Inhibitory Effects Of Bromine Phenol (BPs) And Hypericin On Its Key Transferases During Sex Hormone Synthesis

ObjectiveIn the process of sex hormone synthesis,sulfotransferase 1family（SULTs）and human carboxylesterase 2（CES2）are involved in multiple metabolic regulation.Our study provides a comprehensive description of the inhibitory effect of BPS on salt subtype and hypericin on carboxylesterase（CES）in the process of sex hormone synthesis,and provides a new idea for clarifying its important role and toxic mechanism in the process of sex hormone synthesis.Method1.The inhibition of BPS on the subtype of SULTs was studied by using PNP as a probe reaction catalyzed by recombinant sults.2.The concentration of IC₅₀was determined by adding different concentrations of BPs（0-100μM）.Inhibition kinetics including K_m（for PNP）and IC₅₀（for BPs）were determined.The Lineweaver-Burk diagram was drawn by using 1/reaction rate（V）and 1/concentration PNP（PNP）to determine the type of inhibition kinetics.A second plot was drawn to determine the inhibition kinetic parameter（K_i）,where the relationship between the slope of the line in the Lineweaver-Burk curve and the BPs concentration was calculated and plotted.3.In vivo inhibition was measured by in vivo extrapolation（IVIVE）.4.Probe reactions of FD and NCEN were analyzed by using Synergy-H1 hybrid multimode microplate（Bio Tek）.FD and NCEN were used as the substrates of h CE2 specific fluorescent probes.The inhibition of h CE2 by FD and NCEN was studied with the advantages of high sensitivity,easy conduction and high throughput screening.The fluorescein production was measured by fluorescein fluorescence value,and the residual enzyme activity of CE2 was measured by using the hybrid multimode microplate （Bio Tek）.5.The inhibition of hypericin as an h CE2 inhibitor was determined by standard method.The semi inhibition concentration(IC₅₀)of hypericin was determined,and the inhibition constant（K_i）was determined with FD,NCEN and CPT-11 of different concentrations.By measuring the intersection of Dixon graph and Lineweaver Burk graph,the inhibition kinetics type was evaluated.6.The potential inhibitory effect of hypericin and h CE2 in vivo was analyzed by estimating AUC in the presence and absence of hypericin.7.The molecular interactions between BPs and SULTs,and between hypericin and CES2 were studied by electronic docking.Using Modeller9v14 program,the structure of salt subtype was constructed by homologous modeling method,and the conformation of h CE2 was simulated.BPs and hypericin were fixed in the active cavity and h CE2 of salt subtype by autodock software（version 4.2）.Lamarckian genetic algorithm（LGA）was used to study the molecular docking of BPs with SULTs and hypericin with h CE2.The interaction of hydrogen bond and hydrophobic contact was analyzed.Result1.All BPs tested showed strong inhibition on these four subtypes,and SULT1A1 and SULT1B1 were the most susceptible subtypes to BPs inhibition.The intersection is located on the vertical axis of the Lineweaver-Burk graph,indicating the competitive inhibition of 2,4,6-TBP on SULT1A3.The inhibition kinetic parameter（K_i）was 16.28μM.In vitro in vivo extrapolation（IVIVE）showed that the threshold concentration of SULT1A3 inhibition induced by 2,4,6-TBP was 1.628μM.The inhibition of hydrogen bond formation on 3,5-DBP was stronger than that on3-BP.2.Hypericin has a strong inhibitory effect on the hydrolysis of FD,NCEN and CPT-11 mediated by h CE2,with IC₅₀values of 0.99μM,26.6μM and 113μM,respectively.The inhibition kinetic analysis showed that hypericin inhibited the hydrolysis of FD and NCEN mediated by h CE2 in a non competitive manner,with K_i values of 1.88μM and 28.5μM,respectively.Hypericin has a competitive inhibitory effect on the hydrolysis of CPT-11,K_ivalue is55.6μM,and hypericin has a significant inhibitory effect on the endogenous h CE2 activity.In the presence of hypericin,AUC of h CE2 with metabolic sites similar to FD and NCEN increased by71%and 5%,respectively.After oral administration of hypericin,CPT-11 exposure in intestinal epithelium is expected to increase by2%-69%.Molecular docking studies showed that hypericin formed hydrogen bonds with OD2 of ASP72 and O of ALA75 in h CE2 binding vesicles.Conclude1.The disturbance of BPs to the important target of SULTs can lead to the damage of target cell synthesis.The metabolic reaction catalyzed by SULTs can reduce the activity of compounds and increase their water solubility,thus promoting the excretion of exogenous substances into bile,urine and feces.BPs has a good inhibitory effect on the subtype of SULTs in the process of sex hormone synthesis.2.Hypericin has a strong inhibitory effect on the hydrolysis of FD,NCEN and CPT-11 mediated by h CE2.The inhibition kinetic analysis shows that hypericin inhibits the hydrolysis of FD and NCEN mediated by h CE2 in a non competitive.Hypericin has a competitive inhibitory effect on the hydrolysis of CPT-11,and hypericin has a significant inhibitory effect on the endogenous h CE2 activity.