1. Introduction

2. CYP1A1: Mechanism of

carcinogenesis and anticancer

activity

3. Therapeutic potential of

CYP1A1: outcome of clinical

drug candidate

4. In silicomethodologies applied

to CYP1A1

5. Conclusion

6. Expert opinion: in silico

perspective for designing

CYP1A1 inducers/substrates

Review

Cytochrome P450 1A1-mediatedanticancer drug discovery: in silicofindingsPrajwal P Nandekar & Abhay T Sangamwar††National Institute of Pharmaceutical Education and Research (NIPER), Department of

Pharmacoinformatics, Punjab, India

Introduction: Target-specific drugs may offer fewer side/adverse effects in com-

parison with other anticancer agents and thus save normal healthy cells to a

greater extent. The selective overexpression of cytochrome P450 1A1 (CYP1A1)

in tumor cells induces the metabolism of benzothiazole and aminoflavone com-

pounds to their reactive species,whichare responsible forDNAadduct formation

and cell death. This review encompasses the novelty of CYP1A1 as an anticancer

drug target and explores the possible in silico strategies thatwould be applicable

in the discovery and development of future antitumor compounds.

Areas covered: This review highlights the various ligand-based and target-

based in silico methodologies that were efficiently used in exploration of

CYP1A1 as a novel antitumor target. These methodologies include electronic

structure analysis, CoMFA studies, homology modeling, molecular docking,

molecular dynamics analysis, pharmacophore mapping and quantitative struc-

ture activity relationship (QSAR) studies. It also focuses on the various approaches

used in the development of the lysyl amide prodrug of 5F-203 (NSC710305) and

dimethanesulfonate salt of 5-aminoflavone (NSC710464) as clinical candidates

from their less potent analogues.

Expert opinion: Selective overexpression of CYP1A1 in cancer cells offers

tumor-specific drug design to ameliorate the current adverse effects associ-

ated with existing antitumor agents. Medicinal chemistry and in vitro driven

approaches, in combination with knowledge-based drug design and by

using the currently available tools of in silico methodologies, would certainly

make it possible to design and develop novel anticancer compounds

targeting CYP1A1.

Keywords: 2-(4-aminophenyl) benzothiazoles, AhR, antitumor, CYP1A1, homology modeling,

in silico, molecular docking, molecular dynamics, QSAR

Expert Opin. Drug Discov. (2012) 7(9):771-789

1. Introduction

Cancer is a disease of multiple accumulating mutations in developing cells, whichinvolves complex interactions between neoplastic cells and the surrounding microenvi-ronment. The changes in current lifestyle and certain environmental disturbances havefurther increased the ratio of cancer patients in the society. According to World HealthOrganization (WHO) report, the worldwide mortality due to cancer is projected to risecontinually with an estimated 11 million deaths up to 2030 [1]. The treatment of cancerincludes chemotherapy, radiation therapy or surgery, depending on the type and stage ofcancer. Chemotherapies based on mechanistic pathways are effective only for a definitetime period, as in auto hormonal treatment of prostate cancer that initially shrinks thetumor but eventually fails when the residual tumor cells become hormone resistant [2].Although a few chemotherapeutic regimens have yielded lasting remission or cure,most of them have reported as failed due to different pharmacological issues. Therefore,

10.1517/17460441.2012.698260 © 2012 Informa UK, Ltd. ISSN 1746-0441 771All rights reserved: reproduction in whole or in part not permitted

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it is clear that new therapeutic options are necessary. The antican-cer drug development has to address issues such as i) improvedand durable anticancer efficacy, ii) reduction of toxicity due tooff-target specificity and iii) prevention of drug resistance, whichis caused by overexpression of P-glycoprotein (Pgp) in tumorcells [3]. It was envisioned that the rapid development in molecu-lar pharmacology, molecular oncology and chemoinformaticscould convert the pattern of new anticancer drug discovery(NADD) from pharmacology mode of ‘drug-receptor-gene’ toretromolecular pharmacology mode of ‘gene-receptor-drugs.’Exploitation of novel molecular mechanisms have provided theinsight into anticancer drug discovery aimed at improved efficacyand reduced toxicity to normal cells. Due to these mechanisms,the trend of anticancer drug studies is oriented toward targetingthe multilink mechanism of tumorigenesis such as targetinginter-related enzymes, interrupting cell signal transduction path-way, cell apoptosis and cell metabolism. Looking at the future,new approaches could be useful in increasing the target selectivityof anticancer drugs. One such approach is change in cancer cellsignaling due to alteration of cell metabolism, which has upraisedthe attentiveness in targeting drug-metabolizing enzymes for can-cer chemotherapy [4,5]. The concept of overexpression of individ-ual forms of cytochrome P450 (CYP450) isoforms in tumor cellshas been recognized and being exploited by some researchers toprove its druggability [6-8]. Several studies highlight the overex-pression of CYP450s, particularly CYP1A1, in tumor cells andhave reported it as a representative novel target for anticancertherapy [9].

1.1 Cytochrome P450 1A1 (CYP1A1)Cytochrome P450s constitute a large superfamily of heme-containing enzymes, whichmetabolizes (either oxidize or reduce)large number of structurally different endogenous and exogenous

compounds, including steroids, fatty acids, prostaglandins,biogenic amines, plant metabolites, chemical carcinogens, muta-gens and other environmental pollutants [10]. Pharmacokineticstudies of several environmental toxicants in CYP1 knockoutmouse models suggest that CYP1A1, CYP1A2 and CYP1B1are the three CYP450 isoforms, identified for the conversionof procarcinogen to carcinogen and carcinogen to genotoxicmetabolites [11-20]. CYP1A1 is one of the three members ofCYP1A family, which is found mainly in extrahepatic tissuesand participates in metabolism of a large number of androgensubstrates. The transcriptional activation of CYP1A1 gene isinitiated by the binding of environmental pollutants and inhala-tion chemicals, notably substrates of CYP1A1, to the cytosolicaryl hydrocarbon receptor (AhR). This is followed by its translo-cation of AhR ligand complex to nucleus and subsequent forma-tion of a dimer with aryl hydrocarbon nuclear translocator(ARNT), which then interacts with corresponding xenobioticresponse elements (XRE) to activate transcription of CYP1A1regulating gene and translation toCYP1A1 enzyme [13,21]. Recentin vivo investigations suggest that CYP1A1 may function asa carcinogen-detoxification enzyme. The CYP1A1-medicatedbioactivation of natural dietary compounds and the resultinganticancer activity provide further insight into the cancer-protecting role of CYP1A1 [22]. The selective expression ofCYP1A1 in a panel of sensitive cell lines as compared withnon-responsive cancer cell lines after exposure to DF-203supports the logic to consider CYP1A1 as a promising anticancerdrug target [23]. CYP1A1 inducers, which are also the substratesof CYP1A1, induce CYP1A1 through activation of arylhydrocarbon receptor and then get metabolized by CYP1A1 toproduce a genotoxic metabolite that forms DNA adduct andsubsequently leads to cell death. Currently, the 2-(4-amino-phenyl) benzothiazole compounds (1--10) show a promisingrole in producing genotoxic metabolite and acting as anticanceragent [24] as shown in Figure 1.

