UDP-glycosyltransferase family in Haemonchus contortus: Phylogenetic analysis, constitutive expression, sex-differences and resistance-related differences

UDP-glycosyltransferases (UGT), catalysing conjugation of UDP-activated sugar donors to small lipophilic chemicals, are widespread in living organisms from bacteria to fungi, plant, or animals. The progress of genome sequencing has enabled an assessment of the UGT multigene family in Haemonchus contortus (family Trichostrongylidae, Nematoda), a hematophagous gastrointestinal parasite of small ruminants. Here we report 32 putative UGT genes divided into 15 UGT families. Phylogenetic analysis in comparison with UGTs from Caenorhabditis elegans, a free-living model nematode, revealed several single member homologues, a lack of the dramatic gene expansion seen in C. elegans, but also several families (UGT365, UGT366, UGT368) expanded in H. contortus only. The assessment of constitutive UGT mRNA expression in H. contortus adults identified significant differences between females and males. In addition, we compared the expression of selected UGTs in the drug-sensitive ISE strain to two benzimidazole-resistant strains, IRE and WR, with different genetic backgrounds. Constitutive expression of UGT368B2 was significantly higher in both resistant strains than in the sensitive strain. As resistant strains were able to deactivate benzimidazole anthelmintics via glycosylation more effectively then the sensitive strain, UGT368B2 enhanced constitutive expression might contribute to drug resistance in H. contortus

The role of UDP-glycosyltransferase in development of drug resistance in parasitic nematodes

Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Mgr. Diana Dimunová Supervisor: doc. Ing. Petra Matoušková, Ph.D. Title of Doctoral Thesis: The role of UDP-glycosyltransferases in drug resistance in parasitic nematodes The diseases caused by parasitic nematodes represent a serious problem, which threatens livestock's health, because pharmacotherapy is complicated by widespread anthelmintic resistance. Understanding of the mechanisms of parasite drug resistance and defense strategies is important to maintaining the effectiveness of currently used anthelmintics and developing new approaches to controlling these infections. The ability of parasites to inactivate anthelmintics through their metabolism, which is provided by biotransformation enzymes, may contribute to the development of drug resistance. The UDP-glycosyltransferases (UGT) superfamily can protect parasites from the toxic actions of anthelmintics by modifying drugs to inactive glycoside metabolites. These metabolites have been identified in benzimidazole metabolism to an increased extent in a resistant strain of H. contortus, which suggests the involvement of UGTs in anthelmintic resistance. In the genome of this parasitic nematode, 32 genes encoding UGTs divided into 15 families have...Univerzita Karlova Farmaceutická fakulta v Hradci Králové Katedra biochemických vied Kandidát: Mgr. Diana Dimunová Školiteľ: doc. Ing. Petra Matoušková, Ph. D. Názov dizertačnej práce: Úloha UDP-glykosyltransferas vo vývoji liekovej rezistencie u parazitických hlístovcov Ochorenia spôsobené parazitickými hlístovcami predstavujú závažný problém ohrozujúci zdravie hospodárskych zvierat, pretože ich farmakoterapiu komplikuje rozšírená anthelmintická rezistencia. Poznanie mechanizmov rezistencie a obranných stratégii parazitov voči liečivám je doležíte pre zachovanie účinnosti súčasných anthelmintík a vývoji nových prístupov ku kontrole týchto infekcii. K rozvoju rezistencie môže prispievať schopnosť parazitov inaktivovať anthelmintika prostredníctvom ich metabolizmu, ktorý je zabezpečený biotransformačnými enzýmami. Nadrodina UDP-glykosyltranferas (UGT) môže chrániť parazity pred toxickým účinkom anthelmintík modifikáciou liečiv na inaktívne metabolity glykosidy. Tieto metabolity boli identifikované v metabolizme benzimidazolov vo zvýšenej miere v rezistentných kmeňoch H. contortus, čo nasvedčuje zapojeniu UGT do anthelmintickej rezistencie. V genóme tejto parazitickej nematody bolo nájdených 32 génov kódujúcich UGT, ktoré sú rozdelené do 15 rodín. Ďalšia charakterizácia spomínanej skupiny enzýmov...Department of Biochemical SciencesKatedra biochemických vědFarmaceutická fakulta v Hradci KrálovéFaculty of Pharmacy in Hradec Králov

