Phaeodactylum tricornutum as a model organism for testing the membrane penetrability of sulphonamide carbonic anhydrase inhibitors

Article abstract of DOI:10.1080/14756366.2018.1559840

Carbonic anhydrases (CAs) are ubiquitous metalloenzymes, which started to be investigated in detail in pathogenic, as well as non-pathogenic species since their pivotal role is to accelerate the physiological CO₂ hydration/dehydration reaction

Full text of DOI:10.1080/14756366.2018.1559840

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Phaeodactylum tricornutum as a model organism
for testing the membrane penetrability of
sulphonamide carbonic anhydrase inhibitors
Alessandra Rogato, Sonia Del Prete, Alessio Nocentini, Vincenzo Carginale,
Claudiu T. Supuran & Clemente Capasso
To cite this article: Alessandra Rogato, Sonia Del Prete, Alessio Nocentini, Vincenzo Carginale,
Claudiu T. Supuran & Clemente Capasso (2019) Phaeodactylumꢀtricornutum as a model organism
for testing the membrane penetrability of sulphonamide carbonic anhydrase inhibitors, Journal of
Enzyme Inhibition and Medicinal Chemistry, 34:1, 510-518, DOI: 10.1080/14756366.2018.1559840
To link to this article: https://doi.org/10.1080/14756366.2018.1559840
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2019, VOL. 34, NO. 1, 510–518
https://doi.org/10.1080/14756366.2018.1559840
RESEARCH PAPER
Phaeodactylum tricornutum as a model organism for testing the membrane
penetrability of sulphonamide carbonic anhydrase inhibitors
a
c
a
c
Alessandra Rogatoa,b , Sonia Del Prete , Alessio Nocentini , Vincenzo Carginale , Claudiu T. Supuran
and
ꢀ
ꢀ
Clemente Capassoa
b
aInstitute of Bioscience and BioResources, CNR, Naples, Italy; Department of Integrative Marine Ecology, Stazione Zoologica Anton Dohrn,
c
Naples, Italy; Neurofarba Department, University of Florence, Polo Scientifico, Sesto Fiorentino, Florence, Italy
ABSTRACT
ARTICLE HISTORY
Received 24 October 2018
Revised 7 December 2018
Accepted 12 December 2018
Carbonic anhydrases (CAs) are ubiquitous metalloenzymes, which started to be investigated in detail in
pathogenic, as well as non-pathogenic species since their pivotal role is to accelerate the physiological
CO2 hydration/dehydration reaction significantly. Here, we propose the marine unicellular diatom
Phaeodactylum tricornutum as a model organism for testing the membrane penetrability of CA inhibitors
(CAIs). Seven inhibitors belonging to the sulphonamide type and possessing a diverse scaffold have been
explored for their in vitro inhibition of the whole diatom CAs and the in vivo inhibitory effect on the
growth of P. tricornutum. Interesting, inhibition of growth was observed, in vivo, demonstrating that this
diatom is a good model for testing the cell wall penetrability of this class of pharmacological agents.
Considering that many pathogens are difficult and dangerous to grow in the laboratory, the growth inhib-
ition of P. tricornutum with different such CAIs may be subsequently used to design inhibition studies of
CAs from pathogenic organisms.
