Transformation of berberine to its demethylated metabolites by the CYP51 enzyme in the gut microbiota / 药物分析学报

Berberine(BBR)is an isoquinoline alkaloid extracted from Coptis chinensis that improves diabetes,hyperlipidemia and inflammation.Due to the low oral bioavailability of BBR,its mechanism of action is closely related to the gut microbiota.This study focused on the CYP51 enzyme of intestinal bacteria to elucidate a new mechanism of BBR transformation by demethylation in the gut microbiota through multiple analytical techniques.First,the docking of BBR and CYP51 was performed;then,the pharma-cokinetics of BBR was determined in ICR mice in vivo,and the metabolism of BBR in the liver,kidney,gut microbiota and single bacterial strains was examined in vitro.Moreover,16S rRNA analysis of ICR mouse feces indicated the relationship between BBR and the gut microbiota.Finally,recombinant E.coli con-taining cyp51 gene was constructed and the CYP51 enzyme lysate was induced to express.The metabolic characteristics of BBR were analyzed in the CYP51 enzyme lysate system.The results showed that CYP51 in the gut microbiota could bind stably with BBR,and the addition of voriconazole(a specific inhibitor of CYP51)slowed down the metabolism of BBR,which prevented the production of the demethylated metabolites thalifendine and berberrubine.This study demonstrated that CYP51 promoted the deme-thylation of BBR and enhanced its intestinal absorption,providing a new method for studying the metabolic transformation mechanism of isoquinoline alkaloids in vivo.

Berberine is a new mechanism drug against energy metabolic disorder / 中国药理学与毒理学杂志

Berberine (BBR) is a natural compound isolated from Coptis chinensis and for decades an over-the-count medicine in China for bacterial-caused diarrhea. We have identified BBR to be an effective drug in treating hyperlipidemia as well as hyperglycemia. Clinical studies showed that oral administration of BBR caused significant reduction of blood cholesterol, LDL-c, triglyceride, as well as glucose and HbA1c in patients with hyperlipidemia or T2D. A small % of patients have minor and transient side- effect in the GI system. The cholesterol- lowering effect was associated with the ERK mediated LDLR mRNA up-regulation; the glucose-lowering effect mainly resulted from PKD-mediated insulin receptor expression as well as activation of AMPK. The clinical efficacy of BBR were verified by a large number of independent clinical groups in and outside China. For BBR absorption mechanism, we showed that BBR was first converted into dhBBR by nitroreductase in the gut microbiota, promoting BBR enter into intestinal well, where it was oxidized back to BBR, and then got into blood. Our recent study showed that BBR could also promote gut microbiota pathways to make SCFAs (such as butyrate), which entered blood and reduced blood lipids and glucose, suggesting a newly identified MOA of BBR. BBR is now in clinical trial in China. We consider it a multiple-target drug for metabolic disorders in clinic.

Establishment and application of high throughput screening model for hepatitis C virus NS3-4A protease inhibitors in vitro / 中国医学科学院学报

<p><b>OBJECTIVE</b>To establish fluorescence resonance energy transfer (FRET) assay method of detecting proteolytic activity of non-structural protein 3-4A (NS3-4A) serine protease of hepatitis C virus (HCV) for high throughput screening inhibitors against HCV in vitro.</p><p><b>METHODS</b>HCV recombinant plasmid pMAL~c2/NS3-4A was transformed into the E.coli strain K12TB1. Maltose-binding-protein (MBP) NS3-4A fusion protein expression was induced by adding isopropyl-β-D-thiogalacto-pyranoside (IPTG) and purified by affinity chromatography. The proteolytic activity of MBP-NS3-4A protease was analyzed by FRET with the special protease substrate. The reaction system in this model was optimized, and the reliability of the model was evaluated.</p><p><b>RESULTS</b>High throughput screening model for HCV NS3-4A protease inhibitors was established, and the best concentrations of enzyme and substrate were optimized. In the model, the Km value of protease was 4.74 μmol/L, Z factor was up to 0.80, and coefficient of variation (CV) was 1.91%. BILN 2061, one of the known HCV protease inhibitors, was measured with the Ki of 0.30 nmol/L.</p><p><b>CONCLUSION</b>The assay model using FRET method for HCV NS3 4A serine protease is stable and reliable, and the model is suitable for high throughput screening for HCV NS3 4A protease inhibitors.</p>