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. 2023 Mar 21;14(4):487–492. doi: 10.1021/acsmedchemlett.3c00032

Evaluation of 1,3,4-Thiadiazole Carbonic Anhydrase Inhibitors for Gut Decolonization of Vancomycin-Resistant Enterococci

Nader S Abutaleb †,‡, Annadka Shrinidhi §, Aloka B Bandara †,‡, Mohamed N Seleem †,‡,*, Daniel P Flaherty §,*
PMCID: PMC10108396  PMID: 37077393

Abstract

graphic file with name ml3c00032_0005.jpg

Vancomycin-resistant enterococci (VRE), Enterococcus faecium and Enterococcus faecalis, are high-priority drug-resistant pathogens in need of new therapeutic approaches. VRE originate in the gastrointestinal tract of carriers and can lead to more problematic downstream infections in the healthcare setting. Having a carrier of VRE admitted into a healthcare setting increases the risk to other patients for acquiring an infection. One strategy to eliminate the downstream infections is decolonization of VRE from carriers. Here, we report the activity of a set of carbonic anhydrase inhibitors in the in vivo VRE gastrointestinal decolonization mouse model. The molecules encompass a range of antimicrobial potency and intestinal permeability, and these factors were shown to influence the in vivo efficacy for VRE gut decolonization. Overall, carbonic anhydrase inhibitors exhibited superior VRE decolonization efficacy compared to the current drug of choice, linezolid.

Keywords: carbonic anhydrase inhibitors, vancomycin-resistant enterococci, antibiotics, gastrointestinal decolonization, in vivo antimicrobial efficacy

Enterococci are common commensals, albeit in a small percentage, within the human gastrointestinal tract (GIT) microbiota.1 These bacteria can acquire resistance genes that reduce susceptibility to common antibiotics, the most problematic being vancomycin-resistant enterococci (VRE) which comprise the two species Enterococcus faecium and Enterococcus faecalis. Exposure to broad-spectrum antibiotics can lead to dysbiosis of the microbiome, allowing VRE to grow and become a dominant species in the GIT up to 2 months post-treatment.2 Due to the rise of drug resistance and VRE being a leading cause of healthcare-associated infections,3,4 the Centers for Disease Control and Prevention (CDC) has designated VRE as a serious threat to public health.5 Currently, the only U.S. Food and Drug Administration (FDA)-approved antibiotic for VRE infections is linezolid.6,7 However, linezolid has well-known serious side effects such as peripheral neuropathy8 and myelosuppression9 due to off-target impairment of mitochondrial protein synthesis in patients.10 Additionally, linezolid treatment in VRE bloodstream infections is unsatisfactory and resulted in 30% mortality in addition to its limited activity in VRE gut decolonization.6 Consequently, there is a clear need for alternative therapeutic options.

VRE colonization is the most important risk factor for patients that acquire more problematic downstream VRE infections, such as urinary tract and bloodstream infections and endocarditis.2 In addition, nosocomial transmission of VRE from one colonized patient to others is common and could lead to these serious downstream infections.11 It was reported that just having a VRE carrier admitted to a healthcare facility is a strong predictor of transmission to others within that same facility.12,13 Since VRE GIT colonization is the origination point and risk factor for all other downstream VRE infections, one possible preventative strategy to mitigate these infections is to decolonize carriers of VRE.14 Previous attempts to utilize this strategy with the non-absorbable antibiotic ramoplanin have proven effective in a clinical setting15 but still can result in dysbiosis of the GIT microbiome due to the broad-spectrum Gram-positive antibacterial activity.16 Ideally, a molecule to be used for this purpose should be potent against VRE, have little intestinal absorption to increase residence time in GIT, and possess a narrow spectrum of activity to spare the normal gut microbiota.

Previously, our group demonstrated the ability of the carbonic anhydrase inhibitor (CAI), acetazolamide (AZM, Figure 1), to outperform the drug of choice, linezolid, in reducing E. faecium bioburden in a VRE colonization reduction mouse model.17 Additionally, we reported optimized AZM derivatives with potent anti-enterococcal properties against a panel of E. faecium and E. faecalis isolates.18 During this optimization our strategy was to develop two potential lead scaffolds: 1) with improved intestinal absorption properties to treat systemic infection and 2) with reduced absorption to increase GIT residence time for decolonization. From these studies we subsequently reported the in vitro inhibition for this scaffold against bacterial carbonic anhydrases from VRE and Neisseria gonorrhoeae as well as human carbonic anhydrases (hCAs) I and II.19,20

Figure 1.

Figure 1

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Structures and MICs of anti-enterococcal carbonic anhydrase inhibitors used in this study first reported in Kaur et al.18 MICs reported against E. faecium HM-952.

