site. by The researchers goals of such to understand studies included the ligand virtual interactions screening, withprediction the residues of the site in theof metabolism, protein active active site. The goals of such studies included virtual screening, prediction of the site of metabolism, and prediction of the potency/selectivity of inhibition of the CYP enzymes. Our group has recently site.and The prediction goals of of such the potency/selectivity studies included virtualof inhibition screening, of the prediction CYP enzymes. of the Our site group of metabolism, has recently and prediction of the potency/selectivity of inhibition of the CYP enzymes. Our group has recently published extensive reviews on these studies [35,38]. Two types of QSAR models have been employed to study the P450 substrates. The first one is a global QSAR model that is used to predict the ADMET (absorption, Molecules 2017, 22, 1143 7 of 19 distribution, metabolism, elimination and toxicity), which is not very useful in terms of understanding the active site properties and prediction of potency/selectivity. The second type is the detailed QSAR for specific P450 enzymes of smaller sets of molecules that are structurally related. These models are very robust and give a more precise representation of the active site interactions and structural features that impart potency and specificity. Several descriptors including electrostatic, hydrophobic, hydrogen bond acceptor/donor, steric, and desolvation free energy have been studied using quantum/molecular mechanical calculations. Many of these models have been verified by docking studies. QSAR studies on CYP1A1 substrates have been performed by several research groups on polyaromatic hydrocarbons, flavonoids, benzoxazoles, benzothiazoles, and several other inhibitors of CYP1A1 [39–46]. These studies have shown that the properties that impact the binding of the substrates are the linear planarity of the molecules, the HOMO energy, π-π stacking with the Phe residues, optimally placed acceptor and donor substituents that can interact with the polar residues in the active site, and desolvation (ClogP). QSAR analysis of CYP1A2 substrates and inhibitors has been widely studied by many researchers as well [39,45–51]. Chemometric tools that employ stepwise multiple linear regression, genetic function approximation, and genetic partial least squares have been used for small sets of structurally similar molecules. Machine learning techniques such as associative neural networks, kappa nearest neighbor random tree, random forest, and decision tree methods have been used to develop predictive models for large sets of molecules with varied structures. Planarity of the molecule (area/depth2 ratio) and molecular mass were found to be the critical requisites for molecules to be CYP1A2 substrates/inhibitors.

7. Protein-Based Studies Using Molecular Docking and Molecular Dynamics The extensive ongoing research worldwide to better understand the oxidation of exogenous/ endogenous substrates by CYP1A enzymes, and the efforts for the development of CYP1A inhibitors have led to the wide use of docking studies and molecular dynamic studies on the enzymes. To better understand the interactions of the substrate with the enzyme and the specificity of ligands for either CYP1A1 or CYP1A2, docking studies using computational molecular modeling tools has been the go-to method. The docked substrate-enzyme features that have been analyzed are the binding free energies, the interactions between the substrate and the residues lining the active site, and the distance between the heme iron and the substrate reacting group. The docking studies described on CYP1A enzymes employed different software including AutoDock, Surflex dock from Tripos SYBYL, the docking module from Molecular Operating Environment (MOE), etc. The binding scores reflect the binding free energy obtained using a functional form of various terms that describe different interactions including hydrogen bond interactions, hydrophobic interactions, ionic interactions, metal ligation, hydrophobic and polar/aromatic interactions, entropy, desolvations, etc. The value of the binding free energy obtained will depend upon the scoring method employed. The docking studies are validated if there is good correlation of the bioactivity to the docking score. Furthermore, the docking studies produce several binding poses. Some researchers employ 2 or more docking methods and find a consensus binding mode which is then used for scoring to obtain the binding free energy. Docking studies on CYP enzymes have used the optimal distance between the atom of oxidation on the ligand to the heme iron atom (4.0 Å to 7.5 Å) as the chief criteria for determining the best binding mode of the ligand. Oxidation of aromatic hydrocarbons and polyaromatic hydrocarbons (PAHs) found in the environmental pollutants to carcinogens by cytochrome P450 enzymes is a major cause of concern. Docking and QSAR studies were performed by Gonzalez et al. on 37 representative compounds that consisted of PAHs, PAH diols, and heterocyclic aromatic compounds onto CYP1A1 [41]. They used a homology model built using SWISS-MODEL for CYP1A1 as the X-ray crystal structure of CYP1A1 was not reported at the time of this study. It was observed that hydrophobic interactions played an important role in ligand binding. The residues involved were Phe123, Phe224 and Phe258. It was also seen that the ligand orientations were favoring π-π interactions in an edge-to-face manner with Phe123, Molecules 2017, 22, 1143 8 of 18 Molecules 2017, 22, 1143 8 of 19

