Inhibition pattern of sulfamide-related compounds in binding to carbonic anhydrase isoforms I, II, VII, XII and XIV

Article abstract of DOI:10.1016/j.bmc.2012.10.048

A set of sulfamides and sulfamates were synthesized and tested against several isoforms of carbonic anhydrase: CA I, CA II, CA VII, CA XII and CA XIV. The biological assays showed a broad range of inhibitory activity, and interesting results were found for several compounds in terms of activity (K_i <1 μm) and selectivity: some aromatic sulfamides are active against CA I, CA II and/or CA VII; while they are less active in CA XII and CA XIV. On the other hand, bulky sulfamides are selective to CA VII. To understand the origin of the different inhibitory activity against each isozyme we used molecular modeling techniques such as docking and molecular dynamic simulations.

Full text of DOI:10.1016/j.bmc.2012.10.048

Bioorganic & Medicinal Chemistry 21 (2013) 1410–1418
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Inhibition pattern of sulfamide-related compounds in binding to carbonic
anhydrase isoforms I, II, VII, XII and XIV
Luciana Gavernet a, , José L. Gonzalez Funes a, Pablo H. Palestro a, Luis E. Bruno Blanch a,
⇑
Guillermina L. Estiu b,c, Alfonso Maresca d, Ivana Barrios a, Claudiu T. Supuran d
a Medicinal Chemistry, Department of Biological Sciences, Faculty of Exact Sciences, National University of La Plata, 47 and 115, La Plata B1900BJW, Argentina
b Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN, USA
c Center for Rare and Neglected Diseases, University of Notre Dame, Notre Dame, IN, USA
d
`
Universita degli Studi di Firenze, Polo Scientifico, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3, 50019 Sesto Fiorentino, Florence, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 29 November 2012
A set of sulfamides and sulfamates were synthesized and tested against several isoforms of carbonic
anhydrase: CA I, CA II, CA VII, CA XII and CA XIV. The biological assays showed a broad range of inhibitory
activity, and interesting results were found for several compounds in terms of activity (Ki <1 lm) and
Keywords:
Carbonic anhydrase
Sulfamides
Docking
Molecular dynamic simulations
Inhibition pattern
selectivity: some aromatic sulfamides are active against CA I, CA II and/or CA VII; while they are less
active in CA XII and CA XIV. On the other hand, bulky sulfamides are selective to CA VII. To understand
the origin of the different inhibitory activity against each isozyme we used molecular modeling tech-
niques such as docking and molecular dynamic simulations.
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
against CA II, and we interpreted this tendency using molecular
modeling techniques.7 Following this research, we report here
The inhibition of Carbonic anhydrases (CAs) constitutes an
attractive objective in medicinal chemistry. In addition to the tra-
ditional role of CA inhibitors as diuretic and antiglaucoma drugs,
several clinical applications have been discovered. Among them,
antiobesity, antiepileptic, antiinfective and antitumor activity can
be mentioned.1–4
the inhibitory profile of a set of sulfamides and sulfamates against
several CA isoforms. The compounds reported have been previ-
ously synthesized and tested in their anticonvulsant activity.8,9
It is well known that a number of CA inhibitors (CAIs) are/were
clinically used to treat epilepsy (i.e. topiramate and acetazolamide
among others). However, the relationship between CA inhibition
and the anticonvulsant properties of these compounds cannot be
directly assumed mainly due to three factors: (1) There is no com-
plete information about the cellular basis of human epilepsy. It is
known that events associated with the pH shifts, the increase in
extracellular potassium concentration and the modification of the
extracellular ionic environment are involved in the etiology of con-
vulsive activity;10–13 but the exact mechanisms that trigger sei-
zures are not entirely elucidated. (2) The anticonvulsant drugs
act through several mechanisms of action and most of them inter-
act with more than one receptor to exert their action.14 Thus, tar-
geting the inhibition of CA as the unique mechanism for the
anticonvulsant action probably will not resolve the problem, but
it will contribute to elucidate the importance of this enzyme in
the development of seizures. (3) There are many isoforms of CA
present in the brain and some of them (such as CAs II, VII and
XIV), have been pointed out for their contributions to epileptiform
activity.15–19
Despite the fact that sixteen
a-isozymes have been isolated in
mammals with different subcellular localization and tissue distri-
bution, all of them have a high degree of active site conservation.
