Discovering a new class of antifungal agents that selectively inhibits microbial carbonic anhydrases

Article abstract of DOI:10.1080/14756366.2018.1516652

Infections caused by pathogens resistant to the available antimicrobial treatments represent nowadays a threat to global public health. Recently, it has been demonstrated that carbonic anhydrases (CAs) are essential for the growth of many pathogens and their inhibition leads to growth defects. Principal drawbacks in using CA inhibitors (CAIs) as antimicrobial agents are the side effects due to the lack of selectivity toward human CA isoforms. Herein we report a new class of CAIs, which preferentially interacts with microbial CA active sites over the human ones. The mechanism of action of these inhibitors was investigated against an important fungal pathogen, Cryptococcus neoformans, revealing that they are also able to inhibit CA in microbial cells growing in vitro. At our best knowledge, this is the first report on newly designed synthetic compounds selectively targeting β-CAs and provides a proof of concept of microbial CAs suitability as an antimicrobial drug target.

Full text of DOI:10.1080/14756366.2018.1516652

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Discovering a new class of antifungal agents that
selectively inhibits microbial carbonic anhydrases
Giannamaria Annunziato, Laura Giovati, Andrea Angeli, Marialaura Pavone,
Sonia Del Prete, Marco Pieroni, Clemente Capasso, Agostino Bruno, Stefania
Conti, Walter Magliani, Claudiu T. Supuran & Gabriele Costantino
To cite this article: Giannamaria Annunziato, Laura Giovati, Andrea Angeli, Marialaura Pavone,
Sonia Del Prete, Marco Pieroni, Clemente Capasso, Agostino Bruno, Stefania Conti, Walter
Magliani, Claudiu T. Supuran & Gabriele Costantino (2018) Discovering a new class of antifungal
agents that selectively inhibits microbial carbonic anhydrases, Journal of Enzyme Inhibition and
Medicinal Chemistry, 33:1, 1537-1544, DOI: 10.1080/14756366.2018.1516652
To link to this article: https://doi.org/10.1080/14756366.2018.1516652
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2018, VOL. 33, NO. 1, 1537–1544
https://doi.org/10.1080/14756366.2018.1516652
RESEARCH PAPER
Discovering a new class of antifungal agents that selectively inhibits microbial
carbonic anhydrases
a
b
c
a
c
a
ꢀ
ꢀ
Giannamaria Annunziato , Laura Giovati , Andrea Angeli , Marialaura Pavone , Sonia Del Prete , Marco Pieroni ,
Clemente Capassod, Agostino Brunoa,e, Stefania Contib, Walter Maglianib, Claudiu T. Supuranc
Gabriele Costantinoa
and
b
aDepartment of Food and Drugs, University of Parma, Parma, Italy; Department of Medicine and Surgery, Ospedale Maggiore di Parma,
c
University of Parma, Parma, Italy; Department of NEUROFARBA, Section of Pharmaceutical and Nutraceutical Sciences, University of Florence,
d
e
Firenze, Italy; National Council of Research (CNR), Istituto di Bioscenze e Biorisorse, Napoli, Italy; Experimental Therapeutics Program, IFOM
the FIRC Institute for Molecular Oncology Foundation, Milano, Italy
ABSTRACT
ARTICLE HISTORY
Received 24 July 2018
Revised 21 August 2018
Accepted 22 August 2018
Infections caused by pathogens resistant to the available antimicrobial treatments represent nowadays a
threat to global public health. Recently, it has been demonstrated that carbonic anhydrases (CAs) are
essential for the growth of many pathogens and their inhibition leads to growth defects. Principal draw-
backs in using CA inhibitors (CAIs) as antimicrobial agents are the side effects due to the lack of selectivity
toward human CA isoforms. Herein we report a new class of CAIs, which preferentially interacts with
microbial CA active sites over the human ones. The mechanism of action of these inhibitors was investi-
gated against an important fungal pathogen, Cryptococcus neoformans, revealing that they are also able
to inhibit CA in microbial cells growing in vitro. At our best knowledge, this is the first report on newly
designed synthetic compounds selectively targeting b-CAs and provides a proof of concept of microbial
CAs suitability as an antimicrobial drug target.
KEYWORDS
Carbonic anhydrase
inhibitors; antimicrobial
resistance; drug design;
drug discovery;
antifungal agents
Introduction
simple, but physiologically relevant, process: the hydration of car-
bon dioxide to bicarbonate and protons. CAs are ubiquitously
expressed in almost all living organisms and they are encoded by
seven evolutionarily unrelated gene families17: a-CAs (present in
vertebrates, bacteria, algae, and in the cytoplasm of green plants);
b-CAs (in bacteria, algae, and chloroplast of monocotyledons and
cotyledons); c-CAs (mainly in Archaea and some bacteria); d-CAs
(present only in marine diatoms); f-CAs (in marine phytoplankton),
g-CAs (identified in protozoa), and the recently discovered h-CAs
in the marine diatom Phaeodactylum tricornutum18. All of them
require metal cations to convert carbon dioxide to bicarbonate.
