| I Regulation and Molecular Mechanisms of Flavonoids on organic anion transporter 3The organic anion transporter 3(OAT3),an uptake drug transporter highly expressed at the basolateral membrane of the proximal tubule cells in human kidneys,plays critical roles in the renal clearance of endogenous substance and therapeutic drugs into the kidneys.It has been reported that the inhibition of OAT3 could reduce the amount of nephrotoxic substrate drugs into kidneys,consequently alleviating drug-induced acute kidney injury(AKI).Thus,screening and identifying effective OAT3 inhibitors with low toxicity would be a new direction and option for alleviating OAT3-mediated AKI in clinic.Flavonoids are one of the most common dietary polyphenols in human diet and possess extensive pharmacological activities with excellent safety profiles.However,in recent years,it has been reported that some of them can cause drug-drug interactions by inhibiting transporters.The present study was designed to systematically test the inhibitory effect on OAT3 of 97 flavonoids,which are rich in the daily diet or frequently used in traditional Chinese medicine prescriptions.Then the inhibitory biological effects of flavonoids were further explored in cells and animal models,including AA-I-generated cytotoxicity,MTX-induced AKI and furosemide pharmacokinetics.Finally,the pharmacophore model was established to elucidate the structure-activity relationships of flavonoids with OAT3,which would provide useful information in predicting the potential interaction between untested flavonoids and OAT3,and help to optimize flavonoid structure to alleviate AKI.The results were shown below:1.The primary screening and inhibition biological effects of flavonoids on OAT31.1 In the primary screening in OAT3-HEK293 cells,17 out of 97 flavonoids exhibited significant inhibition(>50%)on OAT3,including calycosin,genistein,diosmetin,naringin,artemisetin,luteolin,hispidulin,herbacetin,kaempferol,biochanin A,isosinensetin,sinensetin,oroxylin A,α-naphthoflavone,β-naphthoflavone,7hydroxyflavone and pinocembrin.Another 49 flavonoids showed weak inhibition(2050%),and the rest 31 flavonoids had little or no inhibitory effects(<20%)on OAT3.1.2 The concentration-dependent inhibition experiments in OAT3-HEK293 cells showed that isosinensetin was the strongest inhibitor of OAT3(IC50=9.60 μM),followed by hispidulin(IC50=12.21 μM),diosmetin(IC50=18.59 μM),α-naphthoflavone(IC50=26.02 μM),7-hydroxyflavone(IC50=38.71 μM).artemisetin(IC50=40.83 μM).luteolin(IC50=42.35 μM),naringin(IC50=42.47 μM),pinocembrin(IC50=43.37 μM),calycosin(IC50=46.92 μM),oroxylin A(IC50=62.17 μM),sinensetin(IC50=70.02μM),β-naphthoflavone(IC50=81.02 μM),herbacetin(IC50=84.62 μM).kaempferol(IC50=88.77 μM),genistein(IC50=90.97 μM)and biochanin A(IC50=93.75 μM).1.3 In OAT3-HEK293 cells,the positive inhibitor probenecid and five flavonoids with the strongest inhibitory activity,like isosinensetin,hispidulin,diosmetin,αnaphthoflavone and 7-hydroxyflavone,could obviously decrease the cytotoxicity of AA-I in OAT3-HEK293 cells with increased cell survival to varying degrees.Among these flavonoid inhibitors,7-hydroxyflavone showed the strongest inhibitory effect,leading to an upward shift of cell viability curves at 10-100 μM.The results suggest that OAT3 flavonoid inhibitors could reduce the influx of AA-I into cells by inhibiting OAT3 activity,thereby lowering its cytotoxicity.1.4 In flavonoid inhibitors pre-treated SD rats,the AUC0-t of furosemide increased by 11.54%-59.08%compared with the control group,yet weaker than that of probenecid(310.48%).The results suggest that OAT3 flavonoid inhibitors may influence the exposure of furosemide in SD rats via modulation of OAT3 activity.1.5 In the MTX-induced AKI rat model,the tested flavonoid inhibitors could significantly reduce the level of serum BUN and CREA.Meanwhile,compared with the model group,the MTX concentration in kidney also decreased by 22.68%to 44.33%after flavonoid treatment,which was consistent with the findings of renal function parameters.The results suggest that co-administration of flavonoids with MTX could alleviate MTX-induced AKI via inhibiting OAT3-mediated methotrexate uptake.2.The structure-activity relationship of flavonoids with OAT3The structure-activity relationship between OAT3 and flavonoids was elucidated by the pharmacophores model,and the results showed that hydrogen bond acceptors in 4,7 positions and hydrophobic groups in 4’-position are the critical pharmacophores of flavonoid inhibitors.When these positions bound with larger steric hindrances such as glucose,the inhibitory effect of flavonoids on OAT3 would significantly decrease or even disappear.In conclusion,1 7 out of tested 97 flavonoids exhibited significant inhibition of OAT3 in OAT3-overexpressing HEK293(OAT3-HEK293)cells.Five strongest flavonoid inhibitors were further evaluated for inhibitory biological effects,which can decrease the cell toxicity of AA-I in OAT3-HEK293 cells,change the pharmacokinetic profile of furosemide and alleviate MTX-induced renal toxicity in rats.Moreover,the established structure-activity relationships model would provide useful information for predicting the potential risks of food/herb-drug interactions in humans,and