The active site comprises a Zn+2 ion coordinated by the imidazole
rings of 3 histidine residues with the fourth position occupied by
a water molecule at acidic pH (<8) and by a hydroxide ion at higher
pH.1
In all the known isozymes, most of the inhibitors exert their ef-
fect by substituting the non-protein ligand in its interaction with
the Zn+2 ion to generate the tetrahedral adduct. The metal ion is lo-
cated in the bottom of a half hydrophilic and half hydrophobic
cleft, through which the ligand reaches the active site. Typically,
strong inhibition was achieved by sulfonamides, but in the last
decade it was demonstrated that their bioisosteres sulfamides
and sulfamates can exert the inhibitory action in the same man-
ner.1,5,6 Recently we reported an investigation about the weaker
inhibition pattern shown by sulfamides as compared to sulfamates
This research is focused in exploring the inhibition of sulfon-
amide bioisosteres on CA I and other four CNS-related CA isoforms:
CA II, CA VII, CA XII and CA XIV. CA I, CA II and CA VII are cytosolic
⇑
Corresponding author. Tel.: +54 0221 423 5333; fax: +54 0221 422 3409.
E-mail address: lgavernet@biol.unlp.edu.ar (L. Gavernet).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.10.048

L. Gavernet et al. / Bioorg. Med. Chem. 21 (2013) 1410–1418
1411
isozymes whereas CA XII and CA XIV are membrane-bound pro-
teins with an extracellular enzyme active site.1 These environmen-
tal divergences, as well as the slightly structural differences in the
active site region for each isozyme, provide the opportunity of
designing new selective inhibitors. For example, it has been re-
cently demonstrated that CA XII (and CA IX) represents an interest-
ing target for anticancer drugs, particularly in diseases associated
with hypoxic tumors.20 Hydrophilic glycosyl sulfonamide inhibi-
tors of CA IX and CA XII have been designed to minimize the diffu-
sion through lipid membranes and to promote the selective
inhibition of transmembrane CAs.21 Similarly, this possibility has
been explored through the synthesis of impermeant sulfonamides,
which were designed to be positively charged in order to minimize
their transport through the lipophilic membrane.22 On the other
hand, the sulfamide analogue of topiramate (anticonvulsant drug)
constitutes an interesting example of selectivity based on struc-
tural differences in the vicinity of the active site. Its weak binding
to CA II has been attributed to the unfavorable Van der Waals con-
tacts between the ligand and one residue of CA II (Ala65).4 This res-
idue is present only in the CA II isozyme among many others
isoforms in which the ligand shows good inhibitory properties.
Following this general idea, we decided to investigate the ef-
fects that may contribute to the differences observed in the activity
of sulfamides/sulfamates in CA isozymes. Important interactions in
the CA active sites were analyzed using molecular modeling
techniques. To this end, a set of 23 compounds (Fig. 1) was pre-
pared according to typical procedures, and their Ki values were
determined for the CA isozymes (Table 1). The set includes sulfa-
mates (compounds 1–2, 23), sulfamides substituted in one N atom
(3–4) and their synthetic precursors (5–6), aryl, alkyl and cyclo-
alkyl N,N0-disubsituted sulfamides (7–12), tetra-substituted sulfa-
mides (13–15) and amino acid-derived sulfamides (16–22).
In order to investigate the effects that may contribute to the differ-
ent binding affinity, we simulated the interaction of the compounds
with CAs using docking and molecular dynamics simulations.