The a-, b-, d-, g-, and perhaps h-CAs use Zn2þ ions at the active
site, whereas the c-CAs are probably Fe2þ enzymes and f-class
uses Cd2þ to perform catalysis reaction. f-CAs bind Cd(II) or Zn(II)
within the active site and are defined as cambialistic enzymes19.
The 3D structural folds for four out of seven CAs were determined
by X-ray crystallographic studies, supporting the notion that their
structural fold as well as their oligomerisation state highly differ
from each other20.
It is widely accepted that the use of antimicrobials allowed the
control of many infectious diseases, while their misuse and over-
use promoted the emergence and spreading of antimicrobial
resistance (AMR)1,2. In a number of relevant pathogens, multidrug
resistance is considered one of the most serious current threats
for global health3,4. The infections caused by resistant microorgan-
isms are difficult to treat, and usually require costly and some-
times toxic alternatives to common therapeutics. Problems related
to AMR are expected to increase unless higher efforts are made to
preserve currently available drugs and to intensify the search for
new antimicrobial agents. In the drug discovery field, different
strategies have been pursued in the past years to identify novel
compounds able to escape existing mechanisms of resistance5.
Cloning and high throughput screening of the genomes of many
pathogens paved the way for the identification of alternative cel-
lular pathways, as reservoir of potentially new antimicrobial tar-
gets considered essential for pathogens metabolism and
replication6–8
.
The conversion of CO2 into bicarbonate and protons is impli-
cated in different physiological and pathological processes21.
Classical CA inhibitors (CAIs) are primary sulphonamides, repre-
sented by the general formula RSO2NH2 (Figure 1), and up to
now there are 30 drugs in clinical use or in clinical development
characterised by the presence of such functional groups. In
addition, it has recently emerged that CAIs have a potential as
In this scenario, we embarked into multidisciplinary activities
aimed at the identification and validation of new targets relevant
for antimicrobial therapy9–13
.
Recently, enzymes belonging to the carbonic anhydrase super-
family (CAs, EC 4.2.1.1)14 emerged as potential drug targets in
antibiotic-resistant pathogens15,16
. Such enzymes catalyse a
CONTACT Giannamaria Annunziato
giannamaria.annunziato@unipr.it
Department of Food and Drugs, University of Parma, Via Parco Area delle Scienze 27/A,
Parma, 43124, Italy
ꢀ
These authors contributed equally.
Supplemental data for this article can be accessed here.
ß 2018 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.

1538
G. ANNUNZIATO ET AL.
anticonvulsant, anti-obesity, anticancer, anti-pain and also as anti- yeast is an important and worldwide distributed human opportun-
infective drugs22.
istic pathogen, and cryptococcosis is associated with significant
In many pathogens, CAs are essential in various steps of
growth and their inhibition leads to growth impairment or growth
defects23. Several of the already clinically approved CAIs were suc-
cessfully tested in vitro against CA enzymes of important human
pathogens24. However, their development as antimicrobial drugs
is hampered by the side effects due to the lack of selectivity
toward the human CA isoforms. In human, 15 different a-class iso-
forms are expressed in many tissues/organs, thus explaining the
side effects, such as hepatic cirrhosis, metabolic acidosis, kidney
stones deposition, and arrhythmia25–28, observed in using CAIs as
therapeutics. Unfortunately, to date, there are no CAIs that select-
ively target only microbial isoforms. The disclosure of highly
selective inhibitors for relevant microbial CAs would thus be of
great value (i) to reduce the off-targeting of the human isoforms,
dramatically reducing the occurrence of side effects; and (ii) to fur-
ther confirm CA enzymes as potential antimicrobial drug targets
in therapeutic interventions against drug-resistant pathogens.
In the effort to identify new chemotypes endowed with the
morbidity and mortality, particularly in immunocompromised
patients29,30; (ii) the spreading of resistant C. neoformans strains
has been reported31, and, furthermore, the antifungal treatment
for cryptococcosis is usually aggressive, toxic, and inefficient32; (iii)
finally, it has been demonstrated that C. neoformans tightly
depends on the activity of the b-CA Can2 for growth, CO2 sensing
and, consequently, pathogenicity and virulence33,34
.
