Carbonic anhydrases from pathogens

C H A P T E R 18

Carbonic anhydrases from pathogens: bacterial carbonic anhydrases and their inhibitors as potential antiinfectives Claudiu T. Supuran1, Clemente Capasso2 1

Department of NEUROFARBA, Section of Pharmaceutical and Nutraceutical Sciences, University of Florence, Firenze, Italy; 2Istituto di Bioscienze e Biorisorse, CNR, Napoli, Italy

18.1 Bacterial carbonic anhydrase as drug targets At present, infectious diseases are the second leading cause of death in the world, especially with the emerging phenomenon of bacterial antibiotic resistance implicating most of the existing clinical drugs [1,2]. This issue has raised tremendous scientific interest expressly for (1) discovering new molecular targets essential for life cycle/growth of the pathogens and (2) developing a feasible plan for the realization of novel antibacterial drugs, possibly effective as next-generation antibiotics. Microbes express their pathogenicity through their virulence, which determines the ability of pathogens to enter a host, evade host defenses, grow in the host environment, counteract its immune responses, assimilate iron or other nutrients from the host, or sense environmental changes [3e5]. The main classes of clinically used antibiotics inhibit bacterial growth by interfering with the biosynthesis of proteins, nucleic acids, folate metabolism, or the formation of the microbe cell wall [6]. Many bacterial infections are treated with antibiotics, such as tetracyclines, macrolides, lincosamides, erythromycin, phenicols, aminoglycosides, and fusidic acid, which are good examples of inhibitors affecting the protein synthesis [6]. These compounds act by inhibiting the elongation of the peptide chain after binding to one of the two subunits of the bacterial ribosomes. The DNA-interfering antimicrobials are a group of drugs able to interfere with DNA replication and transcription [7]. These molecules include quinolones, novobiocin, ansamycins, imidazole, nitrofurans, benzylpyrimidines, and sulfonamides. Antimicrobials, such as b-lactams, glycopeptides, lipoglycopeptide, and fosfomycin are the principal families of antiinfectives acting on bacterial cell wall synthesis. These molecules interfere with the synthesis of peptidoglycans, the macromolecules of the bacterial cell walls incorporating N-acetylmuramic acid [8]. One strategy for fighting antibiotic resistance Carbonic Anhydrases. https://doi.org/10.1016/B978-0-12-816476-1.00018-6 Copyright © 2019 Elsevier Inc. All rights reserved.

387

388 Chapter 18 Table 18.1: List of enzymes used as drug targets, which are traditionally used as molecular targets and the clinically used drugs against these molecular targets. Enzyme

Clinical drug

Carbonic anhydrase

Acetazolamide

Neuraminidase

Oseltamivir

Angiotensin-converting enzyme

Captopril

HIV-1 protease

Saquinavir, Indinavir

Dihydrofolate reductase

Methotrexate

Guanine phosphoribosyltransferase

Allopurinol

0

Inosine 5 -monophosphate dehydrogenase

Tiazofurin

Thymidylate synthase

Tomudex

Cyclooxygenase

Diclofenac, Indometacin

Dihydropteroate synthase

Sulfanilamide, Sulfathiazole

is represented by the upgrade of the current clinical drugs for generating novel antibiotics [9,10] but, as a limitation, the newly created drugs could have a limited lifespans for the possible resistance they would develop sooner or later. An interesting strategy to overcome antibiotic resistance is to discover and use novel enzymes essentially involved in the central bacterial metabolism. As shown in Table 18.1, numerous enzymes are responsible for the pathogen virulence, acting against host components and contributing to the damage of host tissues. In this scenario, a crucial physiologic reaction for the survival of microbes, as well as for all living organisms, is a pivotal reaction of the central bacterial metabolism: the CO2 hydration/dehydration reaction. This reaction is connected with numerous metabolic pathways, such as photosynthesis and carboxylation reactions, and biochemical pathways including pH homeostasis, secretion of electrolytes, transport of CO2 and bicarbonate, and so on [11,12]. Moreover, the interconversion of CO2 and HCO3
is spontaneously and precisely balanced from the living organisms to maintain the equilibrium between dissolved
inorganic carbon
dioxide (CO2), carbonic acid (H2CO3), bicarbonate HCO3
, and carbonate CO3 2
[13e16]. Thus, the survival of the microbe is compromised by restricting the access of the pathogen to these metabolites, which are essential for microbe biosynthesis and energy metabolism [17]. The CO2 hydration/dehydration is catalyzed by a superfamily of metalloenzymes, known as carbonic anhydrases (CAs, EC 4.2.1.1) [18e22], which are categorized into seven genetically distinct families (or classes), named a-, b-, g-, d-, z-, h-, and ɵ-CAs [18e22], and only three of these classes are encoded by the bacterial genome: a-, b-, and g-CA [22e28]. In Fig. 18.1, the simple but physiologically crucial interconversion of carbon dioxide and water into bicarbonate and protons is shown

Bacterial carbonic anhydrases and their inhibitors as antibiotic agents 389

Figure 18.1 A representative scheme illustrating the role of carbonic anhydrases (CAs) in Gram-negative bacteria. The a-CA has a periplasmic localization and can convert the diffused CO2 inside the periplasmic space into bicarbonate. The feeding of CO2 and bicarbonate to the central bacterial metabolism depends on the action of the cytosolic b- and g-CAs.

(CO2 þ H2 O#HCO3
þ Hþ [18,22,29e34]), as well as the transport of carbon dioxide and bicarbonate assisted by bacterial CAs. The bicarbonate indispensable for the bacterial metabolic processes cannot be provided through the uncatalyzed naturally occurring CO2 hydration/dehydration reaction because, at physiologic pH, the rate is too low (kcat hydration ¼ 0.15 s
1 and kcat dehydration ¼ 50.0 s
1), whereas the catalyzed reaction has a rate of 104e106 s
1 [26,35]. As shown in Fig. 18.1, it has been speculated that in Gram-negative bacteria, the a-CA is able to convert the atmospheric and/or metabolic CO2 diffused in the periplasmic space, whereas b- or g-classes have a cytoplasmic localization and are responsible for CO2 supply for carboxylase enzymes, pH homeostasis, and other intracellular functions, ensuring the survival and/or satisfying the metabolic needs of the microorganism [36,37]. It is interesting to note that bacteria show a very variegated distribution pattern of the CA classes. In fact, it is true that the bacterial genome encodes for the three CA classes (a, b, and g), but bacteria are frequently identified whose genome encodes only for one or two CA classes and, rarely, for any CAs [26,38]. Moreover, a common feature of all bacterial a-CAs known to date is the presence of an N-terminal signal peptide, which suggests a periplasmic or extracellular location (see Fig. 18.1). Recently, using the program available as a Web tool at http://www.cbs.dtu.dk/services/

390 Chapter 18 SignalP/ [39], it has been demonstrated that the primary structure of the b-CAs identified in the genome of the pathogenic Gram-negative bacteria, such as such Helicobacter pylori, Vibrio cholerae, Neisseria gonorrhoeae, and Streptococcus salivarius present at the N-terminal part a secretory signal peptide of 18 or more amino acid residues [26,35]. Intriguingly, the CAM enzyme, which is a g-CA, contained a short putative signal peptide at its N-terminus, too. Because the signal peptide is essential for the translocation across the cytoplasmic membrane in prokaryotes, probably the b- and/or g-CAs characterized by the presence of a signal peptide might coexist in the periplasmic space together with the a-CAs [26,35].