important role in ligand binding. The residues involved were Phe123, Phe224 and Phe258. It was also andseen offset that stacked the ligand manner orientations with Phe224 were and favoring Phe258. π Heterocyclic-π interactions aromatic in an edge carbon-to- PAHface manner diols exhibited with bindingPhe123, orientations and offset stacked similar manner to those with of PAHs Phe224 with andπ- Phe258.π interactions Heterocyclic and hydrophobic aromatic carbon interactions PAH diols with theexhibited Phe residues binding and orientations with the polar similar groups to those forming of PAHs hydrogen with bond π-π interactions interactions and with hydrophobic polar amino interactions with the Phe residues and with the polar groups forming hydrogen bond interactions acids in the active site such as Asn221, Ser122, or Asp320, respectively. with polar amino acids in the active site such as Asn221, Ser122, or Asp320, respectively. Arylacetylenes were developed by our group as time-dependent inhibitors of CYP1A1 and Arylacetylenes were developed by our group as time-dependent inhibitors of CYP1A1 and CYP1A2 [52]. These molecules are members of the PAHs with an acetylenic moiety as a substituent, CYP1A2 [52]. These molecules are members of the PAHs with an acetylenic moiety as a substituent, which imparts the time-dependent inhibition via a reactive ketene intermediate. These molecules which imparts the time-dependent inhibition via a reactive ketene intermediate. These molecules did didnot not possess possess any any polar polar groups groups and andwere were incapable incapable of hydrogen of hydrogen bonding bonding interactions interactions with the withactive the π π activesite site polar polar residues. residues. All All of the of the arylaceylenes arylaceylenes made made the the critical critical edge edge-to-face-to-face and and offset offset stacked stacked π-π - interactionsinteractions with with the the Phe Phe residues residues in in the the activeactive site of t thehe CYP1A CYP1A enzyme enzyme (Figure (Figure 6A,6A,B).B). For For CYP1A1, CYP1A1, thethe distance distance between between the the heme heme iron iron atom atom andand thethe triple bond of of the the acetylene acetylene moiety moiety determined determined the the potencypotency of of the the inhibitors inhibitors (Figure (Figure6 C).6C). In In the the case case ofof CYP1A2,CYP1A2, the ππ-π-π stackingstacking interactions interactions were were found found to beto be the the most most important important determinants determinants of of thethe inhibitioninhibition potency (Figure (Figure 6D).6D).

Figure 6. Docking studies showing the π-π interactions between the arylacetylenes and the enzymes Figure 6. Docking studies showing the π-π interactions between the arylacetylenes and the enzymes (A) CYP1A1 and (B) CYP1A2. The docking studies also showed (C) the direct relationship between (A) CYP1A1 and (B) CYP1A2. The docking studies also showed (C) the direct relationship between the inhibitory activity and the heme-acetylene moiety distance for CYP1A1 and (D) the relationship the inhibitory activity and the heme-acetylene moiety distance for CYP1A1 and (D) the relationship between the inhibitory activity and distance between centroids of aromatic rings of Phe residues and between the inhibitory activity and distance between centroids of aromatic rings of Phe residues and arylacetylenes for CYP1A2. [(A) and (B) reprinted from reference 52 with permission]. arylacetylenes for CYP1A2. [(A) and (B) reprinted from reference 52 with permission]. Benzoxazole and benzothiazole anticancer agents are known to inhibit CYP1A1. Surflex docking studiesBenzoxazole and CoMFA and benzothiazoleanalysis of 48 derivatives anticancer agentsof benzoxazoles are known and to benzothiazoles inhibit CYP1A1. were Surflex performed docking studiesby Pan and et al CoMFA. [53] on analysis the homology of 48 derivatives model of CYP1A1. of benzoxazoles Arg106 and and Ile386 benzothiazoles of CYP1A1 played were performed important by Panroles et al. in [forming53] on the hydrogen homology bonds model with ofthe CYP1A1. oxygen atoms Arg106 of these and Ile386molecules. of CYP1A1 They concluded played importantthat the roles in forming hydrogen bonds with the oxygen atoms of these molecules. They concluded that the hydrogen bond interactions of these molecules with Ile386 are critical in determining the potency MoleculesMolecules2017 2017, 22, ,22 1143, 1143 9 of9 18 of 19

hydrogen bond interactions of these molecules with Ile386 are critical in determining the potency of ofinhib inhibitionition for for CYP1A1 CYP1A1 (Figure (Figure 7). 7The). Thebinding binding free energy, free energy, CScores CScores and the andantitumor the antitumor activity (pGI50) activity (pGI50)were compared were compared for the for docked the docked structures structures of all 48 of benzoxazole all 48 benzoxazole and benzothiazole and benzothiazole derivatives. derivatives. The ThepGI50 pGI50 values values ranged ranged from from 4.37 4.37 to to8.68, 8.68, and and the the CScores CScores varied varied from from 4.47 4.47 to to7.54. 7.54. The The free free energy energy of of bindingbinding ranged ranged from from− 6.07−6.07 kcal/mol kcal/mol to −10.3810.38 kcal/mol. kcal/mol. The The authors authors found found that that the the activity activity of ofthe the compoundscompounds corresponded corresponded well well with with the the CScores.CScores.

FigureFigure 7. 7Adapted. Adapted from from reference reference [[5353]] ModelModel of 5-fluoro-2-(3,4,5-trimethoxyphenyl)benzo[d]thiazole5-fluoro-2-(3,4,5-trimethoxyphenyl)benzo[d]thiazole (a)( anda) and 4-(benzo[d]thiazol-2-yl)benzene-1,2-diol 4-(benzo[d]thiazol-2-yl)benzene-1,2-diol (b) docked(b) docked into the into binding the binding site of CYP1A1. site of CYP1A1. Important residuesImportant are shownresidues as are sticks. shown as sticks.