help to optimize flavonoid structure and obtain promising inhibitors of OAT3 to alleviate AKI.II Regulation and Molecular Mechanisms of Alkaloids on glucose transporter 1Glucose transporter 1(GLUT 1)is an important membrane protein encoded by the solute carrier family 2A1(SLC 2A1)gene,which is closely related to glucose uptake and energy metabolism.Normally,GLUT1 is expressed at low levels in various tissue,but in many cancer cells such as lung,breast and liver cancers,where it is abnormally highly expressed,for providing sufficient energy for cell proliferation and migration.A number of studies have found that inhibition of GLUT1 may not only suppress tumor growth by reducing glucose uptake,but also increase the sensitivity of anticancer drugs to resistant tumors.Therefore,GLUT 1 inhibitors have become a global research hotspot in recent years.Natural products,as an important source of anticancer medicines,some of which,such as curcumin,quercetin,gossypol and so on,were reported to exert antitumor effects by inhibiting GLUT 1-mediated glucose uptake.Alkaloids,a large group of nitrogenous natural products with outstanding anticancer activity,have been widely used in the clinical treatment of various cancers,like paclitaxel and hypericin.Therefore,the search for GLUT 1 inhibitors among alkaloids may provide a new direction for the discovery of efficient GLUT1-target anticancer drugs.In this study,we investigated the inhibitory effects of 130 alkaloids widely existing in plants on GLUT1 using GLUT1-HEK293t cells to screen out the potential inhibitors,and futher evaluated their biological effects in human hepatoma HepG2 cells.Then,the inhibitory molecular mechanism was assessed by CDOCKER molecular docking.Finally,a pharmacophore model was constructed to elucidate the conformational relationship between alkaloids and GLUT1 inhibition,which can be useful to predict the potential inhibitory effect of untested alkaloids on GLUT1 and provide valuable information for the optimization and design of GLUT1 inhibitors.The results were shown below:1.The primary screening and inhibition biological effects of alkaloids on GLUT11.1 In the primary screening in GLUT1-HEK293t cells.five alkaloids exhibited significant inhibition(>50%)on GLUT 1,including cepharanthine,dihydroberberine.catharanthine hemitartrate,berbamine hydrochloride and(+)-Corydaline.Another 46 alkaloids showed weak inhibition(20-50%).and the rest 79 alkaloids had little or no inhibitory effects(<20%)on GLUT1.1.2 The concentration-dependent inhibition experiments in GLUT1-HEK293t cells showed that dihydroberberine was the strongest inhibitor of GLUT1(IC50=39.78μM),followed by berbamine hydrochloride(IC50=44.06 μM),(+)-corydaline(IC50=50.68 μM),catharanthine hemitartrate(IC50=59.10 μM)and cepharanthine(IC50=67.28 μM).1.3 In wild-type HepG2 cells,the inhibitory biological effects of alkaloids on GLUT 1 were assessed.The results demonstrated that dihydroberberine,(+)-corydaline and catharanthine hemitartrate could significantly increase the cytotoxicity of sorafenib by inhibiting GLUT 1.Among them,dihydroberberine exhibited the strongest inhibitory effect,resulting in a decrease in the IC50 value of sorafenib in HepG2 cells from 14.38μM to 5.43 μM,which was much stronger than the natural positive inhibitor quercetin(IC50=10.87 μM).These results suggested that alkaloid inhibitors of GLUT1 may enhance the sensitization of HepG2 cells to sorafenib,exerting a synergistic antitumor effect,and better than that of the positive inhibitor quercetin.2.The inhibition mechanisms and structure-activity relationships of alkaloids with GLUT12.1 The results of molecular docking of alkaloids with GLUT1 showed that the amino acid residues including Gln283、Asn288、Asn317、Asn411、Trp412 and Ile164 may play key roles in the binding of alkaloids.In addition,the inhibitory effect of alkaloids on GLUT1 might be related to conventional hydrogen bond and pi-alkyl interaction,the numbers of which may be positively linked to the inhibitory intensity.2.2 The structure-activity relationship between alkaloids and GLUT1 was elucidated by pharmacophore calculation.The results showed that the critical pharmacophores of GLUT 1 inhibitors are the hydrogen bond acceptor and the hydrophobic group.Combined with the results of the primary screening in vitro,the hydrophobic groups,such as hydrocarbon,methoxy,ester and amide groups,play a key role in the inhibitory activity of alkaloids against GLUT1.In conclusion,five of tested 130 alkaloids exhibited significant inhibition of GLUT1 in GLUT1-overexpressing HEK293t(OAT3-HEK293t)cells.Among them,dihydroberberine,(+)-corydaline and catharanthine hemitartrate could remarkably enhance the sensitivity of HepG2 cells to sorafenib by inhibiting GLUT1.Molecular docking indicated that the inhibitory effect of alkaloids might be related to conventional hydrogen bonds and pi-alkyl interaction.The constructed pharmacophore model suggested that hydrophobic groups play a key role in the inhibition of alkaloids on GLUT 1.Our findings would provide valuable information in optimizing alkaloid structure and developing GLUT1 inhibitors with high-efficiency and low toxicity for cancer treatment. |
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