Seeking new approach for therapeutic treatment of cholera disease via inhibition of bacterial carbonic anhydrases: experimental and theoretical studies for sixteen benzenesulfonamide derivatives

Article abstract of DOI:10.1080/14756366.2019.1618292

A series of sixteen benzenesulfonamide derivatives has been synthesised and tested as inhibitors of Vibrio cholerae carbonic anhydrase (CA) enzymes, belonging to α-CA, β-CA, and γ-CA classes (VchCAα, VchCAβ, and VchCAγ). The determined K_i value

Full text of DOI:10.1080/14756366.2019.1618292

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Seeking new approach for therapeutic
treatment of cholera disease via inhibition of
bacterial carbonic anhydrases: experimental
and theoretical studies for sixteen
benzenesulfonamide derivatives
Rosaria Gitto, Laura De Luca, Francesca Mancuso, Sonia Del Prete, Daniela
Vullo, Claudiu T. Supuran & Clemente Capasso
To cite this article: Rosaria Gitto, Laura De Luca, Francesca Mancuso, Sonia Del Prete, Daniela
Vullo, Claudiu T. Supuran & Clemente Capasso (2019) Seeking new approach for therapeutic
treatment of cholera disease via inhibition of bacterial carbonic anhydrases: experimental and
theoretical studies for sixteen benzenesulfonamide derivatives, Journal of Enzyme Inhibition and
Medicinal Chemistry, 34:1, 1186-1192, DOI: 10.1080/14756366.2019.1618292
To link to this article: https://doi.org/10.1080/14756366.2019.1618292
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
019, VOL. 34, NO. 1, 1186–1192
2
https://doi.org/10.1080/14756366.2019.1618292
RESEARCH PAPER
Seeking new approach for therapeutic treatment of cholera disease via inhibition
of bacterial carbonic anhydrases: experimental and theoretical studies for sixteen
benzenesulfonamide derivatives
a
ꢀ
a
a
b
c
c
Rosaria Gitto
, Laura De Luca , Francesca Mancuso , Sonia Del Prete , Daniela Vullo , Claudiu T. Supuran
b
ꢀ
and Clemente Capasso
a
Department of Chemical, Biological, Pharmaceutical and Environmental Sciences (CHIBIOFARAM), University of Messina, Messina, Italy;
Department of Biology, Agriculture and Food Sciences, Institute of Biosciences and Bioresources- CNR, Napoli, Italy; NUROFARBA Department,
b
c
University of Florence, Sesto Fiorentino, Italy
ABSTRACT
ARTICLE HISTORY
Received 4 April 2019
Revised 6 May 2019
Accepted 8 May 2019
A series of sixteen benzenesulfonamide derivatives has been synthesised and tested as inhibitors of Vibrio
cholerae carbonic anhydrase (CA) enzymes, belonging to a-CA, b-CA, and c-CA classes (VchCAa, VchCAb,
and VchCAc). The determined K values were compared to those of selected human CA isoforms (hCA I
i
and hCA II). Structure-affinity relationship analysis highlighted that all tested compounds proved to be
active inhibitors of VchCAa at nanomolar concentration. The VchCAb activity was lower to respect inhibi-
tory efficacy toward VchCAa, whereas, these benzenesulfonamide derivatives failed to inhibit VchCAc.
Interestingly, compound 7e combined the best activity toward VchCAa and VchCAb. In order to obtain a
model for binding mode of our inhibitors toward bacterial CAs, we carried out docking simulations by
using the available crystal structures of VchCAb.
KEYWORDS
Carbonic anhydrase
inhibitors (CAIs);
benzenesulfonamides; Vibrio
cholera; molecular docking
GRAPHICAL ABSTRACT
1
. Introduction
V. cholerae preferentially colonises. Therefore, bicarbonate is likely
3
,4
The gram-negative bacterium Vibrio cholerae is responsible for the an important chemical signal during the cholera disease
.
secretory diarrheal disease cholera that infects millions of people Generally, bacteria increase cytosolic bicarbonate levels through
worldwide and might cause high mortality if left untreated. While bicarbonate transporter proteins and carbonic anhydrases (CAs,
2
the rehydration therapy is an effective treatment approach as EC 4.2.1.) . It has been demonstrated that V. cholerae can
well as the use of antibiotics in severe infections, there is a press- increase the cytosolic bicarbonate levels through the hydration
ing need for agents that prophylactically could prevent the injuri- of CO
2
by the action of CAs since V. cholerae lacks bicarbonate
3
ous effects of a cholera infection.
