Sulphonamide inhibition studies of the β-carbonic anhydrase from the bacterial pathogen Clostridium perfringens

Abstract The β-carbonic anhydrases (CAs, EC 4.2.1.1) from the pathogenic bacterium Clostridium perfringens (CpeCA) was recently characterised kinetically and for its anion inhibition profile. In the search of effective CpeCA inhibitors, possibly useful to inhibit the growth/pathogenicity of this bacterium, we report here an inhibition study of this enzyme with a panel of aromatic, heterocyclic and sugar sulphonamides/sulphamates. Some sulphonamides, such as acetazolamide, ethoxzolamide, dichlorophenamide, dorzolamide, sulthiame and 4-(2-hydroxymethyl-4-nitrophenyl-sulphonamido)ethylbenzenesulphonamide were effective CpeCA inhibitors, with KIs in the range of 37.4–71.6 nM. Zonisamide and saccharin were the least effective such inhibitors, whereas many other aromatic and heterocyclic sulphonamides were moderate – weak inhibitors with KIs ranging between 113 and 8755 nM. Thus, this study provides the basis for developing better clostridial enzyme inhibitors with potential as antiinfectives with a new mechanism of action.

Bacteria encode CAs belonging to three classes, the a-, band c-CAs 7,34-40 . These enzymes seem to be involved in crucial metabolisms, which probably explains both their wide distribution in Gram-negative and Gram-positive bacteria, as well as their generally very effective catalytic properties for the hydration of CO 2 to bicarbonate and protons [34][35][36][37][38][39][40][41][42] . Thus, ultimately, inhibition of bacterial CAs has been proposed as an alternative approach for obtaining antibiotics with an alternative mechanism of action compared to the classical drugs that interfere with bacterial cell wall biosynthesis, DNA-gyrase or similar such targets, which led to an extensive drug resistance phenomenon 7,12,34,36 .
In previous work from our groups, we have reported the cloning and characterisation of a new b-CA from the bacterial pathogen Clostridium perfringens, CpeCA [41] that has also been investigated for its interaction with anions and other small molecules known to interact with metalloenzymes such as CA 42 . We previously observed that most anions are millimolar CpeCA inhibitors, whereas sulphamate, sulphamide, phenylboronic acid and phenylarsonic acid are the most effective inhibitors, with K I s in the range of 7-75 lM. Thus, no highly effective CpeCA inhibitors were detected so far and this is the reason why we investigated the interaction of this enzyme with sulphonamides and sulphamates, the class of CAIs which usually leads to effective antimicrobial agents.

Chemistry
Compounds 1-24 and AAZ-HCT were commercially, highest purity available derivatives from Sigma-Aldrich (Milan, Italy) and were used without further purification or were prepared as reported earlier by our group 43-51 .

Carbonic anhydrase assay
An applied photophysics stopped-flow instrument has been used for assaying the CA catalysed CO 2 hydration activity [52]. Phenol red (at a concentration of 0.2 mM) has been used as indicator, working at the absorbance maximum of 557 nM, with 20 mM TRIS (pH 8.3) as buffer, and 20 mM Na 2 SO 4 (for maintaining constant the ionic strength), following the initial rates of the CA-catalysed CO 2 hydration reaction for a period of 10-100 s. The CO 2 concentrations ranged from 1.7 to 17 mM for the determination of the kinetic parameters and inhibition constants. For each inhibitor, at least six traces of the initial 5-10% of the reaction have been used for determining the initial velocity. The uncatalysed rates were determined in the same manner and subtracted from the total observed rates. Stock solutions of inhibitor (0.1 mM) were prepared in distilled-deionised water and dilutions up to 0.01 nM were done thereafter with the assay buffer. Inhibitor and enzyme solutions were pre-incubated together for 15 min at room temperature prior to assay, in order to allow for  the formation of the E-I complex. The inhibition constants were obtained by non-linear least-squares methods using PRISM 3 and the Cheng-Prusoff equation, as reported earlier [23][24][25][26][27] and represent the mean from at least three different determinations. All CA isofoms were recombinant ones obtained in-house as reported earlier 41,42 .