The selected compounds were therefore tested against C.
neoformans strains in different conditions (air or CO2-rich environ-
ment), revealing that our compounds are able to penetrate micro-
bial cells and to engage Can2-CA thus inhibiting yeast growth.
The results highlight that it is possible to identify compounds
selectively targeting different classes of CAs. The ability of our
CAIs to selectively inhibit microbial growth provides a robust
proof of concept that CAs can be suitable targets for drugs specif-
ically acting on microbial pathogens, including fungi.
desired pharmacological profile, we previously reported10 the Results and discussion
identification of a class of CAIs characterised by the presence of a
new potential Zinc Chelating Group (ZCG), which preferentially
Synthesised compounds
Compounds 4 and 5 were obtained from commercially available
interact with microbial CA active sites over the human ones. The
identified compounds showed an interesting degree of selectivity 2-aminonicotinic acid 2, via prior conversion into their methyl
but were characterised by low mM affinity, highlighting the need ester 3 by reaction with trimethylsilyl (TMS)-diazomethane in tolu-
to be optimised in classical medicinal chemistry cycles. We herein
report the optimisation of our class of inhibitors. The previously
identified hit compound (1, Figure 2)10 was used as a starting
point for the hit expansion. All the newly synthesised compounds
were tested against a large panel of different classes of CAs,
allowing us to identify a small sub-set of compounds that inhibit
b- and g-CAs with a selectivity fold greater than 80 over the
human a-CAs.
ene/methanol, followed by ethyltrioxorhenium(VII) (MTO)-catalysed
oxidation of the pyridine nitrogen obtaining compound 4. The
reaction of 4 with a solution of ammonium hydroxide led to
desired compound 5 (Scheme 1).
In some cases, due to the lack of useful pyridine-based starting
materials from readily available commercial sources, the de novo
construction of the pyridine ring, with appropriate substituents,
was necessary. A reported microwave-accelerated, one pot, multi-
component reaction (MCR) was exploited for the synthesis of com-
pounds 12–23, with slight modifications to the original procedure
(Scheme 2). A fast and versatile MCR, already optimised in our lab-
oratories, was followed35, which allowed to obtain, in very few
steps, a series of pyridine N-oxide derivatives with the selected
substituents at the desired positions. In this way, we also afforded
an improved knowledge of the chemical space of the active site.
Irradiating a tube by a microwave oven (MW) containing malono-
nitrile (6), ammonium acetate (7), as ammonia source, an appro-
priate aldehyde (aromatic or aliphatic) (8), and an enolizable,
aliphatic ketone (9), with neither solvent nor catalyst, for 15 min at
100 ꢁC, provided key pyridine products 10a–i in 37–65% isolated
yields. Oxidation of pyridine nitrogen of MCR-derived intermedi-
ates (11a–i) followed by hydrolysis of the cyano group, afforded
compounds 12–23 in good yields.
The most promising compounds were investigated against
Cryptococcus neoformans cells growing in vitro. Such pathogen
was chosen accordingly to the following key observations: (i) this
Figure 1. Commercially available carbonic anhydrase inhibitors.
Compounds 28–31 were obtained from commercially available
compounds 24 and 25, via prior conversion into their methyl
esters 26 and 27 by reaction with TMS-diazomethane in toluene/
methanol, and MTO-catalysed oxidation of the pyridine nitrogen
to obtain desired compounds 28 and 29. The reaction of these
Figure 2. New pyridine N-oxide derivatives and modification sites.
Scheme 1. Reagents and conditions. (a) TMS-diazomethane, toluene/methanol, 0 ꢁC, 30 min, 95%; (b) MTO, 35% aqueous H2O2, EtOH, RT, 3 h, 78%; (c) NH4OH,
RT, 3 h, 98%.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1539
Scheme 2. Reagents and conditions. (a) appropriate aldehyde, appropriate ketone, malononitrile, ammonium acetate, MW, 100 ꢁC, 15 min, 37–65%; (b) MTO 35%
aqueous H2O2, EtOH, RT, 3 h, 78%; (c) 10% KOH, 100 ꢁC, 3 h, 98%.
Scheme 3. Reagents and conditions. (a) TMS-diazomethane, toluene/methanol, 0 ꢁC, 30 min, 95%; (b) MTO, 35% aqueous H2O2, EtOH, RT, 3 h, 78%; (c) NH4OH, RT,
3 h, 98%.
compounds with a solution of ammonium hydroxide led to
Table 1. Synthesised compounds.
desired compounds 30 and 31, respectively (Scheme 3).
In vitro characterisation of CAIs and structure–activity
relationship (SAR) analysis
In a previous work10, we reported the identification of a new che-
motype as prototype of new selective inhibitors for microbial CAs.