transporter proteins in its genome . Thus, the CAs represent an
There is evidence that protein ToxT is one of the transcription attractive molecular target for the development of innovative
3
factors activating the V. cholerae virulence gene expression; anti-infective agents . The metalloenzyme CAs are grouped into
therefore, ToxT is a target for the development of cholera thera- seven genetically distinct families, named a-, b-, c-, d-, f-, g-, and
1
,2
peutics . ToxT activity is (a) negatively controlled by the unsat- Õ-CAs, with different structures and active site architecture
urated fatty acids (UFAs) of the bile and (b) enhanced by as well as metal ions in the catalytic region (zinc, iron, cobalt,
5
the presence of bicarbonate in the upper small intestine where and cadmium) . Vibrio cholerae encodes carbonic anhydrase
CONTACT Rosaria Gitto
Messina, Viale Palatucci 13, I-98168 Messina, Italy; Clemente Capasso
rosaria.gitto@unime.it
Department of Chemical, Biological, Pharmaceutical and Environmental Sciences (CHIBIOFARAM), University of
clemente.capasso@ibbr.cnr.it Department of Biology, Agriculture and Food Sciences,
Institute of Biosciences and Bioresources-CNR, Via P. Castellino 111, I-80131, Napoli, Italy
ꢀ
These authors equally contributed to the work.
Supplemental data for this article can be accessed here.
ß 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1187
belonging to a-CA, b-CA, and c-CA classes (VchCAa, VchCAb, decided to further study their CA inhibitory profile thus exploit-
6
and VchCAc) , that could represent potential targets for the ing their capability to act as potential anti-infective agents.
development of anti-infectives targeting V. cholerae colonisa- Therefore, the primary purpose of our work is to provide new
7
–9
tion . The a- and b-CAs are Zn(II) metalloenzymes, whereas information about structure-affinity relationship relative to the
c-CAs use Fe(II) as metal ion; however they are also active with inhibition of the three classes from V. cholerae, VchCAa, VchCAb,
Zn(II) or Co(II). The metal ion is coordinated by three His residues
and VchCAc . Particularly, our idea was to study in depth the
role of hydrophobic interactions within VchCA cavity.
in the a- and c-classes, and one His and two Cys residues in the
10,11
b-class. Several classes of CA inhibitors (CAIs)
have been
identified; among them, sulfonamide derivatives have been
shown to anchor the metal-coordinated water molecule/hydrox- 2. Materials and methods
ide ion within catalytic site thus inducing CA inhibition. The
prototype sulfonamide inhibitor acetazolamide (Figure 1, AAZ, 1)
2.1. Chemistry
is clinically used as diuretic and anticonvulsant agent in humans; All reagents were used without further purification and bought
additionally, AAZ displays inhibitory effects toward bacterial iso- from common commercial suppliers. Microwave-assisted reac-
zymes. Moreover, as confirmation of the role of bicarbonate in tions were carried out in a Focussed Microwave TM Synthesis
virulence and colonisation processes, the carbonic anhydrase System, Model Discover (CEM Technology Ltd Buckingham, UK).
inhibitor ethoxzolamide (EZA, 2) proved to decrease the viru- Melting points were determined on a Buchi B-545 apparatus
7
lence gene expression and to reduce the growth of pathogen .
(BUCHI Labortechnik AG Flawil, Switzerland) and are uncor-
The principal drawback using CAIs as antiinfectives agents is rected. By combustion analysis (C, H, N) carried out on a Carlo
the lack of selectivity towards the pathogenic versus human iso- Erba Model 1106-Elemental Analyser we determined the purity
7
forms . For this reason, many research groups are continually of synthesised compounds; the results confirmed a ꢁ 95% purity.
