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A High Throughput Assay for Discovery of Bacterial β-Glucuronidase Inhibitors.

Ahmad S, Hughes MA, Lane KT, Redinbo MR, Yeh LA, Scott JE - Curr Chem Genomics (2011)

Bottom Line: To identify chemical inhibitors of GUS activity, we employed and validated a high throughput, fluorescence-based biochemical assay and used this assay to screen a compound library.Novel inhibitors of GUS were identified with IC(50) values ranging from 50 nM to 4.8 µM.Our results demonstrate that this high throughput assay can be used to identify small molecule inhibitors of GUS.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Sciences, Biomanufacturing Research Institute and Technology Enterprise, North Carolina Central University, 1801 Fayetteville Street, Durham, NC 27707, USA.

ABSTRACT
CPT-11 is a widely-used anti-cancer drug that is converted in vivo to its active metabolite, SN-38. In the liver, enzymes detoxify SN-38 by coupling it to a glucuronidate moiety and this inactive compound (SN-38G) is excreted into the gastrointestinal tract. In the intestine, commensal bacteria convert the SN-38G back to the active and toxic SN-38 using bacterial β-glucuronidase enzyme (GUS). This intestinal SN-38 causes debilitating diarrhea that prevents dose-intensification and efficacy in a significant fraction of patients undergoing CPT-11 treatment for cancer. This CPT-11 metabolic pathway suggests that small molecule inhibitors of GUS may have utility as novel therapeutics for prevention of dose-limiting diarrhea resulting from CPT-11 therapy. To identify chemical inhibitors of GUS activity, we employed and validated a high throughput, fluorescence-based biochemical assay and used this assay to screen a compound library. Novel inhibitors of GUS were identified with IC(50) values ranging from 50 nM to 4.8 µM. These compounds may be useful as chemical probes for use in proof-of-concept experiments designed to determine the efficacy of GUS inhibitors in altering the intestinal metabolism of drugs. Our results demonstrate that this high throughput assay can be used to identify small molecule inhibitors of GUS.

No MeSH data available.


Related in: MedlinePlus

Schematic of the GUS high throughput enzyme assay.
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Figure 1: Schematic of the GUS high throughput enzyme assay.

Mentions: Bacterial GUS activity can readily be measured using the substrate 4-methylumbelliferyl glucuronide (4MUG) [26]. Upon hydrolysis of 4MUG by GUS, the fluorescent compound 4-methylumbelliferone (4MU) is generated. We used this substrate to detect GUS activity in an end point assay that is amenable for high throughput screening for inhibitors of GUS. The assay was developed with a final volume of 50 µl using the 384-well plate format to match the intended screening format. The general assay set-up included the addition of diluted GUS enzyme to the well followed by the addition of 4MUG to initiate the reaction (Fig. 1). The reaction was terminated with the addition of a 1 M Na2CO3 solution and the fluorescence measured in a plate reader. The final assay buffer consisted of 50 mM HEPES pH 7.4 and 0.01% TX-100. The Km for 4MUG was experimentally determined to be 125 μM (data not shown) and therefore this was the concentration of 4MUG used in this assay. Stopping of the reaction was confirmed by time course studies following termination of the assay (data not shown).


A High Throughput Assay for Discovery of Bacterial β-Glucuronidase Inhibitors.

Ahmad S, Hughes MA, Lane KT, Redinbo MR, Yeh LA, Scott JE - Curr Chem Genomics (2011)

Schematic of the GUS high throughput enzyme assay.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3106358&req=5

Figure 1: Schematic of the GUS high throughput enzyme assay.
Mentions: Bacterial GUS activity can readily be measured using the substrate 4-methylumbelliferyl glucuronide (4MUG) [26]. Upon hydrolysis of 4MUG by GUS, the fluorescent compound 4-methylumbelliferone (4MU) is generated. We used this substrate to detect GUS activity in an end point assay that is amenable for high throughput screening for inhibitors of GUS. The assay was developed with a final volume of 50 µl using the 384-well plate format to match the intended screening format. The general assay set-up included the addition of diluted GUS enzyme to the well followed by the addition of 4MUG to initiate the reaction (Fig. 1). The reaction was terminated with the addition of a 1 M Na2CO3 solution and the fluorescence measured in a plate reader. The final assay buffer consisted of 50 mM HEPES pH 7.4 and 0.01% TX-100. The Km for 4MUG was experimentally determined to be 125 μM (data not shown) and therefore this was the concentration of 4MUG used in this assay. Stopping of the reaction was confirmed by time course studies following termination of the assay (data not shown).

Bottom Line: To identify chemical inhibitors of GUS activity, we employed and validated a high throughput, fluorescence-based biochemical assay and used this assay to screen a compound library.Novel inhibitors of GUS were identified with IC(50) values ranging from 50 nM to 4.8 µM.Our results demonstrate that this high throughput assay can be used to identify small molecule inhibitors of GUS.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Sciences, Biomanufacturing Research Institute and Technology Enterprise, North Carolina Central University, 1801 Fayetteville Street, Durham, NC 27707, USA.

ABSTRACT
CPT-11 is a widely-used anti-cancer drug that is converted in vivo to its active metabolite, SN-38. In the liver, enzymes detoxify SN-38 by coupling it to a glucuronidate moiety and this inactive compound (SN-38G) is excreted into the gastrointestinal tract. In the intestine, commensal bacteria convert the SN-38G back to the active and toxic SN-38 using bacterial β-glucuronidase enzyme (GUS). This intestinal SN-38 causes debilitating diarrhea that prevents dose-intensification and efficacy in a significant fraction of patients undergoing CPT-11 treatment for cancer. This CPT-11 metabolic pathway suggests that small molecule inhibitors of GUS may have utility as novel therapeutics for prevention of dose-limiting diarrhea resulting from CPT-11 therapy. To identify chemical inhibitors of GUS activity, we employed and validated a high throughput, fluorescence-based biochemical assay and used this assay to screen a compound library. Novel inhibitors of GUS were identified with IC(50) values ranging from 50 nM to 4.8 µM. These compounds may be useful as chemical probes for use in proof-of-concept experiments designed to determine the efficacy of GUS inhibitors in altering the intestinal metabolism of drugs. Our results demonstrate that this high throughput assay can be used to identify small molecule inhibitors of GUS.

No MeSH data available.


Related in: MedlinePlus