molecules Article Quantitative Structure–Activity Relationships for the Flavonoid-Mediated Inhibition of P-Glycoprotein in KB/MDR1 Cells Mengmeng Xia , Yajing Fang, Weiwei Cao, Fuqiang Liang , Siyi Pan and Xiaoyun Xu * Key Laboratory of Environment Correlative Dietology (Huazhong Agricultural University), Ministry of Education, Wuhan 430070, China; [email protected] (M.X.); [email protected] (Y.F.); [email protected] (W.C.); [email protected] (F.L.); [email protected] (S.P.) * Correspondence: [email protected] ; Tel.: +86-27-87671056; Fax: +86-27-87288373 Received: 16 April 2019; Accepted: 24 April 2019; Published: 27 April 2019 Abstract: P-glycoprotein (P-gp) serves as a therapeutic target for the development of inhibitors to overcome multidrug resistance (MDR) in cancer cells. In order to enhance the uptake of chemotherapy drugs, larger amounts of P-gp inhibitors are required. Besides several chemically synthesized P-gp inhibitors, flavonoids as P-gp inhibitors are being investigated, with their advantages including abundance in our daily diet and a low toxicity. The cytotoxicity of daunorubicin (as a substrate of P-gp) to KB/MDR1 cells and the parental KB cells was measured in the presence or absence of flavonoids. A two-dimensional quantitative structure–activity relationship (2D-QSAR) model was built with a high cross-validation coefficient (Q2) value of 0.829. Descriptors including vsurf_DW23, E_sol, Dipole and vsurf_G were determined to be related to the inhibitory activity of flavonoids. The lack of 2,3-double bond, 30-OH, 40-OH and the increased number of methoxylated substitutions were shown to be beneficial for the inhibition of P-gp. These results are important for the screening of flavonoids for inhibitory activity on P-gp. Keywords: flavonoids; P-gp; inhibitory activity; QSAR 1. Introduction P-glycoprotein (P-gp) is a member of the ATP-binding cassette (ABC) family, which is encoded by the ABCB1 gene. The overexpression of P-gp has been associated with multidrug resistance (MDR) in cancer cells, a limiting factor in the success of cancer chemotherapy. Given the role of P-gp in influencing cancer chemotherapy, methods to overcome P-gp-mediated efflux have been investigated. It is generally believed that the mechanisms of P-gp inhibition mainly comprise four aspects: competitively, non-competitively or allosterically blocking the drug binding site; interfering with the ATP hydrolysis process; altering the integrity of cell membrane lipids; decreasing the P-gp expression [1]. Four generations of P-gp inhibitors have been identified in recent years. The first-generation inhibitors, including verapamil [2] and cyclosporine A [3], were found to possess high toxicity at their effective doses [4]. The derivatives of the first-generation inhibitors, dexverapamil and VX710, are termed the second-generation inhibitors. However, due to their impact on P450 and drug interaction profiles, these inhibitors were not used clinically [5]. Elacridar, tetrandrine, and zosuquidar, the third-generation inhibitors, are limited due to their low survival [6]. Therefore, high-potency and low-toxicity P-gp inhibitors are urgently required for chemotherapy treatment. Compounds from natural products belonging to the fourth-generation P-gp inhibitors are of great significance [7]. Flavonoids are a class of compounds based on the diphenylpropane (C6–C3–C6) skeleton, which are widespread in our common diet, including in fruits and vegetables. Flavonoids have been Molecules 2019, 24, 1661; doi:10.3390/molecules24091661 www.mdpi.com/journal/molecules Molecules 2019, 24, x FOR PEER REVIEW 2 of 12 Compounds from natural products belonging to the fourth-generation P-gp inhibitors are of great Molecules 2019significance, 24, 1661 [7]. 2 of 11 Flavonoids are a class of compounds based on the diphenylpropane (C6–C3–C6) skeleton, which are widespread in our common diet, including in fruits and vegetables. Flavonoids have been shown shown to be beneficial to human health for their antioxidant, anti-inflammatory, anticarcinogenic and to be beneficial to human health for their antioxidant, anti-inflammatory, anticarcinogenic and antiviral activitiesantiviral activities [8]. Several [8]. Several studies studies have have suggested suggested that that flavonoids flavonoids can can inhibit inhibit P-gp P-gp in in order order toto enhance theenhance bioavailability the bioavailability and uptake and uptake of anticancer of anticancer drugs drugs [9]. [9]. Flavonoids Flavonoids (biochanin (biochanin A, A, morin morin [[10],10], silymarinsilymarin [10,11], quercetin [10,11], quercetin [11–13], [11–13], kaempferol kaempferol [11–14 [1]1–14] and and tangeretin tangeretin [15 [15])]) have have been been demonstrated demonstrated to