Structure activity relationships and quantitative structure activity relationships for the flavonoid-mediated inhibition of breast cancer resistance protein

Abstract

Breast cancer resistance protein (BCRP) is a newly identified ABC transporter, which plays an important role in drug disposition and represents an additional mechanism for the development of MDR. Flavonoids, a major class of natural compounds widely present in foods and herbal products, have been shown to be BCRP inhibitors. The objective of the present study was to elucidate the SAR and derive a QSAR model for flavonoid–BCRP interaction. The EC₅₀ values for increasing mitoxantrone accumulation in MCF-7 MX100 cells for 25 flavonoids, from five flavonoid subclasses, were determined in this study or obtained from our previous publication [Zhang S, Yang X, Morris ME. Combined effects of multiple flavonoids on breast cancer resistance protein (ABCG2)-mediated transport. Pharm Res 2004;21(7):1263–73], and ranged from 0.07 ± 0.02 μM to 183 ± 21.7 μM. We found that the presence of a 2,3-double bond in ring C, ring B attached at position 2, hydroxylation at position 5, lack of hydroxylation at position 3 and hydrophobic substitution at positions 6, 7, 8 or 4′, are important structural properties important for potent flavonoid–BCRP interaction. These structural requirements are similar but not identical to those for potent flavonoid–NBD2 (P-glycoprotein) interaction, indicating that inhibition of BCRP by flavonoids may involve, in part, the binding of flavonoids with the NBD of BCRP. In addition, a QSAR model consisting three structural descriptors was constructed, and both internally and externally validated, suggesting the model could be used to quantitatively predict BCRP inhibition activity of flavonoids. These findings should be useful for predicting BCRP inhibition activity of other untested flavonoids and for guiding the synthesis of potent BCRP inhibitors for potential clinical application.

Introduction

Flavonoids (Fig. 1) are a large class of polyphenolic compounds, which are ubiquitously present in the green plant world, and they are also integral components abundant in our common diet, such as vegetables, fruits and plant-derived beverages. Over 6500 flavonoids have been described and the average intake of total flavonoids from the Western diet was estimated to be 200 mg to 1 g per day [2]. A number of studies [3], [4], [5], [6] have suggested that flavonoids may play a protective role in the prevention of cancer, coronary heart diseases, bone loss and many other age-related diseases. Due to these perceived health beneficial activities and their low toxicity [4], [7], hundreds of herbal preparations containing flavonoids are available in the market as over-the-counter dietary supplements, and the consumption of these products are becoming more and more widespread, along with the burgeoning public interest in alternative medicine and in disease prevention. However, since these products are classified as dietary supplements and their marketing does not require FDA approval, the potential interactions of these herbal preparations with conventional drugs, in general, have not been carefully evaluated, leading to a serious concern about the safety of using these products. These concerns are relevant because significant or even life-threatening pharmacokinetic interactions of flavonoids or flavonoid-containing food/herbal products with conventional drugs have been observed in animals or patients [8], [9], [10]. Therefore, elucidating the interactions of flavonoids with molecular determinants that are important for drug disposition, such as drug transporters and drug metabolizing enzymes, as well as an understanding of the potential clinical consequences of these interactions, is very important.

One of the drug transporters for which flavonoid–drug interactions have been described is breast cancer resistance protein (BCRP, MXR, ABCP, ABCG2) [11], [12], a newly identified membrane efflux transporter belonging to the ABC (ATP binding cassette) transporter superfamily [13], [14], [15]. This transporter extrudes its substrates out of the cells by using the energy derived from ATP hydrolysis. Since the transporter only has six transmembrane domains and one ATP binding site, distinct from other ABC proteins, which typically have a core structure of 12 transmembrane domains and two ATP binding sites, BCRP is considered an ABC half transporter and its transport activity may require the formation of a homodimer [16]. Many important drugs have been shown to be BCRP substrates including anthracyclines, topoisomerase I inhibitors, mitoxantrone (MX), methotrexate, flavopiridol, and nucleoside HIV reverse transcriptase inhibitors [17]. The expression of BCRP has been detected in a number of human tumors [18], [19], [20], [21], and may represent an additional multidrug resistance (MDR) mechanism [22], [23]. Therefore, potent inhibitors of BCRP may have the potential to reverse MDR in the treatment of cancer. High levels of BCRP are also present in excretory organs and tissues with barrier functions, such as liver canalicular membranes, the luminal surface of intestine, blood brain barrier, and human placenta [24], [25], [26]. This normal tissue localization profile indicates that BCRP may have an important role in drug disposition. This indication was subsequently confirmed by the observation that co-administration of GF120918 (a BCRP inhibitor) altered the disposition of topotecan (a BCRP substrate), including increase in bioavailability and decrease in biliary excretion, in mdr1a/1b (−/−) mice and in humans [27], [28]. Therefore, BCRP inhibitors could potentially alter the pharmacokinetics of the drugs that are BCRP substrates, resulting in beneficial or adverse drug interactions.

We [11] and other investigators [12], [29] have demonstrated that many naturally occurring flavonoids can inhibit BCRP. The EC₅₀ values of the flavonoids chrysin, biochanin A and apigenin for BCRP inhibition (measured as the concentration of flavonoids for producing 50% of the maximal increase in MX (a BCRP substrate) accumulation in BCRP-overexpressing MCF-7 MX100 cells) were shown to be within the sub- or low micromolar range (0.39 ± 0.13, 1.62 ± 1.02 and 1.66 ± 0.55 μM, respectively) [1]. These EC₅₀ values are likely much lower than the intestinal concentrations of these flavonoids after ingestion of food or herbal preparations [1]; thus, clinically relevant flavonoid–drug interactions may occur through a BCRP-mediated mechanism. In addition, flavonoids have little toxicity, and therefore, flavonoids with potent BCRP inhibition activities could be potentially used as reversal agents, or as the lead compounds for developing reversal agents, for BCRP-mediated MDR.

The objective of the present study was to elucidate the structure-activity relationship (SAR), and to derive a quantitative structure-activity relationship (QSAR) of flavonoid–BCRP interactions, in order to predict the BCRP inhibition activities of many other untested flavonoids, and to direct the synthesis of flavonoid compounds with higher potency for potential clinical application. To achieve this goal, a panel of 25 flavonoids, covering five flavonoid subclasses (flavones, isoflavones, chalcones, flavonols and flavanones), were selected and their EC₅₀ values for BCRP inhibition were obtained from our previous report [1], or measured in the present study. The structural features of flavonoids important for BCRP inhibition were identified by comparing the EC₅₀ values of flavonoids with and without a particular structural element. In addition, a QSAR model was constructed using a genetic algorithm coupled with multiple linear regression, and the model could be used to predict BCRP inhibition activities of flavonoids.