Advances on structure-activity relationship of NQO1-targeting antitumor quinones

Chinese Journal of Natural Medicines 2012, 10(3): 01700176

Chinese Journal of Natural Medicines

doi: 10.3724/SP.J.1009.2012.00170

Advances on structure-activity relationship of NQO1-targeting antitumor quinones LIAO Ke, NIU Fang, HAO Hai-Ping*, WANG Guang-Ji* State Key laboratory of Natural Medicines, Key Lab of Drug Metabolism and Pharmacokinetics, China Pharmaceutical University, Nanjing 210009, China Available online 20 May 2012

[ABSTRACT] NAD(P)H: quinone oxidoreductase 1 (NQO1) is an obligate two-electron reductase that is involved in chemoprotection and can also bioactivate certain antitumor quinones. Levels of NQO1 expression are elevated in tumors particularly in those of the lung, colon and breast in relation to the surrounding normal tissues. The high levels of NQO1 in solid tumors in combination with the ability to reduce many quinone-containing antitumor drugs has drawn our attention to NQO1 as a potential molecular target in cancer treatment. NQO1-targeting drugs are thus expected to achieve high selectivity and specificity to kill tumor cells. This review focuses on discussing the structure-activity relationships of NQO1-targeting antitumor quinones, from which possible future prospectives in this area are presented, in order to ignite and promote the development of NQO1-targeting antitumor drugs. [KEY WORDS] NQO1; Structure-activity relationship; Antitumor quinones

[CLC Number] R965

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[Document code] A

[Article ID] 1672-3651(2012)03-0170-07

Introduction

A general goal in anti-cancer drug development is to achieve highly selective toxicity against tumor cells while largely sparing the normal cells. NQO1 is such a desirable target that is attributed to a combination of overexpression in tumor cells compared with normal cells and the ability to activate a broad range of cytotoxic quinone anti-cancer drugs. For this reason, NQO1 has been proposed to be a promising anti-cancer drug target. A huge volume of research concerning the design and development of novel NQO1-targeting antitumor quinones has been reported. Two types of NQO1-targeting drug design strategies have been proposed: one is to design a nontoxic prodrug that can be reduced to an active form selectively in the NQO1 highly expressed tumor tissues [1-4]; the other is to design a typical quinone substrate [Received on] 05-Mar.-2012 [Research funding] This project was supported by the Special Fund for the Author of National Excellent Doctoral Dissertation of China (No. 200979) [ Corresponding author] HAO Hai-Ping: Prof., Tel: 86-25 -83271179, Fax: 86-25-83271060, E-mail: hhp_770505@yahoo. com.cn; WANG Guang-Ji: Prof., Tel: 86-25-83271128, Fax: 86-25-83302827, E-mail: [email protected] These authors have no any conflict of interest to declare. Copyright © 2012, China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.

that can be bioactivated by NQO1 reduction producing a futile redox cycle which can lead to the cell death via multiple mechanisms. Although dozens of NQO1 substrates including clinically used drugs, currently developed agents, and some natural products have been reported, there is still a long way to go before developing an ideal NQO1-targeting drug. The currently known NQO1 substrates either lack sufficient specificity or cannot be translated to a promising anti-cancer drug because of the largely unknown causes. Thus, the development of NQO1-targeting drugs has not yet exhibited a promising prospect as originally expected from its unique properties in tumor tissues. Much work remains to be done to uncover the mechanisms underlying cell death initiated by NQO1 bioactivation. Analysis of the structure-activity relationship of currently known NQO1 substrates would be expected to be helpful for the future design of promising NQO1-targeting drugs. This review aims at providing an insight into the NQO1-targeting drug design strategy based on an intensive summary and discussion of the structure-activity relationship of known substrates, with the hope to ignite and stimulate new ideas for the rational design of ideal NQO1-targeting antitumor quinones.