2. Materials and methods
2.1. Synthesis of sulfamides and sulfamates
The compounds presented here were obtained from different meth-
odologies according to the nature and position of the subtituents of the
sulfamide group.8,9,23 Their syntheses was previously reported and it
was performed following the methodologies described below:
Symmetric N,N0-disubstituted sulfamides and tetra-substituted
sulfamides were prepared in concordance with the typical proce-
dures by condensation of an excess of amine with sulfuryl chloride
(compounds 7 to 15).24–29 Similarly, N,N0-di-substituted aminoacid
derived sulfamides 16–18 were prepared by reaction of the corre-
sponding salts of amino esters with sulfuryl chloride in presence of
triethylamine.30
Mono-substituted sulfamides (compounds 3–4) were synthe-
sized following the standard method that involves the preparation
of N-alkoxycarbonyl sulfamides as intermediates of the
O
S
O
O
S
O
O
S
O
S
O
H2N
N
H
H2N
N
H2N
O
H2N
O
O
3
2
1
4
O
S
O
S
O
O
O
O
S
O
O
S
N
H
N
H
O
N
N
H
O
N
H
N
H
N
H
N
H
O
O
7
6
5
8
O
S
O
O
O
S
O
O
S
S
S
O
N
N
O
N
N
N
N
H
N
N
H
N
H
N
H
H
H
H
H
O
O
10
O
12
O
11
13
9
O
O
O
O
O
O
S
O
O
H
O
S
O
S
O
N
S
N
H
N
N
N
N
O
N
H
N
H
O
O
O
17
O
14
16
15
O
S
O
O
O
CH(CH3
O
)
2
O
O
S
O
H
N
O
H
N
O
S
N
H
N
H
N
O
N
H
O
H
O
O
O
18
O
CH(CH3
)
19
2
20
O
O
S
O
O
O
S
O
H
H
O
O
N
O
O
N
N
O
O
N
N
S
H
H
O
O
O
O
O
OH
21
23
22
Figure 1. Chemical structures of the tested compounds.

1412
L. Gavernet et al. / Bioorg. Med. Chem. 21 (2013) 1410–1418
Table 1
CO2 hydration reaction for a period of 10–100 s. The CO2 concen-
trations ranged from 1.7 to 17 mM for the determination of the ki-
netic 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 ob-
served rates. Stock solutions of inhibitor (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 at room temperature for 15 min prior to assay,
in order to allow for the formation of the E-I complex. The inhibi-
tion constants were obtained by non-linear least-squares methods
using PRISM 3, whereas the kinetic parameters for the uninhibited
enzymes were obtained from Lineweaver–Burk plots, as reported
earlier,38–40 and represent the mean from at least three different
determinations.
Inhibition of CA isozymes hCA I, II, VII, XII and XIV by sulfamates/sulfamides
(compounds 1–23)
Compound
hCA I
hCA II
Kia
(l
M) hCA VII
hCA XII
hCA XIV
1
2
3
4
5
6
7
8
3.66
2.98
0.13c
24.9
8.84
6.88
>200
>200
6.64
4.69
>200
6.64
>200
9.35
>200
>200
>200
>200
>200
>200
62.2
>200
88.7
2.82b
1.92
0.70
4.30
0.30
>200
0.17
0.32
0.38
>200
0.41
>200
0.16
0.32
0.47
>200
>200
>200
>200
>200
>200
>200
>200
>200
68.7
0.73
0.89
0.94
3.53
0.89
0.91
81.5
>200
0.69
>200
0.77
0.70
9.10
>200
>200
70.8
>200
73.0
61.2
56.3
65.4
54.5
65.0
0.82
0.86
0.84
7.10
40.4
52.6
35.5
>200
9.79
>200
8.79
9.94
48.8
>200
>200
8.40
7.66
7.97
8.37
5.92
8.09
5.38
8.53
0.12c
60.3
161
>200
0.65c
>200b
66.3b
124b
>200b
100.1b
135b
>200b
>200b
>200
>200
>200
>200
55.7
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
2.3. Computational
The binding of the compounds to CA I, CA II and CA VII, was ana-
lyzed using AutoDockTools 1.5.0 and AutoDock 4.0 docking pro-
grams.41 The CA I starting protein was prepared from the crystal
structure of the complex deposited by Christianson and coworkers
(PDB code 2NN7).42 For CA II we used the 0.99 Å resolution crystal
structure of the CA II-sulfonamide complex (2FOU),43 as previously
done when studying CA II sulfamide inhibition.7 We also modeled
the conformational mobility of the side chain of His64 residue in
the docking, which is likely associated with its role as a proton
shuttle in catalysis.44 To this end, the docking was performed with
the two different ‘in’ and ‘out’ conformations44 of His64 for CA II
(Fig. S1). The out conformation was taken from the experimental
structure (PDB code 2FOU), whereas the ‘in’ conformations were
obtained from snapshots of molecular dynamics simulations
(MD) for CA II (see below).