18.1.1 In vivo carbonic anhydrase druggability It has been demonstrated in vivo that the pathogenic and nonpathogenic Gram-negative bacteria need functional CAs for their survival or for manifesting their virulence in the host. Good examples are represented by the two nonpathogenic Gram-negative bacteria, Ralstonia eutropha and Escherichia coli [40,41], and pathogenic bacteria, such as H. pylori [42e45], V. cholerae [20,32,34,46], Brucella suis [47e50] Mycobacterium tuberculosis [47e50], B. suis [15,47,51,52], N. gonorrhoeae [53,54], and Neisseria sicca [55,56]. Here, two striking examples highlight the role of CAs in bacterial growth and virulence. H. pylori, a pathogen which colonizes the human stomach, uses two CAs (a and b) and the urease for its acid acclimatization within the human stomach. The inhibition of these two/three enzymes, in fact, led to the death of the bacteria and a possible eradication of H. pylori from the stomach and has been used clinically for the treatment of gastric ulcers [42,43]. V. cholerae, the causative agent of cholera [57e59], uses all its three CAs (a, b, and g) for producing sodium bicarbonate, which triggers the cholera toxin (CT) expression [15,25,42,44,45,59e62]. CA inhibitors (CAIs) caused a significant reduction in virulence gene expression. From these salient evidences, it is readily apparent that the bacterial CAs might be considered as excellent drug targets because they are essential for the life cycle of the pathogens [63,64]. The main focus of the present chapter is inhibition of the bacterial CAs, which were prepared by our groups in sufficient amounts for biologic assays and/or X-ray analysis with the recombinant DNA technology. However, the main drawbacks of this research are connected to the fact that many bacterial CAs were not yet validated in vivo as drug targets.

18.2 Set of carbonic anhydrase inhibitors The CAIs can be clustered into several different groups dependent on their binding mode to the enzyme active site [36,65]. The four groups are schematically represented in

Bacterial carbonic anhydrases and their inhibitors as antibiotic agents 391

Figure 18.2 Schematic representation of the four known carbonic anhydrase (CA) inhibition mechanisms. (A) zinc-binding (sulfonamides, dithio-/monthiocarbamates, carboxylates, boronic acids, borols, etc.); (B) anchoring to the metal ion coordinated water (phenols, polyamines, sulfocoumarins, carboxylates); (C) occlusion of the active site entrance (coumarins); (D) out of the active site binding [2-(benzylsulfonyl)-benzoic acid]. Legend: AG, anchoring group; SG, sticky group; ZBG, zinc-binding group.

Fig. 18.2 as: (A) the metal ion binders (anion, sulfonamides and their bioisosteres, dithiocarbamates [DTCs], xanthates, etc.); (B) compounds that anchor to the zinccoordinated water molecule/hydroxide ion (phenols, polyamines, thioxocoumarins, sulfocoumarins); (C) compounds occluding the active site entrance, such as coumarins and their isosteres; (D) compounds binding out of the active site, such as an aromatic carboxylic acid derivative [36]; and also inhibitors with an unknown binding mechanism, such as secondary/tertiary sulfonamides, protein tyrosine kinase inhibitors, and fullerenes, for which the X-ray crystallographic structure is unavailable [36].

392 Chapter 18

18.2.1 The most investigated carbonic anhydrase inhibitors 18.2.1.1 Sulfonamides The most investigated CAIs are the anions and sulfonamides [36,37,66,67]. Antimicrobial sulfonamides were discovered by Domagk in 1935 [68], and they were the first antimicrobial drugs to be widely used in clinical settings. The first sulfonamide showing effective antibacterial activity was Prontosil, a sulfanilamide prodrug, which is isosteric/ isostructural with p-aminobenzoic acid (PABA), the substrate of dihydropteroate synthase [69]. In the following years, a range of analogs constituting the so-called sulfa drug class of antibacterials entered into clinical use, and many of these compounds are still widely used, despite significant drug resistance problems. A library of 40 compounds, 39 primary sulfonamides and one sulfamate, was used as CAIs (Fig. 18.3) [20,30,33,34,50,62,70e79]. Derivatives 1e24 and AAZ-HCT are either simple aromatic/heterocyclic sulfonamides widely used as building blocks for obtaining new families of such pharmacologic agents, or they are clinically used agents, among which acetazolamide (AAZ), methazolamide (MZA), ethoxzolamide (EZA), and dichlorophenamide (DCP) are the classical, systemically acting antiglaucoma CAIs. Dorzolamide (DZA) and brinzolamide (BRZ) are topically acting antiglaucoma agents; benzolamide (BZA) is an orphan drug belonging to this class of pharmacologic agents. Moreover, zonisamide (ZNS), sulthiame (SLT), and the sulfamic acid ester topiramate (TPM) are widely used antiepileptic drugs. Sulpiride (SLP) and indisulam (IND) were also shown by our group to belong to this class of pharmacologic agents, together with the COX2 selective inhibitors celecoxib (CLX) and valdecoxib (VLX). Saccharin (SAC) and the diuretic hydrochlorothiazide (HCT) are also known to act as CAIs. Sulfonamides, such as the clinically used derivatives AAZ, MZA, EZA, DCP, DZA, and BRZ, bind in a tetrahedral geometry to the Zn(II) ion in the deprotonated state, with the nitrogen atom of the sulfonamide moiety coordinated to Zn(II) and an extended network of hydrogen bonds, involving amino acid residues of the enzyme, also participating in the anchoring of the inhibitor molecule to the metal ion [36,37,65,80]. The aromatic/heterocyclic part of the inhibitor interacts with the hydrophilic and hydrophobic residues of the catalytic cavity [36,37,66,67]. 18.2.1.2 Anions Anions, such as inorganic metal-complexing anions or more complicated species such as carboxylates, are also known to bind to CAs (Fig. 18.4) [36,65]. These anions may bind either the tetrahedral geometry of the metal ion or as trigonal-bipyramidal adducts (see Fig. 18.3). Anion inhibitors are important both for understanding the inhibition/catalytic mechanisms of these enzymes fundamental for many physiologic processes and for designing novel types of inhibitors, which may have clinical applications for the management of a variety of disorders in which CAs are involved [36,65].