Several reviews published on CYP1A1 focused oncatalytic mechanisms and role of CYP1A1 in chemicalcarcinogenesis and cancer chemotherapy [11,21,22,25-27];this review will focus on the current state of informationregarding computational methodologies used in designand development of new structural scaffold with antican-cer activity by exploiting CYP1A1-mediated pathway. Itdescribes the involvement of CYP1A1 in cancer regulationand suppression through reactive metabolite generation. Itelucidates the development of lysyl amide prodrug of5F-203 (NSC710305) from benzothiazole skeleton anddevelopment of dimethanesulfonate salt of 5-aminofla-vone (NSC710464). The efficiency and side effects ofclinical drug candidates NSC710305 and NSC710464are still uncertain [28]. The application of current in silicomethodologies such as homology modeling, moleculardocking, molecular dynamics analysis, quantum chemicalstudies, QSAR, pharmacophore mapping and the bio-transformation of cancer chemotherapeutic agents byCYP1A1 enzyme are also discussed.

Article highlights.

. CYP1A1 mediated drug metabolism, generation ofreactive metabolite and DNA adduct formation thatleads to cancerous cell death, which proves druggabilityof CYP1A1.

. CYP1A1 acts as potential drug target for anticancerdrug designing.

. NSC710305 and NSC710464 are leading clinical drugcandidates acting on CYP1A1 as an anticancerdrug target.

. In silico methodologies such as homology modeling,pharmacophore mapping, QSAR, molecular docking,molecular dynamics simulation studies, quantumchemical studies and virtual screening of databaseswould be efficiently used to design new chemical entityas CYP1A1 inducer/substrate.

. In silico methodologies coupled with medicinal chemistrydriven and in vitro approaches would be helpful fordesigning better anticancer compoundstargeting CYP1A1.

This box summarizes key points contained in the article.

P. P. Nandekar & A. T. Sangamwar

772 Expert Opin. Drug Discov. (2012) 7(9)

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2. CYP1A1: Mechanism of carcinogenesis andanticancer activity

2.1 Mechanism of CYP1A1-mediated carcinogenesis2.1.1 AhR-mediated CYP1A1 inductionThe CYP1A1 enzyme induction is mediated through binding ofsmall inducer molecules to a specific cytosolic aryl hydrocarbonreceptor (AhR) [29-31]. AhR is present as one of the part of acytosolic protein complex, which is composed of two Hsp-90 (heat-shock proteins) units, co-chaperone p23 and an immuno-phillin-like protein XAP2 (Hepatitis B virus X-associatedprotein) [29,31-33]. The binding of an exogenous inducer ligand,such as B[a]P or the industrial byproduct 2,3,7,8-tetrachlorodi-benzo-p-dioxin (TCDD), translocates the AhR complex withinducer to the nucleus, where heterodimerization takes placewith another protein ARNT (Figure 2) [32-37]. The heterodimerformed binds to consensus regulatory sequences known as arylhydrocarbon response elements (AhREs) and xenobiotic responseelements (XREs) or dioxin response elements (DREs), which arelocated in the promoter region of AhR target genes, such asCYP1A1 and initiates their transcription by recruiting RNA poly-merase II (Figure 2) [38]. CYP1A1mRNA transcription is inhibitedby aryl hydrocarbon receptor repressor (AhRR). AhRR is localizedin nucleus in the form of dimeric protein along with ARNT(Figure 2). AhRR competes with AhR for the heterodimer forma-tion with ARNT. The AhRR--ARNT heterodimer, when formed,acts as a repressor by stopping transcription, which is initiated atXREs. The AhR, ARNT and AhRR are members of the bHLH(basic helix-loop-helix) PAS (Per-ARNT-Sim) family of pro-teins [13,21,29,31,39,40]. The same mechanism of CYP1A1 inductionalso applies to certain Phase II xenobiotic metabolizing enzymes,

such as NQO1, UGT1A6, ALDH3A1 and several glutathioneS-transferases [13]. The AhR activation pathway is also involvedin expression of several other enzymes. The inhibition of AhRactivity leads to malfunctioning of other enzyme systems [29,41-43].Hence, AhR inducers or inhibitors do not always act as an antican-cer agent [44]. It is important to note that xenobiotics, which areAhR inducers and get selectively metabolized by CYP1A1, offertheir candidature as anticancer agents.

2.1.2 CYP1A1 expressionCYP1A1 is the primary extrahepatic enzyme involved in themetabolism of carcinogens. Tobacco smoking is associatedwith CYP1A1 methylation in the lungs [45] and it is the maincause of lung cancer [46]. In addition, expression of CYP1A1and AhR in small-cell lung carcinoma has been proposed as aputative diagnostic marker and has also been correlated withthe history of cigarette smoking [47,48]. The higher level ofCYP1A1 in malignant tissues as compared with the normalbreast tissues was determined by mRNA level expression [15,16].Also the estradiol C-2 hydroxylase activity, which is also amarker of CYP1A1 expression, was observed in neoplastic mam-mary tissue [49]. Enhanced level of CYP1A enzymes is also pres-ent in neoplastic samples of esophageal tissue, stomach tumorsand urinary bladder tumors [50-52]. Two different polymorphsare observed in human CYP1A1 gene, which are associatedwith an increased risk for tobacco-related lung cancer [53]. Suchfindings support the well-established carcinogen-activating roleof CYP1A1, since a higher expression of this enzyme would beexpected in pre-malignant or malignant tissues due to the con-tinuous exposure and subsequent metabolism by polyaromatichydrocarbons (PAHs) and related compounds. CYP1A1 is

Uptake 4 3

2

S 2

R

N

R1 NH2

1′ 4′

3′17

6

5

AhR ligand

AhR translocation

Induction of CYP1A1

Reactive intermediate

DNA adducts anddamage

1 R = Me, R1 = 4 2 R = Me, R1 = 6-OH 3 R = Me, R1 = 4-F 4 R = Me, R1 = 5-F 6 R = Me, R1 = 6-F 7 R = Me, R1 = 7-F 8 R = Me, R1 = 5,6-di-F 9 R = Me, R1 = 4,5-di-F10 R = Me, R1 = 5,7-di-F

Cell death

Figure 1. 2-(4-Aminophenyl) benzothiazoles class of compounds [96] and mechanism of action [61].

Cytochrome P450 1A1-mediated anticancer drug discovery: in Silico findings

Expert Opin. Drug Discov. (2012) 7(9) 773

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actively present in human placenta obtained from smokers,whereas in microsomes prepared from non-smokers, CYP1A1activity was considerably low. In human choriocarcinoma cellline, CYP1A1 is readily induced using PAHs (3-methylcholan-threne, 1,2-benzanthracene, a-naphthoflavone), as reported bywestern immunoblotting and EROD assay (7-ethoxyresorufin-O-deethylase) while CYP1A1 activity is minimal in the absenceof any inducer [47,54]. The observed selective overexpression ofCYP1A1 in neoplastic cell lines further supports the druggabilityof CYP1A1 as an anticancer target [23].