KEYWORDS
Carbonic anhydrase;
metalloenzymes; sulphona-
mide inhibitors; marine
diatom; membrane
penetrability
1. Introduction
pyrimidine pathway and necessary for DNA/RNA synthesis during
the exponential growth and replication or the parasite4,21. Again,
the growth of two non-pathogenic bacteria Ralstonia eutropha (its
genome encodes for one periplasmic A-CA, two b-CAs, and one
ÇCA) and Escherichia coli (its genome contains one b-CA and one
Ç-CA) is strictly correlated to the CA activity22,23. The two CAs (A
and b) encoded in the genome of the pathogen Helicobacter
pylori, are essential for the acid acclimation and the survival of the
pathogen within the human stomach24–26; Vibrio cholerae uses its
CAs (A, b and Ç) for producing sodium bicarbonate, which is an
inducer of the cholera toxin gene expression27–32. The two patho-
genic bacteria, Brucella suis and Mycobacterium tuberculosis, need
functional CAs for growing33–38. It is readily apparent that CAs
from pathogens are potential drug targets and their inhibition
leads to growth impairment or growth defects of the microorgan-
ism. Fortunately, many CA inhibitors (CAI) exists, which could be
classified as inhibitors binding the metal ion (anion, sulphona-
mides and their bioisosteres, dithiocarbamates, xanthates); inhibi-
tors anchoring to the water molecule/hydroxide ion coordinated
to the metal (phenols, polyamines, thioxocoumarins, sulphocumar-
ins); inhibitors occluding the active site entrance (coumarins and
their isosteres) and inhibitors binding out of the active site39. As
described in the literature, the principal drawback using CAIs as
antiinfectives agents is the lack of selectivity towards the patho-
genic versus human isoforms40–43. For this reason, many research
groups are continually involved in the synthesis of new CAIs or in
the modification/optimisation of the existing inhibitors, which are
The physiologic/biosynthetic processes requiring CO2 or HCO3ꢁ
(respiration, photosynthesis/gluconeogenesis, lipogenesis, urea-
genesis, carboxylation) and biochemical pathways involving pH
homeostasis, secretion of electrolytes, calcification, bone resorp-
tion, transport of CO2, and bicarbonate, etc., are connected with
the interconversion of CO2 to bicarbonate and protons (CO2
þ
H2O ꢀ HCOꢁ3 þ Hþ)1,2. In all living organism, the CO2 hydration/
dehydration reaction is catalysed by a superfamily of ubiquitous
metalloenzymes, known as carbonic anhydrases (CAs, EC
4.2.1.1)3–10, which catalyse these reactions at very high rates, with
a pseudo-first order kinetic constant (kcat) ranging from 104 to
106 sꢁ1 for the CO2 hydration11,12. At the intracellular concentra-
tions of CO2, the uncatalysed CO2 hydration/dehydration reaction
has a too low rate with an effective kcat of 0.15 sꢁ1 for the hydra-
tion reaction, and a rate constant of 50 sꢁ1 for the reverse reac-
tion11,12
. CAs have thus the physiologically pivotal role to
accelerate the CO2 hydration/dehydration reaction significantly, in
order to support the fast processes involving dissolved inorganic
carbon, which otherwise would be impaired without these
enzymes13. In this context, CAs started to be investigated in detail
in pathogenic, as well as non-pathogenic organisms recently, since
it has been demonstrated that CAs are essential for the life cycle
of bacteria, fungi and protozoa4,9,13–20. For example, fungal b-CAs
plays a critical role in the CO2-sensing and in the regulation of
sexual development of many fungal organisms20. Plasmodium fal-
ciparum, which is a protozoan, uses its g-CA for producing HCOꢁ3 ,
which is the substrate of the first enzyme involved in the commonly tested on CAs from mammalian and pathogenic
CONTACT Claudiu T. Supuran
claudiu.supuran@unifi.it
Neurofarba Department, University of Florence, Polo Scientifico, Sesto Fiorentino Florence, Italy;
Clemente Capasso
These authors contributed equally to this work.
ß 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
clemente.capasso@ibbr.cnr.it Institute of Bioscience and BioResources, CNR, Naples, Italy
ꢀ
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
511
organisms11,13–15,20,44–46. Another critical issue is related to the diluting stock solutions in a mixture of mQ water:acetonitrile 1:1
inhibitor penetrability into microbial cells. Many CAIs are very effi- (v/v) up to a concentration of 1.0 mg mLꢁ1 The mass spectra of
each analyte were acquired by introducing, via syringe pump at
10 mL minꢁ1, of the its working solution. Raw-data were collected
and processed by Varian Workstation Vers. 6.8 software.
