Synthesis and biological evaluation of sulfonamide‐based compounds as inhibitors of carbonic anhydrase from Vibrio cholerae

This study reports our continued efforts to identify inhibitors capable of targeting carbonic anhydrases (CAs) expressed in bacteria. Based on previously identified chemotypes, we designed and synthesized new analogs that were screened toward the α, β, and γ classes encoded in Vibrio cholerae (Vch). The Ki values measured in the stopped‐flow hydrase assay revealed that very simple structural modifications might induce a relevant impact on the inhibitory effects as well as the selectivity profile over ubiquitous human isozymes (hCA I/II). Unfortunately, the best active VchCA inhibitors demonstrated a dramatic loss of hCA II selectivity when compared to previously reported compounds. Among the new series of sulfonamides, several molecules proved to be about sevenfold more potent against VchCAγ than the reference compound acetazolamide, thus furnishing new insights for further development of inhibitors targeting CAs expressed in bacteria.

active monomeric or dimeric structure, whereas the β-CA class is active as dimeric, tetrameric, or octameric structures. Finally, the the γ-CA class is characterized by an active trimeric organization. For VchCAα, VchCAβ, and VchCAγ classes, the X-ray crystallography furnished structural information exclusively for VchCAβ, whereas no crystal structures of VchCAα and VchCAγ proteins are available so far. As expected, VchCAβ assumes a tetrameric structure with four active sites composed of two cysteines and one histidine residue (namely, Cys42, Cys101, and His98) coordinating a zinc ion.
Moreover, the dyad composed of aspartate and arginine residues (Asp44 and Arg46) regulates the opening and closing of the catalytic site. This process is finely tuned by pH value related to bicarbonate concentration. The pH value higher than 8.0 generates the salt bridge between Arg46 and Asp44, thus generating the active form type-I, for which the zinc ion results coordinated by water/hydroxide ion, whereas the type-II form is characterized by the zinc coordination mediated by aspartate in place of water/hydroxide ion at pH lower than 8.0. Different from VchCAβ, in the other two classes, VchCAα and VchCAγ, the zinc ion is classically coordinated by three histidine residues in combination with a fourth histidine, which assumes the well-known pivotal role of the proton shuttle, thus assisting the CO 2 hydration process.
To reduce the cholera illness and treat the spreading antimicrobial resistance, emerging therapeutic approaches recognize the inhibition of VchCAs as an intriguing opportunity to impair the cholera toxin expression as well as the bacterial adaption in the intestinal environment. To date, several CA inhibitors (CAIs) have been screened for VchCAα, VchCAβ, and VchCAγ. [13] Different chemotypes demonstrated efficacy in the nanomolar range and relevant selectivity over human carbonic anhydrases (hCAs) belonging to the α-class. Sulfonamides are the most investigated CAIs; in the deprotonated form the sulfonamide moiety acts as a metal binder against the zinc ion of VchCAs, thus preventing the catalytic cycle of CAs as demonstrated for acetazolamide (AAZ, 1) and ethoxzolamide (EZA, 2) displayed in Figure 1. [9,14,15] The abovementioned binding mode has been demonstrated by several co-crystal adducts of sulfonamides in complex with α-classes [16][17][18][19][20] ; moreover, docking simulations suggested the network of interactions for sulfonamides with VchCAα, VchCAβ, and VchCAγ. [21][22][23] Several benzoxaboroles [24] (e.g., compound 3, Figure 1) also proved to be potent inhibitors against VchCAγ over VchCAα, VchCAβ, and hCA I/II isozymes (3). Finally, dithiocarbamates, coumarins, and carboxylic acids inhibited bacterial CAs through a distinct mode of interaction. [25,26] In our previous study, we have reported a computational approach to identify VchCA inhibitors culminating in the discovery of N-(4sulfamoylbenzyl)biphenyl-4-carboxamide ( Figure 1, compound 4) as a small molecule capable of reducing CA activity of all three classes expressed in V. cholerae in the nanomolar range in stopped-flow hydrase assay [22] and marked selectivity over hCA I/hCA II isoforms. To collect information about its binding mode, we have also described its hypothetical orientation into the VchCAβ cavity through docking simulations ( Figure 2). As a result, we have visualized the canonical contacts displayed by sulfonamide-based CAIs for which R-SO 2 NH 2 functionality is anchored to the zinc ion coordinated by Cys42, His98, and Cys101 residues, whereas the aromatic ring of the benzenesulfonamide fragment established π-π interaction with Tyr83. Notably, the -NHgroup of the amide spacer appeared to make an H-bond interaction with the oxygen atom of the Gly102 backbone. The best affinity measured for this inhibitor (K i = 96.5 nM) has been rationalized by the additional hydrophobic interactions with a cluster of aminoacidic residues Thr105, Ala106, Ile108, Pro111, and Ala139 that were localized in the sub-pocket beyond the middle portion of the CA cavity. These data were consistent with previous molecular/dynamic studies on various sulfonamides targeting VchCAs. [27] Based on the study mentioned above, we decided to extend here the structure-affinity relationships (SARs) introducing new moieties able to occupy hydrophobic/hydrophilic pocket subsites. Therefore, we first designed a small series of N-[ (4-sulfamoylphenyl)  and 4-(aminomethyl)benzenesulfonamide (10, n = 1) or 4-(aminoethyl) benzenesulfonamide (11, n = 2) in alkaline medium following a similar procedure employed for 4. [22] Keeping in mind the crucial role of the amide functionality as an anchoring group within the backbone and a polar side chain of pivotal residues, in the third round of our SAR exploration, we moved our

| CA inhibition
The inhibitory effects of the newly synthesized compounds were measured for VchCAα, -β, and -γ classes by means of a stopped-flow CO 2 hydrase assay as previously reported for prototype 4. [22] In Table 1, we collected the K i values for target compounds 12-20, 29, 31-33, 36, and 39-40 as well as prototype 4 and acetazolamide (AAZ, 1), which were used as reference compounds. The K i values measured for the physiologically ubiquitous hCA I and hCA II were included for comparison purposes, thus leading to information on the selectivity profile over human isoforms. The following SAR consideration may be recovered.   [22] 3 | CONCLUSION In conclusion, a small series of sulfonamide-based compounds was designed and synthesized. By collecting the data on VchCA inhibition, we found that binding requirements for these classes of CAs are extremely mandatory to produce inhibitory effects and/or selectivity over hCA I/II isoforms

| Preparation of the CAs from bacteria
The α, β, and γ-CAs were obtained accordingly to the previously reported by our groups. [33] The GeneArt Company (Invitrogen), which were prepared in distilled-deionized water, then dilutions up to 0.01 nM were done with distilled-deionized water. Inhibitor and enzyme solutions were preincubated together for 15 min at room temperature before assay, to allow for the formation of the E−I complex. The inhibition constants (K i ) were obtained by nonlinear least-squares methods using PRISM 3 and represent the mean from at least three different determinations. CA isoforms were recombinant ones, as reported earlier by this group. [34]