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1 1. CynT2 was heterologously overexpressed to investigate the kinetic constants and its inhibition profile with substituted benzene-sulfonamides. 2. The compounds 22, 24, and the clinically used SLP were the best CynT2 inhibitors sowing KIs= 82–97 nM + 1213 word(s) 1213 2020-06-15 08:16:29

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Del Prete, S.; De Luca, V.; Bua, S.; Nocentini, A.; Carginale, V.; Supuran, C.T.; Capasso, C. The Catalytic Activity and the inhibition profile of the carbonic anhydrase CynT2. Encyclopedia. Available online: https://encyclopedia.pub/entry/1096 (accessed on 15 June 2024).
Del Prete S, De Luca V, Bua S, Nocentini A, Carginale V, Supuran CT, et al. The Catalytic Activity and the inhibition profile of the carbonic anhydrase CynT2. Encyclopedia. Available at: https://encyclopedia.pub/entry/1096. Accessed June 15, 2024.
Del Prete, Sonia, Viviana De Luca, Silvia Bua, Alessio Nocentini, Vincenzo Carginale, Claudiu T. Supuran, Clemente Capasso. "The Catalytic Activity and the inhibition profile of the carbonic anhydrase CynT2" Encyclopedia, https://encyclopedia.pub/entry/1096 (accessed June 15, 2024).
Del Prete, S., De Luca, V., Bua, S., Nocentini, A., Carginale, V., Supuran, C.T., & Capasso, C. (2020, June 15). The Catalytic Activity and the inhibition profile of the carbonic anhydrase CynT2. In Encyclopedia. https://encyclopedia.pub/entry/1096
Del Prete, Sonia, et al. "The Catalytic Activity and the inhibition profile of the carbonic anhydrase CynT2." Encyclopedia. Web. 15 June, 2020.
The Catalytic Activity and the inhibition profile of the carbonic anhydrase CynT2
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CAs catalyze the physiologically crucial reversible reaction of the carbon dioxide hydration to bicarbonate and protons. Herein, we investigated the sulfonamide inhibition profile of the recombinant β-CA (CynT2) identified in the genome of the Gram-negative bacterium, Escherichia coli. This biocatalyst is indispensable for the growth of the microbe at atmospheric pCO2. CynT2 was strongly inhibited by some substituted benzene-sulfonamides, and the clinically used inhibitor sulpiride (KIs in the range of 82–97 nM). This study may be relevant for identifying novel CA inhibitors, as well as for another essential part of the drug discovery pipeline, such as the structure-activity relationship for this class of enzyme inhibitors.

 