Results and discussion
We investigated the inhibition of CpeCA with a panel of sulphonamides of type 1-24, which include both aromatic and heterocyclic derivatives, employed extensively for the design of various classes of CAIs with interesting physicochemical properties 43-51 (Figure 1).  [9] were also included in this study as they incorporate the sulphonamide/sulphamate zinc-binding function and act as potent CAIs against many b-CAs investigated earlier [7]. The inhibition observed with these derivatives against CpeCA and the human (h) off-target a-class enzymes hCA I and II, are shown in Table 1.
The following structure-activity relationship (SAR) can be drawn from the data of Table 1 regarding CpeCA inhibition with these compounds i. The least effective CpeCA inhibitors were zonisamide and saccharin, which did not affect the enzyme activity up to 100 mM (Table 1). ZNS is in fact the only aliphatic sulphonamide, whereas SAC the only secondary, acylated sulphonamide among the investigated compounds. ii. Moderate-weak inhibitory action, in the micromolar range, was observed for the following sulphonamides: 5, 6, 10-12 and 18, which had K I s in the range of 1.268-8.755 mM. These compounds belong to the aminoalkyl-benzenesulphonamide (5 and 6) and tetrasubstituted benzenesulphonamide/disulphonamide (10-12) series. Probably, the large number of substituents on the phenyl ring for the last type of derivatives is detrimental to their efficient binding to the enzyme. iii. More effective but moderate CpeCA inhibitors were the following compounds: 1-4, 7-9, 13, 14, 17, 19, 21-23, BZA, TPM, SLP-CLX and HCT, which had K I s in the range of 160-713 nM. It is obvious that these derivatives belong to a variety of different classes, with both aromatic, heterocyclic and sugar derivatives among them. Thus, a real SAR is difficult to draw, but it is important to note that many structural variations in the scaffold of aromatic/heterocyclic sulphonamides are tolerated without a significant loss of the CpeCA inhibitory action. iv. The most effective CpeCA inhibitors were 15, 16, 20, 24, AAZ, MZA, EZA, DCP, DZA, BRZ and SLT, which showed K I s in the range of 37.4-145 nM (Table 1). Again many different chemotypes led to quite effective CAIs, among which the most notable are dorzolamide, a rather bulky bicyclic sulphonamide (the best inhibitor with a K I of 37.4 nM), acetazolamide (the second best inhibitor with a K I of 49.1 nM) as well as the aromatic compound 4-(2-hydroxymethyl-4-nitrophenyl-sulphonamido)ethylbenzenesulphonamide 24, with a K I of 51.2 nM ( Table 1). All of them are highly different structurally, which is of extreme importance for the possible design of even better CpeCA inhibitors belonging to the sulphonamide class. v. The off-target isoforms hCA I and II have a very different inhibition profile with the compounds investigated here (Table 1), whereas hCA I has generally a lower affinity for most of these inhibitors, hCA II is highly inhibited by most of them, usually in the low nanomolar range, which makes it quite difficult to obtain CpeCA-selective inhibitors form this class of agents.

Conclusions
Species belonging to the genus Clostridium, such as Clostridium tetani, C. botulinum, C. barati, C. butirycum, C. hystolyticum and C. perfringens among others, are strictly anaerobic pathogens that provoke serious human disease, such as tetanus, botulism, gas gangrene, bacterial corneal keratitis and other infections 53,54 .
Although some progress has been achieved ultimately for designing pharmacological agents effective against these diseases, such as for example protease inhibitors targeting various metalloproteases essential for the life cycle of these pathogens, there is a constant search for novel drug targets that may lead to new classes of such agents, considering the serious antibiotic drug resistance problems emerging worldwide with the clinically used drugs 53,54 . In the search of effective compounds interfering with the metabolism of these pathogens, in this paper, we investigated potential CpeCA inhibitors, possibly useful to inhibit the growth/ pathogenicity of this bacterium. A panel of aromatic, heterocyclic and sugar sulphonamides/sulphamates were employed for the inhibition of this bacterial b-class enzyme. Some sulphonamides, such as acetazolamide, ethoxzolamide, dichlorophenamide, dorzolamide, sulthiame and 4-(2-hydroxymethyl-4-nitrophenyl-sulphonamido)ethylbenzenesulphonamide were effective CpeCA inhibitors, 219 Ã Errors in the range of 5-10% of the shown data, from three different assays. a Human recombinant isozymes, stopped flow CO 2 hydrase assay method, from reference [3][4][5]. b Recombinant bacterial enzyme, stopped flow CO 2 hydrase assay method, this work.
with K I s in the range of 37.4-71.6 nM. Zonisamide and saccharin were the least effective inhibitors, whereas many other aromatic and heterocyclic sulphonamides were moderate-weak inhibitors with K I s ranging between 113 and 8755 nM. This study thus provides the basis for developing better clostridial enzyme inhibitors with potential as antiinfectives with a new mechanism of action.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
This work was supported in part by Person Endowment to JGF.