Non-steroidal anti-inflammatory drugs (NSAIDs) are one of the most frequently used classes of medications in the world. Unfortunately, NSAIDs induce an enteropathy associated with high morbidity and mortality. Although the pathophysiology of this condition involves the interaction of the gut epithelium, microbiota, and NSAIDs, the precise mechanisms by which microbiota influence NSAID enteropathy are unclear. One possible mechanism is that the microbiota may attenuate the severity of disease by specific metabolite-mediated regulation of host inflammation and injury. The microbiota-derived tryptophan-metabolite indole is abundant in the healthy mammalian gut and positively influences intestinal health. We thus examined the effects of indole administration on NSAID enteropathy. Mice (n D 5 per group) were treated once daily for 7 days with an NSAID (indomethacin; 5 mg/kg), indole (20 mg/kg), indomethacin plus indole, or vehicle only (control). Outcomes compared among groups included: microscopic pathology; fecal calprotectin concentration; proportion of neutrophils in the spleen and mesenteric lymph nodes; fecal microbiota composition and diversity; small intestinal mucosal transcriptome; and, fecal tryptophan metabolites. Co-administration of indole with indomethacin: significantly reduced mucosal pathology scores, fecal calprotectin concentrations, and neutrophilic infiltration of the spleen and mesenteric lymph nodes induced by indomethacin; modulated NSAID-induced perturbation of the microbiota, fecal metabolites, and inferred metagenome; and, abrogated a pro-inflammatory gene expression profile in the small intestinal mucosa induced by indomethacin. The microbiota-derived metabolite indole attenuated multiple deleterious effects of NSAID enteropathy, including modulating inflammation mediated by innate immune responses and altering indomethacininduced shift of the microbiota.

Intestinal microbial dysbiosis contributes to the dysmetabolism of luminal factors, including steroid hormones (sterones) that affect the development of chronic gastrointestinal inflammation and the incidence of sterone-responsive cancers of the breast, prostate, and colon. Little is known, however, about the role of specific host sterone nucleoreceptors, including estrogen receptor ␤ (ER␤), in microbiota maintenance. Herein, we test the hypothesis that ER␤ status affects microbiota composition and determine if such compositionally distinct microbiota respond differently to changes in diet complexity that favor Proteobacteria enrichment. To this end, conventionally raised female ER␤ ؉/؉ and ER␤ ؊/؊ C57BL/6J mice (mean age of 27 weeks) were initially reared on 8604, a complex diet containing estrogenic isoflavones, and then fed AIN-76, an isoflavone-free semisynthetic diet, for 2 weeks. 16S rRNA gene surveys revealed that the fecal microbiota of 8604-fed mice and AIN-76-fed mice differed, as expected. The relative diversity of Proteobacteria, especially the Alphaproteobacteria and Gammaproteobacteria, increased significantly following the transition to AIN-76. Distinct patterns for beneficial Lactobacillales were exclusive to and highly abundant among 8604-fed mice, whereas several Proteobacteria were exclusive to AIN-76-fed mice. Interestingly, representative orders of the phyla Proteobacteria, Bacteroidetes, and Firmicutes, including the Lactobacillales, also differed as a function of murine ER␤ status. Overall, these interactions suggest that sterone nucleoreceptor status and diet complexity may play important roles in microbiota maintenance. Furthermore, we envision that this model for gastrointestinal dysbiosis may be used to identify novel probiotics, prebiotics, nutritional strategies, and pharmaceuticals for the prevention and resolution of Proteobacteria-rich dysbiosis.