cient inhibitors when tested in vitro on the purified enzymes
(inhibitors with a nanomolar KI) but showed ineffective in vivo
results when tested on the microorganisms38,47,48. Since it is very
complicated to obtain specific control measures and containment
levels for activities with pathogenic organisms, in this article, we
propose the marine unicellular diatom Phaeodactylum tricornutum
as a model organism for testing the membrane penetrability of
the CAIs. P. tricornutum is a eukaryotic organism characterised by
fusiform cells with a cell wall poor in silica49,50. The genome of
the P. tricornutum encodes for nine CAs: five a-CAs confined in
the matrices of the four-layered plastid membranes, two b-CAs
(PtCA1 and PtCA2) located in the pyrenoid and two mitochondrial
c-CAs49. Recently, in the lumen of the pyrenoid-penetrating thyla-
koid a new class of CAs, named h-CA, has been identified49. The
CA inhibition of marine diatoms, as well as of other microalgae,
lead to growth impairment or defects in the microalga because
these enzymes have a pivotal role in ensuring the supply of inor-
ganic carbon (Ci) to RuBisCO and phosphoenolpyruvate
2.1.1. Synthesis of 4-(t-butyl)-N-(3-methyl-5-sulphamoyl-1,3,4-thia-
diazol-2(3H)-ylidene)benzamide 1 740
O
Cl
O
H2NO
2S
S
H2NO
2S
S
B
NH
N
N
N
N
N
Pyridine
CH3CN dry
1
A
4-t-Butylbenzyl chloride B (1.2 eq) was added to a solution of
5-imino-4-methyl-4,5-dihydro-1,3,4-thiadiazole-2-sulphonamide
A
(0.3 g, 1.0 eq) and pyridine (3.0 eq) in dry acetonitrile (5 ml) at 0 ꢂC
under a nitrogen atmosphere. The solution was stirred at r.t. until
the starting material was consumed (TLC monitoring), then
quenched with HCl 1 M (15 ml). The formed precipitate was fil-
tered-off and purified by silica gel column chromatography elut-
ing with 40% EtOAc in n-hexane to afford the title compound 1
as a white solid. 52% yield; m.p. 295–6 ꢂC; silica gel TLC Rf 0.47
(EtOAc/n-hexane 60% v/v); dH (400 MHz, DMSO-d6): 1.32 (s, 9H,
3 ꢃ CH3), 4.08 (s, 3H, NCH3), 7.56 (d, J ¼ 8.8, 2H, Ar–H), 8.20 (d,
J ¼ 8.8, 2H, Ar–H), 8.46 (s, 2H, exchange with D2O, SO2NH2); dC
(100 MHz, DMSO-d6): 31.6, 35.8, 39.3, 126.3, 130.1, 133.3, 156.7,
159.0, 166.3, 174.3; m/z (ESI negative) 353.0 [M-H]ꢁ.
carboxylase49,51
.
Here, seven inhibitors belonging to the sulphonamide types,
such as the AAZ, MZA, which are clinically used agents, and com-
pounds 1–5 have been explored for their in vitro inhibition of the
diatom CAs and in vivo inhibitory effect on the growth of the P.
tricornutum cell. Our results demonstrate that the growth of the P.
tricornutum cells is affected by the CAIs and the unicellular diatom
represents a good model for verifying the CAIs membrane
penetrability.
2. Material and methods
2.1.2. Synthesis of t-butyl (2–(4-sulphamoylbenzamido)ethyl)carba-
mate 2 484
2.1. Chemistry
Compounds 3–5 used in the work were reported earlier by our
groups52,53. AAZ and MZA were commercially available from
Sigma-Aldrich (Milan, Italy). All the chemicals and solvents were
purchased from Sigma-Aldrich (Milan, Italy). All reactions involving
air- or moisture-sensitive compounds were performed under a
nitrogen atmosphere using dried glassware and syringes techni-
ques to transfer solutions. Nuclear magnetic resonance (1H-NMR,
13C-NMR) spectra were recorded using a Bruker Advance III
400 MHz spectrometer in DMSO-d6. Chemical shifts are reported in
parts per million (ppm) and the coupling constants (J) are
expressed in Hertz (Hz). Splitting patterns are designated as fol-
lows: s, singlet; d, doublet; t, triplet; q, quadruplet; m, multiplet;
bs, broad singlet; dd, double of doubles. The assignment of
exchangeable protons was confirmed by the addition of D2O.