Flavonoids are natural products that have antioxidative and antimutagenic properties thereby Flavonoids are natural products that have antioxidative and antimutagenic properties thereby preventing several debilitating diseases including cancer, heart disease, and bone loss. Flavonoids preventing several debilitating diseases including cancer, heart disease, and bone loss. Flavonoids are well known to inhibit several cytochrome P450 enzymes such as P450s 1A1, 1A2, 1B1, 2C9 and are well known to inhibit several cytochrome P450 enzymes such as P450s 1A1, 1A2, 1B1, 2C9 and 3A4. Docking and QSAR studies were performed for several PAHs and flavonoids on the X-ray 3A4.crystal Docking structure and of QSAR CYP1A2 studies and the were homology performed model for of several CYP1A1 PAHs by Shimada and flavonoids et al. [54].on Several the X-ray of crystaldocked structure flavones of hadCYP1A2 hydroxyl and the groups homology at various model positions of CYP1A1 of the by Shimadaaromatic rings.et al. [54 The]. Several ligand- of dockedinteraction flavones energies had hydroxyl varied between groups at−9.5 various and 255 positions (U values of, the kcal/mol). aromatic A rings. clear Thecorrelation ligand-interaction between energiesthe U values varied and between the IC−50 9.5values and for 255 inhibition (U values, of kcal/mol).CYP1A1, CYP1A2 A clear and correlation CYP1B1 betweenwas not found. the U valuesThe andbindin the ICg orientations50 values for of inhibitionthese flavonoids of CYP1A1, differed CYP1A2between CYP1A1 and CYP1B1 and CYP1A2. was not In found. the case The of strong binding orientationsinhibitors, of the these flavonoids B-rings differed docked between close CYP1A1 to the heme and CYP1A2. iron center In ofthe CYP1A2, case of strong and the inhibitors, π-π thestacking flavonoid of the B-rings favorably docked oriented close to flavonoid the heme hydroxyl iron center group of CYP1A2, with Phe2 and26 was the evidenced.π-π stacking Weak of the favorablyinhibitors oriented favored flavonoid an orientation hydroxyl that was group different with Phe226 from that was of evidenced. the strong inhibitors Weak inhibitors for CYP1A2, favored in an orientationwhich the that π-π was stacking different with from Phe226 that seen of the in strong strong inhibitors inhibitors was for CYP1A2,absent for in weak which inhibitors. the π-π stackingIn the withcase Phe226 of CYP1A1, seen in all strong of the inhibitors flavonoids was docked absent in for similar weak orientations inhibitors. Inin thethe caseactive of site. CYP1A1, The studies all of the flavonoidsindicated docked that thein presence similar of orientations a 5,7-dihydroxyl in the group active in site. the The A ring studies and indicateda 3-hydroxyl that group the presence in the C of a 5,7-dihydroxylring of the flavone group increased in the A the ring inhibition and a 3-hydroxyl potency for group these in enzymes. the C ring These of the studies flavone also increased showed the inhibitionthat the potencypresence forof 3 these′,4′-dihydroxyl enzymes. group These in studies the B- alsoring showedof the flavone that the could presence increase of 3the0,4 0inhibition-dihydroxyl grouppotency. in the B-ring of the flavone could increase the inhibition potency. Citrus fruits such as oranges, mandarins, and grapefruits are rich in flavonoids and coumarins Citrus fruits such as oranges, mandarins, and grapefruits are rich in flavonoids and coumarins that that potently inhibit several CYP enzymes including CYP 1A1, CYP1A2, and CYP3A4. The potently inhibit several CYP enzymes including CYP 1A1, CYP1A2, and CYP3A4. The components of components of grapefruit juice have been extensively studied for their interactions with several drugs grapefruit juice have been extensively studied for their interactions with several drugs and their role and their role as chemoprotective agents. Furanocoumarin monomers, bergamottin and 6′,7′- as chemoprotective agents. Furanocoumarin monomers, bergamottin and 60,70-dihydroxybergamottin dihydroxybergamottin (DHB) are potent inhibitors of CYP1A1 and CYP1A2 [55–57]. The flavonoid (DHB)naringenin are potent (NAR) inhibitors isolated of CYP1A1 from grapefruit and CYP1A2 juice [ is55 a– 57 potent]. The competitiveflavonoid naringenin inhibitor (NAR) of CYP1A1. isolated fromEspinosa grapefruit-Aguirre juice et is ala potent. have competitivestudied the inhibitorbinding of of DHB CYP1A1. and Espinosa-Aguirre NAR to human and et al. rat have forms studied of theCYP1A1 binding through of DHB kinetic and NAR analysis to human and docking and rat formsstudies of [58] CYP1A1. Kinetic through analysis kinetic revealed analysis species and-related docking studies [58]. Kinetic analysis revealed species-related differences in the DHB and NAR inhibition of Molecules 2017, 22, 1143 10 of 19