41.1
>200
>200
a
b
c
From three different assays, errors 10% of the reported value.
Data taken from Ref. 7.
Data taken from Ref. 5.
reaction.5,31 The reaction starts with the one-pot synthesis of
N-alkoxycarbonyl sulfamides (compounds 5 and 6) from chlorosul-
fonyl isocyanate, tert-butanol, and the corresponding amine in
presence of triethylamine. The tert-butoxy carbonyl group was
then removed via acidic hydrolysis. The N-alkoxycarbonyl sulfa-
mides 5 and 6 obtained from the first step of the reaction were also
tested against CAs. We considered that the measure of the inhibi-
tory activity of these compounds is interesting to arrive to new
structure–activity relationships. For the same reason we included
the product of the reaction of chlorosulfonyl isocyanate, tert-buta-
nol and the corresponding amino ester in presence of triethyl-
amine (compounds 20 and 22).32
CA VII was prepared from the complex provided by Opperman
et al (PDB code 3MDZ).45 Similarly to CA II, both ‘in’ and ‘out’ con-
formations of His64 were considered. In this case, Opperman et al
distinguished the alternative conformations for the residue in the
X-ray experiment (Fig. S1), and we were able to prepare both con-
formations from the experimental data.
Compound 21 is the product of the alkylation reaction in Mits-
unobu conditions33 of the reaction described before by using the
methyl ester of B-Ala as reactant: Initially the chlorosulfonyl isocy-
anate and tert-butanol were added to the amino ester in presence
of triethylamine; then the product (the N-terbutoxycarbonyl sulf-
amide derivative) was allowed to react with the Mitsunobu tan-
dem triphenylphosphine and diisopropylazodicarboxylate.32
Sulfamide 19 was obtained from the reaction of catechol sulfate
(prepared with catechol and sulfuryl chloride)34 and the corre-
sponding amino ester under controlled reaction conditions to yield
a sulfamate ester derivative (compound 22); which reacts with
buthylamine yield the nonsymmetric sulfamide.35
Compound 22 was also tested as a sulfamate derivative. The
other sulfamates 1 and 2 were prepared by adding formic acid to
chlorosulfonyl isocyanate to generate sulfamoyl chloride,36 which
was then allowed to react in situ with the corresponding alcohol
in basic conditions.36
In all cases, the crystallographic water molecules, the ligand,
and any cocrystallized molecule/ion were stripped. Hydrogen
atoms were added using the leap module of AMBER11.46 Special
attention was given to the protonation state of the Zn-bound His
residues of the actives site, which were defined as two histidines
type Hid and one histidine type Hie in all the isozymes.