The reported compounds relied on the presence of N-oxide pyri-
dine ring as new potential ZCG, showing an encouraging activity,
in the low mM range, and combining an interesting selectivity pro-
file toward the human isoforms. The identified compounds repre-
sented excellent hits to be further optimised in classical medicinal
chemistry optimisation cycles. Prompted by these encouraging
results, we decided to start a hit-to-lead campaign aimed at
improving the activity profile of our class of inhibitors, while pre-
serving the selectivity profile toward the human CA isoforms. The
main sites of scaffold modifications, reported in Figure 2, can be
summarised as follow: (i) retention of the 2-amino N-oxide-pyri-
dine core as main scaffold of most of synthesised compounds; (ii)
modification of the ester moiety in position R with a more meta-
bolically stable functional group; (iii) introduction of alkyl or aro-
matic groups of different nature in position R1; (iv) introduction of
alkyl in position R2 and R3; (v) introduction of a nitrogen atom in
position 4 (X), since the 1-N-oxide pyrazine ring is a scaffold
described in several approved drugs (e.g. Minoxidil36, Acipimox37).
In Table 1, all the synthesised compounds are reported, which
were characterised in vitro for their ability to inhibit different
Compounds
X
R
R1
–H
–H
R2
–H
–H
–i-Pr
–Et
–Me
–Me
–i-Pr
–Et
–Me
–Me
–i-Pr
–Et
–Me
–Me
–H
R3
R4
4
5
C
C
C
C
C
C
C
C
C
C
C
C
C
C
N
N
N
N
–OEt
–NH2
–NH2
–NH2
–NH2
–NH2
–NH2
–NH2
–NH2
–NH2
–NH2
–NH2
–NH2
–NH2
–OEt
–OEt
–NH2
–NH2
–H
–H
–H
–H
–Me
–H
–H
–H
–Me
–H
–H
–H
–Me
–H
–H
–H
–H
–H
–NH2
–NH2
–NH2
–NH2
–NH2
–NH2
–NH2
–NH2
–NH2
–NH2
–NH2
–NH2
–NH2
–NH2
–NH2
–H
12
13
14
15
16
17
18
19
20
21
22
23
28
29
30
31
–i-Pr
–i-Pr
–i-Pr
–i-Pr
–Et
–Et
–Et
–Et
–Ph
–Ph
–Ph
–Ph
–
–
–
–
–Me
–H
–Me
–NH2
–H
Plasmodium falciparum, PfCA); and (iii) 3 b-CAs (Vibrio cholerae,
VchCA; Malassezia globosa, MgCA; C. neoformans, Can2-CA). The
aforementioned CAs were chosen in order to compare the activity
classes of CAs. The panel of CAs is composed as follows: (i) 4 of the synthesised compounds on human isoforms and microbial
a-CAs (three humans CA I, CA II, and CA IX and one bacterial from CAs representing already validated targets for antimicro-
Sulfurihydrogenibium yellowstonensis, SspCA); (ii) 1 g-CA (from bial drugs38,39
.

1540
G. ANNUNZIATO ET AL.
Table 2. Inhibition constants (Ki) of investigated compounds (Cmpd) and selectivity index toward human (CA I, CA II and CA IX) and microbial (Ssp, Pf, Vch, Mg,
Can2) CA isoforms.
Inhibition constant, Ki (lM)
a-family
g-family
b-family
Selectivity index, SI
Compounds
CAI
CAII
CAIX
Ssp
Pf
Vch
Mg
Can2
Ssp
Pf
Vch
Mg
Can2
4
5
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
0.25
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
0.012
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
n.a
46.9
83.6
>100
>100
68.0
59.0
58.5
42.2
75.0
>100
64.4
69.1
9.2
56.6
29.0
9.1
6.0
66.4
5.5
78.2
6.7
6.6
9.2
54.6
8.1
>100
2.1
6.8
2.8
3.2
8.6
3.4
4.7
>100
2.2
>100
52.8
47.9
>100
1.7
9.5
15.4
7.3
7.0
>100
17.9
>100
>100
>100
>100
>100
>100
1.1
2.13
1.68
1
10.86
1.76
3.45
11
1
1
1.89
2.09
1
1
1
1
1
1
58.82
10.53
6.49
13.70
14.28
1
1
1
1
1
47.62
14.70
35.71
31.25
11.63
29.41
21.28
1
45.45
2.86
29.41
1.32
1
12
13
14
15
16
19
20
21
22
23
28
29
30
31
AAZ
>100
>100
>100
>100
>100
46.2
>100
>100
>100
7.1
1
1.47
1.69
1.71
2.37
1.33
1
1.55
1.45
2.07
1
16.67
1.51
18.18
1.28
14.92
15.15
10.87
1.83
12.34
1.71
13.33
11.63
–
2.16
1
1
34.9
3.4
1
1
1
1
48.3
75.9
>100
38.4
62.0
0.451
14.08
2.86
14.92
1
>100
>100
60.8
58.3
7.5
8.6
34.9
6.7
>100
74.0
1
1.65
–
2.60
1.61
–
90.91
76.92
–
1.3
0.01
0.005
0.17
–
AAZ: acetazolamide.
