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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

IC50 value determination for two screen hits. Compounds were serially diluted in 100% DMSO then transferred to assay plates for the GUS activity assay. Concentration response curves are shown for two representative hits from the screen.
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Figure 8: IC50 value determination for two screen hits. Compounds were serially diluted in 100% DMSO then transferred to assay plates for the GUS activity assay. Concentration response curves are shown for two representative hits from the screen.

Mentions: A diverse collection of 11,520 small organic molecules were purchased from Asinex Corporation and screened at 10 µM compound concentration for activity in this GUS assay. The plate Z’-factors for the screen were all ≥0.82. A total of 583 actives (≥ 50% inhibition) were obtained for an initial active rate of 5.1%. We sought to limit follow-up confirmatory IC50 determinations to 120 actives, so the active cut-off was raised to ≥ 89% inhibition to obtain the most potent actives. Of these 120 compounds tested in IC50 assays, 93 of them (77.5%) confirmed with an IC50 of <20 µM. The Hill slopes generated from such data can be used to triage hits since steep slopes, i.e. significantly greater than 1.0, can be an indication of compound aggregation which is a non-specific inhibition mechanism displayed by some compounds [32-33]. Of the 93 confirmed hits, 26 had Hill slopes between 0.8 and 1.2. The potencies of these hits ranged from 50 nM to 4.8 µM. The structures and activities of 4 of these 26 hits have already been published [29]. Here we show the structures of 8 more novel GUS inhibitors as representative examples of the hits obtained from the screen with IC50 values ranging from 1.7 to 4.8 µM (Table 1). The concentration response data used to calculate IC50 values for two compounds are provided as representative curves (Fig. 8). Examination of the structures from Table 1 indicated that 3 of these (compounds 1, 4 and 5) are structurally related to the quinolinone thiourea scaffold previously published [29]. However, compounds 2, 3 and 6 - 8 are novel and structurally unique inhibitors of GUS.


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)

IC50 value determination for two screen hits. Compounds were serially diluted in 100% DMSO then transferred to assay plates for the GUS activity assay. Concentration response curves are shown for two representative hits from the screen.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 8: IC50 value determination for two screen hits. Compounds were serially diluted in 100% DMSO then transferred to assay plates for the GUS activity assay. Concentration response curves are shown for two representative hits from the screen.
Mentions: A diverse collection of 11,520 small organic molecules were purchased from Asinex Corporation and screened at 10 µM compound concentration for activity in this GUS assay. The plate Z’-factors for the screen were all ≥0.82. A total of 583 actives (≥ 50% inhibition) were obtained for an initial active rate of 5.1%. We sought to limit follow-up confirmatory IC50 determinations to 120 actives, so the active cut-off was raised to ≥ 89% inhibition to obtain the most potent actives. Of these 120 compounds tested in IC50 assays, 93 of them (77.5%) confirmed with an IC50 of <20 µM. The Hill slopes generated from such data can be used to triage hits since steep slopes, i.e. significantly greater than 1.0, can be an indication of compound aggregation which is a non-specific inhibition mechanism displayed by some compounds [32-33]. Of the 93 confirmed hits, 26 had Hill slopes between 0.8 and 1.2. The potencies of these hits ranged from 50 nM to 4.8 µM. The structures and activities of 4 of these 26 hits have already been published [29]. Here we show the structures of 8 more novel GUS inhibitors as representative examples of the hits obtained from the screen with IC50 values ranging from 1.7 to 4.8 µM (Table 1). The concentration response data used to calculate IC50 values for two compounds are provided as representative curves (Fig. 8). Examination of the structures from Table 1 indicated that 3 of these (compounds 1, 4 and 5) are structurally related to the quinolinone thiourea scaffold previously published [29]. However, compounds 2, 3 and 6 - 8 are novel and structurally unique inhibitors of GUS.

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