involved in the synthesis of new CAIs or in the modification/
optimisation of the existing inhibitors, which are commonly tested
Merck Silica Gel 60 F254 plates were used for analytical TLC
(
Merck KGaA, Darmstadt, Germany). For detection, iodine vapour
10,11
on CAs from mammalian and pathogenic organisms
. Most of
and UV light (254 nm) were used. Flash Chromatography (FC)
was carried out on a Biotage SP1 EXP (Biotage AB Uppsala,
these inhibitors represent interesting leads towards the optimisa-
tion of new antibiotic agents showing excellent inhibitory effi-
ciency and selectivity for the target CAs over the human (h) off-
1
Sweden). H NMR spectra were measured in dimethylsulfoxide-
d6 (DMSO-d
6 3
) and CDCl with a Varian Gemini 300 spectrometer
1
0,11
target isoform hCA I
. In the course of our efforts to identify
(
Varian Inc. Palo Alto, California, USA); chemical shifts are
novel selective CAIs, we have synthesised and tested a series of
quinolone/isoquinoline-arylsulfonamides showing high affinity
expressed in d (ppm) and coupling constants (J) in hertz. All
exchangeable protons were confirmed by addition of D O. Rf
2
1
2–22
toward human CAs
. Specifically, we have reported the discov-
values were determined on TLC plates using a mixture of DCM/
MeOH (96/4) as eluent. All compounds have been re-synthesised
following the previously reported procedure, that is briefly
ery of a set of heteroaryl-N-carbonylbenzenesulfonamides as a
class of potent inhibitors of druggable CA isoforms (hCA II, hCA
15,17,18,20–22
VII, hCA IX, and hCA XIV)
.
23
described below .
Recently, we have studied a new small library of CAIs contain-
ing azepine/piperidine/piperazine nucleus linked to benzenesul-
fonamide fragment and disclosed several compounds that
demonstrated inhibitory effects in low nanomolar range toward
2
.1.1. Synthetic procedures for benzenesulfonamides 5a–e, 6a,
a–f, 8a–d
7
2
3
hCAs . This series of compounds has been rationally designed
by using the cycloalkylamine nucleus as the core for binding
contact in the middle area of catalytic site (Figure 1). The visual
inspection of their binding mode in co-crystal adducts with hCA
II and hCA VII confirmed that the sulfonamide portion is crucial
Pathway i: To a solution of 4-(aminosulfonyl)benzoic acid (4)
(
0
6 mmol) in THF (15 ml) the carbonylimidazole (6 mmol) (CDI) at
C was added. The obtained mixture was stirred at room tem-
ꢂ
perature for 3 h and then the appropriate amine derivative
(15 mmol) in DMF (5 ml) was added dropwise. The reaction mix-
ture was stirred at room temperature for 2 h. The solvent was
23
for CA recognition process . By considering the high inhibitory
effects of this series of potent sulfonamide compounds, we
removed in vacuo; by adding of aqueous solution of NaHCO
3
(5 ml) we obtained compounds 5a and 5d as crude products,
which were purified through crystallization by a mixture of Et
and EtOH (1:1)
2
O
N N
O
N
HN
S
O
O
S
Pathway ii: A mixture of 4-(aminosulfonyl)benzoic acid (4)
(2 mmol) and N,N,N,N-tetramethyl-O-(1H-benzotriazol-1-yl)uranium
hexafluorophosphate (HBTU) (2 mmol) in DMF(2 ml) was stirred at
room temperature for 1 h. Then, a solution of the appropriate
amine derivative (2 mmol) in TEA (2 mmol) was added dropwise.
The reaction mixture was left overnight, then quenched with
water (10 ml) and extracted with EtOAc (3 ꢃ 10 ml). The organic
phase was dried with Na SO and the solvent was removed in
S
NH2
O
O
NH2
S
O
AAZ, 1
EZA, 2
O
R
N
2
4
NH2
S
O
vacuo. The residue was purified by flash chromatography (DCM/
R
MeOH 96:4), crystallized by treatment with a mixture of Et O and
2
EtOH (1:1) to give the desired final compounds 5b, 5c, 6a, 7a–f,
and 8a–d as white powders; the chemical characterisation of all
re-synthesised compounds was in good agreement with literature
(see Supporting Material).
O
Figure 1. Chemical structures of well-known CAIs acetazolamide
AAZ, 1) and ethoxzolamide (EZA, 2) and designed 4-(cycloalkyl)-1-
carbonylbenzenesulfonamides.
