Inhibition of pathogenic bacterial carbonic anhydrases by monothiocarbamates

Abstract Carbonic anhydrases (CAs) from the pathogenic bacteria Nesseria gonorrhoeae and vancomycin-resistant enterococci (VRE) have recently been validated as antibacterial drug targets. Here we explored the inhibition of the α-CA from N. gonorrhoeae (α-NgCA), of α- and γ-class enzymes from Enterococcus faecium (α-EfCA and γ-EfCA) with a panel of aliphatic, heterocyclic and aryl-alkyl primary/secondary monothiocarbamates (MTCs). α-NgCA was inhibited in vitro with KIs ranging from 0.367 to 0.919 µM. The compounds inhibited the α-EfCA and γ-EfCA with KI ranges of 0.195–0.959 µM and of 0.149–1.90 µM, respectively. Some MTCs were also investigated for their inhibitory effects on the growth of clinically-relevant N. gonorrhoeae and VRE strains. No inhibitory effects on the growth of VRE were noted for all MTCs, whereas one compound (13) inhibited the growth N. gonorrhoeae strains at concentrations ranging from 16 to 64 µg/mL. This suggests that compound 13 may be a potential antibacterial agent against N. gonorrhoeae.


Introduction
The emergence of drug resistance to most antibacterials used clinically over the last decades 1-6 , coupled with the climate change which exacerbates this problem 7,8 , makes the finding of novel approaches to fight bacterial infections as well as agents with lower rates of resistance a crucial task for medicinal chemists and microbiologists 1,2,9 .Inhibition of bacterial carbonic anhydrases (CAs, EC 4.2.1.1)is one such approach, already proposed more than a decade ago 10 .CAs play crucial roles in bacterial metabolism and pH regulation [10][11][12][13][14] .Many bacterial CAs have been investigated over the last decade, and they have been validated as a bacterial target [11][12][13][14] , for several bacteria such as Neisseria gonorrhoeae 15 , vancomycin-resistant enterococci (VRE) 16 , Helicobacter pylori 17 and Vibrio cholerae 18 .Most such studies have been performed with sulphonamide CA inhibitors (CAIs), one of the most investigated class of inhibitors for these enzymes.However, other classes of CAIs have recently been investigated for their inhibitory potential against bacterial CAs, such as coumarins 19 , dithiocarbamates 20 and phenols 21 .Monothiocarbamates (MTCs) represent another class of zinc-binding CAIs 22 , which have not yet been investigated for their interaction with bacterial CAs.In this work, we report the in vitro inhibitory activity of a panel of MTCs against CAs of N. gonorrhoeae and VRE, which were reported before to be inhibited by CAIs.We do this in the search for non-sulphonamide leads which might possess antibacterial activity.

Enzymology and CA activity and inhibition measurements
CO 2 hydration activity of CAs from N. gonorrhoeae (a-NgCA) and E. faecium (a-EfCA and c-EfCA) as well as their inhibition in the presence of MTC inhibitors has been assessed by a stopped-flow method reported by Khalifah 23 .The experiments were performed at the pH of 7.4 for the a-class enzymes, and the pH of 8.3 for the c-CA, as reported earlier 15,16 .The bacterial enzymes were recombinant proteins obtained as reported earlier by our group 15,16 .

The minimum inhibitory concentrations (MICs) determination
The MICs were assessed using the broth microdilution method against clinically-relevant N. gonorrhoeae and VRE strains, as described in previous reports 15,16,[24][25][26] .Briefly, serial dilutions of the test agent were incubated with bacteria at 37 � C either aerobically or in the presence of 5% CO 2 .MICs were determined as the Table 1.Inhibition data of hCA I and II and bacterial a-NgCA, a-EfCA and c-EfCA, using AAZ as a standard drug, by a stopped-flow CO 2 hydrase assay 22  lowest concentration that completely inhibited bacterial growth as observed visually.

