Inhibition studies with simple and complex (in)organic anions of the γ-carbonic anhydrase from Mammaliicoccus (Staphylococcus) sciuri, MscCAγ

Abstract The γ-carbonic anhydrase (CA, EC 4.2.1.1) from the pathogenic bacterium, Mammaliicoccus (Staphylococcus) sciuri (MscCAγ) was recently cloned and purified by our groups. Here we investigated inhibition of this enzyme with (in)organic simple and complex anions, in the search of inhibitors with potential applications. The most effective inhibitors (KIs in the micromolar range) were peroxydisulfate and trithiocarbonate, whereas submillimolar inhibition was observed with N,N-diethyldithiocarbamate and phenylboronic acid (KIs of 0.5–0.9 mM). Thiocyanate, hydrogensulfide, bisulphite, stannate, divanadate, tetraborate, perrhenate, perruthenate, hexafluorophosphate, triflate and iminodisulfonate showed KIs of 1.0–13.7 mM. Cyanate, cyanide, azide, carbonate, nitrate, tellurate, selenocyanide, tetrafluoroborate, sulfamide, sulphamic acid and phenylarsonic acid were weaker inhibitors, with KIs in the range of 25.2–95.5 mM, whereas halides, bicarbonate, nitrite, sulphate, perchlorate and fluorosulfonate did not show inhibitory action up until 100 mM concentrations in the assay system. Finding more effective MscCAγ inhibitors may be helpful to fight drug resistance to antibiotics.


Introduction
Carbonic anhydrases (CAs, EC 4.2.1.1) are present in prokaryotes and eukaryotes, with a large number of genetic families encoding them 1-4 . By efficiently catalysing the hydration of CO 2 to bicarbonate and protons, these enzymes participate in many crucial physiologic processes, such as pH regulation 1-4 , metabolism (as they are involved in several biosynthetic pathways) 5 , photosynthesis (in plants and cyanobacteria) 6 , electrolyte secretion (in vertebrates) 7 , respiration and CO 2 /bicarbonate homeostasis 8 , just to mention the essential ones. Bacteria encode for at least 4 CA classes (a-, b-, cand i-CAs) 2,4 and in many pathogenic ones (but also in fungi or protozoans) the CAs are also involved in virulence, acclimation in specific tissues/organs and pathogen survival/fitness in environments possessing highly variable pH, bicarbonate and CO 2 levels 2,9 . Thus, recently, inhibition of bacterial CAs started to be considered as a valid approach for obtaining antibiotics with a novel mechanism of action 2,10,11 , and at least for Helicobacter pylori 12 , Neisseria gonorrhoeae 11,13 and vancomycin-resistant Enterococci 14 , detailed in vitro, in vivo and animal model proof-ofprinciple studies provided the experimental evidence that the approach is feasible and provides a powerful antibacterial effect 2,10-14 . Up until now, most such studies have been performed with sulphonamide CA inhibitors (CAIs) targeting these bacterial enzymes 10-14 . There are, however, many other less investigated chemotypes, such as the (in)organic anions, many of which act as zinc binders, as well as compounds possessing different inhibition mechanisms (e.g. anchoring to the zinc-coordinated water, occlusion of the active site entrance or out of the active site binding) which should be taken into consideration 15 . Apart natural products, which offer interesting structures for designing structurally novel enzyme inhibitors, including CAIs 16 , the investigation of simple (in)organic anions (or similar small molecules) afforded the discovery of new CAI classes, such as mono-and dithiocarbamates 15 , selenols 17 , ninhydrins 18 and many other chemotypes 15 . Many such new types of reported CAIs were among the derivatives showing relevant CA isoform-selective inhibition profiles 15 .
Recently, we reported 19 the cloning, purification and initial characterisation of a c-class CA from the Gram-positive bacterium Mammaliicoccus sciuri (previously known as Staphylococcus sciuri), MscCAc, which is responsible of infections in humans and various other wild/domestic animals, but also of gene transfer phenomena to other bacteria, that confer drug resistance to multiple antibiotics 19 . We hypothesised 19 that inhibition of this enzyme may lead to a novel strategy for limiting the spread of multidrug resistance and investigated a range of sulphonamides and sulfamates as MscCAc inhibitors. Some sulphonamides were indeed rather effective inhibitors, but compounds with an optimal selectivity profile for inhibiting the bacterial over the human enzymes have not yet been detected so far. Thus, here we report the investigation of (in)organic anions and structurally related small molecules as MscCAc inhibitors, in the search of compounds with interesting inhibition profiles.