Figure 18.3 A library of 40 compounds, of which 39 sulfonamides and 1 sulfamate.

394 Chapter 18

Figure 18.4 List of 37 anions/small molecules used as carbonic anhydrase inhibitors.

18.2.1.3 Dithiocarbamates Another class of CAIs recently investigated is constituted by the DTCs [81e86]. These were discovered by considering the inorganic anion trithiocarbonate (TTC, CS3 2
) as a lead compound, as an X-ray crystal structure of this weak inhibitor with the human isoform hCA II was available [87]. DTCs, as TTC coordinate through one sulfur atom to the Zn(II) ion from the enzyme active site, and also interact with the conserved Thr199 amino acid residue. DTCs are micromolarelow nanomolar CAIs against many CA isoforms as their organic scaffold participates in supplementary interactions with the enzyme active site. These compounds were also investigated for the inhibition of some pathogenic CAs such as those of M. tuberculosis, Porphyromonas gingivalis, etc. [83,88e90]. Some DTCs effective against several bacterial CAs are shown in Fig. 18.5.

18.3 In vitro inhibition of the bacterial carbonic anhydrases Bacterial CA classes (a, b, and g) were extensively investigated by our groups reporting the kinetic parameters and the in vitro anion and sulfonamide inhibition profiles. Most of these studies were carried out on pathogenic bacteria, such as Francisella tularensis, Burkholderia pseudomallei, V. cholerae, Streptococcus mutans, P. gingivalis, Legionella pneumophila, Clostridium perfringens, Mycobacterium tuberculosis, etc. [20,34,73,91,92]. Our results indicated the following: (1) the sulfonamides and their bioisosteres (Fig. 18.3) were able to highly inhibit most of the CAs identified in the genome of the aforementioned bacteria [18,76,93e96] and (2) simple and complex anions as well as small molecules (see Fig. 18.4) showed that the most efficient inhibitors detected so far are sulfamide, sulfamate, phenylboronic acid, and phenylarsonic acid [25,76,97]. Generally, halide, cyanide, bicarbonate, nitrite, selenate, diphosphate, divanadate, tetraborate, peroxodisulfate,

Bacterial carbonic anhydrases and their inhibitors as antibiotic agents 395 O S NH O

O SO2NH2

n

32; n= 1 33; n= 2

34

O

S

S S

SO2NH2

HN

S

NH N

N

NH

O

HN

S

N

H N

K O

S S

S

35

36

37

38

H2NO2S N

N N R

NH H2 N

N H

SO3Na

39; R= H 40; R= Me

O

H N

H2N

O

H N NH

SO2NH2

41

NH O

NH

H2NO2S O H N

O O

42

NH O

NH

H2NO2S 43

Figure 18.5 Sulfonamides and dithiocarbamates 32e43 showing selective bacterial carbonic anhydrase inhibitory properties.

396 Chapter 18 Table 18.2: Pathogen, CA class, disease, and infection caused by the microbes investigated by our groups. Pathogen

CA class

Disease

Infections

Porphyromonas gingivalis

b and g

Periodontitis Rheumatoid arthritis

An infection of the tissues that surround and support the teeth. An infection of articular cartilage

Streptococcus mutans

b and g

Dental caries

An infection of the dental hard tissues

Vibrio cholerae

a, b, and g

Cholera

An infection causing severe watery diarrhea with dehydration and even death if untreated

Francisella tularensis

b and g

Tularemia

A potentially debilitating febrile illness

Burkholderia pseudomallei

b and g

Melioidosis

Septicemia and pneumonia in susceptible individuals

Legionella pneumophila

b

Legionellosis

Fatal form of pneumonia

hexafluorophosphate, and triflate exhibit weak inhibitory activity against the bacterial CAs [23,25,26,35,97]. Moreover, AAZ and MZA were shown to effectively inhibit the bacterial growth in cell cultures [98]. In this chapter, a collection of the kinetic parameters and the inhibition profiles with sulfonamides and their bioisosteres, and (in)organic anions of the CAs identified in the genome of bacterial pathogens is reported. These studies highlighted the recent developments in the search for new antibacterial agents able to interfere with the growth and virulence of the microorganisms by inhibition of CAs encoded in their genomes. As the physiologic role of bacterial b-CAs is poorly understood for the virulence/lifecycle of these pathogens, the present study is a starting point in the design of effective pathogenic bacteria CAIs with potential use as antiinfectives. The pathogenic bacteria reported in this chapter are shown in Table 18.2, whereas Table 18.3 shows the kinetic parameters of the CAs encoded by the genome of these pathogens.

18.3.1 Porphyromonas gingivalis Periodontal disease is a general term describing the inflammatory pathologic states of the supporting tissues of teeth, which can be grouped into two major categories, gingivitis and periodontitis. Gingivitis is defined as an inflammation of gingival tissues without affecting the attachment of teeth, whereas periodontitis involves the destruction of the connective tissue attachment to the tooth and the adjacent alveolar bone [99,100]. Chronic adult periodontitis is the most common form of advanced periodontal disease. The microbes involved are extremely diverse and may be composed of more than 150 different

Bacterial carbonic anhydrases and their inhibitors as antibiotic agents 397 Table 18.3: Kinetic parameters of the recombinant pathogenic carbonic anhydrases (CAs) for the CO2 hydration reaction determined by the stopped-flow technique. Pathogen

CA class

kcat (s¡1)

kcat/KM (M¡1 s¡1)

Porphyromonas gingivalis

b g

2.8  105 4.1  105

1.5  107 5.4  107

Streptococcus mutans

b g

4.2  105 e

5.8  107 e

Vibrio cholerae

a b g

8.2  105 3.3  105 7.3  105

7.0  107 4.1  107 6.4  107

Francisella tularensis

b g

9.8  105 e

8.9  107 e

Burkholderia pseudomallei

b g

1.6  105 5.3  105

3.4  107 2.5  107

Legionella pneumophila

b1 b2

3.4  105 8.3  105

4.7  107 8.5  107

e, not detected.

