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

Syed Ahmad1, Mark A Hughes1, Kimberly T Lane2, Matthew R Redinbo2, 3, 4, Li-An Yeh1, John E Scott*, 1
1 Department of Pharmaceutical Sciences, Biomanufacturing Research Institute and Technology Enterprise, North Carolina Central University, 1801 Fayetteville Street, Durham, NC 27707, USA
2 Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA
3 Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA
4 Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA

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© Ahmad et al.; Licensee Bentham Open.

open-access license: This is an open access article licensed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited.

* Address correspondence to this author at 1801 Fayetteville St, BRITE Bldg, Rm 1019, North Carolina Central University, Durham, NC 27707, USA; Tel: 919-530-7569; Fax: 919-530-6600; E-mail:


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 IC50 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.

Keywords: β-glucuronidase, CPT-11, screen, inhibitor.