The structures of sulfamides and sulfamates were docked in
their deprotonated form, since it is believed that these compounds
coordinate to the active site as negative species. The anions of
mono-substituted structures 1–4 were prepared by removing one
H atom of the NH2 group. For N,N0-di-substituted sulfamide 5
two different deprotonated forms can be constructed. Both anions
were considered, and the data associated with the most stable
complex (lower binding energy) is reported. Exceptions were com-
pound 13–15 bearing no H attached to the binding N. They were
docked in their neutral form.
We used the default Autodock parameters for all the variables
but the charges of the ligands, for which AM1-BCC charges were
calculated using quacpac.47 This was found to perform better than
the default Gasteiger charges in the CA II docking.7
2.2. CA inhibition
An Applied Photophysics stopped-flow instrument has been
used for assaying the CA catalysed CO2 hydration activity.37 Phenol
red (at a concentration of 0.2 mM) has been used as indicator,
working at the maximum absorbance of 557 nm, with 10–20 mM
Hepes (pH 7.5) as buffer, and 20 mM Na2SO4 for maintaining con-
stant ionic strength, following the initial rates of the CA-catalyzed
The structures were docked using the Lamarckian genetic algo-
rithm (LGA) in the ‘docking active site’, defined through a grid cen-
tered on the CG1 atom of Val59 residue for CA I, the ND2 atom of
Asn67 residue for CA II and the ND2 atom of Asn64 for CA VII. In all
isozymes we used 60, 50, and 60 grid points in X, Y, Z dimensions
respectively, with the default grid spacing (0.375 Å); and

L. Gavernet et al. / Bioorg. Med. Chem. 21 (2013) 1410–1418
1413
performed 50 docking runs. The docking active site was treated as
a rigid molecule and the ligands as flexible, that is, all non-ring tor-
sions were considered active.
MD simulations were carried out for compound 11 in CA II and
CA VII, using the PMEMD version included in the AMBER11 suite of
programs46 after careful relaxation of the system using minimiza-
tion and equilibration protocols. The conformations predicted by
Autodock for the CA II /11 and CA VII/11 complexes were used as
starting points for MD simulations. In both complexes His64 was
selected in the ‘out’ conformation.
The initial geometries were minimized (1000 cycles for the
water molecules followed by 2500 cycles for the entire systems).
After a 20 ps NTV equilibration period with a weak restraint
(10 kcal/mol Å2) for the complex and a NTP 200 ps without re-
straint, production runs larger than 12 ns were computed for each
complex, for the coordinates saved every 1000 time steps.
The ionizable residues were set to their normal ionization states
at pH 7, except for the His residues coordinating the Zn metallo-
center, which were modeled as Hid94, Hid96, and Hie119 (num-
bers relative to CA II). The protein atoms as well as all the water
molecules of the crystal structure were surrounded by a periodic
box of TIP3P32 water molecules that extended 10 Å from the pro-
tein. Counterions were placed by LEaP26 to neutralize the system.
The ff03 version of the all-atom AMBER force field was used to
model the protein, and the GAFF force field was used for the organ-
ic ligand.48 Atom-centered partial charges were derived by using
the AMBER antechamber program (restrained electrostatic poten-
tial [RESP] methodology),49,50 after geometry optimization at the
B3LYP/6-31G⁄⁄ level.51
The weak inhibitory activity found in CA I compared with other
isoforms was expected: The active site cavity of CA I is smaller than
other isozymes, due to the presence of two characteristic His resi-
dues (His200 and His67) that are no conserved in other isoforms
(Fig. 2).
For the case of CA II, derivatives bearing benzyl substituents 3
and 7 are the most promising inhibitors. As stated in previous
investigations, we have found good CA II affinity with these aro-
matic compounds, and the origin of their inhibition has been ana-
lyzed earlier by means of molecular modeling techniques.7 The
modeling has found favorable pi-stacking interactions between
the phenyl rings and aromatic residues that can certainly contrib-
ute to the enhancement of activity.7 Structure 5 also has a benzyl
substituent, but it is 247 times less active than 7 and 1341 times
less active than 3, what can be attributed to the negative steric hin-
drance originated by the Boc-subtituent (see Section 3.1).