2.1.3 Activation of procarcinogen by CYP1A1The CYP1A1 is actively involved in the metabolic activationof environmental toxicants to their highly reactive product,which will have deleterious effects on cells [14,22,55-57]. In theprocess of metabolic activation, carcinogens are converted intoits epoxide intermediate, which are further converted to diolepoxides by enzyme epoxide hydrolase. Thewidely accepted pat-tern is metabolic activation of benzo[a]pyrene (B[a]P), which ispresent in cigarette smoke [58]. It involves oxidation of B[a]P to B[a]P-7,8-oxide, and subsequent hydrolysis to B[a]P-7,8-dioland their two enantiomers (+)-B[a]P-7,8-diol and (-)-B[a]P-7,8-diol. Then, each of these metabolites after oxidationproduces two diol epoxides namely (+)-B[a]P-7,8-diol-9,10-epoxide-1, (-)-B[a]P-7,8-diol-9,10-epoxide-2, (-)-B[a]P-7,8-diol-9,10-epoxide-1 and (+)-B[a]P-7,8-diol-9,10-epoxide-2.The final oxidized products have an ability to cause oncogenicmutations in specific parts of DNA; hence, they work as bayregion epoxides. The metabolite, (+)-B[a]P-7,8-diol-9,10-epox-ide-2, is considered as the ultimate carcinogenic conversionproduct of B[a]P, because of its level of carcinogenicity,which is parallel to that of B[a]P and (-)-B[a]P-7,8-diol. Thestructures of B[a]P and its carcinogenic metabolites are shownin Figure 3 [14,59].

2.2 Mechanism of CYP1A1-mediated anticancer

activityThe enzyme CYP1A1 is also associated with many vital physio-logical and cell growth regulatory processes, which are mediatedby involvement of AhR with multiple pathways other than thexenobiotic activating pathway [26]. The antitumor compound,NSC710305, which entered into the Phase I clinical trials inthe year 2004 induces CYP1A1 and thus forms DNA adductand causes cell cycle arrest in breast cancer-sensitive cell lines,such as MCF-7, T-47D and IGROV-1 (IC50 < 10 nM) with afully functional AhR signaling pathway [60]. This mechanisminvolves the binding of 5F-203 to AhR, followed by complexa-tion with ARNT and translocation to the nucleus, where it acti-vates XREs and induces CYP1A1. This mechanism is similar topreviously describedmechanism of benzo[a]pyrene. These com-pounds were assayed using intact MCF-7 and AHR100 (AHRdeficient) cells by measuring EROD activity [61,62]. The com-pounds were 4500-fold less active in MCF-7 AhR-null mutantcells, thus confirming the requirement of AhR for CYP1A1induction. Apart from this, aminoflavone also follows the samemechanism, that is, induction of CYP1A1 and its subsequentactivation to genotoxic metabolite, which was tested inMCF7 cell lines (IC50 = 0.1 nM) [63]. The aminoflavone isconverted to its bioactive metabolite by induced CYP1A1,which causes breaking of DNA double-strand, therebyinhibiting DNA synthesis in the S phase [64]. The molecularpathway of CYP1A1-mediated aminoflavone activation isshown in Figure 4.

Recently, some naturally occurring phytochemicals such asflavonoids, which act as anticancer agents, have been identifiedas CYP1A1 substrates. Eupatorin was found to be active againstMDA-MB-468 human breast cancer cells that express CYP1A1,but it is inactive in normal breast MCF-10A cells, devoid of anyCYP1A1 activity [65]. Isoflavone component of soya beans, that

AhR

AhR

AhR

PAHs, HAH,exogeneous andendogenous AhR

ligand

Translocateto nucleus

AhR

AhREs and XREs or DREs

Nucleus

CYP1A1

mRNA

CytoplasmP23

hsp90

hsp90

XAP2

ARNT AhRR

Other factorsARNT

Figure 2. AhR-ligand-mediated activation of CYP1A1 gene and enzyme [29].

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is, daidzein gets metabolized by aromatic hydroxylation at 3’position. In vitro enzyme and cell-based assays on MCF-7 cellsconfirmed the antiproliferative activity of the formed activemetabolite [17]. Similarly, the flavone diosmetin, found in oliveleaves, is activated to the flavone luteolin, mainly by CYP1A1-mediated metabolism, which was studied in MDA-MB-468and TCDD-induced MCF-7 cells [66,67]. It has been evidencedthat CYP1A1 has the highest rate of metabolism, as comparedwith hepatic CYP450s 1A2, 3A4, 2C9 and 2D6 for methylatedanticancer flavonoids such as tangeretin, which is an activeconstituent of orange peel [68]. Furthermore, the other flavonols

such as galangin and kaempferide have also been reported assubstrates of CYP1A1. The kaempferol present in black andgreen tea is a hydroxylated product of galangin, which hasbeen found to possess anticancer properties [69]. It is observedthat theCYP1A1-catalyzedmetabolism of dietary anticancer fla-vonoids produces compounds that also possess strong anticanceractivity.Dietary constituents such as stilbene resveratrol suppresscancer progression by inhibiting the CYP1A1-catalyzed meta-bolic activation to carcinogenic product and AhR-mediatedCYP1A1 enzyme induction. It occurs either by blockage ofAhR binding to the inducer or by prevention of AhR-ARNT

O

HO

OH

HO

OH

O

B[a]P B[a]P-7,8-Oxide

Epoxide hydrolase

CYP1A1

[-]-B[a]P-7,8-dioland

[+]-B[a]P-7,8-diol

[-]-B[a]P-7,8-diol-9,10-epoxideand

[+]-B[a]P-7,8-diol-9,10-epoxide

CYP1A1

DNA mutation

Carcinogenicity

Figure 3. The structures of B[a]P and its carcinogenic metabolites [14].

O

F

H3C

F

H3C

F

NH2 O

F

NH2

Inactive prodrug

N

S

F

NH2

Me

O

F

NH2 O

FHN

S

NF

Me

OH

NH

OH

Drug-AhR

Drug-AhR-ARNT

XREs Genotoxic metabolite

Aminoflavone

Translation

Translation

Active drug

5F 203

CYP1A1

CYP1A1

Figure 4. Model explaining the mechanism of aminoflavone or 5F-203-mediated CYP1A1 induction and their anticancer

effect [61,64,95,96].

Cytochrome P450 1A1-mediated anticancer drug discovery: in Silico findings

Expert Opin. Drug Discov. (2012) 7(9) 775

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binding to XREs as shown in Figure 5 [70]. Most of thecompounds are observed to possess CYP1A1 inhibitory activitybut still these models do not show adequate confidence inexplaining why CYP1A1 is induced and further inhibited.More importantly, in the in vitro or in vivo models where celllines with a malignant phenotype are used, the inhibition ofCYP1A1 does not alter the tumorigenic state of the cells, asthey have already lost the ability to control their growth. Inthis sense, the mechanism of action of dietary flavonoids andphytochemicals as CYP1A1 inhibitors need to be subjected forfurther consideration [71]. CYP1A1 induction in cancerous cellsby these compounds and their subsequent metabolism to moreactive compounds is an alternative model that can explain thecancer-preventive properties of these compounds [72].