For this study, we selected three leads from our previous report that represent each type of scaffold: CAI0019, CA10028, and CAI0031 (Figure 1). CAI0019 and CA10028 are the most potent for anti-enterococcal activity (MIC = 0.03 μg/mL). Using human colon carcinoma (Caco-2) permeability as an in vitro surrogate to estimate intestinal epithelial permeability,21 CAI0019 displayed a higher rate of permeability compared to AZM.18 Alternatively, CAI0031 is equipotent compared to AZM and linezolid but exhibited much reduced Caco-2 permeability, which was below the limit of quantitation.18 Due to the varying potencies and in vitro absorption properties, these analogs were ideal candidates to evaluate for VRE GIT decolonization. Here, we report the in vivo efficacy for the three leads in the VRE GIT decolonization mouse model as compared to AZM and linezolid as well as discuss the influence of antimicrobial potency paired with Caco-2 permeability on the in vivo results.

The molecules were assessed for their efficacy in reducing the VRE bioburden in a GIT decolonization mouse model (Figure 2) as described previously.17,22−25 The animal experiment was conducted following protocols approved under guidelines from the Virginia Tech Animal Care and Use Committee. Briefly, C57BL/6 mice microbiomes were sensitized by addition of ampicillin to the drinking water (0.5 g/L) for 7 days, to simulate microbiota depletion that results in dysbiosis of the GIT in the clinical setting. After 7 days mice were inoculated with 5.39 × 108 CFU/mL of E. faecium HM-952 via oral gavage. The pathogen was then allowed an additional 7 days to establish infection, at which point the mice were randomly allocated into six groups (n = 7/group). One group was treated with the vehicle (dimethylacetamide:20% propylene glycol:40% polyethylene glycol 400:30% PBS) as the negative control, while the other groups were treated with each molecule or AZM at a dose of 20 mg/kg by oral gavage quaque die (q.d.) for eight consecutive days. One group was treated with linezolid as a control antibiotic. The E. faecium HM-952 bioburden was quantified in the fecal pellets at day 0 (prior to treatment) as well as on days 3, 5, and 7 after start of treatment. At day 9, mice were euthanized and the E. faecium bioburdens were quantified from the cecal and ileal tissues.

Figure 2.

Figure 2

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VRE GIT decolonization model. Mice were sensitized with ampicillin for 7 days followed by inoculation with E. faecium HM-952 by oral gavage. A week later, mice were dosed with each molecule (20 mg/kg) by oral gavage once daily for 8 days. E. faecium bioburden was quantified was quantified in fecal pellets at days 0 (prior to treatment), 3, 5, and 7. Bioburden was also quantified in the intestinal tissues at day 9.

The results indicate that mice treated with CAI molecules showed significantly reduced E. faecium fecal bioburden as compared to both the vehicle and linezolid control groups (Figure 3 and tabular results in Table S1). On day 3, AZM and each CAI analog showed at least 1-log10 CFU (∼90%) reduction as compared to the vehicle control, while linezolid exhibited little change. The burden of VRE continued to significantly decrease with CAI molecules treatment, resulting in a CFU reduction ranging between 1.13- and 2.18-log10 (92.6–99.3%), with AZM providing the largest decrease in VRE bioburden, after 5 days of treatment. Linezolid still lagged with 0.87-log10 (86.5%) reduction. Day 7 samples showed even further decolonization for AZM and the CAI analogs ranging from 2.16- to 2.81-log10 (99.3–99.8%) reduction of E. faecium load in the fecal pellets. The results of bacterial burden in the fecal pellets indicated CAI0031 and AZM were the most effective in reducing VRE bioburden, resulting in nearly a 3-log10 reduction, followed by CAI0028 and CAI0019. For comparison, linezolid exhibited a 1.07-log10 (91.5%) reduction of E. faecium bioburden on day 7 in the fecal pellets (tabular data presented in Table S1).

Figure 3.

Figure 3

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E. faecium HM-952 bioburden in fecal contents of colonized mice dosed with each molecule at 20 mg/kg p.o. q.d. for 8 days. The CFU data were analyzed via a two-way ANOVA with post hoc Dunnett’s test for multiple comparisons. **** indicates significant difference (P < 0.0001) of bioburden in mice treated with compounds compared to vehicle control. ^ indicates significant difference (P < 0.05) of bioburden in mice treated with CAI0019 compared to linezolid-treated mice at day 5. # indicates significant difference (P < 0.001) of bioburden in mice treated with the respective CAI compared to mice treated with linezolid at the same time point.

At day 9, mice were euthanized and VRE bioburden was quantified from the ileum (small intestine) and cecum (junction between small and large intestine) (Figure 4). The VRE bioburden reduction trends were similar to those of the fecal samples except for linezolid, which displayed reduced efficacy for tissue bioburden reduction compared to the fecal sample experiments. For example, CAI0019 displayed 1.13–1.57-log10 (94.1–97.3%) reduced E. faecium CFU in the ileum and cecum, respectively, while CAI0028 exhibited 1.98–2.36-log10 (98.9–99.6%) reduction in the same tissues. CAI0031 reduced VRE burden by 1.62–1.92-log10 (97.6–98.8%) CFU in the ileum and cecum, respectively (Figure 4 and Table S1).

