Inhibition of bacterial α-, β- and γ-class carbonic anhydrases with selenazoles incorporating benzenesulfonamide moieties

Abstract A series of benzenesulfonamides incorporating selenazoles with diverse substitution patterns were investigated as inhibitors of six bacterial carbonic anhydrases (CAs, EC 4.2.1.1) from bacterial pathogens, such as Helicobacter pylori (hpCAα was the investigated enzyme), Vibrio cholerae (all the three CAs from this pathogen were considered, VchCAα, VchCAβ and VchCAγ) and Burkholderia pseudomallei (with its two CAs, BpsCAβ and BpsCAγ). All these sulfonamides were effective CA inhibitors, with potencies in the low micromolar or submicromolar range, making them attractive as lead compounds for designing antibacterials with a novel mechanism of action, which could counteract the extensive resistance problem observed with many clinically used antibiotics.


Introduction
Carbonic anhydrases (CAs, EC 4.2.1.1) are a superfamily of ubiquitous metalloenzymes which catalyze the simple but physiologically crucial interconversion of carbon dioxide and water, with the formation of bicarbonate and protons: CO 2 þ H 2 O HCO 3 À þ H þ1-11 . The hydration/dehydration of CO 2 is a key physiological reaction for the life cycle of most organisms, including bacteria, since it is connected with numerous metabolic pathways, such as the biosynthetic processes requiring CO 2 or HCO 3 À (photosynthesis and carboxylation reactions) and biochemical pathways including pH homeostasis, secretion of electrolytes, transport of CO 2 and bicarbonate, etc. 12, 13 . Figure 1 shows the transport of carbon dioxide and bicarbonate assisted by bacterial CAs denoting their pivotal role in the bacterial metabolism. In fact, bacteria encode such enzymes belonging to three different genetic families, the a-, band c-CAs 11-13 . Inhibition of one or more of the CAs present in bacteria was shown to interfere with the growth and/or pathogenicity of many such organisms [1][2][3][4][5]13 . The reason for that is explained as follows: these enzymes participate in tightly controlled metabolic processes, or in the pH homeostasis, which are highly relevant in all organisms, including bacteria 1- 13 . It has been demonstrated that the two CAs (a and b) encoded by the genome of the pathogen Helicobacter pylori, a Gram-negative bacterium colonizing the human stomach, are essential for the acid acclimatization of the pathogen within the human stomach and thus, for the bacterial survival in the host 14 . On the other hand, Vibrio cholerae (a Gramnegative bacterium provoking cholera) uses its CAs (a, b and c) for producing sodium bicarbonate, which induces cholera toxin expression 15 , and for colonizing the host 16 . Once more, the causative agent of brucellosis, Brucella suis, a non-motile Gram-negative coccobacillus, and the Mycobacterium tuberculosis, an obligate pathogenic bacterium responsible for tuberculosis, were needed of functional CAs for growing [17][18][19] . Indeed, a large number of interesting studies were dedicated in the last decade for finding effective in vitro CA inhibitors (CAIs) targeting these pathogenic enzymes [8][9][10][11][12][13][14][17][18][19] , and possibly translating such results to in vivo and eventually clinical activity 1 . However, CAIs are not yet seriously considered as potential anti-infectives to date, mainly due to the fact that no relevant drug discovery program has been yet started, although these compounds may show indeed a great promise for fighting drug resistant microbes, such as bacteria, fungi and protozoa 20 .
(for maintaining constant ionic strength), following the CA-catalyzed CO 2 hydration reaction for a period of 10 s at 25 C. 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 uncatalyzed rates were determined in the same manner and subtracted from the total observed rates. Stock solutions of inhibitors (10 mM) were prepared in distilled-deionized water and dilutions up to 1 nM were done thereafter with the assay buffer. Enzyme and inhibitor solutions were pre-incubated together for 15 min (standard assay at room temperature) prior to assay, in order to allow for the formation of the enzyme-inhibitor complex. The inhibition constants were obtained by non-linear least-squares methods, using GraphPad PRISM 3 and the Cheng-Prusoff equation, as reported earlier 23 . All CAs were recombinant proteins produced as reported earlier by our groups 9-14,24 .