The role of UDP-glycosyltransferase in development of drug resistance in parasitic nematodes

Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Mgr. Diana Dimunová Supervisor: doc. Ing. Petra Matoušková, Ph.D. Title of Doctoral Thesis: The role of UDP-glycosyltransferases in drug resistance in parasitic nematodes The diseases caused by parasitic nematodes represent a serious problem, which threatens livestock's health, because pharmacotherapy is complicated by widespread anthelmintic resistance. Understanding of the mechanisms of parasite drug resistance and defense strategies is important to maintaining the effectiveness of currently used anthelmintics and developing new approaches to controlling these infections. The ability of parasites to inactivate anthelmintics through their metabolism, which is provided by biotransformation enzymes, may contribute to the development of drug resistance. The UDP-glycosyltransferases (UGT) superfamily can protect parasites from the toxic actions of anthelmintics by modifying drugs to inactive glycoside metabolites. These metabolites have been identified in benzimidazole metabolism to an increased extent in a resistant strain of H. contortus, which suggests the involvement of UGTs in anthelmintic resistance. In the genome of this parasitic nematode, 32 genes encoding UGTs divided into 15 families have..

The role of UDP-glycosyltransferase in development of drug resistance in parasitic nematodes

Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Mgr. Diana Dimunová Supervisor: doc. Ing. Petra Matoušková, Ph.D. Title of Doctoral Thesis: The role of UDP-glycosyltransferases in drug resistance in parasitic nematodes The diseases caused by parasitic nematodes represent a serious problem, which threatens livestock's health, because pharmacotherapy is complicated by widespread anthelmintic resistance. Understanding of the mechanisms of parasite drug resistance and defense strategies is important to maintaining the effectiveness of currently used anthelmintics and developing new approaches to controlling these infections. The ability of parasites to inactivate anthelmintics through their metabolism, which is provided by biotransformation enzymes, may contribute to the development of drug resistance. The UDP-glycosyltransferases (UGT) superfamily can protect parasites from the toxic actions of anthelmintics by modifying drugs to inactive glycoside metabolites. These metabolites have been identified in benzimidazole metabolism to an increased extent in a resistant strain of H. contortus, which suggests the involvement of UGTs in anthelmintic resistance. In the genome of this parasitic nematode, 32 genes encoding UGTs divided into 15 families have..

The role of UDP-glycosyltransferase in development of drug resistance in parasitic nematodes

Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Mgr. Diana Dimunová Supervisor: doc. Ing. Petra Matoušková, Ph.D. Title of Doctoral Thesis: The role of UDP-glycosyltransferases in drug resistance in parasitic nematodes The diseases caused by parasitic nematodes represent a serious problem, which threatens livestock's health, because pharmacotherapy is complicated by widespread anthelmintic resistance. Understanding of the mechanisms of parasite drug resistance and defense strategies is important to maintaining the effectiveness of currently used anthelmintics and developing new approaches to controlling these infections. The ability of parasites to inactivate anthelmintics through their metabolism, which is provided by biotransformation enzymes, may contribute to the development of drug resistance. The UDP-glycosyltransferases (UGT) superfamily can protect parasites from the toxic actions of anthelmintics by modifying drugs to inactive glycoside metabolites. These metabolites have been identified in benzimidazole metabolism to an increased extent in a resistant strain of H. contortus, which suggests the involvement of UGTs in anthelmintic resistance. In the genome of this parasitic nematode, 32 genes encoding UGTs divided into 15 families have...Univerzita Karlova Farmaceutická fakulta v Hradci Králové Katedra biochemických vied Kandidát: Mgr. Diana Dimunová Školiteľ: doc. Ing. Petra Matoušková, Ph. D. Názov dizertačnej práce: Úloha UDP-glykosyltransferas vo vývoji liekovej rezistencie u parazitických hlístovcov Ochorenia spôsobené parazitickými hlístovcami predstavujú závažný problém ohrozujúci zdravie hospodárskych zvierat, pretože ich farmakoterapiu komplikuje rozšírená anthelmintická rezistencia. Poznanie mechanizmov rezistencie a obranných stratégii parazitov voči liečivám je doležíte pre zachovanie účinnosti súčasných anthelmintík a vývoji nových prístupov ku kontrole týchto infekcii. K rozvoju rezistencie môže prispievať schopnosť parazitov inaktivovať anthelmintika prostredníctvom ich metabolizmu, ktorý je zabezpečený biotransformačnými enzýmami. Nadrodina UDP-glykosyltranferas (UGT) môže chrániť parazity pred toxickým účinkom anthelmintík modifikáciou liečiv na inaktívne metabolity glykosidy. Tieto metabolity boli identifikované v metabolizme benzimidazolov vo zvýšenej miere v rezistentných kmeňoch H. contortus, čo nasvedčuje zapojeniu UGT do anthelmintickej rezistencie. V genóme tejto parazitickej nematody bolo nájdených 32 génov kódujúcich UGT, ktoré sú rozdelené do 15 rodín. Ďalšia charakterizácia spomínanej skupiny enzýmov...Department of Biochemical SciencesKatedra biochemických vědFarmaceutická fakulta v Hradci KrálovéFaculty of Pharmacy in Hradec Králov

