An Aniline-Substituted Bile Salt Analog Protects both Mice and Hamsters from Multiple Clostridioides difficile Strains

Abstract

Clostridioides difficile infection (CDI) is the major identifiable cause of antibiotic-associated diarrhea. The emergence of hypervirulent C. difficile strains has led to increases in both hospital- and community-acquired CDI. Furthermore, CDI relapse from hypervirulent strains can reach up to 25%. Thus, standard treatments are rendered less effective, making new methods of prevention and treatment more critical. Previously, the bile salt analog CamSA was shown to inhibit spore germination in vitro and protect mice and hamsters from C. difficile strain 630. Here, we show that CamSA was less active at preventing spore germination of other C. difficile ribotypes, including the hypervirulent strain R20291. Strain-specific in vitro germination activity of CamSA correlated with its ability to prevent CDI in mice. Additional bile salt analogs were screened for in vitro germination inhibition activity against strain R20291, and the most active compounds were tested against other strains. An aniline-substituted bile salt analog, (CaPA), was found to be a better anti-germinant than CamSA against eight different C. difficile strains. In addition, CaPA was capable of reducing, delaying, or preventing murine CDI signs in all strains tested. CaPA-treated mice showed no obvious toxicity and showed minor effects on their gut microbiome. CaPA’s efficacy was further confirmed by its ability to prevent CDI in hamsters infected with strain 630. These data suggest that C. difficile spores respond to germination inhibitors in a strain-dependent manner. However, careful screening can identify anti-germinants with broad CDI prophylaxis activity.

The Design, Synthesis, and Inhibition of Clostridioides difficile Spore Germination by Acyclic and Bicyclic Tertiary Amide Analogs of Cholate

“…This demonstrates that a secondary amide was not necessary for potent antigerminant activity, and that the NH was not required. Unfortunately, 5a was not active in preventing CDI in a mouse model of infection 21 . Increasing the ring size to the quinoline (5h) resulted in a 22-fold loss of activity (IC50 = 22 µM), indicating that either the compound was too large for the binding site or the decrease in coplanarity of the aromatic ring, due to the larger ring, was detrimental to binding.…”

“…These results indicate that this orientation should be preserved in future compound designs to enhance potency of antigerminants. Unfortunately, the unsubstituted indoline compound 5a did not prevent CDI in a mouse model 21 . The exact reason for this is unknown, but we speculate that 5a is rapidly metabolized in the gut via amidases or dehydroxylation to render the compound inactive.…”

“…Given the connection between spore germination and bile salts, we and others have shown that bile salt analogs can inhibit germination and prevent the development of C. difficile colitis 12,[15][16][17][18][19][20][21][22] . Several natural bile salts inhibit germination in vitro; however, their use to treat patients has not been successful 19 .…”

“…CamSA, however, displayed poor activity against hypervirulent strains. Additional investigation revealed that the simple phenyl substitution, CaPA (3b, Figure 1) gave potent inhibition (IC50 = 1.8 µM) against a range of C. difficile strains and could prevent C. difficile infections in mouse and hamster models 15,21 . We have further shown that the cholic acid nucleus gives the most potent inhibition of germination; however, the CDCA moiety also inhibits but with less potency 22 .…”

Mechanism of germination inhibition ofClostridioides difficilespores by an aniline substituted cholate derivative (CaPA)

“…We have developed cholates containing m-sulfanilic acid (CamSA), or aniline (CaPA) sidechains that inhibit C. difficile spore germination at micromolar concentrations even when the taurocholate germinant is present at saturating millimolar levels [11,[33][34][35][36]. As our first CDI prophylactics, CamSA and CaPA have been extensively characterized in vitro and in vivo [27,[33][34][35][36][37][38].…”