Identification of a 1,2,4-Oxadiazole with Potent and Specific Activity against Clostridioides difficile, the Causative Bacterium of C. difficile Infection, an Urgent Public Health Threat

The discovery of compound 57, a new, totally synthetic 1,2,4-oxadiazole antibacterial agent, is described. This oxadiazole displays highly selective, bactericidal killing of Clostridioides (Clostridium) difficile, the bacterium that causes C. difficile infection (CDI) in both hospital and community settings. The narrow spectrum of activity exhibited by 57 should avoid any disruption of commensal anaerobic bacteria in the gut microbiome, minimizing chances for recurrent CDI.


■ INTRODUCTION
Clostridioides dif ficile, formerly known as Clostridium diff icile, is a toxin-producing, Gram-positive anaerobic bacterium that causes colitis (inflammation of the colon) and diarrhea in both hospital and community settings.C. diff icile infection (CDI) has been the most common healthcare-associated bacterial infection for many years and was identified by the Centers for Disease Control and Prevention (CDC) as an urgent public health threat in 2013, and again in 2019. 1 As might be expected, the COVID-19 pandemic caused a significant disruption in CDI surveillance data collection from 2020 onward, but the most comprehensive recent case reports from 2019 remain disturbing.It is estimated that there were 202,600 hospital cases of CDI in the United States in 2019, with 11,500 deaths. 2 The morbidity and mortality associated with CDI are largely the result of C. dif ficile's production of toxins A (TcdA) and B (TcdB), which cause the observed colitis, diarrhea, and dehydration. 3he recommended first-line therapeutic options for treating CDI include orally administered vancomycin or fidaxomicin (metronidazole is no longer recommended when vancomycin or fidaxomicin is available). 4Fortunately, C. dif f icile resistance to vancomycin and fidaxomicin is reported only rarely. 5nfortunately, all available CDI antibacterial agents suffer from three primary deficiencies: 1) varying degrees of disruption of the normal gut microbiome, 2) recurrent CDI (rCDI) in roughly 25% of patients within several months of discontinuation of therapy, and 3) a lack of activity against C. diff icile spore germination, which contributes to rCDI. 3 Recurrent CDI is associated with a significantly higher risk of downstream death.This recurrence phenomenon is a unique aspect of C. diff icile, resulting from its existence in both active (vegetative) and dormant (spore) forms.Marketed antibacterial agents only target the vegetative form of C. dif ficile, leaving the spores to eventually germinate and reactivate CDI.Because of the abovenoted limitations, there exists a real need for the identification of new treatment modalities that address some or all of these real-world shortcomings.