Figure 4.

Figure 4

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Molecules GIT decolonization of E. faecium HM-952 in ileum (A) and cecum (B) after mice were dosed with each molecule at 20 mg/kg p.o. q.d. for 8 days. The CFU data were analyzed via a one-way ANOVA with post hoc Dunnett’s test for multiple comparisons. For statistical comparisons, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Our previous report18 provided the solubility and in vitro ADME properties for CAI0019 and CAI0031 but not for CAI0028. Thus, to fill in these gaps, and to better evaluate the in vivo efficacy results, we determined the solubility and Caco-2 permeability for CAI0028, as well as the experimental LogD for all AZM analogs. All values from our previous manuscript and those newly determined are provided in Table 1. Solubility was assessed in phosphate-buffered saline (PBS) at pH 7.4 (the pH value of the blood). CAI0019 had the lowest measured solubility at 33 μM, followed by CAI0031 with 127 μM solubility and CAI0028 at >200 μM solubility (200 μM was the highest concentration on the calibration curve). By comparison, AZM has a reported solubility of 3,200 μM26 and that of linezolid is 8,800 μM.27 LogD values were determined at two different pH values, 7.4 and 6.5 (the pH values of the blood and intestinal compartments, respectively).28 As expected, the alkyl tails of CAI0019 and CAI0028 contribute to greater lipophilicity compared to AZM at each pH. Alternatively, the morpholine moiety on CAI0031 made the molecule more polar than the previous two analogs and AZM (Table 1).

Table 1. In Vitro Solubility, LogD, and Caco-2 Permeability Properties of the Analogs.

        Caco-2 Pappb
  Sola LogD7.4 LogD6.5 A→B . ERg
AZM 3,200c –0.52 –0.27 0.19 0.77 4.05
Lin 8,800d 1.12e – 6.9f 9.0f 1.30
CAI0019 33 2.26 2.47 6.5h 14.1h 2.17
CAI0028 >200 1.36 1.55 3.7 8.2 2.22
CAI0031 127 –1.38 –0.97 <0.09h <0.09h n.a.
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a

Sol = solubility (in μM) in PBS at pH 7.4.

b

Caco-2 Papp = apparent permeability (×10–6 cm/s). A = apical side, B = basolateral side.

c

From Merck Index.26

d

From FDA review for linezolid.27

e

From Zyvox Safety Data Sheet.29

f

From Nti-Addae et al.30

g

ER is the efflux ratio = B → A/A → B. n.a. = not applicable because Papp values were below the limit of quantitation.

h

Papp values previously reported by Kaur et al.18

While there are many factors that may contribute to the in vivo efficacy, such as solubility in the GIT and non-specific protein binding in the GIT (i.e., hCAs), we sought to evaluate the in vivo efficacy results in the context of the data available for the molecules in terms of the in vitro potency and/or Caco-2 permeability. While linezolid displayed reduced E. faecium bioburden in the fecal pellets, there was no significant difference between the VRE counts in linezolid-treated mice as compared to the vehicle control group (Figure 4 and Table S1), in agreement with previous reports.17,31,32 This lack of efficacy is hypothesized to be related to the rapid absorption from the GIT, as linezolid is well absorbed, with Caco-2 Papp values of 6.9 × 10–6 cm/s from the apical to basolateral direction and 9.0 × 10–6 cm/s from the basolateral to apical direction30 (Table 1) resulting in low concentration in the stool.32 Another possible explanation may be the limited activity against high inoculum (108 CFU) of VRE isolates.31 In this case, linezolid exhibited an MIC of 1 μg/mL against E. faecium HM-952 and was the most rapidly absorbed across Caco-2, two factors that may have combined to result in low efficacy in intestinal bioburden reduction.

Analog CAI0019 is 33-fold more potent (MIC = 0.03 μg/mL) than linezolid and is comparable in terms of Papp from the apical to basolateral direction (Papp = 6.5 × 10–6 cm/s) to linezolid, with a bit of efflux in the opposite direction (Table 1) that would, in theory, increase molecules in the GIT. Taking into account these factors, it is likely that CAI0019 and linezolid may have similar GIT residence times post oral dose. Consequently, the improved efficacy for CAI0019 to reduce E. faecium bioburden compared to linezolid could be likely driven by the superior potency against E. faecium.

