Selenides bearing benzenesulfonamide show potent inhibition activity against carbonic anhydrases from pathogenic bacteria Vibrio cholerae and Burkholderia pseudomallei

Article abstract of DOI:10.1016/j.bioorg.2018.05.015

A series of selenides bearing benzenesulfonamide moieties was evaluated as carbonic anhydrase (CA, EC 4.2.1.1) inhibitors against the pathogenic bacteria Vibrio cholerae (VchCAα and VchCAβ) and Burkholderia pseudomallei (BpsCAβ) enzymes. The molecules represent an interesting lead for antibacterial agents with a possibly new mechanism of action showing excellent inhibitory action and selectivity for inhibiting VchCAα and BpsCAβ over the human (h) off-target isoforms hCA I and II. Identification of potent and possibly selective inhibitors of bacteria CAs over the human counterparts may lead to pharmacological tools useful for understanding the physiological role(s) of these under-investigated proteins.

Full text of DOI:10.1016/j.bioorg.2018.05.015

Accepted Manuscript
Selenides bearing benzenesulfonamide show potent inhibition activity against
carbonic anhydrases from pathogenic bacteria Vibrio cholerae and Burkholderia
pseudomallei
Andrea Angeli, Ghulam Abbas, Sonia del Prete, Clemente Capasso, Claudiu T.
Supuran
PII:
S0045-2068(18)30388-2
DOI:
https://doi.org/10.1016/j.bioorg.2018.05.015
YBIOO 2368
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
21 April 2018
15 May 2018
16 May 2018
Please cite this article as: A. Angeli, G. Abbas, S. del Prete, C. Capasso, C.T. Supuran, Selenides bearing
benzenesulfonamide show potent inhibition activity against carbonic anhydrases from pathogenic bacteria Vibrio
cholerae and Burkholderia pseudomallei, Bioorganic Chemistry (2018), doi: https://doi.org/10.1016/j.bioorg.
2018.05.015
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1
Selenides bearing benzenesulfonamide show potent inhibition activity against carbonic anhydrases
from pathogenic bacteria Vibrio cholerae and Burkholderia pseudomallei
Andrea Angeli,a Ghulam Abbas,a,b Sonia del Prete,c Clemente Capasso,c Claudiu T. Supurana*
a Università degli Studi di Firenze, NEUROFARBA Dept., Sezione di Scienze Farmaceutiche, Via Ugo Schiff 6, 50019 Sesto Fiorentino
(Florence), Italy.
b Department of Biological Sciences and Chemistry, University of Nizwa, Birkat Al-Mauz, P.O.Box 33, Nizwa-616, Sultanate of Oman.
cIstituto di Bioscienze e Biorisorse, CNR, Via Pietro Castellino 111, 80131, Napoli, Italy
Abstract. A series of selenides bearing benzenesulfonamide moieties was evaluated as carbonic
anhydrase (CA, EC 4.2.1.1) inhibitors against the pathogenic bacteria Vibrio cholerae (VchCAα and
VchCAβ) and Burkholderia pseudomallei (BpsCAβ) enzymes. The molecules represent an interesting
lead for antibacterial agents with a possibly new mechanism of action showing excellent inhibitory
action and selectivity for inhibiting VchCAα and BpsCAβ over the human (h) off-target isoforms hCA I
and II. Identification of potent and possibly selective inhibitors of bacteria CAs over the human
counterparts may lead to pharmacological tools useful for understanding the physiological role(s) of
these under-investigated proteins.
Keywords: carbonic anhydrase; inhibitors; metalloenzymes; selenium; Vibrio cholera; Burkholderia
pseudomallei
______
Corresponding author. Tel./fax:+39-055-4573729; e-mail: claudiu.supuran@unifi.it (CTS)