No good inhibition pattern was found against CA XII and CA XIV,
for which the best compounds have Ki near the
lM value. Con-
versely, a larger number of structures were found active in CA VII
(brain-specific isoform). Several N,N0-di-subsituted sulfamides
(such as 5 and 6) show good inhibitory activity (in the range of
0.2–0.5 lM) against this isozyme. Additionally, aromatic and ali-
phatic compounds (i.e. 7 and 9) present a comparable inhibitory
profile in CA VII. In order to gain insight into the facts that might
be relevant for the binding, we investigated in some detail the dif-
ferent effects that can contribute to the trend observed in the
inhibitory activity by means of molecular modeling approaches.
3.1. Molecular modeling analysis
As in previous investigations,7 nonstandard force fields have
been derived for the Zn active site by means of geometry minimi-
zation (B3LYP/6-31G⁄⁄, G03),52 followed by calculation of the sec-
ond derivatives and RESP charges for the active site
supermolecule defined by three His residues, the Zn ion, and the
sulfamide ligand.
During the simulation, the ‘out’ conformation of His64 side
chain changes to ‘in’ for CA II complex. Snapshots of this new con-
formation were captured and used to perform a new docking sim-
ulation with compounds 1–7 and 9–13.
We simulated the binding modes of compounds 1–7 and 9–13
against isozymes I, II and VII, since the other complexes did not
show good inhibitory activity. Docking scores for isozymes I, II
and VII are given in Table 2. They point to compound 3 as the most
active one against CA I. Figure 2 shows the docking conformation
found for the CA I-3 complex. The deprotonated N interacts with
the Zn atom of the enzyme. This interaction is reinforced by two
H bond interactions comprising the backbone N and carbonyl O
atoms of the Thr196 residue with the O and the amide H atom of
the sulfamide group, respectively (Fig. 2). These hydrogen bond
interactions were previously reported for the experimental com-
plex of CA I and a benzene sulfonamide derivative (PDB code
2NN7). The measured distance between the amine N atom of the
sulfonamide and the Thr196 OG atom in the experimental struc-
ture is almost the same that the one found for compound 3 in com-
plex with CA I (2.68A vs. 2.63A, respectively, Fig. 2); whereas the
distance between the backbone N atom of Thr196 and the sulfom-
anide O atom of the ligand is very close to experiment (2.87A for
the sulfonamide and 2.92A for compound 3).
The MD simulation of the compound 7-CA II complex is also
mentioned in this research. We are using the results of previous
investigations, whose computational details are given in Ref. 7.
3. Results and discussion
Table 1 reports the biological activity determined for the com-
pounds shown in Figure 1. A broad range of inhibitory activities
were observed for the compounds of the set. A weak binding (Ki
>1 lM) was observed for amino acid-derived compounds 16–23
The interaction of the sulfamides with the Thr residues close to
the active site was previously described for the complexes of
mono-substituted sulfamides and the CA II isoform.7 In CA II, two
consecutive Thr residues (Thr199 and Thr200) close to the active
site stabilize the coordination of the sulfamide end of the ligands
with the Zn atom through similar H-bond interactions: the NH
and OG atoms of the Thr residues acts as hydrogen donors/accep-
tors of the O and the NH atom of the sulfamides.7
In addition to the previously described interactions, docking
studies show the aromatic scaffold of compound 3 oriented in a
way that can stabilize a pi-stacking interaction with the Phe88 res-
idue in CA I (Fig. 2). This effect, together with the small size and the
mono-substitution of the molecule (that minimize the negative
steric effects in this small active site relative to other CAs) can ex-
plain the better inhibitory activity of compound 3 against CA I in
contrast to the other structures of the set.
against all the tested isoforms. This can be explained by the polar
nature of the substituents, which cannot make lipophilic stabiliz-
ing interactions with residues in the active site pocket, or by the ef-
fect of sizeable substituents on the N atom responsible for the
coordination with the Zn. It has been previously discussed that
the replacement of H atoms of the coordinating N atom by alkyl
(or aryl) substituents impairs the activity of single substituted sul-
famides analogs in CA II.7 In the same line, bulky tetra-substituted
sulfamides 14–15 as well as compound 8 were inactive against all
the tested isozymes. Compound 13, being also tetra-substituted,
shows selectivity against CA VII.
