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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

Screen of the Prestwick collection with the GUS assay. Each point represents a compound. The percent inhibition values were calculated relative to controls on the plates.
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Figure 7: Screen of the Prestwick collection with the GUS assay. Each point represents a compound. The percent inhibition values were calculated relative to controls on the plates.

Mentions: As part of assay validation, a small collection of 1,120 compounds purchased from the Prestwick Chemical company was screened to assess the performance of the assay in the presence of diverse compounds using the HTS protocol (Fig. 7). The Prestwick collection of compounds was screened at 10 μM compound concentration. For this assay, the inhibitor cut-off was defined as 50% inhibition based on plate controls. Due to solubility limitation of inhibitor, we obtained the min controls on screening plates by leaving the enzyme out of these wells (buffer alone was added). The fluorescence obtained by leaving out the enzyme was the same as that of completely inhibited enzyme (data not shown). We obtained 40 actives (actives were defined as those compounds demonstrating ≥50% inhibition) from this test set screening resulting in a hit rate of 3.6%. The Z’-factors of the controls for each of the four plates were all ≥0.8.


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)

Screen of the Prestwick collection with the GUS assay. Each point represents a compound. The percent inhibition values were calculated relative to controls on the plates.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: Screen of the Prestwick collection with the GUS assay. Each point represents a compound. The percent inhibition values were calculated relative to controls on the plates.
Mentions: As part of assay validation, a small collection of 1,120 compounds purchased from the Prestwick Chemical company was screened to assess the performance of the assay in the presence of diverse compounds using the HTS protocol (Fig. 7). The Prestwick collection of compounds was screened at 10 μM compound concentration. For this assay, the inhibitor cut-off was defined as 50% inhibition based on plate controls. Due to solubility limitation of inhibitor, we obtained the min controls on screening plates by leaving the enzyme out of these wells (buffer alone was added). The fluorescence obtained by leaving out the enzyme was the same as that of completely inhibited enzyme (data not shown). We obtained 40 actives (actives were defined as those compounds demonstrating ≥50% inhibition) from this test set screening resulting in a hit rate of 3.6%. The Z’-factors of the controls for each of the four plates were all ≥0.8.

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