3. Therapeutic potential of CYP1A1: outcomeof clinical drug candidate

Certain cancers such as lung, breast, rectal and colon cancers areinitiated by a cascade of molecular events by modulation of AhRand induction of CYP1A1 by environmental pollutants andendogenous compounds containing polyaromatic hydrocarbonring system such as benzo-(a)-pyrene (11), 2,3,7,8-tetrachlorodi-benzo-p-dioxine (12), 3-methylcholanthrene (13) [30]. Thesecompounds get metabolized by CYP1A1 to form carcinogenic

metabolites, which lead to progression of cancer [73-75].However, the compounds belonging to 2-(4-aminophenyl) ben-zothiazole structural class (1 -- 10) can induce expression ofCYP1A1 as well as get metabolized by CYP1A1 and producea genotoxic metabolite or reactive intermediate, which formsDNA adduct and causes lethal damage to DNA leading todeath of oncogenic cells. The CYP1A1 is overexpressed incancerous cells in comparison with normal cells and henceselective targeting of cancerous cells is possible. A number ofacademic and industrial groups are working in this area ofmulti-target-directed mechanisms in cancer management, butso far no drug candidate has reached the market.

3.1 Clinical drug candidate -- NSC710305Bradshaw and research groups at School of Pharmaceutical Sci-ences, University of Nottingham, are actively involved in thedesigning of CYP1A1 substrates, which generate a genotoxicproduct. NSC710305 is one such compound designed by themthat is under clinical trials [61,76]. They have synthesized anddeveloped the 2-(4-aminophenyl) benzothiazole structural classof compounds, and as an outcome they reached up to a clinicalcandidate prodrug of 5F-203, that is, NSC710305 (15) fromthe lead compoundDF-203 (14) [24,61]. Research findings associ-ated with this project have been extensively published in theprimary literature (‘Antitumor Benzothiazoles’ series), and active

11 Benzo(a)pyrene

O

O Cl

Cl

Cl

Cl

12 2,3,7,8-Tetrachlorodibenzo-p-dioxine 13 3-methylcholanthrene

S

N

Me

NH2

S

N

Me

NH

F

NH2

(CH2)4NH2

O

14 DF 203 15 Phortress

O

F

H3C

F

NH2 O

F

NH2

16 Aminoflavone

5

6

7 84′

3′

H3C

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compounds from the series are subjected to patent protectionthrough Cancer Research Technologies, U.K. (International Pat-ent Number, WO 01/14354 A1) [77]. However, inspection of theNCI (National Cancer Institute) cancer cell line selectivity pat-terns for polycyclic aromatic hydrocarbons reveal one strikingfeature bywhich the exquisitely selective 2-(4-aminophenyl) ben-zothiazole series differ dramatically from CYP1A1-inducing car-cinogens [61]. Preclinical toxicology studies indicate low ormanageable toxicity for NSC710305 and have further reinforcedthe view that this novel series of antitumor agents differedmarkedly in mechanistic and toxicological terms to knowncarcinogens [28,78].

3.1.1 Synthesis of antitumor benzothiazolesA series of polyhydroxylated 2-phenylbenzothiazoles has beensynthesized by Stevens et al. in 1994 as tyrosine kinase inhibitors.The synthesized compounds were found to inhibit WiDr humancolon tumor cells and MCF-7 human mammary tumor cellsin vitro with IC50 values in the low micromolar range. Onlymarginal inhibitory activity was found for EGF receptor-associated protein tyrosine kinase from a membrane preparationof A431 cells, but these compounds inhibited DNA synthesiswhen added to cells during S phase. The 4,6-dihydroxy-2-(4-hydroxyphenyl) benzothiazole was found to be the mostactive compound of this series [79]. Using this as a parent com-pound, several series of compounds were synthesized by variousresearch groups, and they found promising antitumor activityagainst sensitive cell lines (e.g., breast MCF-7 and MDA468 cells) [80]. The structure--activity relationships (SAR) byShi et al. revealed that the activity for heterocyclic rings follow

this sequence: benzothiazole > benzoxazole > benzimidazole.The 2-(4-aminophenyl) benzothiazoles bearing a 3’-methyl, 3’-bromo, 3’-iodo, and 3’-chloro substituent were found to bepotent and their activity extends to ovarian, lung and renal celllines [81,82]. Chua et al. confirmed N-acetylation and oxidationas the main metabolic transformations for N-acyl derivatives of2-(4-aminophenyl) benzothiazole, with the predominant processbeing dictated by the nature of 3’-substitution [83]. The role of C-and N-hydroxylated metabolites was studied in vitro byKashiyama et al., which showed that these metabolites are devoidof selective antitumor activity [84]. The new series of quinol etherand ester-substituted benzothiazoles were reported byWells et al.[85]. Hutchinson et al. suggested the synthetic routes to a series ofmono- and difluorinated 2-(4-amino-3-substituted-phenyl) ben-zothiazoles and reported that substitution at 3’-position by cyanoor alkynyl groups was found to have potent cytotoxic activity(GI50 < 1 nM) in sensitive human breast MCF-7 (ER+) andMDA 468 (ER-) cell lines [86,87]. As a prodrug approach, a seriesof sulfamate salt derivatives of 2-(4-aminophenyl) benzothiazoleantitumor agents were prepared and evaluated for parenteraladministration by Shi and his research group. These salts weresparingly soluble under aqueous conditions (pH of 4 -- 9) anddegraded to active free amines under strong acidic conditionsand were markedly less active than their parent amines [88]. Asan amino acid prodrug, a series of water-soluble L-lysyl- and L-ala-nyl-amide prodrugs were synthesized by Hutchinson et al. Theseprodrugs displayed good aqueous solubility (in weak acid) andstability at ambient temperature and also degraded to free basein in vivo studies. The lysyl-amide prodrug of 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (NSC710305) has been

HO

OH

OH

Natural flavonoid

AhR AhR ARNT

CYP1A1

HO

OH

O

Carcinogenic metabolite

Procarcinogen

AhR ARNT

XREs

B[a]P

B[a]PmRNA translation

Figure 5. Anticancer action of natural products, such as the stilbene resveratrol, is based on the inhibition of benzo[a]pyrene

binding to AhR to generation of carcinogenic metabolite notably benzo[a]pyrene-7,8-diol-9,10-epoxide (crossed arrow

indicates inhibition of process at these steps) [70].

Cytochrome P450 1A1-mediated anticancer drug discovery: in Silico findings

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selected for Phase I clinical evaluation [61,78,89,90]. Mortimer et al.synthesized a series of new 2-phenylbenzothiazoles based on2-(3,4-dimethoxyphenyl)-5-fluorobenzothiazole (5F-203, GW610). However, this new series of compounds contrasts with thepreviously reported 2-(4-aminophenyl) benzothiazoles, as com-pounds are not reliant on induction of CYP1A1 expression forits antitumor activity [91]. Vasselin et al. designed and synthesizeda series of fluoro-, methoxyl- and amino-substituted 3-phenyl-4H-1-benzopyran-4-ones derivatives based on quercetin, genis-tein and 2-phenylbenzothiazole structural similarities. Significantgrowth inhibitory activity against MDA-MB-468 cell lineswas found for some compounds but not for methylene-bridged derivative [92]. Furthermore, a novel series of antitumorbenzothiazoles such as 8-chloro-3-cyano-4-imino-2-methylthio-4H-pyrimido[2, 1-b][1, 3]benzothiazole and its 2-substitutedderivatives was synthesized by Labhsetwar et al. and evaluated atNCI. These displayed remarkable anticancer activity againsthuman cancer cell lines [93].