■ DISCUSSION
In this issue, Qian and co-workers report the identification of a new, totally synthetic antibacterial agent which, minimally, inhibits cell-wall peptidoglycan biosynthesis in C. dif f icile. 6his project had its genesis in earlier seminal work reported by the Mobashery lab wherein in silico screening led to the identification of the novel anti-staphylococcal 1,2,4-oxadiazole class of penicillin-binding protein (PBP) 2a inhibitors. 7The lead oxadiazole identified at that time, compound 1 (Figure 1), exhibited good in vitro activity against methicillin-resistant Staphylococcus aureus (MRSA), along with commensurate in vivo efficacy in murine MRSA infection models.Subsequently, Janardhanan and co-workers reported the interesting phenol derivative 2, which also exhibits good anti-staphylococcal activity. 8Fortuitously, both 1 and 2 were also found to have good in vitro activity against C. dif f icile ATCC 43255, with minimum inhibitory concentrations (MICs) of 4 and 2 μg/mL, respectively. 8After screening the team's existing library of 75 oxadiazoles for activity against C. diff icile ATCC 43255, some preliminary structure−activity relationships (SARs) were identified for this series that led to the syntheses of an additional 58 analogs.The culmination of this effort was the identification of compound 57, a potent and very selective 1,2,4-oxadiazole derivative with a MIC of 0.25 μg/mL against C. dif f icile ATCC 43255.Compound 57 is the primary focus of the current work.
Figure 2 presents a high-level summary of the oxadiazole derivatives targeted in this campaign, with compound 2 being the starting point for conceptual changes to the overall structure.The design plan looks to have been largely empirical, with a heavy focus on isosteric replacements.Both the lefthand side (LHS) and right-hand side (RHS) were systematically modified, as shown in the figure.Finally, the central 1,2,4oxadiazole core was also investigated via the incorporation of various acyclic and cyclic subunits.The targeted 1,2,4oxadiazole antibacterial agents were rapidly assembled via an efficient three-step, modular process passing through a key amidoxime intermediate. 6Condensation/cyclization to give the central oxadiazole ring system was achieved by reacting an activated carboxylic acid with the amidoxime under one of three sets of reaction conditions.
The synthesized oxadiazole analogs (133 total) were initially screened for activity against C. dif f icile ATCC 43255.Compound 57 quickly emerged as one of the most potent compounds in the oxadiazole collection (MIC = 0.25 μg/mL).
compound was also shown to be rapidly bactericidal against C. dif f icile in time−kill studies and developed resistance much more slowly than the marketed comparator vancomycin.
A scanning electron microscopy (SEM) study of vegetative C. dif f icile treated with 57 indicated significant damage to the bacterial cell wall, consistent with the expected 1,2,4-oxadiazole mode of action (MOA) involving inhibition of peptidoglycan biosynthesis.Analog 57 also exhibited an in vitro therapeutic index of >512 in a relevant cytotoxicity screen (lactate dehydrogenase assay with THP-1 human monocyte cells), suggesting good selectivity for prokaryotic organisms such as C. dif f icile.It should be noted that the aromatic nitro group of 57 is a structural alert and may eventually require further investigation into possible isosteric replacements.On the other hand, this functional group may be perfectly acceptable for an antibiotic focused on intestinal infections, where oral delivery, high fecal concentrations, and (hopefully) low oral bioavailability are typically the norm.
An evaluation of oxadiazole 57 against an expanded panel of both aerobic and anaerobic bacteria was conducted.This extensive study revealed a remarkably selective and unique spectrum of antibacterial activity for compound 57.Importantly, the lead compound exhibited very good activity against both sensitive and multi-drug-resistant (MDR) strains (n = 101) of C. diff icile, with a MIC 90 of 1 μg/mL.Oxadiazole 57 was totally inactive against aerobic Gram-positive and Gramnegative bacteria.This is in contrast to the results obtained for its progenitor oxadiazoles 1 and 2, which exhibited good activity against Gram-positive aerobes such as the staphylococci, streptococci, and enterococci.The extremely narrow spectrum of activity for oxadiazole 57, encompassing just C. dif f icile, was further supported by its total lack of activity against other common anaerobic gut bacteria.No data was provided to clarify whether 57 retains the ability to inhibit C. dif f icile spore germination, as was demonstrated both in vitro and in vivo for its predecessor, oxadiazole 2. 8 Obviously, prevention of spore germination would further help prevent rCDI.
The reasons for the unprecedented, C. dif f icile-specific activity of 57 remain unclear at this point and will require additional investigation.Certainly, 57 was shown (SEM study) to inhibit cell wall biosynthesis, suggesting that it is an inhibitor of PBP and peptidoglycan biosynthesis.Perhaps 57 expresses a unique selectivity for one of the C. dif f icile PBPs?Perhaps more likely is the possibility that oxadiazole 57 exhibits more than one MOA.Metronidazole (see Figure 3), as at least part of its MOA, relies on cellular reductases to reduce its nitro group to a nitroso radical, which then goes on to inhibit DNA synthesis and repair.This begs the question as to whether 57, which bears some structural resemblance to metronidazole, is also a substrate for these reductases and if this reactive manifold might contribute to the compound's overall antibacterial effect(s).The study under discussion did examine the hydroxyethyl-substituted congener 58, which was found to be totally inactive (MIC = > 128 μg/mL), as well as a regioisomeric N-methylimidazole analog (compound 59 in ref 6), which exhibited a weak MIC of 32 μg/mL.However, the direct oxadiazole comparator to metronidazole, regioisomer 3, was not described.Regardless, more investigation is needed to further clarify the MOA of oxadiazole 57.Some insights into the MOA of 57 might be gleaned from its study in a macromolecular synthesis assay, where the incorporation of radiolabeled precursors is monitored.This would help identify the biosynthesis pathway(s) (cell wall synthesis, protein synthesis, DNA synthesis, RNA synthesis, etc.) being inhibited by 57 in a concentration-dependent manner.In addition, does 57 also exhibit the ability to inhibit C. dif f icile spore germination, like its parent oxadiazole 2, and so reduce the likelihood of rCDI? 8 No indication of this capability was provided in the work under discussion.

■ CONCLUSION
Building on the past discovery of the totally synthetic 1,2,4oxadiazole class of antibacterial agents, which target PBP 2a in Gram-positive pathogens, the work in ref 6 reports the identification of a structurally new oxadiazole, compound 57, with remarkable selectivity for the anaerobe C. dif f icile, the causative bacterium of CDI, currently designated as the most urgent bacterial threat in United States hospitals. 1,2The importance of this pre-clinical agent should not be underestimated.Despite the availability of marketed agents such as vancomycin and, more recently, fidaxomicin, there remains an urgent need for new treatment regimens utilizing a unique MOA that will avoid target-based cross-resistance with other antibiotics.An oxadiazole exclusively targeting C. dif f icile has the potential to avoid disturbing/killing commensal gut bacteria, which contributes to C. diff icile overgrowth and rCDI.It is also hoped that compound 57, like its progenitor 2, will inhibit C. diff icile spore germination and so help break the cycle of rCDI that plagues patients suffering from this medical condition.

Figure 2 .
Figure 2. Modular strategy and targets identified for profiling as next-generation oxadiazole analogs.