Analytical thin-layer chromatography (TLC) was carried out on
Sigma Aldrich silica gel F-254 plates. Flash chromatography purifi-
cations were performed on Sigma Aldrich Silica gel 60 (230–400
mesh ASTM) as the stationary phase and ethyl acetate/n-hexane
or MeOH/DCM were used as eluents. Melting points (mp) were
H
N
O
H2N
O
NH
SO2
D
2
SO2NH2
HOAt
EDCI, DMAP
H
N
O
O
N
DMF dry
O
OH
H
O
C
2
HOAt (1.2 eq) was added to a solution of 4-sulphamoylbenzoic
acid C (0.2 g, 1.0 eq) and N-boc-ethylenediamine D (1.2 eq) in dry
DMF (3 ml) under a nitrogen atmosphere, followed by DMAP (0.03
eq) and EDCI (1.2 eq). The solution was stirred at r.t. until the
starting material was consumed (TLC monitoring), then quenched
with slush (15 ml) and extracted with EtOAc (2 ꢃ 20 ml). The
organic layers were washed with HCl 0.5 M (2 ꢃ 15 ml) and brine
(2 ꢃ 15 ml), dried over Na2SO4, filtered-off and concentrated under
vacuo. The obtained residue was purified by silica gel column
chromatography eluting with 10% MeOH in DCM to afford the
title compound 2 as a white solid. 73% yield; m.p. 198–199 ꢂC; sil-
ica gel TLC Rf 0.35 (MeOH/DCM 10% v/v); dH (400 MHz, DMSO-d6):
1.41 (s, 9H, 3ꢃ CH3), 3.17 (q, J ¼ 5.6, 2H, CH2), 3.34 (q, J ¼ 5.6, 2H,
CH2), 6.95 (m, 1H, exchange with D2O, CONH), 7.50 (s, 2H,
exchange with D2O, SO2NH2), 7.93 (d, J ¼ 8.4, 2H, Ar–H), 8.02 (d,
J ¼ 8.4, 2H, Ar–H), 8.66 (m, 1H, exchange with D2O, CONH); dC
(100 MHz, DMSO-d6): 29.2, 40.4, 40.4, 78.7, 126.5, 128.8, 138.4,
measured in open capillary tubes with
a
Gallenkamp
MPD350.BM3.5 apparatus and are uncorrected. The solvents used
in MS measures were acetone, acetonitrile (Chromasolv grade),
purchased from Sigma-Aldrich (Milan-Italy), and mQ water 18 MX,
obtained from Millipore’s Simplicity system (Milan-Italy). The mass
spectra were obtained using a Varian 1200 L triple quadrupole sys-
tem (Palo Alto, CA) equipped by Electrospray Source (ESI) operat-
ing in both positive and negative ions. Stock solutions of analytes
were prepared in acetone at 1.0 mg mLꢁ1 and stored at 4 ꢂC.
Working solutions of each analyte were freshly prepared by 147.2, 156.7, 166.3; m/z (ESI negative) 341.9 [M-H]ꢁ.

512
A. ROGATO ET AL.
2.2. Cell culture
present in the sample. The Wilbur-Anderson units (WAU) were
calculated according to the following definition: one WAU of CA
activity is defined as the ratio (T0 ꢁ T)/T, where T0 (the time
needed for the pH indicator colour change for the uncatalysed
reaction) and T (the time needed for the pH indicator colour
change for the catalysed reaction) are recorded as the time (in
seconds) required for the pH to drop from 8.3 to the transition
point of the dye (pH 6.8) in a control buffer and the presence of
enzyme, respectively.
The CCMP632 strain of P. tricornutum (Pt1) Bohlin was obtained
from the Provasoli-Guillard National Centre for Culture of
Marine Phytoplankton. Cultures were grown in f/ꢁ2-1Si medium54 at
18 ꢂC under white fluorescent lights (70 lmol m
s
ꢁ1), 12 h:12 h
dark–light cycle as described by De Riso and co-workers55.
Analyses of the wild-type Pt1 have been performed on cells in
exponential phase of growth and collected 4 h after the beginning
of the light period.
2.7. CA inhibition determination
2.3. Spot test analysis
An Applied Photophysics stopped-flow instrument was used for
assaying the total CAs catalysed CO2 hydration activity of the
algal homogenates. Phenol red was used as pH indicator
(0.2 mM) in 20 mM Hepes as buffer (pH 7.5) working at the
absorbance maximum of 557 nm or, alternatively, bromothymol
blue (0.2 mM in 20 mM Tris, pH 8.3) working at an absorbance
maximum of 602 nm. 20 mM Na2SO4 was added to maintain con-
stant the ionic strength in both media. The initial rates of the
CA-catalysed CO2 hydration reaction were followed for a period
of 10–100 s. For each inhibitor at least six traces of the initial
5–10% of the reaction are used for determining the initial vel-
ocity. The uncatalysed rates are determined in the same manner
and subtracted from the total observed rates. Stock solutions of
inhibitor (0.1 mM) were prepared in distilled-deionised water and
dilutions up to 0.01 nM were done thereafter with the assay buf-
fer. The inhibitor and enzyme solutions were pre-incubated
together at room temperature prior to assay, in order to allow
for the formation of the E-I complex. The rates of the CA-cata-
lysed reaction were followed by measuring the absorbance at
the kmax of the proper pH indicator, with the inhibition constants
obtained by non-linear least-squares methods using PRISM 3 and
the Cheng–Prusoff equation as the mean from at least three dif-
ferent determinations.