different substituents in position R1, R2, and R3 seem to be coopera-
tive, where R2 and R3 seem to play a predominant role (5 vs.
12–19); (iii) the introduction of bulkier substituents in position R1
are in general tolerated (20–23). The cooperative effects observed
for R1, R2, and R3 can be ascribed to electronic effects of the differ-
ent substituents or to small changes in the ability of the different
derivatives to accommodate inside the PfCA active site, both affect-
ing the ability of the N-oxide moiety to chelate the metal ion.
Lacking of any X-ray crystal structure for PfCA, it is difficult to draw
firm conclusions but of great interest is the possibility to introduce
bulkier substituents in position R1 (20–22), paving the way for fur-
ther rounds of medicinal chemistry optimisation cycles.
On the contrary, for the b-CAs considered in this study three
X-ray crystal structures (Can2-CA, PDB codes 2W3N and 2W3Q;
VchCA, PDB code 5CXK) are reported and can be used to rational-
ise the SAR profile of compounds reported in Table 2. The analysis
of the X-ray crystal structures reported so far for b-CAs highlights
that this sub-family can adopt two different conformations at the
active site level (Figure S2)41: an open conformation likely prone
to host small molecules and a closed conformation which is not
accessible to small molecules and not catalytically active43.
Docking studies were performed on Can2-CA (PDB code: 2W3N),
since it is the only one showing an open conformation and the
binding mode of compound 5 is depicted in Figure 3(A,B). The fol-
lowing key interactions can be observed: (i) the N-oxide group
chelates the Zn2þ metal ion; (ii) the amino group in position 2 H-
bonds the backbone oxygen atom of Gly126; (iii) the amide func-
tional group in position 3 establishes two H-bond with Gln59 and
Asn113, thus explaining why compounds bearing an ester in this
position are inactive (5 vs. 4, 28 and 29); (iv) the other side of the
pyrimidine aromatic ring protrudes toward a roomier hydrophobic
pocket defined by Ala69, Phe87, Val92 and Tyr109, thus explaining
why only small or medium substituents are well tolerated in pos-
ition R2 and R3 (12–14, 19 vs. 20–23, 30, 31, Figure 3(B)); and
finally (v) bulkier substituents in position R1 are not well tolerated
in Can2CA, probably it can be due to the limited size of the roof
of the pocket (Figure 3(B)). The conversion of the pyridine ring
into its aza derivative pyrazine has no effects on the ability of the
compounds to bind Can2-CA.
Figure 3. (A) Binding mode of 5 into the Can2-CA active site, depicted as pink
cartoon and sticks for one Can2-CA monomer and in blue cartoon and sticks for
the other monomer; (B) surface representation of the residue surrounding com-
pound 5, showing that small-medium substituents can be accommodated. The
surface is colour-coded accordingly to (A).
Differently, from our previous work, hCA III was discharged
from the analysis since our class of inhibitors proved to be com-
pletely inactive against this isoform. We introduced hCA IX since it
is the human isoform characterised by the highest binding site
similarity with SspCA (Figure S1), for which our class of com-
pounds showed good inhibitory activity.
In Table 2, the inhibition constant (Ki) values for the different
CAs are reported. Accordingly to our initial hypothesis, all the
investigated compounds are inactive towards the tested hCA iso-
forms. At the same time, the modifications introduced in our main
scaffold led to a drop in activity against SspCA, which is a micro-
bial a-CA. As expected and in agreement with our previous data,
the activity and selectivity are conserved for PfCA, which belongs
to the g-class38 and represents an already identified target for
treating malaria. Most importantly, our compounds showed a
good activity profile toward three CAs belonging to the b-class40
from the following human pathogens V. cholerae41, M. globose42,
and C. neoformans34.
Such results highlight that it is possible to obtain class selectiv-
ity inside the CA super-family. Indeed, most of the modifications
introduced on the main scaffold have dramatically reduced the
propensity of our inhibitors to bind the a-class (the only one
expressed in human) in favour to the g- and b-class.
