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.

Gut microbiota mediates the absorption of FLZ, a new drug for Parkinson's disease treatment

The gut microbiota plays an important role in regulating the pharmacokinetics and pharmacodynamics of many drugs. FLZ, a novel squamosamide derivative, has been shown to have neuroprotective effects on experimental Parkinson's disease (PD) models. FLZ is under phase Ⅰ clinical trial now, while the underlying mechanisms contributing to the absorption of FLZ are still not fully elucidated. Due to the main metabolite of FLZ was abundant in feces but rare in urine and bile of mice, we focused on the gut microbiota to address how FLZ was metabolized and absorbed.

Detection of Azole Drug Resistance of the Aspergillus Species Cyp51a Gene by PCR

Allergic bronchopulmonary aspergillosis (ABPA) is a hypersensitivity pulmonary disease occurring in individuals with asthma or cystic fibrosis. In these patients, it is characterized by transient pulmonary infiltrates, reversible airway obstruction, eosinophilia

Development in research of CYP51 as the target of triazoles / 药学实践杂志

Triazoles are the most widely used antifungal drugs in clinic with broad spectrum and high efficacy,which targets sterol 14α-demethylase (CYP51),an enzyme expressed by the gene EGR11,which is a key enzyme in the fungi ergos-terol biosynthesis.On the one hand,the CYP51 belongs to a transmembrane protein.It is difficult to get the exact functional structure conformation which becomes a big challenge for the development of new drugs.On the other hand,it becomes con-sensus that EGR11 exon mutation cause CYP51 structural change is one of the major reasons for antifungal drugs resistance. Therefore,study of the structural changes toward the antifungal drug resistance is quite important.The review authors have summarized the research progress on CYP51 over the recent years.

Molecular mechanisms of cross-resistance to azole antifungal agents in a clinical isolate of Aspergillus fumigatus: a preliminary study / 中华皮肤科杂志

Objective To investigate the molecular mechanisms of cross-resistance to azoles in a clinical isolate of Aspergillus fumigatus. Methods A. fumigatus was isolated from a patient with invasive aspergillosis.Clinical Laboratory Standard Institute M38-A2 broth microdilution method and E-test method were used to determine the minimum inhibitory concentrations (MICs) or minimum effective concentration (MEC) of itraconazole, voriconazole, amphotericin B, posaconazole and caspofungin for the A. fumigatus isolate. DNA was extracted from the isolate and subjected to the amplification of cyp51A gene encoding the target enzyme of azole antifungal agents followed by sequence analysis. Results The broth microdilution test showed that the MEC of caspofungin was 0.5 mg/L, and MICs of itraconazole, voriconazole and amphotericin B were ≥ 16 mg/L,8 mg/L and 1 mg/L, respectively, for this isolate; while E-test assay revealed that the MICs of caspofungin,itraconazole, voriconazole, amphotericin B and posaconazole were 0.047 mg/L, ≥32 mg/L,≥32 mg/L, 12 mg/L and ≥32 mg/L, respectively. Sequence analysis showed an insertion of a 34-bp tandem sequence in the promoter region of the cyp51A gene as well as a T364A point mutation causing the substitution of leucine 98 (L98H). In addition, there were some other mutations in the cyp51A gene of this isolate, such as A137T,G585A, C814A, G836C, T991C and A1350G, which could result in corresponding amino acid substitutions.Conclusions An A. fumigatus strain with cross-resistance to azole antifungal agents is isolated. There is an insertion of a 34-bp tandem sequence into the promoter region as well as a T364A point mutation in the cyp51A gene, which contribute to the cross resistance to azole antifungal agents including itraconazole, voriconazole,and posaconazole. In addition, other mutations causing amino acid substitutions have also been detected in the cyp51 A gene of this isolate.