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1. Introduction
The carbonic anhydrases (CAs, EC 4.2.1.1) are a superfamily of metalloenzymes which catalyse the
−
interconversion reaction of CO2 and H2O with HCO3 and H+ to perform a pivotal role in pH regulation.1
It is an essential enzyme in all life kingdoms, from Archaea to Bacteria and Eukaryotes.2 To date, was
discovered seven genetically distinct families, named α- (present in vertebrates, protozoa, algae,
cytoplasm of green plants, and in many gram-negative Bacteria), β- (in both gram-negative and -positive
Bacteria, mono and dicotyledons plants, fungi and Archaea), γ- (Bacteria, cyanobacteria and Archaea),
δ-, ζ-, θ- (in marine diatoms) and ɳ- CAs (protozoa belonging to the Plasmodium spp ).1-3 Recently, the
inhibition of bacterial CAs was demonstrated to influence both growth and pathogenicity of
microorganisms,4-6 thus, could be a new approach for obtaining anti-infective agents with a new
mechanism of action compared to classical antibiotics.7 Discover and develop new anti-infective agents,
at present, is inevitable because infectious diseases are the second-leading cause of death in the world. In
this particular contest, Burkholderia pseudomallei and Vibrio cholerae are Gram-negative bacteria that
cause different endemic diseases. The first is a saprophytic bacterium responsible for melioidosis, an
endemic disease present in tropical and sub-tropical areas of the world.8,9 B. pseudomallei is highly
virulent by inhalation and also acquired resistance to penicillin, ampicillin, first-generation and second-
generation cephalosporins, macrolides, quinolones and most aminoglycoside antibiotics.8,9 The second
Gram-negative bacterium, Vibrio cholerae, is causative of cholera, a human disease that is characterized
by massive loss of water and electrolytes, which leads to severe dehydration and hypovolemic shock if
the condition is not treated.10 This study may be of interest for designing new organoselenium inhibitors
of VchCAα, VchCAβ and BpsCAβ that may have clinical applications.11
2. Results and discussion

3
2.1. Chemistry
Commercially available sulfanilamide 1, has been used for preparing the corresponding selenocyanate
derivative 2 and diselenide 3, as reported earlier12 (scheme 1).
Scheme 1. Synthesis of selenocyanate 2 and diselenide 3 derivative.
Thereafter, 3 was reduced with NaBH4 to the corresponding selenolate, which in turn was treated in situ
with the appropriate halo-alkyl moieties 4a-j, affording the selenides 5a-j in good yield (Table 1).13

4
Table 1: Synthesis of selenides bearing benzenesulfonamide moiety 5a-j.

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Finaly, the selenide 6 was obtained from reduction of diphenyldiselenide with NaBH4 and in situ was
added 4-(bromomethyl)benzenesulfonamide to afford in good yield as outlined in Scheme 2.13
Scheme 2. Synthesis of 4-((phenylselanyl)methyl)benzenesulfonamide 6
Our main interest was to investigate structure-activity relationship related to the inhibition of the two
pathogenic bacteria from Vibrio cholerae (VchCAα, VchCAβ) and, Burkholderia pseudomallei
(BpsCAβ).
2.2. Carbonic anhydrase inhibition
The inhibition studies of VchCAα, VchCAβ and BpsCAβ with compounds 2, 3, 5a-j and 6 were
performed in order to detect possible candidates for anti-infective studies. We tested in vitro these
compounds for their inhibitory activity against the two off-targets physiologically relevant hCA
isoforms I, II and the bacterial enzymes VchCAα, VchCAβ and BpsCAβ by means of the stopped-flow
carbon dioxide hydration assay.14 These activities were compared to those of the standard, clinically
used CAI acetazolamide (AAZ) (Table 2).
Table 2. Inhibition data against human (h) isoforms hCA I, II and bacterial enzymes VchCAα, VchCAβ
and BpsCAβ of derivates 2, 3, 5a-j, 6 and acetazolamide (AAZ) by a stopped flow CO2 hydrase assay.14
KI (nM)*
Cmp
VhCAα
VhCAβ
BpsCAβ
hCAI
hCAII
2
3
5a
4.6
5.7
5.9
2172.5
6804.3
7597.7
75.3
83.6
754.2
95.6
1522.7
338.3
53.1
7.9
355.3