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Biology of NQO1

NAD(P)H: quinone oxidoreductase 1 (NQO1, DT-diaphorase, EC 1.6.99.2) is a widely-distributed FAD-dependent cytosolic flavoenzyme which was rst

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detected in and isolated from the soluble fraction of rat-liver homogenates by Ernster and Navazio [5]. NQO1 plays an important role in various physiological and pharmacological areas. Major biochemical properties and functions of NQO1 can be summarized as follows [6]: 1) NQO1 is a two-electron reduction and detoxication enzyme, playing an antioxidant role via bypassing the formation of reactive oxygen species derived from one-electron reduction: 2) NQO1 can also bioactivate certain antitumor quinones such as mitomycin C (MMC), EO9, and RH1, in the case when the reduced hydroquinone intermediate is highly unstable and can auto-oxidize back the parent quinones, thus producing a futile redox cycle; 3) NQO1 is responsible for the maintenance of some endogenous lipid-soluble antioxidants -tocopherol and ubiquinone in their reduced and active forms; 4) NQO1 may directly scavenge superoxide anion radicals; 5) NQO1 can regulate the stability of the tumor suppressor protein p53 and several other short-lived proteins including p73 and ornithine decarboxylase, and indeed NQO1 is believed to be a gatekeeper of the 20S proteasome pathway. The crystal structure of human NQO1 has been resolved by X-ray crystallography (Fig. 1) [7]. Structural studies have shown that hNQO1 has a molecular weight of about 60 kDa, and is a homodimer of two interlocked monomers of 274 amino acids. Each subunit consists of two domains, a catalytic domain (residues 1–220) and a smaller C-terminal domain that forms part of the binding site for the hydrophilic regions of NAD(P)H and contains a non-covalently bound molecule of avin adenine dinucleotide (FAD) [8]. FAD is orientated such that its isoalloxazine ring forms the oor of the NQO1 active site cavity [9].

and it acts through competitive binding with NAD(P)H, thereby preventing the two-electron transfer to FAD from occurring [10]. NQO1 is localized primarily in the cytosol but lower levels have been detected in the nucleus. In human tissues, NQO1 is expressed at high levels in kidney, stomach, respiratory epithelial cells, and vascular endothelial but at low levels in liver, colon, and breast. Humans, unlike most other mammals, do not express NQO1 in normal liver hepatocytes but NQO1 expression is seen in preneoplastic lesions and liver cancers. NQO1 is expressed at high levels in most human solid tumors including tumors from colon, breast, pancreas, and lung. NQO1 is highly inducible by a wide variety of chemical inducers, which is mediated through the Keap1/Nrf2/ARE pathway (Fig. 2). Nuclear factor erythroid 2-related factor-2 (Nrf2) binding to antioxidant response elements (AREs) and arylhydrocarbon receptor (AhR) binding to xenobiotic response element (XRE) are crucially involved in the induction of NQO1 gene expression by chemical inducers. Nrf2/ARE and AhR/XRE may coordinatein the regulation of NQO1 expression in mammalian tissues [11].

Fig. 2 Role of Nrf2 and AhR on the transcriptional regulation of NQO1 gene expression. In response to activation by chemical inducers, Keap1 loses its ability to target Nrf2 for degradation; Nrf2 is then stabilized and becomes available for translocation (solid arrow) to the nucleus, followed by dimerization with a Maf protein, binding to AREs and triggers the expression of NQO1. The mechanism by which AhR ligands induce NQO1 is not well understood at present. Nrf2/ARE and AhR/XRE may coordinate in the regulation of NQO1 expression in mammalian tissues

3 Mechanism of Bioreductive Activation by NQO1 Fig. 1 X-Ray crystal structure of human NQO1 dimer (PDB accession code 1d4a). The non-covalently bound FAD is shown at both sites

NQO1 is an obligate two-electron reductase that is characterized by its capacity for utilizing equal NADH and NADPH efficiently as the hydride donor. The catalytic cycle of NQO1 functions via a “ping-pong” mechanism. Dicoumarol is the most commonly used inhibitor of NQO1,