Carbonic anhydrase sulfonamides inhibitors
The first wholly sequenced microbial genomes were obtained in 1995 from two pathogenic bacteria, Haemophilus influenzae and Mycoplasma genitalium [1,2]. From 1995 onward, genomes belonging to 11,691 eukaryotes, 247,392 prokaryotes, and 34,747 viruses have been sequenced (Data from National Center for Biotechnology Information, May 2020). The extensive DNA sequencing has opened a new era to contrast human, animal, and plant diseases [3]. Two main reasons support this. The first is that most of the sequenced genomes belong to pathogens, and the second is that the knowledge of the genome of harmful microbes offers the possibility to identify gene encoding for protein targets, whose inhibition might impair the growth or virulence of the prokaryotic and eukaryotic pathogens [4,5]. Proteins as drug targets are prevalent. Among them, enzymes represent a significant group, since most of them catalyze reactions essential for supporting the central microbe metabolism and, as a consequence, the vitality of the pathogen [6]. The basis of the drug target approach is supported by the following criteria: (a) to identify metabolic pathways which are absent in the host and indispensable for the survival of the pathogen; (b) to recognize enzymes of the metabolic pathway whose inhibition compromise the microbe lifecycle; and, finally, (c) to find compounds which, in vitro (as the first investigation), can interfere with the activity of the identified enzymes [7]. In this context, the genome exploration of pathogenic and non-pathogenic microorganisms has revealed genes encoding for a superfamily of metalloenzymes, known as carbonic anhydrases (CAs, EC 4.2.1.1) [8,9,10,11,12]. CAs catalyze the physiologically crucial reversible reaction of the carbon dioxide (CO2) hydration to bicarbonate (HCO3) and protons (H+) according to the following chemical reaction: CO2 + H2O ⇋ HCO3 + H+ [13,14,15]. Many CA inhibitors (CAIs) exist and efficiently inhibit, in vitro, the activity of the CAs encoded by the genome of several pathogens [13,16,17,18]. It has been demonstrated that CAIs are also effective in vivo, impairing the growth and virulence of several pathogens responsible of human diseases, such as Helicobacter pylori [19,20,21], Vibrio cholerae [22], Brucella suis [23,24,25,26], Salmonella enterica [27], and Pseudomonas aeruginosa [28]. Considering the three major criteria typifying the drug-target approach, it is evident that CAs meet the criteria (b) and (c) entirely. Instead, the criterion (a) is satisfied partly because CAs are ubiquitous metalloenzymes involved in the balance of the equilibrium between dissolved CO2 and HCO3 in all living organisms. Even if CAs are not species-specific enzymes, they are considered promising drug targets because they offer the possibility to design specific and selective inhibitors for the microbial CAs [13,16,17,18]. For example, the enzyme dihydrofolate reductase (DHFR), although it is ubiquitously expressed in all kingdoms, is a target of several drugs, such as the antibacterial trimethoprim [29]. This enzyme is responsible for the nicotinamide adenine dinucleotide phosphate (NADPH)-dependent reduction of 5,6-dihydrofolate (DHF) to 5,6,7,8-tetrahydrofolate (THF), an essential cofactor used in the biosynthetic pathways of purines, thymidylate, methionine, glycine, pantothenic acid, and N-formyl-methionyl tRNA. The bacterial DHFR amino acid sequence has an identity of 30% with the corresponding human protein [29]. Nevertheless, trimethoprim selectively inhibits the bacterial enzyme but not the human DHFR [29].
The CA superfamily is grouped into eight genetically distinct families (or classes), named with the Greek letters α, β, γ, δ, ζ, η, θ, and ι [13,14,15,30,31]. In mammalian, for example, 15 CAs are expressed, 12 of which are catalytically active, and all belong to the α-class [9,16,32,33,34,35,36,37]. It is interesting to stress that the genome of most pathogens does not encode for a α-CA [12,13,14,34,38,39]. This is a unique advantage in finding inhibitors with no inhibitory effect on the CAs from humans and animals. However, when the genome of a pathogen encodes for a α-CA, such enzyme (amino acid sequence identity of about 35% respect to the mammalian protein) shows structural differences in the amino acid residues surrounding the catalytic pocket, offering the possibility to tune the CA inhibitors and, hence, a higher probability to inhibit selectively the α-CA identified in the pathogen [40,41,42]. Recently, our groups focused on the in vitro inhibition of recombinant β-CA (CynT2) from Escherichia coli because this CA, localized in the cytoplasm, is indispensable for the growth of the microbe at atmospheric pCO2 [43,44]. E. coli is a Gram-negative bacterium that, as a commensal microorganism, colonizes the lower intestine of warm-blooded organisms [45,46,47]. In some cases, E. coli can act as a severe pathogen able to generate disease outbreaks worldwide [48,49,50], or, as an opportunistic pathogen, which can cause diseases if the host defenses are weakened [51]. Surprisingly, although this enzyme was reported and crystallized two decades ago [43], no inhibition study with any class of CAIs was reported so far. Here, we compare the inhibition profiles of CynT2 with those determined for the β-CA from Vibrio cholerae and the two human α-CA isoforms (hCA I and hCA II), using the sulfonamides and their bioisosteres, which, among the groups of the classical CAIs, generally inhibit the other CAs in the range of nanomolar and have been clinically used for decades as antiglaucoma [29], diuretic [35], antiepileptic [32], anti-obesity [52,53], and anticancer [37] agents.
The goal of the present manuscript is to identify putative compounds, which can eventually go through the other phases of the drug discovery pipeline, such as the structure–activity relationship (SAR), in vitro cell based-tests, in vivo studies, and, finally, the clinical trials, leading to the discovery of new antibacterials.
 