(

1
188
R. GITTO ET AL.
2
.2. Preparation of the bacterial CAs
and Arg46 of b-CA from V. cholerae using as template the con-
formation of Asp49 and Arg51 of b-CA from Coccomyxa. The
modified structure of b-CA from V. cholerae was minimised using
the conjugate gradient algorithm of NAMD. Minimisation was
performed for 500 steps keeping all atoms fixed except for the
backbones of residue 43–47 and the side-chains of Asp44 and
Arg46: in detail, to maintain the correct orientation of carboxyl-
ate moiety of Asp44 and guanidinium group of Arg46 also these
groups were kept fixed.
The three bacterial enzymes were prepared accordingly to the
24
procedure reported by our groups . Briefly, the GeneArt
Company (Invitrogen), specialised in gene synthesis, designed the
genes encoding for the bacterial a, b, and c- CAs. The BL21 DE3
competent cells (Agilent) were transformed with the expression
vector pET15-b containing the gene encoding for one of the
three CA-classes. Subsequently, bacterial cells were induced with
1
mM IPTG and, after 30 min, treated with 0.1 M ZnCl
2
. After 4 h,
ꢂ
The atom types were assigned using force field CHARMM v22
and the atomic charges according to Gasteiger–Marsili method
cells were harvested and disrupted by sonication at 4 C. After
centrifugation at 12,000ꢃg for 45 min, the supernatant was incu-
bated with His Select HF nickel affinity gel resin (Sigma) equili-
brated in lysis buffer for 30 min. The protein was eluted with the
wash buffer containing 200 mM imidazole. Collected fractions
were dialysed against 50 mM Tris/HCl, pH 8. At this stage of puri-
fication, the protein was at least 95% pure.
3
2
by Vega . Furthermore, in the obtained structure, assuming a
similar binding mode of the AAZ in both of the b-CAs, we
inserted in the catalytic site of b-CA from V. cholerae the X-ray
conformation of the acetazolamide retrieved from b-CA from
Coccomyxa. The side-chains of the residues around the inhibitor
3
3
AAZ have been minimised by LigandScout .
2
.3. Carbonic anhydrase inhibition assay
An Applied Photophysics stopped-flow instrument (Leatherhead,
Surrey (UK)) has been used for assaying the CA catalysed CO
2
.5. Docking studies
The complex obtained was used to perform docking studies by
2
3
4
2
5
GOLD software and the AAZ was discarded. The ligand struc-
tures were constructed by Vega 3.1.1 and the energy was mini-
mised by using the Conjugate Gradient method (1000 steps). The
minimised ligands were docked in their corresponding proteins
hydration activity . Phenol red (at a concentration of 0.2 mM)
has been used as an indicator, working at the absorbance max-
imum of 557 nm, with 20 mM TRIS (pH 8.3) as buffer, and 20 mM
NaClO4 (for maintaining constant the ionic strength), following
the initial rates of the CA-catalysed CO hydration reaction for a using Gold Suite 5.0.1. The region of interest, which was used by
2
period of 10–100 s. The CO
7 mM for the determination of the kinetic parameters (by within 10 Å of the original position of the acetazolamide in the
Lineweaver–Burk plots) and inhibition constants. For each inhibi- model structure.
2
concentrations ranged from 1.7 to the Gold programme, was defined as containing the residues
1
tor, at least six traces of the initial 5–10% of the reaction have
A scaffold constraint (penalty ¼ 5.0) was used to restrict the
been used for determining the initial velocity. The un-catalysed solutions in which the sulfonamide moiety was able to coordinate
rates were determined in the same manner and subtracted from the metal within the catalytic binding site. ChemPLP was chosen
the total observed rates. Stock solutions of inhibitor (10–100 mM) as the fitness function. The standard default settings were used in
were prepared in distilled-deionized water, and dilutions up to all the calculations and the ligands were submitted to 100 genetic
0
.01 mM were done after that with the assay buffer. Inhibitor and algorithm runs. The “allow early termination” command was deac-
enzyme solutions were preincubated together for 15 min at room tivated. Results differing by less than 0.75 Å in the ligand ꢄ all
temperature before assay, to allow for the formation of the E–I atom RMSD were clustered together. The conformations with the
complex or the eventual active site-mediated hydrolysis of the highest fitness values were chosen for both analysis and
inhibitor. The inhibition constants were obtained by non-linear representation.
least-squares methods using PRISM 3 and the Cheng-Prusoff
All obtained complexes were minimised keeping the Zn ion
26–28
35
equation, as reported earlier
, and represent the mean from fixed and using the conjugated gradients algorithm by NAMD .
at least three different determinations. The human CA isoforms The results were displayed using the PyMOL software (https://
were recombinant ones obtained in-house. All salts/small mole- pymol.org).
cules were of the highest purity available, from Sigma-Aldrich
(Milan, Italy).