presentto inhibitory present inhibitory activity activity on P-gp. on KitagawaP-gp. Kitagawa [16], [1 by6], studying by studying the the structure–activity structure–activity relationships relationships (SARs) of(SARs) flavonoids, of flavonoids, found thatfound the that planar the planar structure structure and and hydrophobic hydrophobic nature nature of of flavonoids flavonoids areare importantimportant for the inhibitoryfor the inhibitory effect effect on P-gp. on P-gp. Quantitative Quantitative structure–activity structure–activity relationships relationships (QSARs) (QSARs) can can be usedbe used for for observing observing the the mechanismsmechanisms between between molecu molecularlar structures structures and various and various biological biological activities activities[17]. [17]. QSAR QSAR has has been been widely widely used to used determine to determine whether whethera compound a compound is an inhibitor is anof P-gp. inhibitor Various of studies have built the three-dimensional quantitative structure–activity relationships (3D-QSAR) P-gp. Various studies have built the three-dimensional quantitative structure–activity relationships model to investigate the inhibitory activity on P-gp [18–20]. The model is limited as it is based on the (3D-QSAR) model to investigate the inhibitory activity on P-gp [18–20]. The model is limited as it is assumption that compounds all act on the same receptor. Furthermore, the 3D-QSAR model is based onaffected the assumption by the quality that compounds of molecular all actalignmen on thets/superimpositions same receptor. Furthermore, and information the 3D-QSARon ligand model is abioactiveffected by conformations the quality of[21]. molecular The two-dimensional alignments/ quantitativesuperimpositions structure–activity and information relationships on ligand (2D- bioactiveQSAR) conformations model does [21 not]. Therequire two-dimensional subjective (or time-consuming) quantitative structure–activity molecular alignment relationships or putative (2D-QSAR)binding model conformation does not require or determination subjective (orof time-consuming)3D structures. Furthermore, molecular 2D-QSAR alignment is orsimple putative and binding conformationrobust but has or been determination rarely reported. of 3D structures. Furthermore, 2D-QSAR is simple and robust but has been rarely reported. The aim of this study was to investigate the quantitative structure–activity relationship for the The aimflavonoid-mediated of this study was inhibition to investigate of P-gp in the KB/MDR1 quantitative cells overexpresse structure–activityd with P-gp. relationship Daunorubicin for the[22] flavonoid-mediatedhas been reported inhibition to be ofan P-gpanticancer in KB drug/MDR1 and cellsthe su overexpressedbstrate P-gp. In withthis study, P-gp. the Daunorubicin inhibitory activity [22] has been reported(IC50 of daunorubicin) to be an anticancer of 31 flavonoids drug and (Table the substrate1) was measured P-gp. Inand this used study, to build the the inhibitory 2D-QSAR activity model (IC50 of daunorubicin)to determine the of relationship 31 flavonoids between (Table flavonoid1) was measured structure and and inhibitory used to buildactivity. the The 2D-QSAR structure model to determinecharacteristics the which relationship interact betweenwith P-gp flavonoid could enhance structure the uptake and inhibitory of chemotherapy activity. drugs. The structure characteristics which interact with P-gp could enhance the uptake of chemotherapy drugs. Table 1. The chemical structures of 31 flavonoids. No FlavonoidsTable 1. The chemicalCAS structuresCore structure of 31 flavonoids. Substructure No1 Flavonoids5-Methoxyflavone 42079-78-7 CAS Core Structure SubstructureR5=OMe 1 5-Methoxyflavone 42079-78-7 R5=OMe 2 5,7-Dimethoxyflavone 21392-57-4 R5, R7=OMe 2 5,7-Dimethoxyflavone 21392-57-4 R5,R7=OMe 3 5,33 0-Dimethoxyflavone5,3′-Dimethoxyflavone R R55, ,RR3’3=OMe0 =OMe 4 5,7,3 -Trimethoxyflavone R ,R ,R =OMe Molecules0 2019, 24, x FOR PEER REVIEW 5 7 30 3 of 12 5 7 3’ 5 5,7,304,4 0-Tetramethoxyflavone5,7,3′-Trimethoxyflavone 855-97-0 R5R,R, R7,R, R30 ,R=OMe40 =OMe 6 Tangertin 481-53-8 R5,R6,R7,R30 ,R40 =OMe 7 14 ChrysinGalangin5,7,3′,4′- 480-40-0548-83-4 R3, R R5,R, R7==OHOH 5 855-97-0 R5, R7, 5R3’, 7R4’=OMe 8 TetramethoxyflavoneBaicalein 491-67-8 R5,R6,R7 =OH 15 Kaempferide 491-54-3 R3, R5, R7 =OH, R4’=OMe 9 Wogonin 632-85-9 R5,R7 =OH, R8=OMe 6 Tangertin 481-53-8 R5, R6, R7, R3’, R4’=OMe 10 16 ApigeninFisetin 520-36-5528-48-3 R3, R R57,R, R73’,R, R44’0 ==OHOH 11 Luteolin 491-70-3 R5,R7,,R3 ,R4 =OH 7 Chrysin 480-40-0 R5, R7=OH0 0 12 17 VitexinQuercetin 3681-93-4117-39-5 RR53,R, R75,R, R470, =ROH,3’, R4’ R=OH8=Cglc 13 8 SchaftosideBaicalein 51938-32-0491-67-8 R5,R7,R4R0 =5, OH,R6,