CYP1A1 by type and potency. DHB exhibited competitive inhibition of CYP1A1 in both species, but with an increased inhibition kinetics Ki for human CYP1A1 (55 µM) than for rat CYP1A1 (1.73 µM). NAR showed species variation in type of inhibition with competitive inhibition of human CYP1A1 (Ki = 489 µM) and mixed type inhibition for rat CYP1A1 (Ki = 0.17 µM and KI = 0.39 µM). Molecular dynamics simulations on the X-ray crystal structure of human CYP1A1 and homology model of rat CYP1A1 showed similar catalytic site fluctuations. Four critical residues in the binding site varied between the species- humanCYP1A1/ratCYP1A1: Ser116/Ala120, Ser122/Thr126, Asn221/Ser225, and Leu312/Phe316, and interactions with these differing residues by DHB and NAR could account for the differences in type and potency of inhibition. The docking scores for NAR and DHB for human CYP1A1 were −6.65 kcal/mol and −8.85 kcal/mol. The docking score for NAR with rat CYP1A1 was lower at −5.77 kcal/mol while the docking score for DHB with rat CYP1A1 was −8.05 kcal/mol, which is comparable to that of the human CYP1A1. The other interesting finding in these docking studies was the presence of two potential binding sites for DHB and NAR, the primary catalytic site and an adjacent secondary binding site below the primary site that was exposed to the solvent. This secondary binding site was much larger than the primary catalytic site in rat CYP1A1. Our research group developed several flavone propargyl ethers that selectively inhibit CYP1A1 and CYP1A2 compared to CYP2A6 and CYP2B1 [45]. In that study, 30-flavone propargyl ether (30-PF) and 7-hydroxyflavone (7-HF) were also proved to be time-dependent inhibitors of CYP1A1. The 0 rate constants of 3 -PF and 7-HF for maximal inactivation at saturation (kinact) were 0.09 and 0.115 min−1, respectively, and the concentrations required to produce one-half of the maximal rates of inactivation (KI) were 0.24 and 2.43 µM, respectively. Docking studies showed that these compounds exhibited multiple π-π interactions with the Phe residues in the active site of both CYP1A enzymes. For CYP1A2 enzyme, all of the compounds except for 5-hydroxyflavone were oriented in the active site in such a way that the hydroxyl group was closest to the heme of the enzyme. Even though the flavone propargyl ethers are longer molecules than the hydroxy flavones, their binding orientation in the enzyme active site showed the triple bond closest to the heme center with the signature π-π interactions with the active site Phe residues still maintained. It has been suggested that the unstable ketene intermediate formed by the oxidation of the triple bond could react with the neighboring Arg residue to form an amide bond, leading to the time-dependent inhibition of CYP1A1. Coumarins are one of the classes of compounds that are known to be metabolized by several CYP enzymes including 1A1, 1A2, 3A4, 2A6 and some from the 2B subfamily. Based on the observation that the key metabolic site on the coumarin core structure is the 7-position, our group developed several substituted derivatives of 7-ethynylcoumarin that exhibit selective inhibition of CYP1A1 and CYP1A2 in a time-dependent manner [59]. Two different orientations were found for these 7-ethynylcoumarin derivatives in the CYP1A2 active site. 3-Phenyl substituted derivatives docked in such a way that the phenyl ring was facing the heme center leading to their behavior as competitive inhibitors. On the other hand, smaller derivatives with just alkyl substituents docked with the ethynyl group facing the heme that could explain the time-dependent inhibition of CYP1A2 by these molecules. The advent of hormone replacement therapies in early/late menopausal women has been associated with a higher risk for breast cancer [60]. This has been attributed to the increased levels of hormones that are known procarcinogens [24,61]. is metabolized by CYP enzymes (CYP1A1, CYP 1A2, CYP 1B1 and CYP 3A4) to its 2,3-dihydroxy and 3,4-dihydroxy forms. The 2,3-dihydroxy form is methylated to its non-carcinogenic form by cathecol-o-methyl [62,63]. The 3,4-dihydroxy form is oxidized to its carcinogenic quinone form, which is a marker for human breast tumors, by peroxidases [24,61]. Yamamoto et al. have performed docking studies of 17β-estradiol on CYP1A1, CYP1A2 and CYP1B1 using Surflex Dock and the docked complexes were scored using an empirical scoring function based on the Hammerhead docking system [64]. They looked for binding modes that positioned the A-ring of the molecule within 5.5 Å of the heme iron atom. The binding mode of 17β-estradiol was identical for CYP1A1 and CYP1A2 and it differed from the one for CYP1B1. The accommodation of the 18-methyl group in the binding pocket was the deciding Molecules 2017, 22, 1143 11 of 19 factor in determining the binding orientations of 17β-estradiol in the active sites of CYP1A1, CYP1A2 and CYP1B1 enzymes. These studies clearly indicate that CYP1B1 preferably oxidizes the 4-position while CYPs 1A1 and 1A2 prefer the 2-position. Docking studies of the keto form of estrogen, namely, estrone on the CYP1A1, CYP1A2, CYP3A4 and CYP1B1 were performed by Olah et al. using AutoDock Vina docking program [65]. Their results also indicated that the 2-position of estrone is primarily hydroxylated by CYP1A1 and CYP1A2, whereas, CYP1B1 preferably hydroxylated the 4-position. During the development of a new class of quinone and anthraquinone inhibitors for CYP 1A enzymes, an ortho-methylaminoanthraquinone was found to be a time-dependent inhibitor of both CYP1A1 and CYP1A2 enzymes [36]. Docking studies on CYP1A1 and CYP1A2 showed that the methyl group was in close proximity to the heme, enabling the enzyme to oxidize the methyl group. Due to the presence of an amino group at the ortho position, the authors proposed that the hydrogen abstraction of the methyl group by an iron-oxo species would lead to the formation of a benzylic carbon radical intermediate. To better understand the nature of this proposed mechanism, the authors explored the inhibition characteristics of several polycyclic ortho-methylheteroarylamines [37]. Most of these compounds inhibited both CYP1A1 and CYP1A2 enzymes, with the linear planar molecules showing a marginal selectivity for CYP1A1. Four of these compounds showed time-dependent inhibition of CYP1A1 and not CYP1A2. Docking studies showed that the linear planar shape of molecules resulted in a better binding orientation in the active site of CYP1A1. Additionally, the molecules that had the aromatic methyl group in close proximity to the heme iron showed time-dependent inhibition of CYP1A1 with a mechanism similar to that of furafylline. Furafylline is a caffeine analog that selectively inhibits CYP1A2 (IC50 = 0.07 µM) over CYP1A1 (IC50 > 500 µM) and other CYP enzymes [66,67]. Furthermore , furafylline was found to be a mechanism-based inhibitor of CYP1A2 with a Ki of 23 µM −1 and a Kinact of 0.87 min . Docking studies have revealed that the furan ring interacts with Phe125 of CYP1A2 resulting in the 8-methyl group being directed towards the heme iron [68]. Isotope effects have clearly shown that the initiating step is the abstraction of a hydrogen from the C-8 methyl group of furafylline by P4501A2 [66]. It has been suggested that the loss of P450 observable spectroscopy could be the result of the formation of a reactive intermediate that may alkylate the heme prosthetic group or an amino acid in close proximity. Glycine scanning studies of the enzyme-ligand complexes for the compounds that were selective CYP1A1 time-dependent inhibitors showed that the ligands were making strong π-π interaction with the Phe123, Phe224 and Phe258 with a sandwich configuration contributing to the stabilization of the protein-ligand complexes [37]. π-π interaction of the ligand with Phe123 was the largest contributor for the complex stability. A similar glycine scanning study was performed for a set of carbazole analogs that are selective inhibitors of CYP1A2. Four residues in the active site contributed the most to the stability of the protein-ligand complex- Glu225, Phe226, Phe260 and Leu497. The ligand π-π interactions with the Phe residues were the strongest contributors for the stability of the complex. Additionally, Leu497 formed strong aromatic-aliphatic interactions by the positioning of this residue above a ligand aromatic ring. While Glu225 did not make any hydrogen bonds with the ligand, nonspecific aliphatic interactions contributed to the binding stability of the ligands. Nitropolycyclic aromatic hydrocarbons (nitro-PAHs) are formed in the environment by the reaction of PAHs with nitrogen oxides in the atmosphere. The nitro-PAHs are metabolically activated by enzymes such as NADPH:quinone (NQO1), NADPH:CYP oxidoreductase (POR), and CYP enzymes 1A1 and 1A2. Using theoretical and experimental approaches Stiborova et al. have shown that CYP1A1 and CYP1A2 efficiently activate the nitro-PAHs under anaerobic conditions to mutagens that bind to the DNA to form DNA adducts [69]. 3-Nitrobenzanthrone (3-NBA), found in diesel exhaust, is reductively activated to 3-aminobenzanthrone (3-ABA) by both NQO1 and CYP1A1/2 enzymes. The reductive activation of 3-NBA and the oxidative activation of 3-ABA, lead to the common intermediate N-hydroxy-3-nitrobenzanthrone (N-OH-3-ABA). To understand the reaction mechanisms of 3-NBA reduction by CYP1A1/2 enzymes, the authors performed docking studies using molecular modeling tools. A step-wise reduction mechanism (e−,H+, e−,H+) was proposed for the reduction of 3-NBA by CYP1A1/2. The binding orientation of 3-NBA to the CYP enzymes Molecules 2017, 22, 1143 12 of 19