3. Results and discussion
3.1. Chemistry
2.4. Modelled structure of the opening of the active site of
b–carbonic anhydrase from V. cholerae
As displayed in Scheme 1, the studied 4-(cycloalkyl)-1-carbonyl-
benzenesulfonamides 5a–e, 6a, 7a–f, 8a–d have been re-
synthesised following a synthetic procedure previously reported
by us ; experimental details can be found in “Materials and
Methods” section as well as in Supporting Material.
The structure used to perform docking studies was built on differ-
ent steps. First of all, the crystal structure of closed b-CA from V.
cholerae was retrieved from the RCSB Protein Data Bank (PDB
2
3
2
9
code 5CXK) . The water molecules were discarded, then hydro-
gen atoms were added to the protein by using Discovery Studio
3
0
2
.5.5 software . In the second step, a superimposition of residues
3.2. Carbonic anhydrase inhibition
Cys42, Asp44, Arg46, His98, Cys101, Zn301 of b-CA from V. chol-
erae on corresponding residues Cys47, Asp49, Arg51, His103, The inhibitory effects of this series of sixteen 4-(cycloalkyl-1-car-
Cys106, Zn228 of chain A of X-ray complex of AAZ with b-CA bonyl)benzenesulfonamide derivatives 5a–e, 6a, 7a–f, 8a–c, and
from the unicellular green alga Coccomyxa (Co-CA, PDB code 8d were evaluated against VchCAa, VchCAb, and VchCAc by using
31
3
UCJ) was performed using PyMOL software (https://pymol.org).
The above-mentioned superimposition was useful to modify results are summarised in Table 1 and compared with the well-
the orientation of the side-chains of the two amino acids Asp44 known inhibitor AAZ (1). Moreover, the K values measured
the stopped-flow carbon dioxide hydrase assay. The obtained
i

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1189
O
OH
R
O
S
O
H N
H
N
2
+
3
R
4
i or ii
O
O
N
R 1
] n
O
H N S
N
N
O
2
[
O
S
H N
8 d
2
O
O
R 2
O
5
6
a-e, n= 1
a, n = 2
N
N
O
H N S
N
R 4
O
2
S
O
R
5
H N
7 a-f
R
3
2
8 a-c
O
R 2
R 3
R
R5
R 1
H
4
H
H
H
Cl
Br
H
a
b
c
d
e
H
a
a
b
c
Ph
4-FC H
Ph
2
-CH3
OH
OH
OH
CN
H
Ph
b
c
6
4
3-CH3
4-CH
4-CH -Ph
d
e
3
2
f
H
COCH3
Scheme 1. Reagents and conditions: (i) carbonyldiimidazole (DCI), THF, r.t., 3 h, then RR’NH, DMF r.t., 2 h; (ii) RR’NH, HBTU, DMF, TEA, r.t., overnight.
Table 1. Inhibition of hCA I, hCA II, VchCAa, VchCAb, and VchCAc for com- functionalities at C–4 position of piperidine nucleus induces a
pounds 5a–e, 6a, 7a–f, 8a–c and 8d and acetazolamide (1, AAZ) by a stopped
0
weak decrease of affinity. The presence of 4 -chlorophenyl ring
flow CO
2
hydrase assay.