Chemistry
Selenazoles are an important class of heterocycles with significant biological effects and considerable pharmacological relevance 21,25 . Moreover, these five-membered selenium heterocycles are easily synthesized from primary selenoamide bearing benzenesulfonamide 1 as starting materials. This compound has been used for the preparation of various 2,5-disubstituted 1,3-selenazoles 4a-f and 5a-c, as reported earlier (Scheme 1) 21 .
In order to extend our library of selenazole derivatives, we report the synthesis of a variety of double-functionalized and ionic 1,3-selenazoles, as shown in Scheme 2.
Our main interest was to investigate structure-activity relationship related to the inhibition of different CAs classes from three different pathogenic bacteria: Helicobacter pylori (hpCAa), Vibrio cholerae (VchCAa, VchCAb and VchCAc) and, Burkholderia pseudomallei (BpsCAb and BpsCAc), relevant pathogens in many diseases for which drug-resistant strains were evidenced 1,9-11 .

Carbonic anhydrase inhibition
Selenazole scaffolds showed promising results as antibacterial activity [26][27][28] . For this reason, we investigated whether such compounds may act as inhibitors of bacterial CAs, thus being possible candidates for anti-infective studies. We tested in vitro compounds 4-11 for their inhibitory activity against six bacterial enzyme: hpCAa, VchCAa, VchCAb, VhCAc, BpsCAb and BpsCAc, by means of the stopped-flow carbon dioxide hydration assay 22 . These activities were compared with those of the standard, clinically used CAI acetazolamide (AAZ) ( Table 1).
As seen from data of Table 1, all bacterial enzymes investigated here showed inhibition in the low micromolar or submicromolar range, with all selenazoles incorporating benzenesulfonamide mioietes included in the study.
(i) HpCAa was effectively inhibited, with K I s ranging between 0.79 and 2.84 mM, thus leaving little space to structure-activity relationship (SAR) discussion due to this flat profile. However, all these sulfonamides were at least two orders of magnitude less effective as HpCAa inhibitors compared to acetazolamide, which is a very potent inhibitor of this enzyme.  (ii) VchCAa was inhibited in the submicromolar range by selenazoles 4-11, with K I s ranging between 0.21 and 0.94 mM, again without any relevant SAR, since the activity was very similar for the entire series of derivatives. However, the band c-class enzymes from the same pathogen had a weaker sensitivity to this class of CAIs, since the K I s ranged between 2.61-9.12 mM for VchCAb, and 3.64-9.29 mM for VchCAc (Table 1). Again, AAZ was a better inhibitor for all these CAs compared to the studied derivatives.
(iii) Some of the selenazole sulfonamides investigated here were, on one hand, more effective BpsCAb inhibitors compared to the standard drug AAZ, as they showed submicromolar inhibitory power. Among them were 10a (K I of 90 nM), 9b (K I of 320 nM), 5b and 5c (K I s of 0.33-0.36 mM), compared to the K I of 0.74 mM of acetazolamide. These data show that small structural differences lead to drastic effects on the CA inhibition. For example, 10b, possessing an extra methyl group compared to 10a, was 52 times a weaker BpsCAb inhibitor compared to 10a. BpsCAc was, on the other hand, less sensitive to these inhibitors compared to the b-class enzyme from the same pathogen, although some compounds (4a-4e, 10c) were submicromolar inhibitors, with K I s ranging between 0.70 and 0.91 mM.

Conclusions
A series of benzenesulfonamides incorporating selenazoles with diverse substitution patterns were investigated as inhibitors of six bacterial CAs from pathogens such as Helicobacter pylori (tested enzyme was hpCAa), Vibrio cholerae (all the three CAs from this pathogen were considered, VchCAa, VchCAb and VchCAc) and Burkholderia pseudomallei (with its two CAs, BpsCAb and BpsCAc). All these sulfonamides were effective inhibitors, with potencies in the low micromolar or submicromolar range, making this class of CA inhibitors attractive as lead compounds for designing antibacterials with a novel mechanism of action, which could counteract the extensive antibiotic resistance problem encountered with most clinically used such drugs.