Molecules 2017, 22, 1143 12 of 18 which allowed fast electron flow from the porphyrin ring of the heme cofactor was studied (Figure8). enzymes which allowed fast electron flow from the porphyrin ring of the heme cofactor was studied This binding orientation was identical for both the CYP1A1 and CYP1A2 enzymes, wherein the nitro (Figure 8). This binding orientation was identical for both the CYP1A1 and CYP1A2 enzymes, groupwherein of 3-NBA the nitro was group in close of proximity3-NBA was to in the close excellent proximity proton to the donor excellent proton residues donor 497threonine and 498 inresidues CYP1A1 497 and and CYP1A2 498 in CYP1A1 which enables and CYP1A2 reduction which of enables the compound. reduction The of the same compound. compound The showedsame a differentcompound orientation showed a different in the active orientat siteion of in CYP1B1 the active in whichsite of CYP1B1 the nitro in groupwhich wasthe nitro oriented group close was to amidicoriented side close chains to of amidic Asn228 side and chains Gln332 of thatAsn228 are inferiorand Gln332 proton that donors. are inferior The estimated proton donors. binding The free energyestimated for CYCP1A1 binding free and energy CYP1A2 for was CYCP1A1−8.04 kcal/moland CYP1A2 and was−8.02 −8.04 kcal/mol, kcal/mol respectively, and −8.02 kcal/mol, which was muchrespect higherively, than which the was one much for CYP1B1 higher (than−7.73 the kcal/mol). one for CYP1B1 (−7.73 kcal/mol).