Two approaches to discovering and developing new drugs for Chagas disease

This review will focus on two general approaches carried out at the Sandler Center, University of California, San Francisco, to address the challenge of developing new drugs for the treatment of Chagas disease. The first approach is target-based drug discovery, and two specific targets, cytochrome P450 CYP51 and cruzain (aka cruzipain), are discussed. A "proof of concept" molecule, the vinyl sulfone inhibitor K777, is now a clinical candidate. The preclinical assessment compliance for filing as an Investigational New Drug with the United States Food and Drug Administration (FDA) is presented, and an outline of potential clinical trials is given. The second approach to identifying new drug leads is parasite phenotypic screens in culture. The development of an assay allowing high throughput screening of Trypanosoma cruzi amastigotes in skeletal muscle cells is presented. This screen has the advantage of not requiring specific strains of parasites, so it could be used with field isolates, drug resistant strains or laboratory strains. It is optimized for robotic liquid handling and has been validated through a screen of a library of FDA-approved drugs identifying 65 hits.

Relationship Between Gene CYP51 and Clinical Azole-resistant Candida albicans Isolates / 中华医院感染学杂志

OBJECTIVE To explore the relationship between the point mutations of gene CYP51 and the azole-resistance mechanism in clinical Candida albicans isolates.METHODS The paper diffusion test and NCCLS M-27 protocols were used to screen the fluconazole-resistant and itraconazole-resistant C.albicans clinical isolates.Gene CYP51 of two azole-resistant C.albicans clinical strains(2007H and 2007T strains) was amplified by three pairs of primers,respectively.The PCR products purified were sequenced,and compared with the nucleotide sequences of C.albicans(accession No.:X13296) to find out the mutation sites.RESULTS The nucleotide sequence analysis showed that there were both significant point mutations and silent mutations in gene CYP51 from two azole-resistant isolates of C.albicans.Seven mutations previously described,F105L,K128T,Y132H,T199I,R267H,G464S,and R467K,were identified in the two strains.The animo acid substitutions of Y132H and R467K,known to contribution to azole resistance,were detected in both 2007H strain and 2007T strain.Four novel mutations,including F71L,W244R,T311N and T352I,were simultanously identified.Nine silent mutations appeared in two isolates.CONCLUSIONS In this survey,the two azole-resistant C.albicans clinical isolates contained more than one mutation in gene CYP51 that is associated with azole resistance.Four novel mutations of CYP51 may be associated with the resistance of C.albicans to azoles.And the mechanisms need to be further studied in detail.

Resistant Mechanisms of Candida albicans to Azoles / 中华医院感染学杂志

OBJECTIVE To investigate the resistant mechanisms of Candida albicans to azoles at molecular level.METHODS NCCLS M-27 protocols were used to test the in vitro susceptibilities of 102 C.albicans strains isolated from the patients with recurrent vulvovaginal candidiasis(RVVC) against fluconazole(FLC) and itraconazole(ITC) to screen the FLC-and ITC-resistant C.albicans isolates;six pairs of primers,A1-A2,B1-B2,C1-C2,D1-D2,E1-E2 and F1-F2 were respectively to amplify gene CYP51 of 4 strains with FLC-and ITC-resistance.The PCR products were sequenced and analyzed to identify the mutation sites by compared with the sequence of gene CYP51 of referenced C.albicans strain in NCBI site of Internet.RESULTS The analysis of full length sequence of CYP51 from 4 FLC-and ITC-resistant strains showed that from total 32 mutation sites there were 4 significant site mutations,where the mutation of GAT to GAC at 116 caused the substitution of D by E(E266D in two strains);GCC to GGT at 117 caused the substitution of A by G(A117G in 1 strain);GAA to GAC at 266 caused the substitution of E by D(E266D in 2 strains);and GTT to ATT at 488 caused the substitution of I by V(V488I in 1 strain).The site mutations of 266 and 488 were tested in 1 strain of 4 strains.CONCLUSIONS The CYP51 total gene of 4 strains has been checked out.Of FLC and ITC-resistant C.albicans alignment in this time,find out 4 significant bp mutations.Causing its amino acide change,among them,A117G has not be interrelated report still now.The details of mechanism need to be further studied.