The effect of nerolidol and farnesol on the selected hepatic biotransformation enzymes

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Diana Dimunová Supervisor: Prof. RNDr. Lenka Skálová, Ph.D. Title of diploma thesis: The effect of nerolidol and farnesol on the selected hepatic biotransformation enzymes Sesquiterpenes represent a class of terpenoids that are mostly produced as secondary metabolites in higher plants. These compounds play an important role as components of many folk medicines and dietary supplements. In recent years, they have been studied in view of their antioxidant, anti-parasitic and anti-carcinogenic activities. On the other hand, sesquiterpenes could change the activity of biotransformation enzymes and by this way affect the metabolism of drugs, the safety and efficiency of pharmacotherapy. The aim of diploma thesis was to find out the influence of three acyclic sesquiterpenes farnesol (FAR), cis- nerolidol (CNER) and trans-nerolidol (TNER) on activity and expresion of reducing enzymes (aldehyd reductase AKR1A1, carbonyl reductase CBR1, NADH-quinone oxidoreductase1 NQO1) and conjugating enzymes (sulfotransferase SULT, glutathione-S-transferase GST, UDP-glucuronosyltransferase UGT). The potential inhibitory effects of sesquiterpenes were tested in hepatic subcellular fractions, while primary..

Vliv nerolidolu a farnesolu na vybrané jaterní biotransformační enzymy

Univerzita Karlova v Praze Farmaceutická fakulta v Hradci Králové Katedra biochemických vied Kandidát: Diana Dimunová Školiteľ: Prof. RNDr. Lenka Skálová, Ph.D. Názov diplomovej práce: Vplyv nerolidolu a farnesolu na vybrané pečeňové biotransformačné enzýmy Seskviterpény sú triedou terpénov, ktoré sú väčšinou produkované ako sekundárne metabolity vo vyšších rastlinách. Tieto látky hrajú dôležitú úlohu v tradičnej medicíne a ako zložky potravinových doplnkov. Seskviterpény sú intenzívne študované z hľadiska ich antioxidačných, antiparazitických a protinadorových účinkov. Na druhej strane, môžu meniť aktivitu biotransformačných enzýmov a tak ovplyvniť metabolizmus liečiv, bezpečnosť a účinnosť farmakoterapie. Cieľom tejto diplomovej práce bolo zistiť vplyv troch acyklických seskviterpénov farnesolu (FAR), cis-nerolidolu (CNER) a trans-nerolidolu (TNER) na aktivitu a expresiu redukčných enzýmov (aldehydreduktasa AKR1A1, karbonylreduktasa CBR1, NADH-chinonoxidoreduktasa1 NQO1) a konjugačných enzýmov (sulfotransferasa SULT, glutathion-S-transferasa GST, UDP-glukuronosyltransferasa UGT). Potenciálny inhibičný efekt seskviterpénov bol testovaný v pečeňových subcelulárnych frakciách, pokým primárne kultúry potkaních hepatocytov slúžili na testovanie ich potenciálneho indukčného efektu. Taktiež bol...Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Diana Dimunová Supervisor: Prof. RNDr. Lenka Skálová, Ph.D. Title of diploma thesis: The effect of nerolidol and farnesol on the selected hepatic biotransformation enzymes Sesquiterpenes represent a class of terpenoids that are mostly produced as secondary metabolites in higher plants. These compounds play an important role as components of many folk medicines and dietary supplements. In recent years, they have been studied in view of their antioxidant, anti-parasitic and anti-carcinogenic activities. On the other hand, sesquiterpenes could change the activity of biotransformation enzymes and by this way affect the metabolism of drugs, the safety and efficiency of pharmacotherapy. The aim of diploma thesis was to find out the influence of three acyclic sesquiterpenes farnesol (FAR), cis- nerolidol (CNER) and trans-nerolidol (TNER) on activity and expresion of reducing enzymes (aldehyd reductase AKR1A1, carbonyl reductase CBR1, NADH-quinone oxidoreductase1 NQO1) and conjugating enzymes (sulfotransferase SULT, glutathione-S-transferase GST, UDP-glucuronosyltransferase UGT). The potential inhibitory effects of sesquiterpenes were tested in hepatic subcellular fractions, while primary...Department of Biochemical SciencesKatedra biochemických vědFarmaceutická fakulta v Hradci KrálovéFaculty of Pharmacy in Hradec Králov

