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Identifying and applying a highly selective probe to simultaneously determine the O-glucuronidation activity of human UGT1A3 and UGT1A4.

Jiang L, Liang SC, Wang C, Ge GB, Huo XK, Qi XY, Deng S, Liu KX, Ma XC - Sci Rep (2015)

Bottom Line: Glucuronidation mediated by uridine 5'-diphospho (UDP)-glucuronosyltransferase is an important detoxification pathway.However, identifying a selective probe of UDP- glucuronosyltransferase is complicated because of the significant overlapping substrate specificity displayed by the enzyme.DACB was well characterized as a probe for simultaneously determining the catalytic activities of O-glucuronidation mediated by UGT1A3 and UGT1A4 from various enzyme sources, through a sensitive analysis method.

View Article: PubMed Central - PubMed

Affiliation: College of Pharmacy, Key Laboratory of Pharmacokinetic and Drug Transport of Liaoning, Academy of Integrative Medicine, Dalian Medical University, Dalian, 116044, China.

ABSTRACT
Glucuronidation mediated by uridine 5'-diphospho (UDP)-glucuronosyltransferase is an important detoxification pathway. However, identifying a selective probe of UDP- glucuronosyltransferase is complicated because of the significant overlapping substrate specificity displayed by the enzyme. In this paper, desacetylcinobufagin (DACB) 3-O- and 16-O-glucuronidation were found to be isoform-specific probe reactions for UGT1A4 and UGT1A3, respectively. DACB was well characterized as a probe for simultaneously determining the catalytic activities of O-glucuronidation mediated by UGT1A3 and UGT1A4 from various enzyme sources, through a sensitive analysis method.

No MeSH data available.


Inhibition assay of 3β- (A) and 16β-O-glucuronidation (B) by UGT inhibitors in HLMs.
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f3: Inhibition assay of 3β- (A) and 16β-O-glucuronidation (B) by UGT inhibitors in HLMs.

Mentions: To confirm the key roles of UGT1A3 and UGT1A4 in the 16-O- and 3-O-glucuronidation of DACB, respectively, chemical inhibition studies were also performed. As shown in Fig. 3, phenylbutazone20, TFP20, fluconazole28 and hecogenin29 were used to inhibit the 3-O-glucuronidation of DACB mediated by UGT1A4. Our results showed that TFP (a specific substrate of UGT1A4) and hecogenin (a specific inhibitor of UGT1A4) can significantly inhibit the 3-O-glucuronidation of DACB. The similar inhibitory concentration for 50% reduction (IC50) values of hecogenin for UGT1A4 and HLMs also strongly suggested that UGT1A4 can catalyze the 3-O-glucuronidation of DACB with satisfactory selectivity (Figs. S6A and B). Similarly, the formation of M-2 (16-O-glucuronate) was significantly inhibited by phenylbutazone20, glycyrrhetinic acid30 and β- estradiol31 at substrate concentration of 60 μM. Additionally, the equal IC50 values for glycyrrhetinic acid (a selective inhibitor of UGT1A3), UGT1A3 and HLMs were observed for 16-O-glucuronidation (Figs. S6C and D). This evidence strongly indicated that the 16-O-glucuronidation of DACB is selectively catalyzed by UGT1A3.


Identifying and applying a highly selective probe to simultaneously determine the O-glucuronidation activity of human UGT1A3 and UGT1A4.

Jiang L, Liang SC, Wang C, Ge GB, Huo XK, Qi XY, Deng S, Liu KX, Ma XC - Sci Rep (2015)

Inhibition assay of 3β- (A) and 16β-O-glucuronidation (B) by UGT inhibitors in HLMs.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4401096&req=5

f3: Inhibition assay of 3β- (A) and 16β-O-glucuronidation (B) by UGT inhibitors in HLMs.
Mentions: To confirm the key roles of UGT1A3 and UGT1A4 in the 16-O- and 3-O-glucuronidation of DACB, respectively, chemical inhibition studies were also performed. As shown in Fig. 3, phenylbutazone20, TFP20, fluconazole28 and hecogenin29 were used to inhibit the 3-O-glucuronidation of DACB mediated by UGT1A4. Our results showed that TFP (a specific substrate of UGT1A4) and hecogenin (a specific inhibitor of UGT1A4) can significantly inhibit the 3-O-glucuronidation of DACB. The similar inhibitory concentration for 50% reduction (IC50) values of hecogenin for UGT1A4 and HLMs also strongly suggested that UGT1A4 can catalyze the 3-O-glucuronidation of DACB with satisfactory selectivity (Figs. S6A and B). Similarly, the formation of M-2 (16-O-glucuronate) was significantly inhibited by phenylbutazone20, glycyrrhetinic acid30 and β- estradiol31 at substrate concentration of 60 μM. Additionally, the equal IC50 values for glycyrrhetinic acid (a selective inhibitor of UGT1A3), UGT1A3 and HLMs were observed for 16-O-glucuronidation (Figs. S6C and D). This evidence strongly indicated that the 16-O-glucuronidation of DACB is selectively catalyzed by UGT1A3.

Bottom Line: Glucuronidation mediated by uridine 5'-diphospho (UDP)-glucuronosyltransferase is an important detoxification pathway.However, identifying a selective probe of UDP- glucuronosyltransferase is complicated because of the significant overlapping substrate specificity displayed by the enzyme.DACB was well characterized as a probe for simultaneously determining the catalytic activities of O-glucuronidation mediated by UGT1A3 and UGT1A4 from various enzyme sources, through a sensitive analysis method.

View Article: PubMed Central - PubMed

Affiliation: College of Pharmacy, Key Laboratory of Pharmacokinetic and Drug Transport of Liaoning, Academy of Integrative Medicine, Dalian Medical University, Dalian, 116044, China.

ABSTRACT
Glucuronidation mediated by uridine 5'-diphospho (UDP)-glucuronosyltransferase is an important detoxification pathway. However, identifying a selective probe of UDP- glucuronosyltransferase is complicated because of the significant overlapping substrate specificity displayed by the enzyme. In this paper, desacetylcinobufagin (DACB) 3-O- and 16-O-glucuronidation were found to be isoform-specific probe reactions for UGT1A4 and UGT1A3, respectively. DACB was well characterized as a probe for simultaneously determining the catalytic activities of O-glucuronidation mediated by UGT1A3 and UGT1A4 from various enzyme sources, through a sensitive analysis method.

No MeSH data available.