species [101e103]. Among the bacteria regularly isolated from periodontal pockets, those producing such pathologic states are generally Gram-negative rods and include mainly P. gingivalis, and others such as Prevotella intermedia, Fusobacterium nucleatum, Actinobacillus actinomycetemcomitans, Capnocytophaga sputigena, and Wolinella recta [104,105]. As with many other bacteria, the genome of P. gingivalis encodes for both a b- and g-CA named with the acronyms PgiCAb and PgiCAg, respectively [106e114], which were cloned, expressed, purified, and characterized by our groups. The kinetic parameters for the physiologic reaction, that is, CO2 hydration to bicarbonate and protons, were determined using the stopped-flow techniques [111,115]. PgiCAb showed a significant catalytic activity, with a kcat of 2.8  105 s
1 and a kcat/KM of 1.5  107 M
1 s
1, whereas PgiCAg had a kcat of 4.1  105 s
1 and a kcat/KM of 5.4  107 M
1 s
1. Interestingly, the comparison of the kinetic parameters of PgiCAb and PgiCAg with those of other CAs belonging to a different family and from different organisms showed that the PgiCAb was about 2.3 times faster than the b-CA isolated from Flaveria bidentis (FbiCA1) and 1.5 times slower when compared with the PgiCAg [111,115]. 18.3.1.1 Inhibition profiles with sulfonamides and anions The two recombinant PgiCAb and PgiCAg were antithetic to the sulfonamide inhibitors because the effective inhibitors of PgiCAb were ineffective toward PgiCAg and vice versa [108,109]. However, the behavior of the pharmacologic inhibitor ZNS with a KI of

398 Chapter 18 345 nM against PgiCAb and 157 nM against PgiCAg was intriguing. In fact, compounds 1e7, MZA and BRZ had inhibition constants ranging between 345 and 818 nM toward PgiCAb but were rather ineffective as PgiCAg inhibitors. Moreover, AAZ and EZA were the most effective PgiCAb inhibitors, whereas compounds 9, 10, 16, ZNS, and IND were very effective against PgiCAg. The anion inhibition profile of PgiCAb was quite different from that of PgiCAg as well as the human isoform hCA II [107,114]. It should be noted that perchlorate, tetrafluoroborate, azide, nitrate, hydrogensulfite, and sulfate were not inhibitors of PgiCAb. For example, azide has an inhibition constant of 73 mM against PgiCAg and of 1.5 mM against hCA II, but it is not at all inhibitory against PgiCAb. Halides, cyanide, bicarbonate, nitrite, selenate, diphosphate, divanadate, tetraborate, peroxodisulfate, hexafluorophosphate, and triflate were weak PgiCAb (KI of 5.4e21.4 mM) [107,114]. Interestingly, for the halogenides, fluoride and chloride had a similar behavior (KI of 7.5e7.8 mM), whereas the heavier halogenides were weaker inhibitors (KI of 15.9e21.4 mM). The most efficient PgiCAb inhibitors detected so far are sulfamide, sulfamate, phenylboronic acid, and phenylarsonic acid, with KI ranging between 60 and 78 mM (Fig. 18.3). It is also interesting to note that these compounds are also effective inhibitors of the PgiCAg being far less efficient hCA II inhibitors. The inhibition profile of the P. gingivalis CAs was very different from that of the human isozymes (hCA I and hCA II). Thus, even this preliminary study was able to detect leads with a good inhibitory power against the two pathogenic bacterium enzymes and also with good selectivity for the pathogenic over the host enzyme inhibition.

18.3.2 Streptococcus mutans Dental caries is a bacterial disease of the dental hard tissues (teeth) associated with dental plaque of smooth coronal surfaces, pits, and fissures [116,117]. The demineralization of teeth is caused by organic acid produced from the bacterial fermentation of carbohydrates present in the diet [118,119]. Among these bacteria, S. mutans is the most cariogenic. Its growth leads to the production of acid at a higher rate, enhancing demineralization of the tooth [118,119]. The genome of S. mutans possesses a gene (SMU_328) encoding for a b-CA indicated with the acronym SmuCA [73,120]. This enzyme was cloned, characterized, and investigated for its inhibition profile with the major class of CAIs, the primary sulfonamides. SmuCA has good catalytic activity for the CO2 hydration reaction, with a kcat of 4.2  105 s
1 and kcat/KM of 5.8  107 M
1 s
1. 18.3.2.1 Sulfonamide and anion inhibition profiles SmuCA is efficiently inhibited by most sulfonamides (KI of 246 nMe13.5 mM). The best SmuCA inhibitors were bromosulfanilamide 9, deacetylated AAZ 13, 4-hydroxybenzenesulfonamide 15, the pyrimidine-substituted sulfanilamide derivative 19, aminobenzolamide 20, and

Bacterial carbonic anhydrases and their inhibitors as antibiotic agents 399 compounds structurally similar to it, as well as AAZ, MZA, IND, and VLX. These compounds showed inhibition constants ranging between 246 and 468 nM [73,120]. The anion inhibition profile of the S. mutans enzyme was very different from the other a- and b-CAs investigated earlier. SmuCA was inhibited by cyanate, carbonate, stannate, divannadate, and diethyldithiocarbamate in the submillimolar range (KI of 0.30e0.64 mM) and more efficiently by sulfamide, sulfamate, phenylboronic acid, and phenylarsonic acid (KI of 15e46 mM). The identification of effective inhibitors of this new enzyme may lead to pharmacological tools useful for understanding the role of S. mutans CAs in dental caries formation, and eventually the development of pharmacological agents with a new mechanism of antibacterial action.

18.3.3 Vibrio cholerae The Gram-negative bacterium V. cholerae is the causative agent of cholera [57e59]. Recently it was reported that sodium bicarbonate induces CT expression [44,45]. It was demonstrated that bicarbonate stimulates virulence gene expression by enhancing ToxT, a regulatory protein that directly activates transcription of the genes encoding CT activity [45]. The addition of CAIs caused a significant reduction in virulence gene expression. Thus, bicarbonate was the first positive effector for ToxT activity to be identified [121]. Because the bicarbonate ion is present at a high concentration in the upper small intestine colonized by V. cholera and the microbe lacks bicarbonate transporter proteins in its genome, it is plausible that the pathogen uses the CAs system to accumulate bicarbonate into the cell to activate its virulence [57e59]. The genome of V. cholerae encodes for putative CAs belonging to each bacterial class: a, b, and g, which were cloned, purified, and characterized from our group. These CAs were designated as VchCA (a-CA), VchCAb (b-CA), and VchCAg (g-CA) [32,34,57e59,122]. VchCA is a CA belonging to the a class and consists of 239 amino acid residues. VchCA shows an identity of about 30% with the two human isoforms and 40% with bacterial CAs. Moreover, VchCA conserved all the characteristics common to other a-CAs, for example, the amino acid residues of the catalytic triad, the proton shuttle, and the gate-keeping residues. VchCAb is a b-CA formed by a polypeptide chain of 222 amino acid residues showing an identity of 40%, when compared with the b-CAs from F. bidentis (FbiCA) and Pisum sativum (PsaCA) and, 30% when compared with the bacterial b-CAs from P. gingivalis (PgiCA) and B. suis (BsuCA). The catalytic triad of b-CAs is perfectly conserved in all these enzymes. Moreover, VchCAb showed all the characteristics of a classical b-CA: the two cysteines and one histidine amino, which are the amino acid residues responsible for the catalytic mechanism. VchCAg is a CA belonging to the g class with a polypeptide chain of 184 amino acid residues. It shows an identity of 25%e26% when compared with the two prototypes of the g-CAs, CAM and CAMH, while presenting an identity of 38% compared with PgiCA (g-CA of P. gingivalis). The three recombinant bacterial CAs were purified by nickel affinity chromatography, with a