and hydroxyl group at 4-position of piperidine nucleus of com-
pound 7c afforded equi-active inhibitor of unsubstituted 4-phenyl
analog 7a. Among the series of benzenesulfonamides 8a–d con-
taining the piperazine core, the 4-(4-phenylpiperazine-1-carbonyl)-
a
K
i
(nM)
b
c
Cmp
hCA I
hCA II
5.7
VchCAa
VchCAb
VchCAc
5
5
5
5
5
6
7
7
7
7
7
7
8
8
8
8
a
b
c
d
e
a
a
b
c
d
e
f
a
b
c
9.2
4.4
187
843.2
296.4
528.8
588.9
241.9
275.4
43.3
193.8
45.1
58.8
89.9
72.1
470.4
95.7
275.5
>10,000
>10,000
>10,000
>10,000
3754
>10,000
3308
3714
3945
2776
806.4
6612
>10,000
5008
4816
4156
>10,000
0.63
6.1
0.79
1.5
3.8
0.5
0.6
0.6
0.7
0.6
0.6
3.0
0.5
5.7
2.0
>10,000 benzenesulfonamide (8d) displayed the best inhibitory effect (K
i
>10,000
>10,000
>10,000
>10,000
value of 47.0 nM), thus confirming that the presence of a bulky
aromatic group anchored to the 4-position of cycloalkylamine
nucleus generally improves the affinity against VchCAa. It is inter-
7.7
75.5
85.0
1.7
9.3
6.6
6.3
6.5
7.7
6.8
>10,000 esting to note the impact of the introduction of a methylene spa-
>10,000
>10,000
>10,000
>10,000
cer between piperazine core and phenyl moiety: it was found a
ten-fold reduction of activity of the compound 4-(4-benzylpipera-
zine-1-carbonyl)benzenesulfonamide (8a) (K
i
¼ 470 nM) when com-
>10,000 pared to parent compound 8d. This evidence might be due to a
>10,000
>10,000
>10,000
>10,000
different binding within the catalytic cavity. Moreover, the intro-
duction of a fluorine atom in the para position of the phenyl ring
of benzylpiperazine-sulfonamide 8b (K value of 95.7 nM) improves
the affinity toward VchCAa. On the contrary, the presence of a
0.69
50.1
91.1
250
d
47.0
6.8
i
AAZ (1)
12.1
451
473
aMean from three different assays (errors were in the range of ±5–10% of the benzhydryl moiety (compound 8c, K
¼ 275.5 nM) seems well toler-
i
reported values).
Data reported in reference .
ated to respect the benzyl-fragment (compound 8a).
b,c
23
Concerning the activity on VchCAb, several compounds
proved to be inhibitors at low micromolar concentration showing
against bacterial CAs were compared with an affinity towards inhibition data ranging from 806.4 nM to 6612 nM. Overall, the
23
selected human CA isoforms hCA I and hCA II .
As reported in Table 1, all studied compounds proved to be effects toward VchCAb. The best affinity was found for 4-(4-
inhibitors of the VchCAa and displayed K s falling in a wide range cyano-4-phenyl-piperidine-1-carbonyl)benzenesulfonamide com-
presence of a 4-aryl moiety resulted advantageous for inhibitory
i
of 43.3 and 843.2 nM. In terms of structure-affinity relationship pound 7e (K ¼ 806.4 nM). The presence of other small substitu-
i
considerations, the data collected in Table 1 suggested that the ents, such as acetyl for compound 7f or hydroxyl group for
unsubstituted 4-(piperidine-1-carbonyl)benzenesulfonamide (5a), compounds 7 b, 7c, and 7d did not significantly influence the
the three methyl-substituted analogues 5b, 5c, 5d as well as inhibitory effects toward VchCAb. Moreover, the replacement of
cyclohomologue compound 6a were less active than correspond- piperidine nucleus with piperazine one resulted in divergent
ing 4-aryl-substituted compounds 7a, 7 b, 7c, 7d, 7e, and 7f. effects: (a) it was found a completely loss of inhibitory activity
i
Compound 7a (K value of 43.3 nM) demonstrated the best activity for unsubstituted compound 8a, whereas compounds 8b, 8c,
having the phenyl ring linked to 4-position of piperidine nucleus. and 8d shared similar potency as moderate inhibitors of
The introduction of an additional hydroxyl group, nitrile or acetyl VchCAb class.

1
190
R. GITTO ET AL.
On the contrary, all studied compounds were totally ineffective
inhibitors against VchCAc enzyme. Despite these compounds
demonstrated interesting selectivity against VchCAa to respect b-
and c-classes, they proved to be active inhibitors of ubiquitously
expressed hCAI and hCA II at low nanomolar concentration, thus
impairing their employment as therapeutics in humans.