On the other hand, interesting results were found for the rest of
the compounds from the set in terms of activity (Ki <1 lm) and
selectivity. Compound 3 measured Ki shows it a promising, with
a value in the nanomolar range making it the most active of the
series for CA I and CA II.

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L. Gavernet et al. / Bioorg. Med. Chem. 21 (2013) 1410–1418
Thr196
2.92
Compound 3
3.75
2.63
His200
1.60
Zn
Phe88
His116
His67
His93
His91
Figure 2. Binding geometry of compound 3 in the CA I active site predicted by the Autodock docking algorithms. Residues of the active site are highlighted in green. The Zn
atom is represented in gray as a nonbonded sphere. The carbon atoms of compound 3 are highlighted in purple. Values of the relevant distances are given in Å.
Table 2
Docking scores of sulfamides and sulfamates from docking against CA I, CAII and CA VII
Compound
Ki
CAI docking score
Ki
CAII docking score
Ki
CAVII docking score
1
2
3
4
5
6
7
9
10
11
12
13
3.66
2.98
0.13
24.9
8.84
6.88
>200
>200
>200
6.64
>200
9.35
ꢀ5.88
ꢀ6.14
ꢀ6.64
ꢀ5.97
ꢀ3.77
NA
ꢀ6.07
ꢀ5.4
ꢀ4.82
ꢀ3.34
NA
2.82
1.92
0.12
60.3
161
>200
0.65
66.3
124
ꢀ6.43 (in)
ꢀ6.9 (in)
ꢀ8.08 (in)
ꢀ6.42 (out)
ꢀ6.29 (in)
NA
ꢀ8.03 (in)
ꢀ4.87 (out)
ꢀ5.37 (out)
ꢀ4.11 (out)
NA
0.7
4.3
0.3
ꢀ6.35 (out)
ꢀ6.56 (out)
ꢀ6.64 (out)
ꢀ5.85 (out)
ꢀ5.70 (out)
NA
ꢀ6.65 (out)
ꢀ5.43 (out)
ꢀ5.84 (out)
ꢀ5.62 (out)
NA
>200
0.17
0.32
0.38
0.41
>200
0.16
0.32
0.47
>200
100.1
135
NA
NA
NA
For CA II and CA VII, the best score between the ‘in’ and ‘out’ conformations is reported and the conformation shown between brackets.
NA: not bound conformation was achieved in the docking process.
Regarding CA II, the scores are able to discriminate between the
most active compounds 3 and 7. As explained in the experimental sec-
tion, we performed docking experiments using two different confor-
mations of the His64 residue of CA II. The best scores for these two
most active sulfamides were obtained using the ‘in’ conformation.
The origin of the activity of compounds 3 and 7 has been previously
analyzed for the out conformation of CA II, which is the one found in
the experimental structure of the enzyme (PDB code 2FOU).7 The li-
gand 3 interacts in both cases with Thr198 and Thr199, but the active
site is smaller for the ‘in’ conformation of His64. The benzyl ring adopts
a different conformation in order to fit in the smaller site, interacting
with the lipophilic cleft of the enzyme, especially with the Leu197
residue (Fig. 3). The lack of interaction of compound 3 with His64
is counterbalanced with the favorable lipophilic interaction with
Leu197 and Val144, as well a pi-stacking interaction between the
phenyl ring of the ligand and His119.
activity than the other di-substituted ligands.7 These interactions
also became evident after 12 ns molecular dynamic simulations
(Fig. 4). Phe131 is a non conserved residue that is present in CA
II and CA VII (the isozymes where compound 7 is active), and it
is replaced by Ala in CA XII and by Leu in CA I and CA XIV.