400 Chapter 18 Std

1

2

3

50 kDa 37 kDa 25 kDa 20 kDa

Figure 18.6 SDS-PAGE after affinity chromatography. Std: standards; 1: VchCA; 2: VchCAb; 3: VchCAg.

yield of 30 mg for VchCA and VchCAb, and 0.7 mg for VchCAg. The recombinant proteins were analyzed by SDS-PAGE as shown in Fig. 18.6. 18.3.3.1 Activity of the three carbonic anhydrases determined by the stopped-flow technique Table 18.4 reports the rate constants (kcat, KM, and kcat/KM) of the three classes of CA identified in the genome of V. cholerae and the inhibition constant (KI) using the inhibitor AAZ. These constants were compared with the kinetic parameters of other CAs belonging to a-, b-, and g-classes identified in different organisms and microorganisms. VchCA showed a kcat of 8.23  105 s
1, a KM of 11.7 mM, and a kcat /KM of 7.0  107 M
1 s
1. VchCA was more active than the human isoform hCAI (kcat ¼ 2.0  105 s
1) and hpaCA (kcat ¼ 2.5  105 s
1), it has an activity of an order of magnitude lower than hCA II and SazCA, an extremophilic CA from the genome of Sulfurihydrogenibium azorense. Furthermore, VchCA was inhibited by AAZ (KI ¼ 6.8 nM) more than the hCA II and hpaCA. VchCAb had a kcat of 3.34  105 s
1 and a KM of 8.1  10
3 s
1and a catalytic efficiency of 4.1  107 M
1 s
1. The enzyme showed a kcat similar to that of the enzyme identified in plant (FbiCA1), and its catalytic efficiency is very similar to that of other bacterial enzymes, but it was two orders of magnitude lower compared with the b-class enzyme identified in the bacterium P. gingivalis (PgiCAb). In addition, AAZ was found to be a less effective inhibitor for VchCAb than VchCA, showing an inhibition constant of 451 nM (Table 18.4). VchCAg showed a kcat of 7.39  105 s
1, an order of magnitude higher than that of gCA (CAM) identified in the thermophilic Archeon Methanosarcina thermophila and, slightly higher than the kcat of the bacterial g-CA PgiCA. Interestingly, VchCAg was more active than VchCAb, while the AAZ shows an inhibition constant of 473 nM.

Bacterial carbonic anhydrases and their inhibitors as antibiotic agents 401 Table 18.4: Determination of the kinetic constants for the hydratase reaction catalyzed by carbonic anhydrases (CAs) identified in the genome of Vibrio cholerae. Class

CA acronym

Species

kcat (S¡1)

KM (M)

kcat/KM (M¡1 s¡1)

K1 (nM) (acetazolamide)

a

hCA I

Homo sapiens

2.00  105

4.0  10
3

5.0  107

250

hCA II

Homo sapiens

1.40  106

9.3  10
3

1.5  108

12

hpaCA

Helicobacter pylori

2.5  10

16.6  10

1.5  10

21

SazCA

Sulfurihydrogenibium yellostonense

4.40  106

12.5  10
3

3.5  108

0.90

SspCA

Sulfurihydrogenibium azorense

9.35  105

85  10
3

1.1  108

4.5

VchCA

Vibrio cholerae

8.23  105

11.7  10
3

7.0  107

6.8

Flaveria bidentis

1.2  10

6

7.5  10

27

Brucella suis

6.4  10

3.9  10

7

63

Brucella suis

1.1  10

7

303

Porphyromonas gingivalis

2.8  10

7

214

Helicobacter pylori

7.1  10

Vibrio cholerae

3.34  10

PgiCA

Porphyromonas gingivalis

4.1  10

CAM

Methanosarcina thermophila

VchCAg

Vibrio cholerae

b

FbiCA1 BsuCA219 BsuCA213 PgiCAb hpbCA VchCAb

g

5

5 5 6 7 5


3


3

1.6  10

16.4  10 1.2  10


3


3

7

8.9  10

18.6  10


3

1.5  10

14.7  10


3

4.8  10


3
3

40

7

4.1  10

451

7.5  10

7

5.4  10

324

6.1  104

7.0  10
3

8.7  105

63

7.39  105

11.5  10
3

6.4  107

473

5

5

8.1  10

7

The results were compared with those obtained for CAs identified in other organisms. The inhibition constant (KI) was obtained for the classical sulfonamide inhibitor, AAZ.

18.3.3.2 Activity of the bacterial carbonic anhydrases determined by protonography The activity of proteolytic enzymes, capable of refolding and acquiring the proteolytic activity after treatment with SDS, can be determined by SDS-PAGE after the removal of the detergent. Such a technique is known as zymography. Another technique to identify the activity of CA hydratase on SDS-PAGE, named protonography, has been developed based on protons produced by the CO2 hydratase reaction, which are then responsible for the change of color that appears on the gel, in correspondence of the CA. In Fig. 18.7 are the protonograms obtained using the commercial bovine bCA (a-CA class) and the recombinant CAs (a, b, and g) from V. cholerae. Protonograms of VchCA, VchCAb, and VchCAg showed different behaviors that the three bacterial CAs assumed during SDS-PAGE. It is known that mammal a-CAs are monomeric, and the protonogram of bCA showed a single

402 Chapter 18 (A)

SDS PAGE

Coomassie stained Protonography Std

bCA VchCA

(B)

bCA VchCA

Std

100 kDa 75 kDa

(C) Std VchCAγ

VchCAβ

50 kDa 50 kDa

50 kDa 30 kDa 25 kDa

25 kDa

25 kDa

Figure 18.7 (A) Protonogram and SDS-PAGE stained with Coomassie compared. Both gels were run in nonreducing denaturing conditions and loaded with 4 mg of bCA and VchCA. The appearance of the band occurred after 5 s of incubation in CO2-saturated water. (B) Protonogram of VchCAb with a molecular weight marker (Coomassie gel not shown). The band appeared after 40 s of incubation in CO2-saturated water. (C) Protonogram of VchCAg with a molecular weight marker (Coomassie gel not shown). The band appeared after 25 s of incubation in CO2-saturated water.

