Diverse but desolate landscape of gut microbial azoreductases: A rationale for idiopathic IBD drug response.

Prodrugs reliant on microbial activation are widely used but exhibit a range of efficacies that remain poorly understood. The anti-inflammatory compound 5-aminosalicylic acid (5-ASA), which is packaged in a variety of azo-linked prodrugs provided to most Ulcerative Colitis (UC) patients, shows confounding inter-individual variabilities in response. Such prodrugs must be activated by azo-bond reduction to form 5-ASA, a process that has been attributed to both enzymatic and non-enzymatic catalysis. Gut microbial azoreductases (AzoRs) are the first catalysts shown to activate azo-linked drugs and to metabolize toxic azo-chemicals. Here, we chart the scope of the structural and functional diversity of AzoRs in health and in patients with the inflammatory bowel diseases (IBDs) UC and Crohn's Disease (CD). Using structural metagenomics, we define the landscape of gut microbial AzoRs in 413 healthy donor and 1059 IBD patient fecal samples. Firmicutes encode a significantly higher number of unique AzoRs compared to other phyla. However, structural and biochemical analyses of distinct AzoRs from the human microbiome reveal significant differences between prevalent orthologs in the processing of toxic azo-dyes, and their generally poor activation of IBD prodrugs. Furthermore, while individuals with IBD show higher abundances of AzoR-encoding gut microbial taxa than healthy controls, the overall abundance of AzoR-encoding microbes is markedly low in both disease and health. Together, these results establish that gut microbial AzoRs are functionally diverse but sparse in both health and disease, factors that may contribute to non-optimal processing of azo-linked prodrugs and idiopathic IBD drug responses.

Metagenomics combined with activity-based proteomics point to gut bacterial enzymes that reactivate mycophenolate.

Mycophenolate mofetil (MMF) is an important immunosuppressant prodrug prescribed to prevent organ transplant rejection and to treat autoimmune diseases. MMF usage, however, is limited by severe gastrointestinal toxicity that is observed in approximately 45% of MMF recipients. The active form of the drug, mycophenolic acid (MPA), undergoes extensive enterohepatic recirculation by bacterial ß-glucuronidase (GUS) enzymes, which reactivate MPA from mycophenolate glucuronide (MPAG) within the gastrointestinal tract. GUS enzymes demonstrate distinct substrate preferences based on their structural features, and gut microbial GUS enzymes that reactivate MPA have not been identified. Here, we compare the fecal microbiomes of transplant recipients receiving MMF to healthy individuals using shotgun metagenomic sequencing. We find that neither microbial composition nor the presence of specific structural classes of GUS genes are sufficient to explain the differences in MPA reactivation measured between fecal samples from the two cohorts. We next employed a GUS-specific activity-based chemical probe and targeted metaproteomics to identify and quantify the GUS proteins present in the human fecal samples. The identification of specific GUS enzymes was improved by using the metagenomics data collected from the fecal samples. We found that the presence of GUS enzymes that bind the flavin mononucleotide (FMN) is significantly correlated with efficient MPA reactivation. Furthermore, structural analysis identified motifs unique to these FMN-binding GUS enzymes that provide molecular support for their ability to process this drug glucuronide. These results indicate that FMN-binding GUS enzymes may be responsible for reactivation of MPA and could be a driving force behind MPA-induced GI toxicity.