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Inflammatory bowel disease is a complex collection of disorders. Microbial dysbiosis as well as exposure to toxins including xenoestrogens are thought to be risk factors for inflammatory bowel disease development and relapse. Bisphenol-A has been shown to exert estrogenic activity in the colon and alter intestinal function, but the role that xenoestrogens, such as bisphenol-A , play in colonic inflammation has been previously described but with conflicting results. We investigated the ability of bisphenol-A to exacerbate colonic inflammation and alter microbiota metabolites derived from aromatic amino acids in an acute dextran sulfate sodium-induced colitis model. Female C57BL/6 mice were ovariectomized and exposed to bisphenol-A daily for 15 days. Disease activity measures include body weight, fecal consistency, and rectal bleeding. Colons were scored for inflammation, injury, and nodularity. Alterations in the levels of microbiota metabolites derived from aromatic amino acids known to reflect phenotypic changes in the gut microbiome were analyzed. Bisphenol-A exposure increased mortality and worsened disease activity as well as inflammation and nodularity scores in the middle colon region following dextran sulfate sodium exposure. Unique patterns of metabolites were associated with bisphenol-A consumption. Regardless of dextran sulfate sodium treatment, bisphenol-A reduced levels of tryptophan and several metabolites associated with decreased inflammation in the colon. This is the first study to show that bisphenol-A treatment alone can reduce microbiota metabolites derived from aromatic amino acids in the colon which may be associated with increased colonic inflammation and inflammatory bowel disease. Impact statement As rates of inflammatory bowel disease rise, discovery of the mechanisms related to the development of these conditions is important. Environmental exposure is hypothesized to play a role in etiology of the disease, as are alterations in the gut microbiome and the metabolites they produce. This study is the first to show that bisphenol-A alone alters tryptophan and microbiota metabolites derived from aromatic amino acids in a manner consistent with autoimmune diseases, specifically inflammatory bowel diseases, regardless of dextran sulfate sodium treatment. These findings indicate a potential mechanism by which bisphenol-A negatively affects gut physiology to exacerbate inflammation.

The interplay between microbiota and host metabolism plays an important role in health. Here, we examined the relationship between age, gut microbiome and host serum metabolites in male C57BL/6J mice. Fecal microbiome analysis of 3, 6, 18, and 28 months (M) old mice showed that the Firmicutes / Bacteroidetes ratio was highest in the 6M group; the decrease of Firmicutes in the older age groups suggests a reduced capacity of gut microflora to harvest energy from food. We found age-dependent increase in Proteobacteria , which may lead to altered mucus structure more susceptible to bacteria penetration and ultimately increased intestinal inflammation. Metabolomic profiling of polar serum metabolites at fed state in 3, 12, 18 and 28M mice revealed age-associated changes in metabolic cascades involved in tryptophan, purine, amino acids, and nicotinamide metabolism. Correlation analyses showed that nicotinamide decreased with age, while allantoin and guanosine, metabolites in purine metabolism, increased with age. Notably, tryptophan and its microbially derived compounds indole and indole-3-lactic acid significantly decreased with age, while kynurenine increased with age. Together, these results suggest a significant interplay between bacterial and host metabolism, and gut dysbiosis and altered microbial metabolism contribute to aging.

M. tuberculosis thymidylate kinase (Mtb TMK) has been shown in vitro to be an essential enzyme in DNA synthesis. In order to identify novel leads for Mtb TMK, we performed a high throughput biochemical screen and an NMR based fragment screen through which we discovered two novel classes of inhibitors, 3-cyanopyridones and 1,6-naphthyridin-2-ones, respectively. We describe three cyanopyridone subseries that arose during our hit to lead campaign, along with cocrystal structures of representatives with Mtb TMK. Structure aided optimization of the cyanopyridones led to single digit nanomolar inhibitors of Mtb TMK. Fragment based lead generation, augmented by crystal structures and the SAR from the cyanopyridones, enabled us to drive the potency of our 1,6-naphthyridin-2-one fragment hit from 500 μM to 200 nM while simultaneously improving the ligand efficiency. Cyanopyridone derivatives containing sulfoxides and sulfones showed cellular activity against M. tuberculosis. To the best of our knowledge, these compounds are the first reports of non-thymidine-like inhibitors of Mtb TMK.

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