3.1.2 Electronic structure analysis of

2-(4-aminophenyl) benzothiazolesLaughton and his research group from School of PharmaceuticalSciences, University of Nottingham, reported frontier molecularorbital (FMO) [94] analysis of these compounds and their metab-olites [95]. When conventional chemical reasoning failed toexplain the comparative biological responses of the fluorinatedbenzothiazoles, the FMO analysis can easily explain theelectronic structures and metabolic activation of this simple anti-tumor pharmacophore. FMO analysis of antitumor 2-(4-amino-phenyl) benzothiazoles can rationalize the propensity ofCYP1A1-generated reactive intermediates, which either undergodeactivation by C-hydroxylation or bind covalently to DNA.Distribution of the highest occupied molecular orbital(HOMO) for the nitrenium species derived from each fluori-nated analogue correlates perfectly with production of an export-ablemetabolite. The results obtained by thismethodwere furtherverified experimentally for related compounds shown in Figure 1.Oxidation of 2-(4-aminophenyl) benzothiazoles at several sitesby CYP1A1 could generate a range of electrophilic intermediatescapable of forming covalent bonds with DNA (Figure 6). Thesesites include i) oxidation of the amino group to a hydroxylamine,which could subsequently generate a nitrenium ion$p-carbocation reactive species, ii) hydroxylation at the 3-methylgroup, followed by dehydration, would generate a quinoneme-thide-imine (however, this is unlikely to be a significant routein benzothiazole activation) and iii) epoxidation/hydroxylationin the benzothiazole ring possibly accompanied by an NIH shiftof hydride or fluoride (fluorine walk), or fluorine displacement,could generate DNA alkylating species [95]. FMO studies high-light the activation of an amino group of benzothiazoles alongthe nitrenium ion$p-carbocation pathway and suggest thatDNA adducts derived from epoxide intermediates are unlikely(Figure 2). In formulating this conclusion, it is important tonote that the in vitro antitumor profiles of active 2-(4-amino-phenyl) benzothiazoles are starkly different from those of

carcinogenic amines and polycyclic aromatic hydrocarbons,which induce CYP1A1, and get metabolized by CYP1A1. Thesestudies are useful to elucidate the structures of DNA adductsformed by these mysterious benzothiazoles and to determinewhy they are so damaging to tumor cells.

Ab initio molecular orbital (MO) calculations wereperformed by Hilal et al. to study the electronic structure,substituent and solvent effects on the singlet--triplet gapsof a series of nitrenium ions [96]. DFT (density functionaltheory) calculations at B3LYP/6-311++G** level of theorypredicted that the nitrenium ion derived from metabolismof 2-(4-aminophenyl) benzothiazoles exists in ground sin-glet state. The counter-intuitive patterns of metabolismcan be explained only by considering the active intermedi-ate to be a nitrenium ion probably via an aryl hydroxyl-amine intermediate formation (Figure 7). Nitrenium ionsare azocations with structure [R--N--R’]+ and are believedto play an important role in carcinogenic processes. Thiscarcinogenic power is attributed to their high electrophilic-ity, which allows these species to readily bind to DNA mol-ecules as shown in Figure 7. Nitrenium ions are generallyproduced in the singlet state, and consequently, they mustundergo an intersystem crossing to the low-lying tripletstate as shown in Figure 7. In the singlet state, the nonbond-ing electrons occupy sp2 orbital leaving an empty p orbitalon the central nitrogen, making these species very reactivetoward nucleophiles. In the triplet state, the nonbondingelectrons have parallel spins and occupy sp2 orbital as wellas an orbital with large p character, usually behaving as dir-adical. Stabilization of the singlet state is favored as com-pared with the triplet state to transfer charge density fromthe phenyl ring to formally electron-deficient nitrogenatom. Fourier transform analysis of the internal rotationpotential energy function, around the exocyclic bond(C--N), has been performed for nitrenium ions. The magni-tude and origin of the potential energy barriers show thatparent nitrenium ion derived from metabolism of antitu-mor 2-(4-aminophenyl) benzothiazoles exists in the groundsinglet state.

3.1.3 Comparative molecular field analysis (CoMFA)

studiesA series of benzoxazole and benzothiazole were analyzedusing comparative molecular field analysis (CoMFA) [97] andmolecular docking by Ju et al. to understand the molecularinteractions of inhibitors with CYP1A1 [98]. They appliedtwo conformer-based alignment strategies to construct reliableCoMFA models. The experimental data of benzoxazolesand benzothiazoles were collected from Westwell’s researchpapers [91,99]. A satisfactory CoMFA model was obtained topredict the activities of test set compounds with predictivecorrelation coefficient (r2-pred) value of 0.809. The alignmentof highly active compound with the CoMFA electrostatic andsteric contour maps are shown in Figure 8. The rectangularregions near the middle of 5- and 6-positions of benzothiazole

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ring and 1’-, 3’-, 4’- and 5’-positions of the phenylgroup suggested that the electronegative groups are beneficialfor their activity (atom numbering as per Figure 1). Thecompounds having an electron-withdrawing substitution at5- or 6-position of a benzothiazole ring show higher activitythan that of compounds having no substitution at thesepositions. The activity may be enhanced by introducing theelectron-rich substituent at both 5- and 6-positions of a ben-zothiazole ring of compounds. A large triangular contourcovering the 4’- and 5’-positions of the phenyl group indicatesthat electropositive groups are favorable for its activity. Thepentagonal contour near the 2’-position of phenyl ring indi-cates the unfavorable region for bulky substitutions fortheir antitumor activity. The optimum steric size of 5’-substituent on phenyl ring is equivalent to the size of methoxygroup for maximum activity of compounds. This was sug-gested by a circular contour that covers 5’-methoxy group ofcompound as shown in Figure 8.

3.2 Clinical drug candidate -- 5-aminoflavone

(AFP464)In search of new antitumor compounds, Akama et al. fromKyowa Hakko Kogyo Co., Ltd. synthesized a series ofnovel amino-substituted flavone derivatives and examinedtheir antitumor activities. The compound 5-aminoflavone(4H-1-benzopyran-4-one, 5-amino-2-(4-amino-3-fluoro-phenyl)-6,8-difluoro-7-methyl; NSC686288) showedremarkable antiproliferative activity against the humanbreast cancer cell line MCF-7. 5-Aminoflavone (5-AF) (USPatent Number US 2010/0260753 A1) [100] showed antitu-mor activity toward the estrogen receptor (ER+)-breast can-cer cell lines and was devoid of any effects against the ER-negative human cancer cell lines [101]. The structure--activityrelationship (SAR) studies performed by Akama et al. sug-gested that the methyl, hydroxymethyl, (acyloxy)methyland aminomethyl substituents at 7-position favors itsantitumor activity (atom numbering as shown in structure

S

N

NH2

REpoxide formation +/- NIH shift(when R1= H or F)

Oxidation and dehydration(when R=Me) to aquinonemethide-imene

Oxidation and conversionto a nitrenium species

R1

Figure 6. The potential sites present in fluorinated 2-(4-aminophenyl) benzothiazoles for oxidation and generation of

electrophilic reactive intermediates [95].

N

H

HRCYP450 1A1

N

OH

HR

NH

RdGNH

R

N

HN

N

NH

NH

O

Deoxyguanosine Nitrenium ion

Aryl hydroxylamine

N

Y

Z

N

Y

Z

Singlet Triplet

Figure 7. Carcinogens derived from the in vivo catabolism of aromatic amines [96].