NQO1 is generally considered to be a phase II detoxification enzyme that catalyzes the two-electron reduction of quinones to hydroquinones, bypassing the formation of the highly reactive semiquinones. Hydroquinones can then be conjugated by glucuronide or sulfate and excreted [12-13]. However, for some hydroquinones, the NQO1 reduction produces a fairly unstable hydroquinone intermediate which can auto-oxidize back to the parent

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quinone producing a futile redox cycle. Because of this property of NQO1 and its distinctive high expression levels in tumor tissues in relation to the surrounding normal tissues, NQO1 becomes a promising target for the development of anti-cancer drugs. Based on the study of several well-characterized substrates activated by NQO1, the mechanism of bioreductive activation by NQO1 has been intensively studied [14] (Fig. 3). Currently proposed NQO1 bioactivation mechanisms in initiating the death of tumor cells can be summarized as follows. First, NQO1 triggers a futile redox cycle producing dramatic reactive oxygen species and the depleting NAD and ATP, all of which may activate various apoptotic and necrotic cell death signals. The well-characterized agent that belongs to this type of mechanism is -lapchone. In the case of MMC , it was proposed that the reduced MMC can rearrange to produce a DNA-reactive metabolite inducing DNA alkylation or crosslinking. NQO1 may serve as a prodrug activation enzyme to produce a much more toxic metabolite selectively in the NQO1 highly expressed tumor tissues, for example, 17-allylamino-demethoxygeldanamycin (17-AAG) hydroquinone binds with greater affinity to Hsp90 as compared with the parent quinone.

poor specificity to NQO1 and the formation of reactive semiquinones produced by the one-electron reducing enzymes. The indoloquinone EO9 (Fig. 4), a synthetic analogue of MMC, was designed as a better NQO1 substrate as compared to MMC, as evidenced from the more effective redox cycle, and not pH-dependent. EO9 entered clinical trials on the basis of its excellent activity in solid tumor animal models without significant bone marrow toxicity in animal toxicology studies. However, EO9 failed to produce any clinical responses and its failure in the clinic was attributed to a combination of rapid plasma clearance,poor tissue penetration, and dose-limiting kidney toxicity [15]. Work is still proceeding on designing better analogs of MMC and EO9. Based on recent SAR studies of indolequinones (Fig. 5), the structure-based design criteria for substituents at key ligand positions are discussed in the following sections.

Fig. 4 Structure of MMC and EO9

Fig. 5 Structure of indolequinones

Fig. 3 Pathways for bioreductive activation of antitumor quinones by NQO1. Solid lines represent major pathways. Dashed lines represent minorpathways

4 Classification and SAR of NQO1-targeting Antitumor Quinones Indolequinones MMC (Fig.4) is a quinone antibiotic isolated by Wakaki et al. from Streptomyces caespitosus in 1958. MMC has been used clinically to treat a wide variety of solid tumors; however, its clinical application now becomes of the cause of its high toxicity. MMC, the prototype bioreductive drug, is a relatively poor substrate for hNQO1, and can also be bioactivated by one-electron reductases. The activation is pH-dependent and can be inuenced by hypoxia. The high toxicity and low selectivity of MMC may be derived from its 4.1

Indolequinones-2 Position Molecular modeling studies have shown that the modifications at the 2-position, located in the binding site entrance, tend to be tolerated, suggesting that alterations at the 2-position may be a breakthrough to generate compounds which are good substrates for NQO1 [16]. Hydrogen bonding and electronic effects appear to be more important than steric effects at C2 to the metabolism of these compounds. The introduction of phenyl [17], hydroxymethyl, electron-withdrawing ester groups, etc., at the 2-position are favorable in terms of forming good substrates for NQO1. Indolequinones-3 Position In general terms, substitutions at the (indol-3-yl)methyl position with bulky leaving groups or a group containing a chlorine atom result in compounds which are not only poor substrates for the two-electron reducing enzyme NQO1 but also inactivate the enzyme. Molecular modeling studies confirmed that this is attributed to a combination of the steric effect and these groups sitting close to the important residues Tyr-156 and His-162, and thus possibly resulting in either alkylation within the active site or the disruption of charge-relay mechanisms. Compounds with the introduction of electron-withdrawing groups such as an ester at the indole