3.3. Modeling
In order to gain more information about the binding interaction
of the most active compounds into the enzymatic catalytic site,
we carried out a theoretical structural study for understanding
and revealing the binding mode of inhibitors toward bacterial CA
classes. The crystal structure of apoenzyme VchCAb has been
29
recently reported by Ferraroni et al. (PDB code 5CXK) , whereas,
the determined structures of VchCAa and VchCAc from V. cholerae
are not currently available. The crystal structures of b-CA are
29,36–39
reported for other species
; these structural data furnished
information about the homology of their 3-D folds as well as the
shape of catalytic site, that displays the zinc coordinated by two
cysteines, one-histidine and one-aspartic amino acid residue (the
so-called “closed active site”). Particularly, for VchCAbs the cata-
lytic zinc ion is coordinated by Cys42, Asp44, His98, and Cys101.
(See schematic representation in Supporting Material). The cata-
lytic site of VchCAbs assumes the so-called “closed active site”
conformation as an inactive state. The pH-tuned movement of
the Asp 44 residue from metal ion converts the “closed active
site” to the “open active site”. Since the available 3 D structure
the b-CA from V. cholerae was solved only in the “closed active
2
9
site” configuration , the rational design of novel inhibitors
through a classical structure-based approach is currently ham-
pered. To overcome the lack of this crucial structural information,
in the first step of our theoretical study, we performed a simula-
tion for the opening of the active site of b-CA from V. cholerae. In
more details, we superimposed the 3 D X-ray structure of “closed
2
9
active site” of b-CA from V. cholerae (PDB code 5CXK) to “open
active site” of available b-CA from Coccomyxa in complex with
31
inhibitor AAZ (1) (PDB code 3UCJ) . Based on the conformation
of side chain of crucial residues Asp49 and Arg51 of b-CA from
Coccomyxa, we successively modified the orientation of the corre-
sponding side-chain of Asp44 and Arg46, that are considered
responsible for proton shuttling and “closed state” of b-CA. By
assuming that inhibitor 1 displays a similar binding mode in these
two b-CAs, we extracted AAZ from b-CA of Coccomyxa and then
docked this inhibitor in the built 3 D structure of b-CA from V.
cholerae; in the last step, we minimised all system. As a result, we
obtained the hypothetical “open active site” of b-CA from V. chol-
erae as the “first in silico model”, useful for mapping the inhibitor
binding interactions; our idea was to translate these data for
understanding the inhibitory effects of the above mentioned six-
teen 4-(cycloalkyl-1-carbonyl)benzensulfonamide derivatives (5a–e,
Figure 2. Docking poses into our “modelled” VchCAb catalytic cavity for acetazo-
lamide (A), sulfonamide derivatives 5a (B) and 7e (C). The interactions between
the VchCAb and inhibitors AAZ, 5a and 7e were examined using PyMOL and
6a, 7a–f, 8a–d) toward VchCAb.
Figure 2 (A) displays AAZ (1) bound directly to the “hypothetic
open” active site of b-CA from V. cholerae. As expected, the
40
LIGPLUS software; residues involved in hydrophobic interactions and hydrogen
sulfonamide moiety anchors the catalytic zinc ion; additionally, the bonds (yellow dashed lines) and Zn ion coordination are shown.
nitrogen of the N-acetamido group forms a hydrogen bond to the
oxygen atom of Gly102, the thiadiazole ring establishes p/p inter-
action with Tyr83 which is reinforced by further hydrogen bond “modelled” VchCAb catalytic cavity for all 4-(cycloalkyl-1-carbonyl)-
contacts between heterocyclic nitrogen atoms and hydroxyphenyl benzensulfonamide derivatives (5a–e, 6a, 7a–f, 8a–d) bearing the
substituent. This network of interactions might explain the affinity sulfonamide as a minimal structural requirement to anchor the zinc
for VchCAb at submicromolar concentration (K
Table 1), thus supporting the reliability of our modelled VchCAb
catalytic cavity. Then we studied the docking poses into our unsubstituted compound 5a showing the ability to create a
i
value of 451 nM, ion in the deep catalytic site of all CAs.