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16) [102]. Introduction of fluorine substitution to putativemetabolic positions suggests its effectiveness in the enhance-ment of in vivo antitumor activity, probably due to the blockof metabolic deactivation [103]. 5-AF can act by increasingtranscription of CYP1A1 mRNA and CYP1A1 protein, fol-lowed by covalent binding of 5-AF metabolite to DNA,phosphorylation and stabilization of p53 and increasedexpression of the p53 transcriptional target p21 [104,105].The mechanism of action of 5-AF is also the same as5F-203 and as shown in Figures 3 and 4. Meng et al. studiedthe induction of 5-AF-mediated DNA-protein cross-links(DPC) and H2AX in MCF-7 human breast cancer cells. Sul-fotransferase 1A1 (SULT1A1) and CYP1A1 expression arealso induced by 5-AF in MCF-7 cells. This study suggeststhe existence of an AhR-mediated positive feedback for5-AF activation by CYP1A1 and SULT1A1 and its antican-cer activity. Furthermore, 5-AF can be metabolized byCYP1A1 at two amino groups to form N-hydroxyl metabo-lites that are substrates for bioactivation by SULTs. N-sul-foxy groups can be further converted to nitrenium ion,which are responsible for forming DNA adduct and thustumor cell death [64,106]. Loaiza-Perez et al. proved the selec-tive expression of CYP1A1 and activity of 5-AF againstMCF-7 human breast cancer cells both in vitro and in vivoas xenografts in athymic mice. Expression of SULT1A1and H2AX are used as biomarkers for prediction of 5-AFactivity during clinical trials monitoring [63]. Meng et al.observed that Mdm2 and p21CIP1/WAF1 protein levelsshowed a biphasic response, as they accumulated atsubmicromolar doses and then decreased with increasing5-AF concentration [107]. 5-AF also inhibited HIF-1a tran-scriptional activity and protein accumulation in MCF-7cells in an AhR-independent fashion [108]. 5-AF was exten-sively metabolized by CYP1A1 and CYP1A2 to severalmetabolites, one of which was a potentially reactive

hydroxylamine [104]. Chen et al. identified the thirteen5-AF metabolites in mouse urine or from microsomalincubations, which included three monohydroxy-AFs, twodihydroxy-AFs and their sulfate and glucuronide conjugatesas well as one N-glucuronide by LC-MS/MS fragmentationpatterns [109]. The prodrug of parent aminoflavone asdimethanesulfonate (NSC710464; AFP464) salt was devel-oped to improve its drug administration properties and cur-rently it is in Phase I clinical trials for further clinicalevaluation [106].

4. In silico methodologies applied to CYP1A1

4.1 Homology modeling and molecular docking

studiesSeveral homology modeling [110] studies of CYP1A1 have beenreported in the literature to gain an insight into ligand-bindingdomain of CYP1A1 with substrates and inhibitors [98,111-121].Subsequently, the protein--ligand complexes were developed byincluding information about known ligands. The moleculardocking studies [122] provide useful information about the non-covalent interactions such as hydrogen bonding, van der Waalsinteractions and so on between substrates or inhibitors andactive-site residues of CYP1A1. Homology models of humanCYP1A1 published before year 2007 were built using the tem-plate crystal structures such as rabbit CYP2C5 (PDB ID:1N6B, 1NR6), human CYP2C8 (PDB ID: 1PQ2) andhuman CYP2C9 (PDB ID: 1OG5, 1R9O) in the absence ofunavailability of CYP1A2 crystal structure in protein data bank(PDB). [111-119] The more reliable homology models of humanCYP1A1 were developed on the basis of single templatecrystal structure of human CYP1A2 having PDB ID 2HI4[98,111,121,123], as the homologymodel developed using single tem-plate with high sequence identity is better than model developedon the basis of multiple templates [124,125]. The homology

HH

H

H HF H

H

H

H

H

H

HH

H

C C

C

C

C

O

C

CC

C

C

SC

C

CC

N C

C

O

O

Figure 8. Contour maps with highly active compound, that is, 2-(3,4,5-trimethoxyphenyl)-5-fluorobenzothiazole. The circular

contour indicates where bulky group favors activity, pentagonal contour indicates where small group favors activity, whereas

triangular and rectangular contour indicates positive and negative charge favors activity [98].

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modeling and molecular dynamics simulations were performedby Ju et al. and Rosales-Hernandez et al. to get a steady conforma-tion of human CYP1A1 homology model [98,126,127] using tem-plate of CYP1A2 (PDB ID: 2HI4) crystal structure. Thecompounds used for molecular docking were selected fromCoMFA study using Surflex software. The analysis of hydrogenbond interactions has confirmed that Arg106 and Ile386 play arelatively important role in the binding potency of the inhibitors.Furthermore, the calculated binding free energies of compoundswere in accordance with their GI50 values. Thus 3D-QSARmodel and molecular docking studies may provide the guidancefor designing new potential CYP1A1 substrate as anticancer com-pounds and also prove the relevance of built homology model ofCYP1A1 [98].

In another study, the homology model of CYP1A1 developedusing human CYP1A2 (2HI4) crystal structure as a template wascompared with i) homology model of CYP1A1 based on thetemplate of rabbit CYP2C5 crystal structure as well as ii)CYP1A1 homology model built using composite templates ofhuman CYP2C8 (1PQ2), CYP2C9 (1OG5, 1R9O) and rabbitCYP2C5 (1N6B, 1NR6) crystal structures. Results showed thatthe homology model of CYP1A1 developed from CYP1A2 crys-tal structure gave energetically and sterically favorable struc-ture [111]. The molecular docking studies of CYP1A1 substratessuch as benzo[a]pyrene, ethoxyresorufin and methoxyresorufinin an active site of CYP1A1 homologymodel has identified inter-acting amino acid residues that are involved in substrate binding.The role of Ser120, Ser122, Phe123, Phe224, Phe258, Tyr259,Asp313, Thr321, Val382 and Ile386 active-site residues in7-ethoxyresorufin binding and turnover were confirmed fromthe experimental site-directed mutagenesis studies on CYP1A1mutants: S122A, F123A, F224A, A317Y, T321G, and I386Gas shown in Figure 9. It confirms the importance of aromatic inter-actions over hydrogen bonding in orientating 7-ethoxyresrufinin a catalytically favorable orientation in an active site cav-ity [111,115,116]. Sangamwar et al. [121] also have identified active-site residues based on amino acid sequence alignment ofCYP1A1 with CYP1A2 crystal structure. It was assumed thatthe ligand-binding modes are similar in the target (CYP1A1)and template CYP1A2 protein structure. The active site containsthe highly conserved residues Ile117(Ile115 in CYP1A1 model),Thr118(Ser116), Ser122(Ser120), Thr124(Ser122), Phe125(Phe123), Phe226(Phe224), Phe260(Phe258). The active-site residues in CYP1A1 -- Ile310, Val311, Gly318, Phe319,Ser380 and Gly495 -- are different from those residues involvedin the active site of CYP1A2. These altered amino acid residuesmay be responsible for substrate specificity between CYP1A1and CYP1A2. Molecular docking confirms the hydrophobicinteractions of an aromatic ring of DF-203 with Ile310 andVal311, while Gly318, Phe319 and Gly495 have van der Waalsinteractions with 2-substituted phenyl ring. Ser380 is involvedin weak hydrogen bonding interactions with amino substitutionat phenyl ring. The heme prosthetic group is at a distance of 4Afrom the amino-phenyl group of ligand, which has a significantrole in the metabolism of substrates. This binding has important

role in the potency of a lead compound. This proved effectivenessof structure-based drug design (SBDD) in the designing ofnew structural scaffolds targeting CYP1A1 [121]. Active-sitecharacterization and the study of involvement of specific aminoacids in enzyme-substrate binding facilitate structure-based drugdesign. In future, availability of experimentally determinedCYP1A1 crystal structure in the protein data bank may behelpful in understanding an exact 3D geometry andenzyme--substrate interactions.