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3-position were excellent substrates for NQO1. It is reported that the rate of metabolism by the enzyme decreased as the 3-substituent was altered in the order: CO2Et> CHO> H> CH2OH> CH3> CH2OCONH2 within the 5-methoxyindolequinones [18]. Indolequinones-5 Position In terms of substrate specificity for NQO1, substitutions at the indol-5-position with basic nitrogen substituents are undesirable: as a result of steric effects, such substituents provide a steric interaction with Trp-106, which forces the quinone oxygen atoms to be unfavorably positioned for reduction. However, aziridinyl group at the 5-position is desirable in terms of conferring both potency and selectivity for NQO1-rich cells in vitro under aerobic conditions, because aziridinyl group at the C5 position can form favorable van der Waals interactions with Trp-105. Indolequinones-6 Position There is a negative effect on substrate specificity for NQO1 by substitutions at the indol-6 position, which is attributed to the steric hindrance between the quinone and the active site of the enzyme [19]. 4.2 Benzoquiones The early representative compounds of NQO1-targeting benzoquiones are diaziquione(AZQ), carbazilquinone, and triaziquone, all of which have entered clinical trials. However, they failed in the clinic, being attributed to either undesirable pharmacokinetic properties, high toxicity, or no better effectiveness than the existing chemotherapeutic agents. Recently, RH1 (Fig. 6), a novel water-soluble analogue of MeDZQ has received considerable attention. MeDZQ, an excellent substrate for NQO1, is the lead compound in the aziridinyl benzoquinone series. Unfortunately the development of MeDZQ was limited by its poor solubility. The production of substitutions at the 3-position with hydroxyl group, RH1, is better soluble in water than MeDZQ. RH1 has entered clinical trials on the basis of its low renal toxicity, appropriate plasma clearance, good solubility, and high selectivity. Its clinical trials are still ongoing [20]; however, it remains unclear whether it can become the first NQO1-targeting drug in the clinic. The above is a class of NQO1-targeting antitumor quinones based on the structure of aziridinyl benzoquinone. In addition, there is a category that is based on the structure of nitrogen mustard-benzoquinone (Fig. 7). Steric effects of the functional groups appear to be more important than electronic effects for changing the rate of reduction by NQO1.

Besides, the steric hindrance at the 6-position is more important than 5-position for modifying the substrate specificity for NQO1. The introduction of chloro, phenyl, and sterically bulky groups on the benzoquinone ring is unfavorable in causing signicant decrease of their cytotoxic and DNA cross-linking activities. On the contrary, electron-donating groups can increase their effects on cytotoxic activity [21]. 4.3 Naphthoquinones -Lapachone (Fig. 8), a natural ortho-pyran naphthoquinone isolated from the bark of the lapacho tree, was originally developed as an inhibitor of topoisomerase I to inhibit the repair of mammalian DNA. However, the intensive studies in uncovering the mechanism of -lapachone strongly indicate that it is a specific NQO1 substrate and its anti-cancer efficacy is predominantly NQO1-dependent [22-24]. -lapachone was found to be highly efficient in killing pancreatic cancer cells [25-26], and many other solid tumors such as lung cancer. -lapachone has entered clinical trials on the basis of its excellent activity in killing solid tumors with high NQO1 expression [27]. In addition, -lapachone-based derivatives design is still ongoing to increase the druggability of NQO1-targeting agents. We summarize the SAR of pyran naphthoquinones and furan naphthoquinones as follows. Pyran naphthoquinones (Fig. 8): Į- and ȕ- pyran naphthoquinone -9 or -7 position The introduction of the hydroxyl group at - and - pyran naphthoquinone -9 or -7 position results in the compounds with more powerful and selective cytotoxicity against mela-