As expected, the sequence analysis comparison between Vch,
The results reported in Table 2 reveal that for PfCA (i) the intro-
duction of the amide moiety is well tolerated; (ii) the effects of the Mg, and Can2-CA reveals that the enzyme active site is highly

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1541
conserved inside this family. Nevertheless, important amino acidic
substitutions can be identified (Figure S3), supporting the different
profile activity observed for our class of inhibitors for these three
b-CAs (Table 2). Relevant amino acidic substitutions are repre-
sented by: (i) Asn113 in Can2-CA is substituted with Ala and Val in
Mg and Vch, respectively, thus explaining why the ester deriva-
tives showed some degree of activity for Mg and Vch (4, 28, 29,
Table 2); (ii) Ala 69 in Can2-CA is substituted with a Ser residue in
Mg, thus explaining why hydrophobic groups in position R2 and
R3 are less tolerated in Mg with respect to Vch and Can2-CA
(12–23, Table 2); (iii) Tyr109 is substituted with a Phe in Mg and
this modification together with the substitution of Asn113 (see
point (i)) largely affects the electronic nature of the enzyme active
site, thus explaining why Mg and Vch CAs less tolerate the pres-
ence of the pyrazine ring (28–31, Table 2); finally (iv) all the b-CAs
considered are characterised by different sequence length, espe-
cially at the N-ter and active site levels, and we cannot firmly
exclude that such differences can have effects on the ligand bind-
ing mechanism of the different derivatives (Figure S3).
Figure 4. Inhibition of C. neoformans ATCC 6895 growth in liquid medium. Yeast
cells (1.5 ꢂ 106 cells/ml) were grown in YNB broth at 30 ꢁC for 72h, 160rpm, in
presence of the investigated compounds (3mM) and ethoxyzolamide (ETZ), as a
positive control. Growth was assessed using optical density measurements at
540nm (OD540) and percent growth inhibition was calculated in comparison with
cells incubated in a medium added with solvent alone (3% DMSO). The results
are presented as the average of six independent experiments, each carried out in
ꢀ
ꢀꢀꢀ
p < 0.001, vs. control, as determined
triplicate, standard deviation ( p < 0.05,
by one-way Anova with Dunnet post hoc).
These structural information can pave the way for further
round of medicinal chemistry optimisation cycles aimed at
improving the affinity of this class of inhibitors while preserving
selectivity. Nevertheless, at this point the main questions which
still remain to be addressed are: (i) if the inhibition of CAs by our
compounds in whole cells has an effect on microbial growth and
survival, and (ii) in case a phenotypic manifestation is observed, if
this is linked to the inhibition of CAs.
Table 3. Inhibition of C. neoformans ATCC 6895 growth in presence of selected
compounds at 37 ꢁC in the aerobic environment or in 5.5% CO2.
Growth inhibition (%) vs. control
Compound (3mM)
Air
5.5% CO2
,
ꢀꢀ
30
12
ETZ
42.9 26.4
4.2 5.9
25.6 15.6
#
13.3 12.4
0.0 0.0
8.2 11.2
ꢀꢀ
ETZ: ethoxyzolamide.
ꢀꢀ
p < 0.01 vs. control (medium added with solvent alone).
#p < 0.05 vs. compound 30 in 5.5% CO2, assessed by Student’s t test.
Biological characterisation and target engagement of CAIs in
C. neoformans cells growing in vitro
the effect of our compounds was mediated by the interaction
with the fungal CA, the growth of C. neoformans was thus
assessed on agar plates supplemented with compounds 30 and
12 and incubated both in ambient air and in 5.5% CO2 atmo-
sphere. In these conditions, compound 12 had no significant
effect on C. neoformans. Interestingly, the number of colonies of
C. neoformans grown in the presence of compound 30 in ambient
air was significantly decreased by 42.9% as compared to control
(Table 3).
The average diameter of the colonies was also significantly
reduced (average diameter 263.18 75.77 and 858.17 99.66 mm
for treated and control colonies, respectively; P < 0.001 as deter-
mined by Student’s t test; Figure 5).
No significant growth inhibition was observed on plates added
with compound 30 incubated in 5.5% CO2 atmosphere, proving
the complementation of growth when CA activity is not essential
and confirming the enzymatic target of the inhibitor.
Representative images of the analysed plates are shown in
Figure 6.
The concentrations used to inhibit the growth of C. neoformans
were higher than those needed for the inhibition of the purified
CA enzyme, probably because of the barrier role of the fungal cell
wall. Although the pharmacokinetic properties of the compounds
should be optimised for therapeutic purposes in vivo, these results
represent, at our knowledge, the first demonstration of the activity
of selective b-CAs inhibitors against microbial whole cells.