band of activity corresponding to a monomer of 30 kDa (Fig. 18.7A). The bacterial a-CAs are generally dimeric enzymes and the protonogram showed three bands of activity: a monomer (25 kDa), a dimer (50 kDa), and a tetramer (100 kDa) (Fig. 18.7A). Therefore, unlike bCA, VchCA is present in three different oligomeric states. The protonogram of VchCAb (Fig. 18.7B) showed two bands of activity: a band corresponding to the monomeric form (29 kDa) and one at the dimeric form (58 kDa). The protonogram of VchCAg showed a band of activity in correspondence with the monomer (24 kDa) and trimer (69 kDa) (Fig. 18.7C). The yellow bands found in correspondence with the inactive monomeric form of VchCAb or VchCAg are due to the fact that at the end of the electrophoretic run, the SDS is removed from the gel. This procedure may lead to the rearrangement of b- or g-CA monomers in the gel and the final result is the reconstitution of the active dimeric (b-CA) or trimeric forms (g-CA). Protonography allowed us to distinguish not only the active enzyme but also oligomeric states of the CA. 18.3.3.3 Sulfonamide inhibition profile The VchCA, VchCAb, and VchCAg inhibition data obtained using the library of 40 compounds (39 sulfonamides and 1 sulfamate) reported in Fig. 18.3 showed the following: TPM, a sulfamate, SLP, a primary sulfonamide, as well as SAC, an acylsulfonamide, were ineffective VchCA inhibitors (KIs > 1000 nM). Moreover, SAC was a bad inhibitor for VchCAb and VchCAg, whereas TPM and SLP were effective inhibitors of VchCAg. These compounds (except SAC) generally act as good inhibitors of other bacterial or mammalian a-CAs. ZNS, an aliphatic primary sulfonamide, was also a very weak inhibitor for the bacterial enzymes but effective toward the human enzymes (KIs  725 nM). A large

Bacterial carbonic anhydrases and their inhibitors as antibiotic agents 403 number of simple aromatic sulfonamides, such as derivatives 1e9, showed moderate VchCA and VchCAg inhibitory properties, with inhibition constants in the range of 125e672 nM. It may be observed that all these derivatives are benzenesulfonamides with one or two simple substituents in ortho-, para- or 3,4-positions of the aromatic ring with respect to the sulfamoyl zinc-binding moiety. Most of these CAIs were ineffective toward VchCAb with KIs  9120 nM. Most of the sulfonamides investigated here showed a potent inhibitory effect against VchCA, VchCAb, and VchCAg with inhibition constants in the range of 23.5e88.5 nM. These derivatives include compounds 13e15. It is interesting to note that compounds 10e13, 16e18, 21e24, DCP, VLX, SLT, and HCT showed a potent inhibitory effect against VchCA. Some of these are effective toward VchCAb or VchCAg but never effective for all three classes identified in the genome of V. cholerae. Several very potent VchCA inhibitors were detected, such as compounds 14, 15, 19, 20, AAZ, MZA, EZA, DZA, BRZ, BZA, and IND, which showed KIs in the range of 0.59e12.1 nM. Most of the sulfonamides used were effective inhibitors of VchCAg, but the KIs was always 69 nM. The inhibition profile of VchCA, VchCAb, or VchCAg was different from that of the other bacterial or mammalian CAs investigated up until now, proving that probably it will be possible to design VchCAa,VchCAb, or VchCAg selective inhibitors using the scaffold of leads detected here. 18.3.3.4 VchCA,VchCAb, or VchCAg anion inhibition profile A set of inorganic anions and small molecules (Fig. 18.4) was investigated for inhibition of these enzymes. The most potent VchCAg inhibitors were N,N-diethyldithiocarbamate, sulfamate, sulfamide, phenylboronic acid, and phenylarsonic acid, with KI values ranging between 44 and 91 mM. 18.3.3.5 Other drugs tested on Vibrio cholerae carbonic anhydrases On the basis of the role played by the bicarbonate ion as a virulence factor of V. cholerae, several other series of inhibitors were reported for the development of new antibacterial drugs: 1. A series of imidazole derivatives that were able to inhibit VchCA at nM concentrations; these compounds were also highly selective because they were inactive toward the human CA isoforms hCA I and II [123]. 2. A series of acyl selenoureido benzensulfonamides was evaluated as inhibitors against VchCA and VchCAb. These compounds showed strong inhibitory action against VchCAa over VchCAb and excellent selectivity over the human (h) off-target isoforms hCA I and II [124,125]. 3. A series of selenides bearing benzenesulfonamide moieties was evaluated as inhibitors against VchCAa and VchCAb. The molecules represent an interesting lead for antibacterial agents with a possibly new mechanism of action showing excellent inhibitory action and selectivity for inhibiting VchCAa over the human (h) off-target isoforms hCA I and II [126].

404 Chapter 18 4. A new series of sulfonamides incorporating ortho-, meta, and para-substituted-benzenesulfonamide moieties were investigated for the inhibition of VchCAa and VchCAb [127]. The compounds were prepared by using the “tail approach” aiming to overcome the scarcity of selective inhibition profiles associated with CAIs belonging to the zinc binders. The built structureeactivity relationship showed that the incorporation of benzhydryl piperazine tails on a phenyl sulfamide scaffold determines rather good efficacies against hCA I and VchCAa, with several compounds showing KI < 100 nM. The activity was lower against hCA II and VchCAb, probably due to the fact that the incorporated tails are quite bulky [127]. 5. A panel of N0 -aryl-N-hydroxy-ureas inhibitors was used to inhibit the three V. cholerae enzymes [128]. VchCAa and VchCAb were effectively inhibited by some of these derivatives, with KI in the range of 97.5 nMe7.26 mM and 52.5 nMe1.81 mM, respectively, whereas VchCAg was less sensitive to inhibition (KIs of 4.75e8.87 mM). As most of these N-hydroxyureas are also ineffective as inhibitors of the human (h) widespread isoforms hCA I and II (KI > 10 mM), this class of derivatives may lead to the development of CAIs selective for bacterial/diatom enzymes over their human counterparts and thus to antiinfectives or agents with environmental applications [128]. 6. Four generations of poly(amidoamine) (PAMAM) dendrimers incorporating benzenesulfonamide moieties were investigated as inhibitors of VchCA [129]. These dendrimers incorporate 4, 8, 16, and 32 sulfonamide moieties. In particular, VchCA was best inhibited by the G1 (KI ¼ 8.1 nM) and G3 (KI ¼ 8.5 nM) dendrimer incorporating 8 and 32 sulfamoyl moieties, respectively. Also in this case, this class of molecules may lead to important developments in the field of antiinfective CAIs [129]. 7. Thirteen novel sulfonamide derivatives incorporating the quinazoline scaffold were tested for their ability to inhibit the a-CA from V. cholerae (VchCA) [130]. The best VchCA inhibitor had a KI of 2.7 nM, and many of these developed compounds showed high selectivity for inhibition of the bacterial over the mammalian CA isoforms with two compounds possessing selectivity ratios of >95 against hCA I and >8 against hCA II [130]. 18.3.3.6 Tridimensional structure of VchCAb ˚ Our subsequent work allowed the crystallization of VchCAb, which was solved at 1.9A resolution by molecular replacement using the coordinates of the b-CA from E. coli. The protein crystal was perfectly merohedrally twinned, revealing a tetrameric type II b-CA with a closed active site in which the zinc is tetrahedrally coordinated to Cys42, Asp44, His98, and Cys101 (Figs. 18.8A and B) [131]. The substrate bicarbonate was bound in a noncatalytic binding pocket close to the zinc ion (Fig. 18.8C), as reported for a few other b-CAs, such as those from E. coli and Haemophilus influenzae. It has already been observed that the binding of the bicarbonate ion to this noncatalytic site enforces a closed conformation of the active site in type II b-CA structures. However, the alternative open configuration has not been observed in any type II b-CA structure, regardless of the

