Shiv Sharma

We previously reported that two bile acid (BA) analogs, CamSA and CA-Quin, demonstrate potent anti-germination activity against Clostridioides difficile (C. difficile) spores, protecting rodents from C. difficile infections. Here, we further evaluated the impact of these analogs on the hepatic transcriptome and BA homeostasis in vivo by focusing BA profiles on the liver, feces, and chyme as well as the hepatic transcriptome after a 7-day treatment. The two compounds demonstrated similar impact on BA profiles among the three samples, with significantly increased BA excretion in feces. This change is aligned with significantly altered expression of genes involved in BA homeostasis in both liver and gut tissues. Also, both compounds increased levels of deconjugated BAs in the feces, possibly suggesting increased activity of gut microbiota. Fecal levels of anti-C. difficile germination chenodeoxycholic acid and pro-germination taurocholic acid are significantly increased and decreased by the treatments, respectively. The hepatic transcriptome showed limited difference in gene expression between the three groups, suggesting a minimal adverse impact of the two compounds on liver function. Overall, our study suggests that in vivo CamSA and CA-Quin treatment demonstrated safe and significantly altered BA homeostasis that inhibits C. difficle germination.

We previously reported that two bile acid (BA) analogs CamSA and CA-Quin demonstrate potent anti-germination activity against Clostridioides difficile (C. difficile) spores, protecting rodents from C. difficile infections. Here we further evaluated the impact of these analogs on the hepatic transcriptome and BA homeostasis in vivo by focusing BA profiles on the liver, feces, and chyme as well as hepatic transcriptome after a 7-day treatment. The two compounds demonstrated similar impact on BA profiles among the three samples, with significantly increased BA excretion in feces. This change is aligned with significantly altered expression of genes involved in BA homeostasis in both liver and gut tissues. Also, both compounds increased levels of unconjugated BAs in the feces, indicating an elevated activity of gut microbiota (GM). Notably, fecal levels of anti-c. difficile germination chenodeoxycholate and pro-germination taurocholate are significantly increased and decreased by the treatments, respectively. While hepatic transcriptome did not show significant toxicity, altered genes are enriched in pathways associated with GM activity and lipid metabolism. Overall, our study suggests that in vivo CamSA and CA-Quin treatment demonstrated minimal hepatoxicity but significantly altered BA homeostasis and potentially favorable improvement for GM profiles that together inhibit C. difficle germination.

Clostridioides difficile infection (CDI) is an anaerobic bacterium that is responsible for most antibiotic-associated cases of diarrhea. CDI begins with the ingestion of an environmentally stable spore that germinates within the GI tract under specific conditions. We have shown that the bile salt analog N-phenyl-cholan-24-amide (1) can inhibit spore germination and prevent CDI in animal models of infection. Unfortunately, 1, was stable in the gut of antibiotic-treated animals but was rapidly degraded by gastrointestinal micoflora in the normal gut preventing its use to prevent CDI. We hypothesized that the source of the instability was the amide group. To explore this hypothesis, we removed the amide by either converting it to an amine or to 5,6-fused heterocycles. We found that reduction of the amide to an amine resulted in a significant loss of activity, but conversion to benzimidazole (6a), benzothiazole (6b), or benzoxazole (6c) gave compounds with good antigermination activity. Exploration of other sterane groups such as chenodeoxycholate and deoxycholate gave active compounds only for the benzimidazole series. Examination of 6b showed that it was stable for 24 hours in the presence of feces taken from healthy mice validating the hypothesis that an amide bioisostere would increase the stability of the compound. Mice treated with 6a and 6b also showed no signs of toxicity up to 300 mg/kg daily for 7 days. Each compound was tested for its ability to prevent CDI in a murine model. Compound 6b but not 6a was able to prevent CDI when given at a dose of 50 mg/kg.

Clostridioides difficile infection (CDI) is a major identifiable cause of antibiotic-associated diarrhea. In our previous study (J. Med Chem, 2018, 61, 6759-6778), we have identified N-phenyl-cholan-24-amide as a potent inhibitor of spore germination. The most potent compounds in our previous work are N-arylamides. We were interested in the role that the conformation of the amide plays in activity. Previous research has shown that secondary N-arylamides exist exclusively in the coplanar trans conformation while tertiary N-methyl-N-arylamides exist in a non-planar, cis conformation. The N-methy-N-phenyl-cholan-24-amide was 17-fold less active compared to the parent compounds suggesting the importance of the orientation of the phenyl ring. To lock the phenyl ring into a trans conformation, cyclic tertiary amides were prepared. Indoline and quinoline cholan-24-amides were both inhibitors of spore germination; however, the indoline analogs were most potent. Isoindoline and isoquinoline amides were inactive. We found that the simple indoline derivative gave an IC50 value of 1 M, while the 5-fluoro-substituted compound (5d) possessed an IC50 of 400 nM. To our knowledge, 5d is the most potent known spore germination inhibitor described to date. Taken together, our results indicate that the trans, coplanar conformation of the phenyl ring is required for potent inhibition.