Screening And SAR Study Of Flavonoids Inhibitors For Intestinal Microflora β-glucuronidase And Human Carboxylesterase 2

Irinotecan is a potent topoisomerase I inhibitor,which is used clinically in the treatment of colon cancer,non-small cell lung cancer,ovarian cancer and other cancers.Delayed diarrhea is a serious side effect of irinotecan treatment,which limits the clinical dosage of irinotecan and reduces its therapeutic effect.It is reported that the accumulation of active metabolite SN-38 in intestinal tract,which is metabolized from irinotecan in vivo,played important role in the diarrhea development.The β-glucuronidase secreted by intestinal microflora in the intestine can convert SN-38 G into SN-38;human carboxylesterase 2 is located in the human intestine,which can hydrolyze irinotecan to SN-38.β-Glucuronidase and human carboxylesterase 2 are two key enzyme targets for irinotecan diarrhea treatment.It is important to screen inhibitors for both enzymes in treatment for irinotecan induced delayed-onset diarrhea.Objective: In this study,the inhibitory effect of a series of natural flavonoids were determined to find potential inhibitors of β-glucuronidase and human carboxylesterase 2;the structure-activity relationship was also observed to reveal the importance of the pharmacophore.Methods: 1)The inhibitory activity of flavonoids on β-glucuronidase and human carboxylesterase 2 were detected with p NPG and FD as substrate,respectively.The inhibition kinetics of potential compounds were characterized carefully to obtain the inhibition constant Ki and inhibition type.2)Molecular docking model was constructed to explore the binding mechanism between inhibitors and target enzymes.Results: 1)Scutellarin and luteolin are competitive inhibitors of beta-glucuronidase with IC50 values of 5.76 μM,8.68μM and Ki values of 2.51μM and 2.89 μM,respectively.Molecular docking showed that scutellarin and luteolin could bind closely to the activity site of beta-glucuronidase and connect with Ser360 and Leu361 via hydrogen bond.2)5,6-dihydroxyflavone has a strong inhibitory effect on human carboxylesterase 2 with IC50 value of 3.50 μM and the Ki value of 4.28 μM.5,6-dihydroxyflavone is a noncompetitive inhibitor of carboxylesterase 2,which closely binds to the regulatory region of carboxylesterase 2.3)The structure-activity relationship of natural flavonoids inhibiting beta-glucuronidase and human carboxylesterase 2 has been clarified clearly.Flavonoids with no glycosides and strong hydrophobicity had stronger inhibition on both beta-glucuronidase and human carboxylesterase 2.Conclusion: Potent inhibitors of beta-glucuronidase and human carboxylesterase 2 were found from natural flavonoids,and the results will be helpful for the development of drugs in clinical treatment of irinotecan induced delayed-onset diarrhea.