6
5b
5c
5d
5e
5f
5g
5h
5i
4.1
6.1
6.5
28.6
6.7
6.0
4.4
4.3
7.5
5.4
6.8
5941.7
8726.9
5264.4
7853.1
2589.9
6900.8
5068.3
5897.1
1852.8
7697.1
451
845.6
529.8
51.9
9.1
21.4
5.4
823.9
876.9
866.6
650.4
745
256.8
352.2
9.7
5.2
7.3
293.5
297.1
21.9
261.7
226.1
250
9.3
73.2
69.9
36.5
9.3
7.6
70.8
6.3
41.2
53.0
12.1
5j
6
AAZ
* Mean from 3 different assays, by a stopped flow technique (errors were in the range of  5-10 % of the
reported values).
The following structure–activity relationship (SAR) can be drawn from the data of this table:
i)
Selenides 2, 5a-j, 6 and diselenide 3 exhibited potent inhibitory activity towards VchCAα with
inhibition constants in the low-nanomolar range, except for compound 5c, which was active in the
medium nanomolar range (overall, the KIs ranged between 4.3 to 28.6 nM). However, small
structural differences in the tail moiety, such as the replacement of methyl moiety (5c) with
different methylene chains (5a-b, 5d-j, 6), did not lead to particular effects on the biological
activity except for selenide 5e where efficacy decreased of about 4 times than the other
compounds.
ii)
All compounds, here considerate, had a poor effects on the VchCAβ with a constant of inhibition
in the micromolar range (KIs ranged between 1.8 to 8.7 μM). This time, diselenide 3 was 3 fold
more potent than selenocyanate derivative 2. Also for this isoform, small structural differences on
the tail did not change significantly the activity except for compounds 5f and 5j that showed an
activity near 2 and 3 times, respectively, more powerful.
iii) The inhibition profile of BpsCAβ differs substantially from the other β-class enzyme (VhCAβ)
investigated here. An interesting inhibition profile was observed for compounds 5e-g where a rigid
scaffold on the tails showed a potent activity against this isoform in low nanomolar range (KIs 5.4

7
and 9.1 nM for 5e and 5g, slightly less efficacy with KIs 21.4 nM for compound 5f). Furthermore,
selenide 2 and diselenide 3 reveal good potency of inhibition in the range of medium nanomolar
(KIs 75.3 to 83.6 nM). On the other hand, scaffolds less rigid lead to drastically decrease the
potency in the range of high nanomolar (KIs spanning between 529.8 to 876.9 nM).
The inhibition profile of the two V. cholerae enzymes and β-class enzyme of B. pseudomallei belonging
to the two different CA families are very diverse from the human isoforms hCA I and II (off-target
isoforms), with the α-class one (VchCA) being much more sensitive to this class of inhibitors compared
to two β-CA investigated here (Table 3).
Table 3. Selectivity ratios for the inhibition of the human isoform hCA II versus the bacterial VchCAα,
VchCAβ and BpsCAβ.
Selectivity ratioa
Cmp
hCA II/ VchCAα
hCA II/ VchCAβ
hCA II/ BpsCAβ
2
3
11.54
1.38
60.22
2.26
12
10.75
1.27
1.38
1.26
16.09
1.46
5.49
9.8
2 10-2
0.7
1 10-3
9.4 10-2
0.47
5a
5b
5c
5d
5e
5f
5g
5h
5i
4.6 10-2
1 10-3
1 10-2
8 10-3
1.3
4
0.43
8 10-3
1.3 10-2
4 10-3
3 10-3
1 10-3
1.4
1.3 10-2
1 10-3
8.6 10-2
7 10-3
4.7 10-2
8.1 10-2
1.6 10-2
5j
6
AAZ
2.2 10-2
7 10-3
1.77
2.6 10-2
a Selectivity as determined by the ratio of Ki for hCA II isoform relative to bacterial isoforms.
A relatively high number of selenides that have been investigated act as effective CAIs against the α-
VhCA. Among them, compound with ethanol moiety (5a) showed the most promising profile for
obtaining selective inhibitors with a selectivity ratio near 60 times upper. Indeed, diselenide 3 had potent