Reagents
Anions and small molecules were commercially available reagents of the highest available purity from Sigma-Aldrich (Milan, Italy). The purity of tested compounds was higher than 99%.

CA activity and inhibition measurements
An Applied Photophysics stopped-flow instrument has been used for assaying the CA catalysed CO 2 hydration activity 20 . Phenol red at a concentration of 0.2 mM was used as pH indicator, working at the absorbance maximum of 557 nm, with 10 mM TRIS (pH 8.3) as buffer, and in the presence of 10 mM NaClO 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 uncatalyzed rates were determined in the same manner and subtracted from the total observed rates. Stock solutions of inhibitors (10-50 mM) were prepared in distilled-deionized water and dilutions up to 0.01 mM were done thereafter with the assay buffer. Inhibitor and enzyme solutions were preincubated together for 15 min 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 PRISM 3 and the Cheng-Prusoff equation, whereas the kinetic parameters for the uninhibited enzymes from Lineweaver-Burk plots, as reported earlier 21-23 , and represent the mean from at least three different determinations. MscCAc was obtained as reported earlier 19 and its concentration in the assay system was of 12.6 nM.

Discussion and conclusions
Anions, both inorganic and organic ones, represent a relevant class of CAIs, which in most cases act as zinc binders 15 . They coordinate to the metal ion from the enzyme active site, frequently as monodentate ligands, with the preservation of the tetrahedral geometry of the active site metal ion, although rarely, an addition to the coordination sphere was observed, which led to trigonal bipyramidal geometries (at least for the zinc ion present in many classes of CAs 1,15 ) Thus, we included in our study a large number of inorganic and organic anions, as well as small molecules structurally related to them (  15,25 . IV. Submillimolar MscCAc inhibitory activity has been evidenced for selenite, pyrophosphate, N,N-diethyldithiocarbamate and phenylboronic acid (K I s in the range of 0.5-0.9 mM), whereas peroxydisulfate and trithiocarbonate were the most effective inhibitors, with inhibition constants of 60 and 70 mM, respectively. V. The inhibition profiles with (in)organic anions of the bacterial and human CAs investigated are very different, which signifies that it might be possible to design inhibitors selective for the pathogenic enzyme, as already demonstrated for other bacterial CAs 2,10-12 .
In conclusion, we report here the first inhibition study with (in)organic anions and small molecules (sulfamide, sulphamic acid, phenylboronic acid and phenylarsonic acid) of MscCAc. The most effective inhibitors (K I s in the micromolar range) were peroxydisulfate and trithiocarbonate, whereas submillimolar inhibition was observed with N,N-diethyldithiocarbamate and phenylboronic acid (K I s of 0.5-0.9 mM). Thiocyanate, hydrogensulfide, bisulphite, stannate, divanadate, tetraborate, perrhenate, perruthenate, hexafluorophosphate, triflate and iminodisulfonate showed K I s of 1.0-13.7 mM. Cyanate, cyanide, azide, carbonate, nitrate, tellurate, selenocyanide, tetrafluoroborate, sulfamide, sulphamic acid and phenylarsonic acid were weaker inhibitors, with K I s in the range of 25.2-95.5 mM, whereas halides, bicarbonate, nitrite, sulphate, perchlorate and fluorosulfonate did not show inhibitory action up until 100 mM concentrations in the assay system. Although we did not find highly effective MscCAc inhibitors among the small molecules and anions investigated here, some of the tested compounds, such as N,N-diethyldithiocarbamate, phenylboronic acid or trithiocarbonate, which showed relevant inhibition, may be developed and used as leads for the design of presumably more effective bacterial CA inhibitors.

Disclosure statement
No potential competing interest was reported by all authors except CTS. 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.