The causative agent of cryptococcosis is a major opportunistic
pathogen, associated with severe meningoencephalitis in
immunocompromised hosts29. Cryptococcus species are inherently
resistant to echinocandins32 and azole-resistance is emerging in C.
neoformans strains4. The current gold standard therapy for crypto-
coccal disease presents significant side effects and is not widely
available in resource-limited healthcare settings32,44,45. The identi-
fication of effective and specific inhibitors of C. neoformans CAs
would thus be enormously beneficial for the development of a
new class of molecules with potential anti-cryptococcal activity.
It has been previously demonstrated that the growth of C.
neoformans can be hindered through supplementation of the
potent Can2-CAIs acetazolamide (AAZ) and ethoxyzolamide
(ETZ)33,34. However, these compounds show stronger effects on
the human cytosolic isoform CA II34 and may lead to toxicity prob-
lems due to the off-targeting.
On the other side, many CAIs that display an excellent selective
activity in vitro against CAs from different bacterial and fungal
pathogens often fail to inhibit microbial growth when challenged
against the whole microorganism, probably because of the diffi-
culty to penetrate inside the cells46. The effect of our inhibitors on
the growth of C. neoformans ATCC 6895 was initially evaluated in
a liquid medium in aerobic conditions. The results showed a stat-
istically significant inhibition exerted by compound 30 (21.5%)
and, to a lower extent, by compound 12 (16.9%) (Figure 4), in line
with the in vitro inhibition of the enzyme (Table 2) and thus con-
firming the activity of these compounds on the whole yeast cells.
In many organisms, including C. neoformans, the deletion of
CA encoding genes or CA inhibition leads to severe growth
defects in ambient air, but CA-deficient microorganisms can grow
Conclusions
The results of this study highlight the possibility to design and
in the atmosphere with higher CO2 concentration because the synthesise compounds that selectively target different classes of
enzyme is dispensable in these conditions40. To demonstrate that CA. Indeed, the newly reported inhibitors preferentially interact

1542
G. ANNUNZIATO ET AL.
Figure 5. Effect of compound 30 on the size of C. neoformans colonies. Phase-contrast microscopy images (4ꢂ) of yeast colonies grown at 37 ꢁC for 120h in aerobic
conditions on YNB agar plates supplemented with (A) 3% DMSO solvent alone, (B) 3 mM compound 30. Bar: 500 mm.
on silica gel-coated aluminium foils (silica gel on Al foils, SUPELCO
Analytical, Sigma-Aldrich) at 254 and 365 nm. Where indicated,
intermediates and final products were purified by silica gel flash
chromatography (silica gel, 0.040ꢃ0.063 mm), using appropriate
1
solvent mixtures. H NMR and 13C NMR spectra were recorded on
a BRUKER AVANCE spectrometer at 400 and 100 MHz, respectively,
1
with TMS as an internal standard. H NMR spectra are reported in
this order: multiplicity and number of protons. Standard abbrevia-
tions indicating the multiplicity were used as follows: s ¼ singlet,
d ¼ doublet, dd ¼ doublet of doublets, t ¼ triplet, q ¼ quadruplet,
m ¼ multiplet, and br ¼ broad signal. HPLC/MS experiments were
performed with an Agilent 1100 series HPLC apparatus, equipped
with a Waters Symmetry C18, 3.5 lm, 4.6 mm ꢂ75 mm column
and an MS: Applied Biosystem/MDS SCIEX instrument, with API
150EX ion source. HRMS experiments were performed with an
LTQ ORBITRAP XL THERMO apparatus.
All compounds were tested at 95% purity or higher (by
HPLC/MS).
Synthetic procedures and compounds characterisation are
reported in Supporting Information.
Figure 6. Inhibition of C. neoformans ATCC 6895 growth on solid medium sup-
plemented with compound 30. Yeast cells (200 cells/plate) were grown in YNB
medium supplemented with compound 30 (3 mM), or solvent alone (control, 3%
DMSO), at 37 ꢁC for 120 h in the aerobic environment or in 5.5% CO2 atmos-
phere. Three plates for each condition were assayed in two independent experi-
ments. Representative images are shown.
Molecular modelling
The X-ray crystal structure of Can2-CA was used to pursue docking
studies by means of Glide47. The protein was prepared with the
protein preparation tool of Maestro47, and the docking grid was
centred on the Zn2þ metal ions. Compound 5 was prepared with
the atom builder tool of Maestro47 each docking run was carried
out using the standard precision (SP) method and the van der
Waals scaling factor of nonpolar atoms was set to 0.8, as previ-
ously reported10. The binding mode having the highest score and
able to recapitulate the reported SAR was selected.
with the b- and g-CAs over the a ones. The most promising com-
pound, in terms of affinity and selectivity, when tested against C.
neoformans, an important human pathogen, revealed its ability to
inhibit microbial growth through CA inhibition, providing a robust
proof of concept that CAs are targets that may be specifically
engaged by molecules lacking host toxicity. Our compounds may
thus represent the prototype of selective CAIs which may be used
for the expansion of the current arsenal of anti-infective drugs.