8
inhibition but lose the selectivity over the human isoforms. The selectivity ratio of BpsCAβ differs
substantially from the other β-class enzyme (VhCAβ) investigated here. Only compound 5e showed a
good selective inhibition against this isoform with 4 fold more potency than the off-target isoforms (hCA
I, II) and 3 fold for VhCAα . Selenides 5d and 5g, on the other hand, exhibit slightly selectivity with 1.3
and 1.4 folds respectively.
3. Conclusions
We have investigated a series of selenides bearing benzenesulfonamide functionalities for their
inhibition against CAs from two pathogenic bacteria, Vibrio cholera (VchCAα and VchCAβ) and
Burkholderia pseudomallei (BpsCAβ). Excellent inhibitory activity, in the nanomolar range, was
observed against VchCAα whereas the β class enzymes were much less inhibited, with KIs spanning
between the high nanomolar to the micromolar range. These selenides were also VchCAα-selective
inhibitors over the human off target enzymes hCA I and II. Thus, this series of compounds might be
considered an important lead compound for obtaining more effective and selective CAIs targeting
VchCAα as pharmacological tools useful for understanding the physiological role(s) of these under-
investigated enzymes.
4. Experimental Part
4.1. Chemistry
Compounds 2, 3, 5a-j, 6 were reported earlier by our group.12,13
4.2. Carbonic anhydrase inhibition

9
An Applied Photophysics stopped-flow instrument has been used for assaying the CA catalyzed CO2
hydration activity.14 Phenol red (at a concentration of 0.2 mM) has been used as indicator, working at
the absorbance maximum of 557 nm, with 20 mM Hepes (pH 7.5 for the α-CAs) or TRIS (pH 8.3 for the
β-CAs) as buffers, and 20 mM Na2SO4 (for maintaining constant the ionic strength), following the initial
rates of the CA-catalyzed CO2 hydration reaction for a period of 10–100 s. The CO2 concentrations
ranged from 1.7 to 17 mM for the determination of the kinetic parameters and inhibition constants. For
each inhibitor at least six traces of the initial 5–10% of the reaction have been used for determining the
initial velocity. The uncatalyzed rates were determined in the same manner and subtracted from the total
observed rates. Stock solutions of inhibitor (0.1 mM) were prepared in distilled-deionized water and
dilutions up to 0.01 nM were done thereafter with the assay buffer. Inhibitor and enzyme solutions were
preincubated together for 15 min at room temperature prior to assay, in order to allow for the formation
of the E-I complex. The inhibition constants were obtained by non-linear least-squares methods using
PRISM 3 and the Cheng–Prusoff equation, as reported earlier,15–18 and represent the mean from at least
three different determinations. All CA isoforms were recombinant ones obtained in-house as reported
earlier.15–20
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Highlights
 selenides bearing benzenesulfonamide moieties were tested as carbonic anhydrase inhibitors
 pathogenic bacteria enzymes Vibrio cholerae (VchCAα and VchCAβ) and Burkholderia pseudomallei
(BpsCAβ) were used.
 interesting leads for antibacterial agents were discovered

13
Graphical abstract
Selenides bearing benzenesulfonamide show potent inhibition activity against carbonic anhydrases
from pathogenic bacteria Vibrio cholerae and Burkholderia pseudomallei
Andrea Angeli, Ghulam Abbas, Sonia del Prete, Clemente Capasso, Claudiu T. Supuran*