FigureFigure 8. 8Adapted. Adapted fromfrom reference [56 [56].]. The The most most favorable favorable binding binding orientations orientations of 3-NBA of 3-NBA docked docked into intothe the active active site site of CYP1A1 of CYP1A1 (A), (1A2A), ( 1A2B) and (B )1B1 and (C 1B1). Hydrogen (C). Hydrogen bonds between bonds between 3-NBA and 3-NBA the amino and the acid residues in active site residues are rendered as dashed black lines. 3-NBA (pink), heme (grey) amino acid residues in active site residues are rendered as dashed black lines. 3-NBA (pink), heme and side chains of important amino acid residues (cyan) are rendered as bold sticks; iron ions as (grey) and side chains of important amino acid residues (cyan) are rendered as bold sticks; iron ions as orange spheres. orange spheres. Aristolic acid I (AAI) is a plant alkaloid procarcinogen metabolized by several cytochrome P450 enzymes,Aristolic with acid CYP1A2 I (AAI) and is a CYP1A1 plant alkaloid being procarcinogenthe major enzymes metabolized involved byin the several O-demethylation cytochrome P450of enzymes,aristolic withacid CYP1A2into the and CYP1A1 8-hydroxyaristolic being the major acid enzymes I. Docking involved studies in by the Stiborova O-demethylation et al. [15] of aristolicrevealed acid that into thethe binding carcinogen free energy 8-hydroxyaristolic of AAI to CYP1A1 acid was I. Docking −7.0 kcal/mol studies and by to StiborovaCYP1A2 was et al.−7.7 [15 ] revealedkcal/mol. that These the binding free free energies energy were of AAI far togreater CYP1A1 than wasthe ones−7.0 for kcal/mol CYP2C9and and toCYP3A4 CYP1A2 (−5.3 was −7.7and kcal/mol −6.0 kcal/mol).. These This binding data clearly free energies showed were the preferred far greater oxidation than the of onesAAI forby CYP1A2 CYP2C9 followed and CYP3A4 by (−CYP1A15.3 and −when6.0 kcal/mol).compared to This other data P450 clearly enzymes. showed the preferred oxidation of AAI by CYP1A2 followedAn by integrated CYP1A1 approach when compared has been toemployed other P450 by Kesharwani enzymes. et al. that applied molecular docking andAn molecular integrated dynamic approach simulations has been of the employed protein-ligand by Kesharwani complexes etof al.a wide that variety applied of P450 molecular 1A dockingsubstrates and [32] molecular. Five classes dynamic of substrates simulations were of chosen the protein-ligand based on their complexes ability to be of ( ai) wide preferably variety ofmetabolized P450 1A substrates by CYP1A1 [32]. (5 Five-aminoflavone classes of and substrates 5F-203); were (ii) metabolized chosen based at on different their ability rates and to be percentages by CYP1A1/1A2/1B1 (17-betaestradiol and ); (iii) metabolized at same rates (i) preferably metabolized by CYP1A1 (5-aminoflavone and 5F-203); (ii) metabolized at different and percentages by CYP1A1/1A2/1B1 (melatonin); (iv) preferably metabolized by CYP1A2 (clozapine rates and percentages by CYP1A1/1A2/1B1 (17-betaestradiol and theophylline); (iii) metabolized and lidocaine); and (v) metabolized by all three enzymes CYP1A1/1A2/1B1 with specificity to at same rates and percentages by CYP1A1/1A2/1B1 (melatonin); (iv) preferably metabolized by CYP1A1 (debrisoquine) (Figure 9). Docking studies were performed using Glide, and the docked CYP1A2 (clozapine and lidocaine); and (v) metabolized by all three enzymes CYP1A1/1A2/1B1 with poses were ranked based on their binding orientation and distance from the heme to the site of specificity to CYP1A1 (debrisoquine) (Figure9). Docking studies were performed using Glide, and the metabolism on the ligand. Further MD simulation analysis of the docked complexes and calculation dockedof the poses Molecular were ranked Mechanics based Poisson on their-Boltzmann binding orientation Surface Area and (MM distance-PBSA) from binding the heme free to energies the site of metabolismprovided atomic on the ligand.level information Further MD on simulation the protein analysis-ligand interactions. of the docked The complexes docking andstudies calculation were in of theaccordance Molecular with Mechanics the in- Poisson-Boltzmannvitro results trend in Surface terms Areaof the (MM-PBSA) enzyme that binding the ligand free was energies most providedactive atomicagainst. level The information MD simulations on the and protein-ligand the MM-PBSA interactions. calculations The matc dockinghed up studieswell for were 5-aminoflavone, in accordance with17- thebetaestradiol, in-vitro results debrisoquine, trend in terms and oftheophylline. the enzyme Khan that theet ligand al. performed was most docking active against. studies The using MD simulationsAutoDock and Tools the 4.0 MM-PBSA followed calculations by MD simulations matched of up the well protein for 5-aminoflavone,-ligand complexes 17-betaestradiol, for several debrisoquine,environmental and polycyclic theophylline. aromatic Khan procar et al.cinogens performed such as docking dibenzo[ studiesa,l]pyrene using (DBP), AutoDock 7,12-dimethyl Tools- 4.0 followedbenz[a]anthracene by MD simulations (DMBA), 2 of-amino the protein-ligand-1-methyl-6-phenylimidazo[4,5 complexes for several-b]pyridine environmental (PhIP) and benzo[ polycyclica]- aromaticpyrene procarcinogens(BP) on several CYP such enzymes as dibenzo[ includinga,l]pyrene CYP1A1 (DBP), and 7,12-dimethyl-benz[ CYP1A2 (Figure 9).a CYP1A1]anthracene was (DMBA),found 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridineto be the predominant enzyme that metabolized BP, (PhIP) DMBA and and benzo[ PhIP witha]-pyrene high binding (BP) on energies several CYPof enzymes−11.50 kcal/mol, including − CYP1A111.24 kcal/mol and CYP1A2 and −9.43 (Figure kcal/mol,9). CYP1A1 respectively. was found The in to-silico be the findings predominant for PhIP enzyme was