Figure 18.8 (A) Ribbon structure showing the tetrameric arrangement of VchCA; (B) the active site of VchCA exhibiting a “closed” configuration; (C) the noncatalytic bicarbonate-binding site.

406 Chapter 18 crystallization pH and the concentration of bicarbonate in the crystallization medium, but the kinetic data suggest that these enzymes adopt this conformation to function in a highly cooperative manner [131]. The active form can be inferred from the crystal structures of type I b-CAs in which Asp44 is detached from the catalytic zinc ion and is hydrogen bonded in a bidentate mode to Arg46.

18.3.4 Francisella tularensis F. tularensis is a Gram-negative coccobacillus and a zoonotic facultative intracellular pathogen of humans and many animals [132,133]. Three species belonging to the genus Francisella has been identified: F. tularensis, Francisella novicida, and Francisella philomiragia [132]. Of these, F. tularensis is highly infectious and causes a potentially debilitating febrile illness known as tularemia [133]. Transmission to humans happens via direct contact, through arthropod or insect vectors, by ingestion of contaminated material, or by inhalation of aerosolized organisms. Human lungs are the main targets, where the microbes can cause the most severe form of the disease known as respiratory tularemia [91,133,134]. Our groups, involved for many years in the study of bacterial CAs from pathogenic organisms, cloned, expressed, and purified the recombinant b-CA (FtuCAb). This enzyme showed a kcat of 9.8  105 s
1 and a kcat/KM of 8.9  107 M
1 s
1 for the CO2 hydration, physiologic reaction, being one of the most effective b-CAs known to date, with a catalytic activity only 1.68 times lower than that of the human (h) isoform hCA II. 18.3.4.1 Inhibition profile with sulfonamides The panel of 39 sulfonamides, as well as the clinically used drugs incorporating sulfonamide/sulfamate zinc-binding groups (Fig. 18.3), was used to investigate the inhibition profile of FtubCA [91]. The enzyme generally showed a weaker affinity for these inhibitors compared with other a- and b-CAs investigated earlier, with only AAZ and its deacetylated precursor having an inhibition constant <1 mM. Indeed, the two compounds AAZ and its deacetylated precursor 13 (KI of 655e770 nM), as well as metanilamide and MZA (KIs of 2.53e2.92 mM) are the best FtubCA inhibitors detected so far [91]. 18.3.4.2 Inhibition profile with anions The inorganic anions and small molecules proposed in Fig. 18.4 were used to test the inhibitory effects of these CAIs against FtubCA [134]. The activity of FtubCA was not inhibited by a range of anions that do not typically coordinate Zn(II) effectively, including perchlorate, tetrafluoroborate, and hexafluorophosphate. Surprisingly, some anions that generally complex well with many cations, including Zn(II), also did not effectively inhibit FtubCA, for example, fluoride, cyanide, azide, nitrite, bisulphite, sulfate, tellurate, perrhenate, perrhuthenate, and peroxydisulfate. However, the most effective inhibitors were

Bacterial carbonic anhydrases and their inhibitors as antibiotic agents 407 in the range of 90e94 mM (sulfamide, sulfamic acid, phenylarsonic, and phenylboronic acid). N,N-diethyldithiocabamate (KI of 0.31 mM) was a moderately potent inhibitor [134].

18.3.5 Burkholderia pseudomallei B. pseudomallei is a Gram-negative saprophytic bacteria responsible for melioidosis, which is an endemic disease of tropical and subtropical regions [135]. The B. pseudomallei genome encodes for only b- and g-CAs. The two recombinant CAc (BpsCAb and BpsCAg) were heterologously expressed as soluble protein in the cytoplasm of the E. coli (DE3) codon plus cells and produced as a fusion protein containing a His-tag tail at its N-terminal amino acid sequence. The rate constants (kcat, KM, and kcat/KM) of BpsCAb and BpsCAg were compared with the kinetic parameters of the a-CA from Homo sapiens (isoforms hCA I and hCA II) [92]. The catalytic activity values of these enzymes were determined using the “stopped-flow” technique. BpsCAb showed a kcat of 1.6  105 s
1 and a kcat/KM of 3.4  107 M
1 s
1. It was slightly less active than the human isoform hCAI (kcat ¼ 2.0  105 s
1). Interestingly, the gCA from B. pseudomallei showed a kcat ¼ 5.3  105 s
1, which is 3.3 times higher than the BpsCAb (kcat ¼ 1.6  105 s
1). This is also evident for the g-CA from V. cholerae, which was 2.21 times more active than VchCAb (kcat ¼ 7.39  105 s
1). 18.3.5.1 Sulfonamide inhibition profile The inhibition profile of BpsCAb was investigated using the wide series of sulfonamides/ sulfamates indicated in Fig. 18.3. The inhibition profile of BpsCAb and BpsCAg was compared with those obtained for the human a-CAs (hCA I and hCAII) [92,136]. This study may be of interest for designing new types of inhibitors that may have clinical applications because B. pseudomallei is fundamentally resistant to penicillin, ampicillin, first-generation and second-generation cephalosporins, macrolides, quinolones, and most aminoglycosides [137]. Many simple sulfonamides and clinically used agents such as TPM, SLP, CLX, VLX, and SLT were ineffective BpsCAb and BpsCAg inhibitors (KI > 50 mM) [92,136]. Other drugs, such as EZA, DZA, BRZ, ZNS, IND, and HCT were moderately potent micromolar inhibitors. The best inhibition was observed with benzene-1,3disulfonamides, which showed for BpsCAb a KI in the range of 185e745 nM. AAZ, BZA, and metanilamide were the most effective (KI of 149e653 nM) inhibitors of BpsCAg activity, whereas other sulfonamides/sulfamates such as EZA, TPM, SLP, IND, sulthiame, and SAC were active in the micromolar range (KI of 1.27e9.56 mM) [92,136]. The inhibition profile of BpsCAb is very different from that of BpsCAg. Thus, identifying compounds that would effectively interact with both enzymes is relatively challenging. However, BZA was one of the best inhibitors of these two CAs with KI of 653 and 185 nM, respectively, making it an interesting lead compound for the design of more effective agents, which may be useful tools for understanding the pathogenicity of this bacterium.