Different dilutions of wild-type Pt1 cells (0.5 ꢃ 107; 0.75 ꢃ 107;
1 ꢃ 107; 1.5 ꢃ 107 and 2 ꢃ 107 cells) were spotted on f/2-Si agar
plates (volume of the spot 5ml). Cells were inoculated with CAIs
(AAZ, MZA, 1–5) diluted with 10% DMSO at two different concen-
trations: 0.4 and 1.0 mM. Plates inoculated in an identical manner
but only with 10% DMSO and plates inoculated only with f2/-Si
were used as control. Cell survival was monitored after 5 days of
growth at 18 ꢂC under white fluorescent lights (70 lmol m–1 s–1),
12 h:12 h dark–light cycle.
2.4. Enzyme purification
All the purification steps were carried out at a temperature of
4 ꢂC. Approximately, 10 g of pelleted diatom culture were homo-
genised in 20 ml of 20 mM Tris-HCl buffer pH 8.3 containing
10ꢁ3 M PMSF, 10ꢁ3 M benzamidine and 2 ꢃ 10ꢁ3 M EDTA. The
homogenate was centrifuged twice for 30 min at 12,000ꢃg, and
the resulting supernatant was centrifuged again for 45 min at
100,000ꢃg. The hydratase activity was detected in the supernatant
fraction. This fraction was dialysed against 20 mM Tris–HCl buffer
pH 7.5 and further purified by affinity chromatography on a p-
aminomethylbenzenesulphonamide agarose resin (pAMBS; Sigma-
Aldrich). 1 ml of pAMBS resin was applied to an empty column
(BioRad) and equilibrated with 0.1 M Tris-HCl, pH 7.5 buffer con-
taining 0.2 M K2SO4, 0.5 mM EDTA. The sample containing about
1 mg of total protein was loaded on the p-AMBS column equili-
brated as aforementioned. Unbound proteins were removed by
washing extensively with the same buffer. The bound carbonic
anhydrase was eluted using 0.4 M KSCN dissolved in 0.1 M
Tris–HCl, pH 7.5 buffer. The CA-containing fractions were pooled,
dialysed and concentrated by ultrafiltration. The CA-containing
sample was subject to SDS-PAGE.
2.8. Lipophilicity determination
Prediction of logP is a method for obtaining information on the
partition coefficient of a compound. The platform SwissADME
(http://www.swissadme.ch/) was used to calculate the logP of the
sulphonamide compounds58.
3. Results and discussion
3.1. Lipophilicity of the sulphonamide CAIs
2.5. SDS-PAGE
Sulphonamides inhibitors with the sulphamoyl group have been
extensively studied as CAIs because of their ability to coordinate
the metal ions of CA with high affinities (e.g. 106–109 Mꢁ1 for the
human isoform, hCA II)39. The zinc is located at the bottom of a
deep conical cavity and is coordinated by three histidine ligands
(e.g., a-CAs) or two cysteines and one histidine (e.g., b-CAs) and a
hydroxide ion with tetrahedral geometry (Figure 1)39. The sul-
Sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis
(PAGE) was carried out according to Laemmli56. Samples were dis-
solved in buffer with 5% b-mercaptoethanol. Gel was stained with
Coomassive blue.
2.6. Colourimetric carbonic anhydrase assay
phonamide inhibitor nitrogen atom displaces
a zinc-bound
hydroxide from the active site to form a stable enzyme-inhibitor
complex, as elucidated through numerous solved crystallographic
structures of various CA-inhibitor adducts39. For an optimal in vivo
activity, a balanced hydrosolubility and liposolubility of the sul-
phonamide inhibitors are necessary, even if some sulphonamides
CA activity assay was a modification of the procedure described
by Capasso et al.57. Briefly, the hydratase assay was performed at
0 ꢂC using CO2 as substrate following the pH variation due to the
catalysed conversion of CO2 to bicarbonate. Bromothymol blue
was used as pH indicator. The production of hydrogen ions dur-
ing the CO2 hydration reaction lowers the pH of the solution characterised by low lipid solubility, such as acetazolamide (AAZ),
leading to a colour transition of the dye. The time required for are used as effective drugs for a long period. In the present paper,
the colour change is inversely proportional to the amount of CA seven sulphonamides inhibitors were investigated for their

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
513
Figure 1. (A) The active site of hCA II as representative of hCA isoforms. The catalytic triad is indicated with the residues: H94, H96 and H119 and (B) The catalytic
site of the b-CA from P. tricornutum (PtLCIB4). The metal (yellow sphere) is coordinated by two Cys (C46 e C126) and one His (H102) residues and one water molecule
(red sphere).