Molecules 2017, 22, 1143 13 of 19 that metabolized BP, DMBA and PhIP with high binding energies of −11.50 kcal/mol, −11.24 kcal/mol Molecules 2017, 22, 1143 13 of 18 and −9.43 kcal/mol, respectively. The in-silico findings for PhIP was different from the published experimentaldifferent from data,the published and this variationexperimental was data, attributed and this to variation the variation was inattributed the pKa to values the variation of certain in ionizablethe pKa values residues of certain in the ionizable binding groove residues of in the the CYP binding enzymes groove [70 ].of Qiuthe CYP et al. enzymes studied the[70] molecular. Qiu et al. interactionsstudied the of mole 12 gingercular components interactions (including of 12 ginger gingerols, components shogaols, (including gingerdiones, gingerols, and isogingerols, shogaols, etc.)gingerdiones, that are and actively isogingerols, metabolized etc.) bythat the are CYP actively enzymes metabolized 1A2, 2C9, by 2C19,the CYP 2D6 enzymes and 3A4. 1A2, Ginger 2C9, is2C19, one 2D6 of the and most 3A4. widely Ginger used is one herbal of the dietary most widely ingredients used andherbal is knowndietary toingredients have several and important is known pharmacologicalto have several important activities includingpharmacological the maintenance activities ofincluding the levels the of maintenance lipid, blood glucose, of the levels thromboxane of lipid, 2+ Bblood and prostaglandinglucose, thromboxane E2; inhibition B and of prostaglandin cytokines and chemokines;E2; inhibition and of blockagecytokines of and Ca chemokines;channel. Nearly and 128blockage compounds of Ca2+ have channel. been isolated Nearly 128 from compound ginger withs have gingerols, been phenylpropanoids isolated from ginger and with sesquiterpenes gingerols, beingphenylpropanoids the most pungent and sesquiterpenes of them. Shogoal being and the gingerol most pungent are partially of them. responsible Shogoal for and the gingerol antiemetic are activity.partially Dockingresponsible studies for the have antiemetic shown that activity. CYP2D6 Docking plays studies a major have role shown in the metabolismthat CYP2D6 of plays ginger a componentsmajor role in whilethe metabolism CYP1A2, CYP2C9, of ginger CYP3A3 components and CYP2C19 while CYP1A2, also metabolize CYP2C9, gingerCYP3A3 components, and CYP2C19 but toalso a lowermetabolize extent. ginger The Discovery components, Studio but 3.1to a CDOCKER lower extent. was The used Discovery for performing Studio the3.1 dockingCDOCKER studies. was Theused CDOCKER for performing interaction the docking energies studies. ranged The from CDOCKER 23.0 to 63.1 interaction kcal/mol. energies Three residuesranged from of CYP1A2 23.0 to (Gly316,63.1 kcal/mol. Phe226, Three and residues Phe260) interactedof CYP1A2 with (Gly316, the ginger Phe226, compounds. and Phe260) 10-Gingerol interacted made with a the hydrogen ginger bondingcompounds. interaction 10-Gingerol with made Gly316 a hydrogen (CDOCKER bonding interaction interaction energy with of 61.3568Gly316 kcal/mol)(CDOCKER and interaction 4 ginger compoundsenergy of 61.3568 (6-shogaol, kcal/mol) 8-shogaol, and 6-gingerdione 4 ginger compounds and methyl-6-isogingerol) (6-shogaol, 8-shogaol, made 6 1-gingerdione to 2 π-π stacking and interactionsmethyl-6-isogingerol) with Phe226 made and/or 1 to 2 Phe260 π-π stacking (CDOCKER interactions interaction with energies Phe226 and/or of 55.9354, Phe260 58.6712, (CDOCKER 59.0853 andinteraction 51.232 energies kcal/mol, of respectively). 55.9354, 58.6712, The 59.0853 CDOCKER and 51.232 interaction kcal/mol, energies respectively). from the dockingThe CDOCKER studies indicatedinteraction that energies hydrogen from bonding the docking plays studies a greater indicated role in the that affinity hydrogen of these bonding compounds plays toa greater the binding role sitein the of affinity CYP1A2 of than these the compoundsπ-π stacking to [the71]. binding site of CYP1A2 than the π-π stacking [71].

Figure 9. Structures of compounds studied by Kesharwani et al. [32] and Kahn et al. [57]. Figure 9. Structures of compounds studied by Kesharwani et al. [32] and Kahn et al. [57]. 8. Photo Affinity Ligands (PALs) as Binding Site Probes In 1962, Singh and Westheimer were the first who applied PALs to biochemical studies. PALs do not require any enzymatic activity to be utilized as active site probe. These ligands are in general very sensitive to the light and mostly are found to be stable in dark [72]. They can bind the chemically inert compound to the protein active site in the dark reversibly, and then irradiate the sample to