Results and discussion
The inhibitory effects of MTC derivatives 1-15 on three bacterial CAs isoforms, a-NgCA from N. gonorrhoeae, a-EfCA and c-EfCA from E. faecium, were investigated (Table 1).The inhibition data of these compounds against the human offtarget isoforms hCA I and II are also given in the Table , for comparison reasons.The K I values of MTCs ranged from the high nanomolar to the low micromolar ranges.The K I values of MTC compounds were in the range of 0.367-0.919mM against a-NgCA, of 0.195-0.959mM against a-EfCA, and of 0.149-1.90mM against c-EfCA.In the case of a-NgCA, MTCs 1-4 and 6-11 were poorly effective inhibitors, showing K I values in the high nanomolar range (0.630-0.919 mM), whereas the piperazine derivatives 5 and 13-15 exhibited better inhibitory effects, displaying K I values of 0.367-0.417mM.The c-EfCA was the most effectively inhibited isoform among the bacterial CAs investigated.For instance, MTCs 7 and 12-15 inhibited c-EfCA with K I values of 0.149-0.357mM, which were comparable with the K I value of the standard drug AAZ (K I ¼ 0.322 mM).Among them, compound 14 (K I ¼ 149 nM), bearing a 4-fluorophenyl tail, resulted in 2-fold better potency than the reference drug, AAZ.On the other hand, the simple piperazine-tailed MTC 6 was the weakest inhibitor of c-EfCA, with a K I value in the low micromolar range (1.90 mM).In the case of a-EfCA, the piperazine derivative 11 proved to be one of the weakest inhibitors showing a K I value of 0.914 mM, whereas MTCs 5, 12, 14 and 15 showed better inhibition constants of 290 nM, 357 nM, 195 nM and 300 nM, respectively.However, compound 14, which was superior to AAZ in inhibiting the c-EfCA, was at least 3-fold less potent than AAZ (K I ¼ 56 nM) in inhibiting the a-EfCA.Only the piperazine derivative 15 (NgCAa: K I ¼ 367 nM; EfCAa: K I ¼ 300 nM; EfCAc: K I ¼ 214 nM) demonstrated a good selectivity against the discussed bacterial isozymes over the human widely expressed hCA II isoform (hCA II: K I > 2 mM).
Next, the antibacterial activity of selected MTCs (1, 6, 9, 12 and 13) was evaluated against a panel of multidrug-resistant strains of N. gonorrhoeae and VRE.Compound 13, which has both a lipophilic trifluoromethyl-phenyl fragment and hydrophilic moieties (piperazine and monothiocarbamate functionality), displayed a modest activity against N. gonorrhoeae in addition to its inhibition of the a-NgCA.The compound inhibited N. gonorrhoeae strains with MIC values ranging between 16 and 64 mg/mL under ambient air conditions, while showing limited activity in the presence of 5% CO 2 (Table 2).This suggests that the antigonococcal activity of 13 could be mediated by CA inhibition.Although other compounds, such as 12, 14 and 15 showed similar in vitro a-NgCA inhibitory properties to 13, only the last compound was antibacterial in vivo, presumably due to its enhanced lipophilicity due to the presence of the trifluoromethyl moiety.All the tested MTCs were inactive against the VRE strains tested (MICs >64 mg/mL) (Table 3).
Since the DTCs investigated earlier 20 showed an intermediate behaviour between the ineffective MTCs and the quite effective sulphonamides 15,16 , we can speculate that the highly hydrophilic nature of the MTCs may interfere with their uptake by the bacterial cells.Thus, effective compounds targeting bacterial CAs should not only be lipophilic enough to cross the bacterial cell wall or traverse the water-filled porins, but also they should incorporate zinc-binding groups that allow a potent coordination to the active site metal ion, which is crucial both for catalysis and inhibition of these enzymes 27 .

Conclusion
We report here the first inhibition study of bacterial CAs with MTCs, a class of CAIs that is less investigated as compared to the most well-known classes of CAIs, sulphonamides and their isosteres.CAs of N. gonorrhoeae (a-NgCA) and E. faecium (a-EfCA and c-EfCA) were inhibited by the panel of 15 MTCs with K I s in the medium-high nanomolar range, with some compounds showing similar activity to the clinically used sulphonamide CAI, acetazolamide.However, the activity of these compounds in inhibiting the growth of these bacteria in vitro was lower as compared to the sufonamides.Thus, the MTCs seem to be less effective as bacterial CAIs, but the investigated series of compounds is too small for concluding that such compounds should not be investigated in the future.

Disclosure statement
CT Supuran is Editor-in-Chief of the Journal of Enzyme Inhibition and Medicinal Chemistry.He was not involved in the assessment, peer review, or decision-making process of this paper.The authors have no relevant affiliations of financial involvement with any organisation or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript.This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. .

Table 1 .
Continued.Mean from 3 different assays, by a stopped-flow technique (errors were in the range of ± 5-10% of the reported values). a