O
N
H2NO2S
O
N
S
H2NO2S
H2NO2S
H2NO2S
S
O
S
NH
O
H
N
N
N
N
N
N
O
N
N
H
O
AAZ
MZA
1
2
H2NO2S
H2NO2S
O
H2NO2S
O
N
N
O
CF3
NH
N
N
S
N
N
N
N
H
O
3
4
5
Figure 2. Sulphonamide compounds investigated for their effect on the growth of P. tricornutum cells.
lipophilicity and effects on the growth of P. tricornutum: acetazola- culture. Using CO2 as a substrate, the activity of PtrCAs in the
extract was determined. Most of the CA activity was recovered in
the soluble fraction of cellular extract after centrifugation and the
calculated specific activity was 200 Wilbur-Anderson units (WAU)
mgꢁ1 of protein. The extract containing the diatom hydratase
activity was further purified by p-aminomethylbenzenesulphona-
mide (pAMBS) affinity chromatography because most of the dia-
tom CA-classes show affinity for this resin as described in the
literature59. In fact, the resin consists of a cyanogen bromide-acti-
vated agarose matrix to which the primary amino group of the
pAMBS has been attached. In Figure 3, the SDS-PAGE acquired
after the diatom culture homogenisation and column affinity chro-
matography is shown. The whole native CAs were purified to
apparent homogeneity from the other proteins, as indicated by
the two single bands evidenced on the SDS-PAGE. The molecular
weight estimated by SDS-PAGE was of about 28.0 and 25.0 kDa
under reducing conditions. Interesting, the determination of the
theoretical molecular weight of the different diatom CA classes,
calculated from their amino acid sequences, showed that most of
them had a Mw corresponding to 28 and 25 kDa. The diatom CAs
were indicated with the acronym PtrCA and are mainly repre-
sented by these two bands, as shown by SDS-PAGE (Figure 3).
mide (AAZ), methazolamide (MZA), and compounds 1–5
(Figure 2)52,53. The partition coefficient (P) in the system octanol/
water, which is one of the essential factors for evaluating the drug
penetrability through a biological membrane was calculated by
using various commercially available programmes. The seven CAIs
showed a lipophilicity consensus ranging from ꢁ0.28 to 2.55
(Table 1). A negative consensus LogP value means the compound
has a higher affinity for the aqueous phase; when the consensus
is 0, the compound is equally partitioned between the lipid and
aqueous phases; a positive consensus LogP value denotes a
higher concentration in the lipid phase (the compound is more
lipophilic). The lipophilicity increases as the consensus LogP values
increase (Table 1). From Table 1, it is possible to note that only
three of the investigated sulphonamides have low lipid solubility
(AAZ, MZA and 5), which all incorporate predominantly hydro-
philic moieties. The remaining sulphonamides also possess various
highly lipophilic moieties (tert-Bu-phenyl; phenylthio, T-Bu, trifluro-
methyl-phenyl, etc) which lead to positive LogP values and thus
an enhanced liposolubility over AAZ and MZA (Table 1).
3.2. CAs from P. tricornutum
3.3. In vitro activity/inhibition of the diatom CAs
The genome of P. tricornutum encodes for nine CAs belonging to
the a-, b-, c-, and h-CA classes49. The native P. tricornutum CAs
The sulphonamide inhibitors described in the previously para-
(PtrCAs) were extracted and purified from 10 g of pelleted diatom graph, acetazolamide (AAZ) and methazolamide (MZA), which are

514
A. ROGATO ET AL.
Table 1. The lipophilicity of the seven sulphonamides used in the pre-
sent study.