Fig. 6 Structure of RH1

Fig. 7 Structure of Nitrogen mustard-benzoquinones

Fig. 8 Structures of pyran naphthoquinones and furan naphthoquinones

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noma cancer cells (MDA-MB435) than - and -lapachone and doxorubicin. It is prompted that modifications on the aromatic ring (A ring) may be a desirable strategy to increase the selectivity and activity of these compounds. In addition, 7-hydroxy--lapachones are more active and selective than 9hydroxy--lapachones. Į- and ȕ- pyran naphthoquinone-2 position Substitutions at the (naphth-2-yl) phenyl position with electron-donating and halogenated based groups increase the selectivity against MDA-MB435. Electronic effects appear to be important at C2 to the selectivity of these compounds [28]. Furan naphthoquinones (Fig. 8): Į- and ȕ- furan naphthoquinone-3 position It is reported that arylamino derivatives of ortho-quinones are more active and selective than their para-isomers. The introduction of electron-withdrawing groups in the 3-arylamino moiety enhanced the activity and selectivity against MDA-MB435 cells. The effect of these electron-withdrawing groups was inuenced by the position of the group on the arylamino moiety, indicating that ortho-substitutions are more desirable [29-30]. 4.4 Quinolinequinones Among the quinolinequinone class, there are two important antitumor antibiotics, streptonigrin and lavendamycin, which have attracted substantial interest from the scientic community. Streptonigrin (SN, Fig. 9), a naturally occurring 7-aminoquinoline-5,8-dione antitumor antibiotic and an excellent substrate for NQO1, was first isolated from the cultures of streptomyces flocculus. Lavendamycin (Fig. 9) is shown to be structurally and biosynthetically related to SN. The use of both in the clinic has been precluded due to their high degree of toxicity. Early SAR studies have demonstrated the 7-aminoquinoline-5, 8-dione moiety as the essential moiety for the cytotoxic activity of SN and lavendamycin [31]. In addition, among quinoline-5,8-diones the lavendamycins are apparently much more selectively toxic toward the cancer cells with high NQO1 expression, suggesting that the -carboline moiety is critically important in the potency and selectivity of the lavendamycins [32]. This evidence suggests that lavendamycins have more promising structures to target NQO1. Therefore, we focus on discussing the SAR of lavendamycin analogues (Fig. 10).

Fig. 9 Structure of streptonigrin and lavendamycin

Fig. 10 Structure of lavendamycins

Quinolinedione-7 Position 1) Substitutions at the quinolinedione-7 position should be moderately small (no large substituents, such as NH2 or NHCOCH3 groups) to avoid producing steric hindrance with the internal wall of the NQO1 active site including Trp-105 and Phe-106, to ensure favorable positioning for hydride ion reception from FAD and quinone reduction; 2) Substitutions, with the capability of forming hydrogen-bonding interactions with FAD cofactor and/or Tyr-126 and -128, are good for increasing substrate specificity for NQO1; 3) A small substituent at the this position should be capable of forming van der Waals interactions with the Trp-105/Phe-106 minipocket, which could increase substrate specificity for NQO1. Quinolinedione-6 Position 6-unsubstituted in this position is highly desirable. It is due to the steric effects caused by substituents that hinder the entrance or proper positioning of lavendamycin analogues toward the active site [33]. Quinolinedione-2 Position Substituents, such as morpholino group [34], at C2 position should potentially form hydrogen bonding or van der Waals interactions with the FAD cofactor and/or key residues of the active site including Gly-149, Phe-232 and Gly-150.

