Abstract 3985: Identifying and drugging glucuronidase targets in the human gut microbiome

Abstract

Abstract Background The gut microbiome is a vast, untapped source of non-bactericidal drug targets with applications in therapeutic oncology. Enteric bacteria express β-glucuronidases (GUS) that contribute to tumorigenesis, neoplastic disease and cancer treatment outcomes. We previously reported in 2010 that a microbial-targeting, drug-like small molecule (SBX-1) protects mice from irinotecan (IRI)-induced diarrhea by blocking bacterial GUS-mediated reactivation of SN-38 from the inactive glucuronide (SN-38G). SBX-1 is >100-fold more potent against E. coli GUS than human GUS. We recently reported (2017) that E. coli GUS is one of nearly 300 distinct microbial GUS enzymes (GUSome) expressed in the human gut microbiome. The objectives of this work are two-fold: (1) Define for the first time the subset of GUS enzymes that can metabolize SN-38G; (2) Evaluate the selectivity of SBX-1 against GUS enzymes that metabolize SN-38G vs those that do not. Methods We exploited differences in the fluorescence properties of SN-38G and SN-38 to develop a high-throughput, cost-efficient assay that kinetically monitors GUS-mediated cleavage of SN-38G to SN-38. SN-38G concentrations were monitored at excitation/emission wavelengths of 230/420 nm, respectively. Cleavage of SN-38G to SN-38 results in a measurable decrease in 420 nm fluorescence. Thirteen distinct bacterial GUS enzymes representative of the structural diversity in the GUSome were expressed, purified, and screened to determine relative catalysis of SN-38G in the absence or presence of SBX-1. Enzymes were tested at a single concentration (20 nM) with a single concentration of SN-38G (15 µM). Initial velocities were calculated and normalized. Results The GUS enzymes tested exhibited a broad range of SN-38G catalysis. Rank-ordered from most efficient to least efficient: Escherichia coli, Eubacterium eligens, Bacteroides fragilis, Bacteroides uniformis (Type 2), Clostridium perfringens, H11G11, Bacteroides uniformis (Type 1), Faecalibacterium prausnitzii, Bacteroides uniformis (Type 3), Lactobacillus rhamnosus, Bacteroides ovatus, Ruminococcus gnavus, and Bacteroides dorei. SBX-1 is highly potent against E. coli, weakly potent against E. eligens, and not potent against other bacterial GUS orthologues. For each GUS orthologue, catalytic efficiencies for SN-38G and Ki values for SBX-1 will be presented. Conclusions In our study, we identified the subset of GUS enzymes that metabolize SN-38G most efficiently. We evaluated the selectivity of SBX-1, showing that it is highly potent against the most efficient enzyme (E. coli GUS), but not potent against other GUS enzymes. Identification of the specific bacterial targets responsible for SN-38G reactivation will guide the design of GUS inhibitors to improve IRI outcomes. Current efforts are underway to characterize a library of novel SBX-1 analogs that show differential selectivity across the GUSome. Citation Format: Jeffrey P. Hymes, Bret D. Wallace, Matthew R. Redinbo, Ward Peterson. Identifying and drugging glucuronidase targets in the human gut microbiome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3985.