Metabolism of albendazole, ricobendazole and flubendazole in Haemonchus contortus adults: Sex differences, resistance-related differences and the identification of new metabolites

Haemonchus contortus (family Trichostrongylidae, Nematoda), a hematophagous gastrointestinal parasite found in small ruminants, has a great ability to develop resistance to anthelmintic drugs. We studied the biotransformation of the three benzimidazole anthelmintics: albendazole (ABZ), ricobendazole (albendazole S-oxide; RCB) and flubendazole (FLU) in females and males of H. contortus in both a susceptible ISE strain and resistant IRE strain. The ex vivo cultivation of living nematodes in culture medium with or without the anthelmintics was used. Ultrasensitive UHPLC/MS/MS analysis revealed 9, 7 and 12 metabolites of ABZ, RCB and FLU, respectively, with most of these metabolites now described in the present study for the first time in H. contortus. The structure of certain metabolites shows the presence of biotransformation reactions not previously reported in nematodes. There were significant qualitative and semi-quantitative differences in the metabolites formed by male and female worms. In most cases, females metabolized drugs more extensively than males. Adults of the IRE strain were able to form many more metabolites of all the drugs than adults of the ISE strain. Some metabolites were even found only in adults of the IRE strain. These findings suggest that increased drug metabolism may play a role in resistance to benzimidazole drugs in H. contortus. Keywords: Drug resistance, Drug metabolism, Anthelmintics, Benzimidazole, Nematod

Nerolidol and Farnesol Inhibit Some Cytochrome P450 Activities but Did Not Affect Other Xenobiotic-Metabolizing Enzymes in Rat and Human Hepatic Subcellular Fractions

Sesquiterpenes, 15-carbon compounds formed from three isoprenoid units, are the main components of plant essential oils. Sesquiterpenes occur in human food, but they are principally taken as components of many folk medicines and dietary supplements. The aim of our study was to test and compare the potential inhibitory effect of acyclic sesquiterpenes, trans-nerolidol, cis-nerolidol and farnesol, on the activities of the main xenobiotic-metabolizing enzymes in rat and human liver in vitro. Rat and human subcellular fractions, relatively specific substrates, corresponding coenzymes and HPLC, spectrophotometric or spectrofluorometric analysis of product formation were used. The results showed significant inhibition of cytochromes P450 (namely CYP1A, CYP2B and CYP3A subfamilies) activities by all tested sesquiterpenes in rat as well as in human hepatic microsomes. On the other hand, all tested sesquiterpenes did not significantly affect the activities of carbonyl-reducing enzymes and conjugation enzymes. The results indicate that acyclic sesquiterpenes might affect CYP1A, CYP2B and CYP3A mediated metabolism of concurrently administered drugs and other xenobiotics. The possible drug–sesquiterpene interactions should be verified in in vivo experiments