5

Conclusion and Perspective

Several clinically used anti-cancer drugs have been tested to be NQO1 substrates and their anti-cancer effects are somewhat NQO1-dependent; however, high toxicity, poor solubility, or undesirable pharmacokinetics properties of such drugs limit their clinical applications. None of these drugs was originally designed to target NQO1, and thus it is not surprising to find that all of these drugs lack NQO1 specificity and can be activated by one-electron reducing enzyme such as cytochrome P450 reductase, which is found at appreciable levels in hematopoietic cells and other organs, via a one-electron reduction resulting in the formation of a semiquinone radical to produce toxic side-effects [21]. It is thus implied that the balance of affinity to NQO1 and P450 reductase is an important factor in designing NQO1-targeting antitumor quinones. Based on the current knowledge about the NQO1 bioactivation property, two types of NQO1-targeting strategies are proposed herein: One is to design a highly specific NQO1 substrate that can be reduced to produce a highly unstable hydroquinone intermediate generating a futile redox cycle; the other is to design a non-or less-toxic prodrug

LIAO Ke, et al. /Chinese Journal of Natural Medicines 2012, 10(3): 170176

that can be selectively released and/or rearranged to produce a highly active metabolite in the NQO1 highly expressed tumor tissues. In recent decades, dozens of NQO1 substrates have been designed and the SAR studies have been performed as discussed in this review, however, much work remains to be done to develop a promising NQO1-targeting drug. First, in the design of NQO1-targeting quinones, it is important to balance the pharmacological and pharmacokinetic properties. Second, the design of NQO1-targeting agents should concurrently consider and test the affinity to various one-electron reducing enzymes, which is important to ensure NQO1 specificity and tumor-specific cytotoxicity. Third, the current understanding of NQO1-mediated biological process is still limited; it deserves intensive research to uncover the role, mechanism, and its link to many other tumor cell signals of NQO1.

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ᄯ‫ࢳྐڳ‬Ӗ༰ྐ๞ӝ໦Վॏ༰ᄷԤಬི೉ “ඟ௶ྐ๞‫ݣ‬໿ᆦ‫ྐူד‬໒”‫ޥڳ‬ᄷԤಬི೉, ઒࠸ 210009 ᨬ 㽕€NAD(P)H: ᱧཾ‫ܮܤ‬ၐਝ 1 (NQO1)ᅥ໿ӂ‫ܤ‬ᱧ०Ԩᆐ‫ܮ‬ၐֱ࿫, ࡮ပ‫ܤ‬༰ͬ‫ۤܙ‬ಓ๞‫ݣݷ‬ᆴဈdNQO1 ၽտᄵ ᄶট຅ͦඋιಾ‫̔׋‬cࠒЫ̔cత຦̔຅ͦᄯԅζӒၙ‫غ‬ဟჾШᆦᄎdဎဟ NQO1 ၽᄶট຅ͦԅ‫غ‬ζӒ‫ރ‬ୣಓ๞‫ܤݣ‬ԅඋ໿, ൑ ΄ఊนಾᄭ९տᄵᄶটԅஎၽ‫ד‬ᆐ̻γd̻຿ NQO1 ྐ๞ပฌಬຣ‫غ‬༪႔໿cඋ࿓໿ొ੕ᄶট຅ͦd·ำᄷԤᆘ೭ॴ‫؞‬फ‫ۦܤ‬ ๞‫ٲ‬໒‫ڑ‬ຂཙࡎ, ωඔѻॴ໭ԅཙࡎ഑া, ᄛၽҼࠩճ̻຿ NQO1 ࢦᄶটᱧԅཙࡎۤࢗ֟d ݇䬂䆡€ NAD(P)Hġᱧཾ‫ܮܤ‬ၐਝ 1; ‫ٲ‬໒‫ڑ‬ຂ; ࢦᄶটᱧ

෎䞥乍Ⳃ€

‫غ‬ԉ༰໎௦‫̈́ڳ‬଩ည༎ϐಶৢำᆴრᅥົᆇࠡ“NQO1 ࠚӽԅಓ๞‫דူܤݣ‬ᆐԸࣅ‫ݯ‬ᄥཙࡎ” (No. 200979)