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    Topic review

    The Catalytic Activity and the inhibition profile of the carbonic anhydrase CynT2

    Subjects: Others
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    Abstract

    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.

     

    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

    -

     

     
     
     
     
     
     

    This entry is adapted from 10.3390/ijms21114175