4.2 Molecular dynamics simulation studiesSchwarz et al. performed molecular dynamics (MD) simulationstudies [128] on the homology model of CYP1A1 to understandthe metabolism of arachidonic acid (AA) and eicosapen-taenoic acid (EPA) [114]. The enzyme--substrate complexeswere adjusted manually to explain fatty acid oxidation at variouspositions. Results suggested that the substrates were stabilized inCYP1A1 active-site cavity, mainly by hydrophobic interactions;the terminal carboxyl group is responsible for forming hydrogenbonds with the amide hydrogen of Gly79, Ser80 and Thr81 andwith the hydroxyl group of Thr81. These interactions persistthroughout the 5ps molecular dynamics simulations for differ-ent productive orientations of AA and EPA. Molecular dynam-ics simulations showed that the most stable binding orientationwas the one leading to oxidation at C19 (-135 kcal/mol energy),which allowed C19 methylene hydrogen atoms to be within 3Afrom the heme oxygen during the 5ps simulation, thus promot-ing substrate hydroxylation. In case of C17 hydroxylation, thedistance between methylene hydrogen and heme oxygen wasabout 5 A, which is longer than C19 hydroxylation. This studysuggests that these hydrogen bonds play a role in stabilizingthe fatty acid molecule in the CYP1A1 active site and ensureits oxidation at hydrocarbon tail of the substrate. The moleculardynamic simulation results are well correlated with experimentalproduct characterization results by high-performance liquidchromatography (HPLC). In another study by Szklarz andPaulsen [115], the molecular dynamics simulations were per-formed to calculate binding free energy [129] for benzo[a]pyrene,ethoxyresorufin and methoxyresorufin. It was observed that thenon-bonded enzyme--substrate interaction energy for ethoxyre-sorufin (-13.19 kcal/mol) was lower than that for benzo[a]pyr-ene (-11.11 kcal/mol) and for methoxyresorufin (-10.88 kcal/mol). Results are consistent with higher activity of CYP1A1toward the ethoxyresorufin as compared with others. Theobtained values were similar to those observed experimentally,suggesting that this approach might be useful for prediction ofbinding constants [115].

4.3 Quantitative structure--activity relationship

(QSAR) studiesQuantitative structure--activity relationship (QSAR) proce-dures [130] are regularly employed in the drug discovery process,especially where physicochemical properties can be modified inorder to improve clearance characteristics of CYP450 substrates.The QSAR studies were performed by Lewis et al. [112] using the

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previously reported biological activities on induced liver micro-somes [131,132]. A dataset of molecular and structural descriptorswas generated for the homologous series of N-alkoxyresorufins(namely methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl andoctyl) using molecular modeling techniques. These includedmolecular orbital (MO) calculations, molecular size, shape,such as planarity (area/depth2 ratio) and rectangularity (length/width ratio). Furthermore, lipophilicity and log P values wereestimated using Pallas software (CompuDrug Ltd, Budapest,Hungary) and Clog P software (BioByte Corp., Pomona, CA,USA). There is a good correlation (R = 0.97) between experimen-tally determined linear physicochemical TLC parameter [133] andlog P values. The decrease in biological activity was observed withincrease in alkyl chain length for N-alkoxyresorufin series. TheCYP1A subfamily appears to have a preference formethyl in con-trast to ethyl for CYP1A2 enzymes. It was observed that thehydrophobic effect is greater than the loss in rotational freedom,although there is decrease in translational energy upon binding.Secondly, the topography that includes size, shape and hydro-phobic character of CYP450 active site plays an important rolein the substrate selectivity. When location of the hydrogenbond donor residues and their orientation with respect to thehydrophobic region in an active site is considered, it gives moreunderstated distinctions between methoxy and ethoxy congenerstoward CYP1A1 andCYP1A2. As the biological data used in thisQSAR study were obtained from a single series of compoundsusing rodent liver microsomes, the conclusions derived are notmuch reliable for predictions of biological activity in human liver

microsomes. Thus, to increase the reliability of predictions,QSAR studies must include multiple series of compounds anddata from human studies. In this way, a combination of homol-ogy modeling, molecular docking with QSAR analysis providedsome crucial insights into the understanding of basic molecularmechanisms of substrate selectivity toward CYP1A1 [112]. Thisfacilitates an understanding of CYP1A1-mediated metabolismat the molecular level that can be employed as a pre-screeningprocess in the safety evaluation of chemicals [134]. Iori et al. haveused highly informative descriptors derived fromQMcalculationand various ligand interaction energies calculated by moleculardynamics simulation for QSAR studies. They have establishedgood correlation between CYP1A1 inhibitory activity by flavo-noids and the desolvation free energy. They were successfullyable to establish relationship between calculated descriptorsand differences in metabolism of anticarcinogens giving riseto bioactive metabolites [135]. The QSAR studies performed byGonzalez et al. stated importance of HOMO energy forCYP1A1 substrates and their activity. Also favorable electrostaticinteractionswere observed with Phe123 and stacking interactionswith Phe224 or Phe258 in CYP1A1 [136].

4.4 Pharmacophore mappingThe pharmacophore mapping [137] gives indirect informationabout the protein active site based on shape, electronic propertiesand conformation of substrates, inhibitors ormetabolic products.For rat CYP1A1, the first pharmacophore model was developedusing benzo[a]pyrene and a variety of other polycyclic aromatichydrocarbons (PAHs) [138]. A hydrophobic cleft that is asymmet-rically oriented relative to hemewas described by thismodel. Thismodel was extended to accommodate several other PAHs [139,140]and a variety of small non-PAH substrates [27]. It indicated thatthe hydrogen bond and aromatic interactions between thesesmall substrates and the CYP1A1 were important for substratebinding. No pharmacophore models have been developed forhuman CYP1A1 so far. As seen from literature, the pharmaco-phore mapping has only just begun to apply computationalmethodologies to CYP1A1 enzyme. 3D-QSAR/Pharmacophoremapping approaches have produced a qualitative, quantitativeand visual approximation of CYP1A1 ligands and offer insightsinto the CYP1A1 active site.

5. Conclusion

The history of CYP1A1 as a novel molecular target for cancermanagement started when the unusual anticancer activity for2-(4-aminophenyl) benzothiazoles and 5-aminoflavone wereoriginally discovered in a program of tyrosine kinase inhibitorscreening. The chemical analogues possessing superior growthinhibitory properties were then synthesized and found activeagainst certain human cancer cell lines by NCI in vitro antican-cer drug screening. The SAR studies indicated that the highestantitumor activity for benzothiazole ring system and aminofla-vone ring system with fluoro substitution. Also the effect ofmethyl, bromo, iodo and chloro substitution was successfully

Figure 9. The key residues involved in formation of the

active-site pocket of CYP1A1 and docked 7-ethoxyresorufin

in productive orientation for hydroxylation [111]. 7-Ethox-

yresorufin and heme are represented in stick view.