In Figure 2(B), we described the best pose of prototype

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1191
network of interactions with the deep area of the catalytic site
near to zinc ion. As expected, the sulfonamide group is engaged
in polar contacts with the CA cavity, whereas the remaining por-
tion of molecule occupies the middle area establishing few
hydrophobic interactions with residues Tyr83, Leu113, and
Ala106. Therefore, we hypothesised that these interactions could
4. Cobaxin M, Martinez H, Ayala G, et al. Cholera toxin expres-
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be not sufficient to inhibit VchCAb ( K
i
>10,000 nM ). This consid-
6. Capasso C, Supuran CT. Bacterial, fungal and protozoan car-
bonic anhydrases as drug targets. Expert Opin Ther Targets
2015;19:1689–704.
7. Modak JK, Liu YC, Machuca MA, et al. Structural basis for
the inhibition of Helicobacter pylori alpha-carbonic anhy-
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inhibitors: a patent and literature review. Expert Opin Ther
Pat 2013;23:693–704.
eration might be applicable to justify the low efficacy demon-
strated by sulfonamide derivatives 5 b–d and 6a structurally
related to parent compound 5a. Interestingly, the best active
inhibitor 7e (K ¼ 806.4 nM, see Figure 2(C)) demonstrated the
i
ability to form additional interactions with a cluster of hydropho-
bic residues Ala106, Pro111, and Leu 113 when compared with
unsubstituted analog 5a, thus suggesting that the 4-phenyl sub-
stituent of compound 7e is a crucial fragment to make more
favourable hydrophobic contacts in the top of the cavity. This
result indicates that the ligands having a good affinity towards
VchCAb are expected to establish additional hydrogen bonding
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Curr Top Med Chem 2017;17:1237–48.
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of the enzymatic cavity.
modes of inhibitors to carbonic anhydrases: how to design
specific drugs targeting 15 different isoforms? Chem Rev
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4
. Conclusions
1
1. Supuran CT. Carbonic anhydrase inhibitors and their poten-
tial in a range of therapeutic areas. Expert Opin Ther Pat
2018;28:709–12.
In conclusions, a small series of benzenesulfonamides has been
screened as inhibitors of V. cholerae carbonic anhydrases. Compound
7
e demonstrated interesting affinity against VchCAa and VchCAb 12. De Luca L, Mancuso F, Ferro S, et al. Inhibitory effects and
with K
i
values of 89.9 and 806.4 nM, respectively. The predicted bind-
structural insights for a novel series of coumarin-based com-
pounds that selectively target human CA IX and CA XII
carbonic anhydrases. Eur J Med Chem 2018;143:276–82.
ing mode of compound 7e in the modelled catalytic site of VchCAb
suggests that the introduction of an extra aromatic ring might
improve the contacts in the top area of the catalytic cavity, thus fur- 13. Bruno E, Buemi MR, Di Fiore A, et al. Probing molecular
nishing suggestions for the rational design of new compounds tar-
interactions between Human Carbonic Anhydrases (hCAs)
geting V. cholerae CAs.
and a novel class of benzenesulfonamides. J Med Chem
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017;60:4316–26.
1
1
4. Bruno E, Buemi MR, De Luca L, et al. In vivo evaluation of
selective carbonic anhydrase inhibitors as potential anticon-
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inhibitors: design, synthesis and structural characterization
of new heteroaryl-N-carbonylbenzenesulfonamides target-
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screening for the discovery of new carbonic anhydrase VII
inhibitors. Eur J Med Chem 2014;71:105–11.
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Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This work was financially supported by Fondo di Ateneo per la
Ricerca [PRA grant number ORME09SPNC – Universit ꢀa degli Studi
di Messina].
1
1
ORCID
Rosaria Gitto
http://orcid.org/0000-0003-0002-2253
Claudiu T. Supuran
Clemente Capasso
http://orcid.org/0000-0003-4262-0323
http://orcid.org/0000-0003-3314-2411
1
1
8. Gitto R, Damiano FM, De Luca L, et al. Synthesis and bio-
logical profile of new 1,2,3,4-tetrahydroisoquinolines as
selective carbonic anhydrase inhibitors. Bioorg Med Chem
2
011;19:7003–7.
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