In vitro CA inhibition assay
An Sx.18Mv-R Applied Photophysics (Oxford, UK) stopped-flow
instrument has been used to assay the catalytic activity of various
CA isozymes for CO2 hydration reaction48. Phenol red (at a con-
centration of 0.2 mM) was used as indicator, working at the
absorbance maximum of 557 nm, with 10 mM Hepes (pH 7.5, for
a- and g-CAs) or TRIS (pH 8.3, for b- and c-CAs) as buffers, 0.1 M
Na2SO4 (for maintaining constant ionic strength), following the
CA-catalysed CO2 hydration reaction for a period of 10 s at 25 ꢁC.
The CO2 concentrations ranged from 1.7 to 17 mM for the deter-
mination of the kinetic parameters and inhibition constants. For
Materials and methods
Chemistry section
All the reagents were purchased from Sigma-Aldrich (Milan, Italy),
Alfa Aesar (Riga, Latvia), and Enamine at reagent purity and,
unless otherwise noted, used without any further purification. Dry
solvents used in the reactions were obtained by distillation of
technical grade materials over appropriate dehydrating agents.
MCRs were performed using CEM Microwave Synthesizer-Discover
model. Reactions were monitored by thin layer chromatography each inhibitor at least six traces of the initial 5–10% of the

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1543
reaction have been used for determining the initial velocity. The
uncatalysed rates were determined in the same manner and sub-
tracted from the total observed rates. Stock solutions of inhibitors
(10 mM) were prepared in distilled–deionised water and dilutions
up to 1 nM were done thereafter with the assay buffer. Enzyme
and inhibitor solutions were pre-incubated together for 15 min
(standard assay at RT) prior to assay, in order to allow for the for-
mation of the enzyme–inhibitor complex. The inhibition constants
were obtained by non-linear least-squares methods using PRISM 3
and the Cheng–Prusoff equation, as reported earlier10,49. All CAs
were recombinant proteins produced as reported earlier by
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The growth inhibition of C. neoformans ATCC 6895 in presence of
compounds 12–15, 28, 30, and 31, and ETZ as a positive control,
was evaluated in YNB medium (2% glucose, 1 ꢂ Difco yeast nitro-
gen base without amino acids), pH 7.0. Yeasts were pre-grown at
37 ꢁC on Sabouraud dextrose agar (SDA) plates for 48 h. The com-
pounds were resuspended in hot DMSO (100 ꢁC) according to
their solubility (concentrations ranging from 300 to 100 mM).
Growth assays in liquid medium were performed on 96-wells
microtiter plates. Yeast cells (1.5 ꢂ 106 cells/mL) were incubated in
200 lL of YNB with 3 mM compounds in ambient air for 72 h at
30 ꢁC, 160 rpm. Three wells for each condition were prepared
together with controls (medium alone and medium with DMSO).
Growth was measured spectrophotometrically, recording the
optical density at 540 nm (OD540) before (t0) and after incubation.
After baseline correction, percent growth inhibition was calculated
using the following formula: 100 – [(OD540 at 72 h – OD540 at t0)/
(OD540 in 3% DMSO at 72 h – OD540 in 3% DMSO at t0) ꢂ 100]. Six
independent experiments were performed. For growth assay in a
solid medium, 200 cells of C. neoformans were spread on YNB
agar supplemented with selected compounds at a concentration
of 3 mM and incubated at 37 ꢁC in air and 5.5% CO2 atmosphere.
After 120 h the colony forming units (CFUs) were counted. Percent
growth inhibition was calculated in comparison to controls as pre-
viously described54. Three plates for each condition were assayed
in two independent experiments. The average colony diameter
per plate was determined from microscope images of 20 ran-
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ware). Statistical analyses were performed by GraphPad Prism 5
software. A p values <0.05 was considered significant.
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Disclosure statement
The authors do not declare conflict of interest.
ORCID
19. Xu Y, Feng L, Jeffrey PD, et al. Structure and metal exchange
in the cadmium carbonic anhydrase of marine diatoms.
Nature 2008;452:56–61.
Claudiu T. Supuran
http://orcid.org/0000-0003-4262-0323
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Biochem J 2016;473:2023–32.
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