CAI0028 is equipotent to CAI0019 against E. faecium HM-952 but exhibits a Caco-2 Papp 1.75-fold slower than that of CAI0019 (Papp = 3.7 × 10–6 cm/s from the apical to basolateral direction). Using the same assumptions, CAI0028 would be predicted to maintain increased GIT residence time compared to both CAI0019 and linezolid. Interestingly, CAI0028 exhibited improved decolonization efficacy, with 2–2.36-log10 (99–99.6%) reduction of E. faecium in the two intestinal tissues compared to the vehicle control. This also accounted for 0.79-log10 CFU improvement over CAI0019 (Figure 4, Table S1). Consequently, given that the molecules display similar activity against the VRE strain tested, the improvement in the decolonizing activity over CAI0019 may be attributed to the decrease in Caco-2 permeability.

CAI0031 exhibited superior efficacy in GIT decolonization of the intestinal tissues, with 1.62–1.92-log10 (97.6–98.8%) reduction as compared to linezolid. Although CAI0031 is equipotent to linezolid against the strain tested, the design of the molecule was meant to yield reduced intestinal permeability. This resulted in CAI0031 being the least permeable among the three molecules in the Caco-2 assay, which may be the contributing factor driving the efficacy compared to linezolid.

AZM was equipotent to both CAI0031 and linezolid, displayed slightly increased Caco-2 permeability compared to CAI0031, and was shown to have the largest effect in VRE GIT decolonization, with 2.59–2.72-log10 (99.7–99.8%) reduction in the intestinal tissue compared to the vehicle cohort. Even though AZM is more effective than CAI0031, it could be argued that CAI0031 may be more beneficial to a patient in need of decolonization due to the potential to dose at higher concentrations and frequency because of the reduced intestinal absorption. This would allow patients to avoid the systemic effects commonly associated with AZM treatment such as diuretic effects or tinnitus.33

Finally, of note for permeability, and likely relevant to the in vivo efficacy, there were observed differences in molecule recovery from the Caco-2 assay between the analogs. For CAI0019, 35–54% was recovered in the assay, depending on the direction of the diffusion, while for CAI0028, 80–88% was recovered. This indicates that CAI0019 may 1) non-selectively bind to the cells and/or 2) become sequestered within the cells. Caco-2 cells have high endogenous expression of hCA XIII,34 which may bind to and limit CAI0019’s diffusion. This is a phenomenon that has been previously described with AZM as it partitions into erythrocytes and is sequestered in these cells over the plasma fraction by a ratio of 4:1 due to binding to hCA I and II.35,36 It is also important to note that this phenomenon would theoretically be relevant to oral dosing when molecules are translated to the in vivo mouse model, as normal mouse GIT expresses high levels of mouse CA I, CA III, and CA XIII.37 We have previously reported that these analogs bind to and inhibit hCA I and hCA II in the low nanomolar range,20 although inhibition against hCA XIII and III has not been determined.

In conclusion, we follow-up our previous optimization of CAIs against enterococci with reporting the in vivo efficacy in a mouse model of GIT decolonization of VRE. The previous analog design aimed at improvement of the anti-enterococcal activity, and selection focused on evaluation of each molecule’s Caco-2 permeability properties. The data suggest that, among similarly potent molecules, the improved VRE decolonization may be attributed to reduced GIT absorption, as quantified by the Caco-2 permeability assay. Moreover, among molecules with similar Caco-2 absorption profiles, the in vivo efficacy was likely influenced by the anti-enterococcal potency. The promising in vivo results support the hypothesis that optimization to increase GIT residence time and antibacterial potency may improve the in vivo efficacy. However, a caveat should be mentioned that antimicrobial potency and intestinal absorption are only two of the many variables that may influence the VRE decolonization efficacy. Further research is being pursued to improve the antibacterial potency of the scaffold while maintaining low Caco-2 permeability and to assess for potential adverse effects on the GIT and on the microbiome. Nonetheless, these studies represent an advancement in the anti-enterococcal drug discovery and in the GIT decolonization as a viable strategy to treat patients afflicted by VRE.

Acknowledgments

We thank BEI Resources for providing the clinical isolates utilized in this work. Figure 2 was created using Biorender.com.

Glossary

Abbreviations

AZM

acetazolamide

Lin

linezolid

VRE

vancomycin-resistant Enterococcus

hCA

human carbonic anhydrase

CA

carbonic anhydrase

CAI

carbonic anhydrase inhibitor

GIT

gastrointestinal tract

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.3c00032.

  • Tabular VRE CFU data for fecal pellets and intestinal tissue, and experimental methods (PDF)

This work was funded by the Purdue College of Pharmacy (D.P.F), the Purdue Institute for Drug Discovery (D.P.F., M.N.S), and the National Institute for Allergy and Infectious Diseases (Grants 5R01AI148523 and 1R01AI153264, D.P.F. and M.N.S.).

The authors declare no competing financial interest.

Supplementary Material

ml3c00032_si_001.pdf (184.4KB, pdf)

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