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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

GUS assay time course. A time course study was performed under final assay conditions in the presence of 1% DMSO. Data points represent the average of three determinations per time point and error bars represent standard deviations. Data are representative of three independent experiments.
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Figure 3: GUS assay time course. A time course study was performed under final assay conditions in the presence of 1% DMSO. Data points represent the average of three determinations per time point and error bars represent standard deviations. Data are representative of three independent experiments.

Mentions: The activity in the assay was directly proportional to the concentration of GUS, up to at least 100 pM enzyme (Fig. 2). We chose to use 50 pM as the final concentration in the assay to provide a robust assay window. This concentration of enzyme resulted in a linear time course in this assay for 40 minutes under final assay conditions in the presence of 1% DMSO (Fig. 3). The enzyme titration and time course data suggested that the assay conditions should be sensitive to compounds that are competitive inhibitors of GUS activity.


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)

GUS assay time course. A time course study was performed under final assay conditions in the presence of 1% DMSO. Data points represent the average of three determinations per time point and error bars represent standard deviations. Data are representative of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: GUS assay time course. A time course study was performed under final assay conditions in the presence of 1% DMSO. Data points represent the average of three determinations per time point and error bars represent standard deviations. Data are representative of three independent experiments.
Mentions: The activity in the assay was directly proportional to the concentration of GUS, up to at least 100 pM enzyme (Fig. 2). We chose to use 50 pM as the final concentration in the assay to provide a robust assay window. This concentration of enzyme resulted in a linear time course in this assay for 40 minutes under final assay conditions in the presence of 1% DMSO (Fig. 3). The enzyme titration and time course data suggested that the assay conditions should be sensitive to compounds that are competitive inhibitors of GUS activity.

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