408 Chapter 18 18.3.5.2 Inhibition profile with anions The inhibition of BpsbCA and BpsgCA with a group of anions and small molecules was also investigated. The best inhibitors were sulfamide, sulfamic acid, and phenylarsonic acid, which showed KI in the range of 83e92 mM, whereas phenylboronic acid, fluoride, cyanide, azide, bisulfite, tetraborate, perrhenate, perruthenate, peroxydisulfate, perchlorate, tetrafluoroborate, fluorosulfonate, and hexafluorophosphate showed KI > 100 mM [138,139]. Other inhibitors of this new enzyme were bicarbonate, trithiocarbonate, some complex inorganic anions, and N,N-diethyldithiocarbamate, which had inhibition constants of 0.32e8.6 mM. The best BpsgCA inhibitors were sulfamide, sulfamic acid, phenylboronic acid, and phenylarsonic acid, which showed KI in the range of 49e83 mM (these inhibitors showed millimolar inhibition constant against hCA II), followed by diethyldithiocarbamate, selenate, tellurate, perrhenate, selenocyanate, trithiocarbonate, tetraborato, pyrophosphate, stannate, carbonate, bicarbonate, azide, cyanide, thiocyanate, and cyanate with KI in the range of 0.55e9.1 mM [138,139]. In our laboratories, work is in progress to resolve the Xray crystal structures of BpsgCA, which may allow the development of small-molecule inhibitors with desired properties for targeting and inhibiting specifically the bacterial over the human CAs, considering the fact that B. pseudomallei is involved in a serious bacterial disease. 18.3.5.3 Other drugs A series of selenides bearing benzenesulfonamide moieties as CAI against the recombinant BpsCAb from the pathogenic bacteria B. pseudomallei has been investigated. The molecules showed an excellent inhibitory action and selectivity for inhibiting BpsCAb over the human off-target isoforms hCA I and II. This is very important because the present study offers the possibility of designing new organoselenium inhibitors of BpsCAb that may have clinical applications.

18.3.6 Legionella pneumophila Discovered 37 years ago, L. pneumophila is a Gram-negative environmental bacterium, which normally infects ameba [140,141]. However, this bacterium provoked life-threatening pneumonia-like disease in many participants to the 58th Annual Convention of the American Legion in Philadelphia, in 1976, an occasion with which it was in fact first discovered. This condition was subsequently called Legionnaires’ disease or legionellosis, and the bacterial pathogen characterized in detail, and it was shown that a large number of its subspecies and serovars are widespread in nature [140]. Nowadays, L. pneumophila and the related species Legionella longbeachae are responsible for legionellosis in humans, their spread being favored by the development of artificial water systems for air conditioning, cooling towers, aerosolizing devices, etc., all over the world [77,142]. It seems that many of the physiologic/

Bacterial carbonic anhydrases and their inhibitors as antibiotic agents 409 pathologic events used by Legionella to infect Ameba are the same as those used when infecting macrophages, thus suggesting a coevolution of these organisms [77,141,142]. Considering our interest in the cloning and characterization of CAs from pathogenic organisms, we identified in the genome of L. pneumophila two b-CAs designated as lpCA1 (locus tag lpg2500, NCBI reference sequence WP_014844650.1), and lpCA2 (locus tag lpg2194; NCBI reference sequence WP_014842179.1) [77]. The two recombinant enzymes were prepared and investigated for their kinetic properties and inhibition profiles with sulfonamides and inorganic anions. The CO2 hydrase activity of the two enzymes LpCA1 and LpCA2 was measured by a stopped-flow assay. The first isoform, LpCA1 showed a moderate degree of activity with a kcat of 3.4  105 s
1 and kcat/KM of 4.7  107 M
1 s
1. This activity is comparable with that of other a- and b-CAs from different species. The second isoform, LpCA2, on the other hand, was more active than LpCA1, with the following kinetic parameters: kcat of 8.3  105 s
1 and kcat/KM of 8.5  107 M
1 s
1. 18.3.6.1 Sulfonamide inhibition profile The two isoforms, LpCA1 and LpCA2, are inhibited by sulfonamides and sulfamates, the main class of CAIs (some of which have been clinically used drugs for 60 years). The best LpCA1 inhibitors were aminobenzolamide and structurally similar sulfonylated aromatic sulfonamides, as well as AAZ and EZA (KI in the range of 40.3e90.5 nM) [77]. The best LpCA2 inhibitors belonged to the same class of sulfonylated sulfonamides, together with AAZ, MZA, and DCP (KI in the range of 25.2e88.5 nM) [77]. As these enzymes may be involved in pH regulation in the phagosome during Legionella infection, their inhibition may lead to antibacterials with a novel mechanism of action. 18.3.6.2 Inhibition profile with anions The inorganic anions and small molecules reported in Fig. 18.4 were used to identify inhibitors of these enzymes [142]. Perchlorate and tetrafluoroborate did not act as inhibitors (KI > 200 mM), whereas sulfate was a very weak inhibitor for both LpCA1 and LpCA2 (KI values of 77.9e96.5 mM) [142]. The most potent LpCA1 inhibitors were cyanide, azide, hydrogen sulfide, diethyldithiocarbamate, sulfamate, sulfamide, phenylboronic acid, and phenylarsonic acid, with KI values ranging from 6 to 94 mM. The most potent LpCA2 inhibitors were diethyldithiocarbamate, sulfamide, sulfamate, phenylboronic acid, and phenylarsonic acid, with KI values ranging from 2 to 13 mM [142].

18.4 Conclusions In the last few years, numerous CAs belonging to the a-, b-, and/or g-CA classes have been detected, cloned, and characterized in many pathogenic bacteria. Many of these enzymes were shown to possess significant catalytic activity for CO2 hydration reaction, and furthermore, it has been demonstrated that in some of these pathogens they are crucial for

410 Chapter 18 the life cycle of the bacterium. In vitro inhibition studies were performed with inorganic anions, small molecules such as boronic acids, phenylarsonic acid, sulfamic acid, sulfamide, sulfonamides, and DTCs. In many cases, effective inhibitors were detected, some of which also inhibited the bacterial growth in vivo. However, very few of the detected inhibitors were also selective for the bacterial isoforms over the human ones, such as the hCA II isoform. This is one of the main challenges in proposing CAIs as novel antiinfectives with a new mechanism of action. In fact, by using structure-based drug design processes, we estimate that it will be possible to achieve the desired selectivity for inhibiting preferentially the bacterial but not the host CA isoforms.

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