Molecules 2017, 22, 1143 14 of 19

8. Photo Affinity Ligands (PALs) as Binding Site Probes In 1962, Singh and Westheimer were the first who applied PALs to biochemical studies. PALs do Moleculesnot require 2017 any, 22, 1 enzymatic143 activity to be utilized as active site probe. These ligands are in general14 veryof 18 sensitive to the light and mostly are found to be stable in dark [72]. They can bind the chemically inert procompoundduce the to reactive the protein moiety. active In site case in of the PALs dark reversibly,with aliphatic and side then chains, irradiate the the aliphatic sample togroup produce can thebe covalentlyreactive moiety. labeled In case via of photogeneration PALs with aliphatic of a side radical chains, from the the aliphatic parent group ligand. can They be covalently do not alwayslabeled viarequire photogeneration nucleophilic amino of a radical acid residues from the to parentbe in close ligand. proximit Theyy. do The not first always example require was nucleophilicreported by Swansonamino acid and residues Dus to where be in they close usedproximity. tritiated The 1first-(4-azidophenyl)imidazole example was reported by (Figure Swanson 10) and[73] Dus to wheresuccessfully they used andtritiated quantitatively 1-(4-azidophenyl)imidazole label P450cam. The (Figurecompound 10)[ 73was] to used successfully as a general and quantitatively probe as N- phenylimidazolelabel P450cam. The binds compound with high was used affinity as a togeneral several probe P450 as N-phenylimidazole enzymes. The earliest binds success with high in photolabelingaffinity to several of P450 P450 1A1 enzymes. came in The 1992 earliest with success the use inof photolabeling compounds having of P450 azo 1A1 moiety came inon 1992 warfarin with (theFigure use of10) compounds [74]. The authors having reported azo moiety that on they warfarin prepared (Figure different 10)[74 ].isomer The authorss of warfarin reported with that azide they preparedgroup which different was isomers placed of at warfarin different with positions. azide group In the which same was year placed Guengerich at different and positions. his coworkers In the reportedsame year that Guengerich they have and successfully his coworkers demonstrated reported thatthe theyuse of have 4-azidobiphenyl successfully demonstrated(Figure 10) as thea PAL use of 4-azidobiphenylrat cytochrome P450 (Figure 1A2 10 [75]) as aand PAL the of covalent rat cytochrome binding P450 of the 1A2 ligand [75] and following the covalent photolysis binding was of shownthe ligand in such following a way photolysis that was PAL was shownconcentration in such- adependent way that wasand PALwhich concentration-dependent increased with irradiation and whichtime. increased with irradiation time.

Figure 10. Structures of photo-affinity ligands used as binding site probes. Figure 10. Structures of photo-affinity ligands used as binding site probes. In 1996, Strobel and coworkers first reported the use of bifunctional crosslinking PAL [76–78]. TheyIn used 1996, a benzphetamine Strobel and coworkers analog having first reported two azido the usegroups of bifunctional which were crosslinkingplaced at opposite PAL [76 ends.–78]. TheyStrobel used and a coworkers benzphetamine used radiolabeled analog having p- twoazidocumene azido groups (Figure which 10) wereto identify placed the at oppositecritical amino ends. acidStrobel residue and coworkersof P450 1A1 used involved radiolabeled in bindingp-azidocumene of cumene hydroperoxide (Figure 10) to identify[76]. The the experiments critical amino and studiesacid residue performed of P450 by 1A1 these involved and many in bindingother research of cumene groups hydroperoxide have provided [76 crucial]. The experimentsdata, which help and instudies understanding performed the by thesebinding and site many of the other CYP research enzymes. groups have provided crucial data, which help in understanding the binding site of the CYP enzymes. 9. Conclusions 9. Conclusions Computational molecular modeling studies have shed important insights into the binding of substratesComputational to CYP1A1 molecular and CYP1A2 modeling enzymes. studies These have studies shed have important led to insightsseveral intoimportant the binding findings of aboutsubstrates the structural to CYP1A1 features and CYP1A2 imparting enzymes. specificity These and studies potency have toward led tothese several enzymes. important Ligand findings-based studiesabout the such structural as QSAR features studies imparting and structural specificity meta and-analysis potency studies toward have these clearly enzymes. shown Ligand-based that planar molecules are the best substrates for these enzymes. CYP1A1 shows a preference for linear planar molecules while CYP1A2 prefers triangular planar molecules. Protein-based studies have included docking studies and molecular dynamic simulations of protein-ligand docked complexes. The docking studies have shown the importance of edge-to-face and offset stacked π-π stacking of the ligand aromatic rings with the various Phe residues in the binding site of the CYP1A1 and CYP1A2 enzymes. These π-π stacking interactions were found to be the strongest contributors to the stable

Molecules 2017, 22, 1143 15 of 19 studies such as QSAR studies and structural meta-analysis studies have clearly shown that planar molecules are the best substrates for these enzymes. CYP1A1 shows a preference for linear planar molecules while CYP1A2 prefers triangular planar molecules. Protein-based studies have included docking studies and molecular dynamic simulations of protein-ligand docked complexes. The docking studies have shown the importance of edge-to-face and offset stacked π-π stacking of the ligand aromatic rings with the various Phe residues in the binding site of the CYP1A1 and CYP1A2 enzymes. These π-π stacking interactions were found to be the strongest contributors to the stable binding of ligands to CYP1A2. Docking studies have also shown that the optimum distance between the heme iron and the position of oxidation on the substrate should be between 4.0 Å and 7.0 Å. The positioning of polar substituents on the ligands in close proximity to polar residues in the binding site also favors good binding free energies for the complexes. The insights obtained by these studies will help researchers to design and develop highly specific inhibitors of CYP1A1 and CYP1A2 enzymes.

Acknowledgments: The authors gratefully acknowledge support from the NIH-BUILD program (TL4GM118968, RL5GM118966, and UL1GM118967), Louisiana Biomedical Research Network- National Institutes of Health grant P20GM103424, the NIH-RCMI grant 2G12MD007595, and Louisiana Cancer Research Consortium (LCRC). Conflicts of Interest: The authors declare no conflict of interest.

References

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