Log Po/w
Compound iLOGP XLOGP3 WLOGP MLOGP SILICOS-IT Consensus
AAZ
MZA
1
2
3
4
5
ꢁ0.21
0.05
2.23
1.78
1.67
2.45
0.87
ꢁ0.26
0.03
ꢁ0.34
2.25
1.67
3.14
4.59
ꢁ0.38
ꢁ2.75
ꢁ1.88
0.96
0.34
1.80
1.88
ꢁ0.99
ꢁ0.33
ꢁ0.70
ꢁ0.40
2.21
0.81
1.91
2.55
ꢁ0.28
0.13
0.02
3.46
0.37
1.95
2.30
2.13
ꢁ0.12
0.99
1.50
0.01
ꢁ0.92
The logP octanol-water partition coefficient (Log Po/w) value has been obtained
using five prediction methods. The consensus is the average of the five predic-
tions methods (iLOGP, XLOP3, WLOGP, MLOGP and SILICOS-IT) and the values
are between ꢁ0.70 and þ2.55.
clinically used agents, and compounds 1–5, were used for deter-
mining the IC50 values relative to the inhibition of the PtrCAs puri-
fied from the marine diatom P. tricornutum as described above
(Table 2). These values were compared with those obtained for
the two human CA isoforms (hCA I and hCA II). All the inhibitors
considered resulted very efficient versus PtrCAs, with IC50 in a
Figure 3. SDS-PAGE of the whole CAs purified by P. tricornutum cellular culture.
Lane STD, molecular markers, MW starting from the top: 50, 37, 25, 20, and
15 kDa; Lane 1, diatom cell extract; Lane 2, purified whole CAs from the affinity
column purification.
Table 2. Inhibition data with the sulphonamides reported here
of the two human isoforms, hCA I and hCA II, and the diatom
CAs, PtrCAs.
range of 8.6–96.6 nM, and the human isoform hCA II (IC50
¼
1.2–42.1 nM). These inhibitors were less efficient for the human
isoform hCA I, and two of them resulted in inefficient inhibitors
(IC50 1 ¼ 1458.9 or IC50 4 > 10,000). It is interesting to note that
among the three CAIs with a low lipophilicity (see Table 1), only
the AAZ has an IC50 of 8.6 nM, which resulted ten times lower
respect to those of the two inhibitors with a negative consensus
(MZA and compound 5) and the four inhibitors with a good lipo-
philicity (compounds 1, 2, 3 and 4) (Table 2).
IC50 (nM)a
Inhibitor
hCA I
hCA II
PtrCAs
AAZ
MZA
1
350
97
1458.9
375.7
21.1
27.5
7.2
28.7
1.5
8.6
92.3
85.0
91.6
78.6
96.7
86.0
2
3b
195.7
>10,000
4b
15.7
42.1
5c
658.9
The CO2 hydratase assay was determined using the stopped-
flow technique.
3.4. In vivo inhibition of growth in the presence of
sulphonamides
aMean from 3 different assays, by a stopped flow technique
(errors were in the range of 5–10% of the reported values).
52
bFrom Reference
cFrom Reference
.
.
The in vivo cell wall penetrability of the sulphonamide inhibitors
AAZ, MZA, 1–5 cannot be easily quantified, and thus we tested
their effects on the cellular growth of P. tricornutum. Different
numbers of diatom cells were spotted on the agar plates contain-
ing two different concentrations of each inhibitor (0.4 and 1 mM)
(Figures 4 and 5). The agar plate free of the inhibitor and the
plate inoculated with the solvent (10% DMSO) were used as con-
trols. Diatom cell survival was monitored for five days under nor-
mal light conditions, at 18 ꢂC. Under laboratory conditions, the
effect of the inhibitors on the diatom growth is evidenced by the
disappearance of the diatom colonies. As evidenced from Figures
4 and 5, all the sulphonamides CAIs, which were characterised in
vitro for their ability to inhibit PtrCAs (Table 2), impaired the
growth of P. tricornutum cells when the plates were supplemented
with these sulphonamides at concentrations of 0.4 and 1.0 mM.