Active-site amino acid residues are represented in lines view.

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elucidated by various SAR studies. Finally, the L-lysyl amide pro-drug of 5F-203 and dimethanesulfonate prodrug of 5-AF weredeveloped to improve their intravenous administration and arecurrently under clinical investigation in clinical trials. Analysiswith western blot and RT-PCR techniques in sensitive andnon-sensitive human cancer cell lines has identified selectiveexpression of CYP1A1 by these chemical analogues. Thebiotransformation of these analogues to reactive metaboliteis responsible for their antitumor potency. The role ofCYP1A1 in such biotransformation is sufficiently elaboratedby several researchers across the globe suggesting druggabilityof CYP1A1 as an anticancer target. Till date, various in silicotechniques were applied by researchers to explore the structureand function of CYP1A1. The homology model of CYP1A1developed on the basis of CYP1A2 as template was found to per-form better than other homology models. The role of Ser120,Ser122, Phe123, Phe224, Phe258, Tyr259, Asp313, Thr321,Val382 and Ile386 amino acid residues are found to be impor-tant for hydrophobic and hydrogen bonding interactions ofsubstrate withCYP1A1.Usingmolecular docking and dynamicssimulation studies, researchers successfully correlated substratebinding energy with their experimentally observed parameters.QSAR studies highlight the importance of lipophilicity,HOMO energy and desolvation free energy of compounds tobe substrates of CYP1A1.Quantummechanically derived distri-bution of HOMO was successfully correlated with reactivemetabolite generation from 2-(4-aminophenyl) benzothiazoleusing FMOanalysis. Also the formation of nitrenium ion speciesfavorable in its singlet state is confirmed by quantummechanicalstudies. The importance of more electronegative and electron-withdrawing substitutions at various positions on benzothiazoleand phenyl ring was investigated from CoMFA studies. Theinformation obtained from in silicomethodologies can be bene-ficial for the designing and development of better antitumorcompounds targeting CYP1A1. From the above discussion, weconclude that the in silico approaches would be helpful to explorethe different modes of physicochemical interactions in the cata-lytic site of CYP1A1 and various properties of compoundsproducing genotoxic metabolite.

6. Expert opinion: in silico perspective fordesigning CYP1A1 inducers/substrates

The enhanced endogenous expression of CYP450s in tumor cellsoffers certain advantages in the cancer chemotherapy by theactivation of prodrugs specifically in tumor cells. The CYP1A1overexpression in certain types of cancer cells offers a novel targetin cancer management because although several CYP1As,CYP2Cs and CYP3As exhibit enhanced expression in sometumor cells, these enzymes also have considerable expression innormal tissue, mainly in liver. Additionally, the less polymorphicactivity of CYP1A1 offers minimum pharmacokinetic variabilityin anticancer drug regimen over the large population. The cur-rent inclusion of NSC710305 and NSC710464 in Phase I/IIclinical trials also validates anticancer potential of CYP1A1.

Currently, the drug discovery studies on CYP1A1 remain con-fined to the group of 2-(4-aminophenyl) benzothiazole and5-aminoflavone compounds, which are inducers as well as sub-strates of CYP1A1. Therefore, designing of novel and potentiallymore effective classes of compounds acting on CYP1A1 mayprove beneficial in anticancer drug discovery. However, the dis-covery and development of above molecules lack insights fromin silicomethodologies. In this context, in silico techniques couldbe efficiently employed in search of novel molecules or structuralscaffolds, which would selectively bind to the CYP1A1 and inturn produce a genotoxic metabolite.

Homology modeling has revealed 3D structure of CYP1A1 inthe absence of its crystal structure and also helped in elucidatingthe structural features of CYP1A1 that are responsible for sub-strate recognition. The determination of crystal structure ofhuman CYP1A1 may offer new insights in CYP1A1--substrateinteraction patterns. The in-depthmolecular docking and molec-ular dynamics simulation studies on benzothiazole and aminofla-vone series of compounds would probably be helpful in exploringexact mechanism of substrate binding to CYP1A1. The crucialfindings obtained from these molecular docking and dynamicssimulation studies may perhaps be used to modify the com-pounds with better molecular interactions and metabolic profileby CYP1A1. Various pharmacophore mapping and QSARapproaches were previously used on series of CYP1A1 inhibitorsbut till date no such methodologies were carried out for the seriesof benzothiazole and aminoflavone compounds. The practice ofthese two methodologies could certainly help in identifying cru-cial structural features in active compounds that are recognizedat CYP1A1 receptor site and are responsible for their antitumoractivity. The pharmacophore maps of active compounds couldbe useful to screen huge library of synthesizable molecules tofind out potential hits against CYP1A1. Additionally, applicationof quantum chemical studies on benzothiazole and aminoflavoneclass of compound can give an idea about some informativedescriptors that would explain the CYP1A1-specific metabolismand generation of reactive metabolites. These ligand-basedapproaches should be coupled with receptor-based approachessuch as molecular docking and dynamics techniques to get valu-able hints for structural modifications to potentiate the antitumoractivity. Furthermore, in silico pharmaceutical profiling of thescreened molecules can reduce the chance of failure of lead com-pounds during drug development phase. Prediction of toxicityand other pharmacokinetic parameters (ADME) could be accom-plished using QSAR models or pharmacokinetic prediction soft-ware such as Pallas, Rebol and so on. Further lead optimizationcould be done by taking account of these toxicity and pharmaco-kinetic properties. It is also acceptable that the in silico predictionsare not accurate until they are coupled with in vitro findings.However, this information would influence the researcher’s deci-sion to proceed with synthesis via medicinal chemistry strategies.The molecules obtained through virtual screening should betested in vitro against various cancer cell lines.

Combined findings from all in silico tools could providevaluable clues to pick up promising hits acting on CYP1A1.

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While addressing the time and money needed to develop a mol-ecule successfully up to the market, one can reduce both byexploring the potential known compounds, which are alreadyreported for their antitumor potential against CYP1A1. Finally,this review sufficiently elaborates the various in silico approaches,which promises to be fruitful if coupled withmedicinal chemistrydriven and in vitro approaches to get holistic picture in discoveryof antitumor compounds active on CYP1A1.

Declaration of interest

The authors acknowledge financial support from Departmentof Biotechnology (DBT), New Delhi, Govt. of India. Authorsare also thankful to the Director of the National Institute ofPharmaceutical Education and Research (NIPER), S.A.S.Nagar for providing financial support and necessary facilitiesto carry out this work.

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AffiliationPrajwal P Nandekar1 & Abhay T Sangamwar†2

†Author for correspondence1Research scholar,

National Institute of

Pharmaceutical Education

and Research (NIPER),

Department of Pharmacoinformatics,

S.A.S. Nagar (Mohali),

Punjab-160062, India2Assistant professor,

National Institute of Pharmaceutical Education

and Research (NIPER),

Department of Pharmacoinformatics,

S.A.S. Nagar (Mohali),

Punjab-160062, India

Tel: +91 172 2214682 87 Ext. 2211;

Fax: +91 172 2214692;

E-mail: abhays@niper.ac.in

Cytochrome P450 1A1-mediated anticancer drug discovery: in Silico findings

Expert Opin. Drug Discov. (2012) 7(9) 789

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