Results:
The recombinant enzyme resulted in an excellent catalyst for the CO2 hydration reaction with a kcat= 5.3 x 105 s-1 and a kcat/KM= 4.1 x 107 M-1 s-1. (Table 1)

 

Table 1. Kinetic parameters for the CO2 hydration reaction catalyzed by the human α-CAs and bacterial CAs (α-, β-, γ- and ι-CAs).

Organism

Acronym

Class

kcat

(s-1)

kcat/Km

 (M-1 x s-1)

KI (acetazolamide)

(nM)

 

Homo sapiens a

hCA I

α

2.0 x 105

5.0 x 107

250

 

hCA II

α

1.4 x 106

1.5 x 108

12

Vibrio cholerae 

VchCAα

α

8.2 x 105

7.0 x 107

6.8

Escherichia coli

CynT2

β

5.3 x 105

4.1 x 107

227

Vibrio cholerae 

VchCAβ

β

3.3 x 105

4.1 x 107

451

Porphyromonas gingivalis 

PgiCAβ

β

2.8 x 105

1.5 × 107

214

Helicobacter pylori 

HpyCAβ

β

7.1 x 105

4.8 x 107

40

Porphyromonas gingivalis 

PgiCAγ

γ

4.1 x 105

5.4 × 107

324

Vibrio cholerae 

VchCAγ

γ

7.3 x 105

6.4 x 107

473

Burkholderia territorii 

BteCAι

ι

3.0 x 105

9.7 × 107

65

 

In Table 2 is reported the inhibition profile of CynT2. The comparative analysis was carried out analyzing the CynT2 inhibitory behavior with those obtained for the enzyme VchCAβ (β-CA form Vibrio cholerae)  and the two human γ-CA isoforms, hCA I and hCA II.

Table 2. Inhibition of the human isoforms hCA I and hCA II and the two bacterial β-CAs (CynT2 and VchCAβ) with sulfonamides 1-24 and the clinically used drugs AAZ-EPA.

Inhibitor

                        KI*(nM)

  hCA Ia    hCA IIa   CynT2       VchCAβa

1

28000

300

705

463

2

25000

240

790

447

3

79

8

457

785

4

78500

320

3015

>10,000

5

25000

170

2840

>10,000

6

21000

160

3321

>10,000

7

8300

60

>10000

>10,000

8

9800

110

>10000

9120

9

6500

40

2712

>10,000

10

7300

54

8561

>10,000

11

5800

63

6246

879

12

8400

75

4385

4450

13

8600

60

4122

68,1

14

9300

19

440

82,3

15

5500

80

6445

349

16

9500

94

2340

304

17

21000

125

502

3530

18

164

46

205

515

19

109

33

416

2218

20

6

2

726

859

21

69

11

473

4430

22

164

46

93

757

23

109

33

322

817

24

95

30

82

361

AAZ

250

12

227

4512

MZA

50

14

480

6260

EZA

25

8

557

6450

DCP

1200

38

>10000

2352

DZA

50000

9

629

4728

BRZ

45000

3

2048

845

BZA

15

9

276

846

TPM

250

10

3359

874

ZNS

56

35

3189

8570

SLP

1200

40

97

6245

IND

31

15

2392

7700

VLX

54000

43

2752

8200

CLX

50000

21

1894

4165

SLT

374

9

285

455

SAC

18540

5959

6693

275

HCT

328

290

5010

87

FAM

922

58

2769

-

EPA 

8262

917

2560

-

 

 
 
 
 
 
 
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