Compounds 4, 3, 1 and 5 showed a noticeable effect on the cellu-
lar growth at 0.4 mM concentrations, compared to the controls,
and this effect was more pronounced at a low number of cells
(Figure 4). At higher concentration of inhibitors (1 mM) it is readily
apparent that MZA, 2 and 3 resulted to be more effective than
the other inhibitors investigated here (Figure 5). However, except
for compound 3, these compounds showed different behaviour
when tested at the two different concentrations. For example,
MZA seems to be not efficient at 0.4 mM, but it interferes effi-
ciently with the diatom growth when used at 1.0 mM (Figures 4
and 5). The stronger effect at a higher concentration of some of
these inhibitors may be due to the toxicity problems caused by
the off-targeting of other proteins than CAs. Interesting, 10%
53
the cellular diatom growth (Figures 4 and 5). Besides, the results
obtained using a concentration of inhibitors of 0.4 mM reflect
those of the lipophilicity. AAZ, MZA and 5, which have low lipo-
philicity, were the less effective inhibitors of the diatom cell
growth because of difficulties to penetrate through the mem-
brane. AAZ has a LogP of ꢁ0.70 and is an excellent in vitro inhibi-
tor when tested on the whole diatom CAs. This inhibitor failed to
show growth impairment of the cells when used at 0.4 mM, while
MZA and 5, with lipophilicity values higher than AAZ, showed a
better inhibition of growth at 0.4 mM (Figure 4).
4. Conclusions
In this study, we proposed the marine unicellular diatom
Phaeodactylum tricornutum as a model organism for testing the
membrane penetrability of CAIs and their effects on the growth of
the organism. P. tricornutum is an eukaryotic organism character-
ised by fusiform cells with a cell wall poor in silica49,50. The scar-
city of silica in the composition of the cell wall makes possible to
consider these diatoms as a good model for testing the mem-
brane penetrability of small molecules, such as the CA sulphona-
mide inhibitors. The seven sulphonamides inhibitors AAZ, MZA,
and compounds 1–5, resulted to be effective inhibitors of the dia-
tom CAs when tested in vitro (IC50–s in the range of 8.6–96 nM).
DMSO, which was the solvent of the inhibitors, had no effects on Some of these inhibitors probably can cross the membrane of P.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
515
Figure 4. Diatom cells were spotted on the agar plates containing 0.4 mM of each sulphonamide inhibitors. Panel containing the agar with the inhibitor has been indi-
cated with the acronym of the inhibitor used. The panel C1 and AAZ are separated by the other groups because obtained from a different set of experiments.
Legend: C and C1, panels with the plate free of the inhibitor; 10% DMSO, panel with the plate inoculated with 10% DMSO; Panels 1, 2, 3, 4, 5, MZA, AAZ, plates with
the sulphonamide inhibitors; Number of cells going from left to right: 2.0 ꢃ 107; 1.5ꢃ 107; 1.0ꢃ 107; 0.75 ꢃ 107 and 0.5ꢃ 107. Diatom cell survival was monitored after
five days under normal light conditions and 18 ꢂC. The disappearance of the diatom colonies evidences the inhibitor effect of the CAIs.
Figure 5. Diatom cells were spotted on the agar plates containing 1.0 mM of each sulphonamide inhibitors. Legend of Figure 5 is the same as Figure 4 except for the
AAZ panel, which, here, is part of the same set of experiments. The disappearance of the diatom colonies evidences the inhibitor effect of the CAIs.

516
A. ROGATO ET AL.
tricornutum possessing both hydrophobic and hydrophilic moieties
in their molecule. The most efficient in vivo inhibitors were com-
pounds 4, 3, 1 and 5 with a noticeable inhibitory growth effect at
a low number of cells. Furthermore, these sulphonamides inhibi-
tors showed a different behaviour when tested at the concentra-
tions of 0.4 and 1.0 mM, which is difficult to rationalise. For
example, MZA was not efficient at 0.4 mM, but it interfered effi-
ciently with the diatom growth when used at 1.0 mM. Probably
this effect might be due to the off-targeting of other proteins
than the CAs considered here. In general, these results demon-
strate that P. tricornutum might be considered as an excellent and
rather simple model for testing the membrane penetrability of
new CAIs and their effects on the growth of the organism.
Considering that many pathogens are difficult and dangerous to
grow in the laboratory, the preliminary results obtained for the
growth inhibition of P. tricornutum with different such CAIs may
be subsequently used to design inhibition studies of CAs from
pathogenic organisms, which may pave the way to novel anti-
infectives with a diverse mechanism of action.
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Disclosure statement
No potential conflict of interest was reported by the authors.
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Funding
Ente Cassa di Risparmio di Firenze, Italy, is gratefully acknowl-
edged for a grant to A.N (ECR 2016.0774).
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ORCID
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the beta-carbonic anhydrase from the pathogenic bacterium
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Chem 2017;25:4800–4.
Claudiu T. Supuran
Clemente Capasso
http://orcid.org/0000-0003-4262-0323
http://orcid.org/0000-0003-3314-2411
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