The previously described stabilizing interactions are not easily
established for compound 8. Although with a similar scaffold as
compound 7, the additional methylene group increases the size of
the compounds precluding the fitting in the active site pocket. The
superimposition of the docking poses for compounds 7 and 8 in CA
II (Fig. 5) shows that compound 8 severely distorts, adopting a con-
formation that are higher in the energy than the more stable one and
increasing the total energy of the CAII-compound 8 complex.
According to the results of the docking, compound 7 defines the
maximum length of the aryl–alkyl substitution compatible with
the fitting in the CAII active site pocket. Similar explanation can be
derived from the docking of compounds 7 and 8 in CA VII (Fig. S2).
The influence of the solvent in CA binding has been previously
discussed.7 Diffusion of water molecules to the active site were
detected and reported for compound 3 in CA II.7 In the case of
Despite the negative effect caused by disubstitution in com-
pound 7, it reinforces its binding through a lipophilic interaction
with Leu197 and a second pi-stacking (T-shape) between the other
benzyl ring and Phe131, which are responsible for the higher

L. Gavernet et al. / Bioorg. Med. Chem. 21 (2013) 1410–1418
1415
Thr198
Thr199
Leu197
2.17
1.80
His64 “in”
1.96
1.96
Compound 3
Val142
Zn
His96
His64 “out”
His119
His94
Figure 3. Binding geometry of compound 3 in CA II with the ‘in’ conformation of His64 obtained from MD simulations. Representative residues of the active site are
highlighted in green and the Zn atom is represented in gray as a nonbonded sphere. The out conformation of His64 detected in the experimental structure is superimposed
and shown in orange. The Carbon atoms of compound 3 are highlighted in purple. Only the H atoms involved in important interactions are shown. Values of representative
distances are given in Å.
Phe131
Leu197
2.22
Compound 7
Zn
His94
Figure 4. Representative snapshot from molecular dynamics that shows the interaction of 7 with water and residues close to the active site (in green). The Carbon atoms of
compound 7 are highlighted in purple and Zn atom is represented in gray as a nonbonded sphere. The value of distance to water is given in Å.
compound 7 the solvent also plays a role. One water molecule acts
as a donor to form a positive H-bond interaction with the H atom of
the non-coordinating N of the sulfamide function (Fig. 4).
has to adopt a very unfavorable conformation in order to enter
the active site, due to the presence of the negative steric hindrance
originated by the Boc-subtituent (Fig. S3).
Compound 5 also has a benzyl substituent, but it is 247 times
less active than 7 and 1341 times less active than 3 in CA II. No
good coordination was found for 5 in the CA II ‘out’ His64 confor-
mation. For the structure of CA II ‘in’, the benzyl group of 5 tends to
interact with the Leu197 residue in the same manner as 3, but it
The docking algorithms do not explain the activity of the com-
pounds in CA VII for either the in or the out conformation: the
binding energies predicted by docking did not evidence an accept-
able correlation with the biological data (Table 2). Attending the
inhibitory activity, the nature of the substituent seems to be less

1416
L. Gavernet et al. / Bioorg. Med. Chem. 21 (2013) 1410–1418
Thr199
Thr198
His64 “in”
Leu197
Phe131
Compound 7
Compound 8
Zn
His96
His119
Val142
His94
Figure 5. Binding geometries of compounds 7 and 8 in the CA II active site predicted by the docking. Residues of the active site are highlighted in green. The Zn atom is
represented as a nonbonded sphere in gray. The Carbon atoms of compound 7 are highlighted in purple and those of compound 8 are highlighted in orange.
CA II
CA VII
Compound 11
Thr198
Compound 11
Thr199
Thr201
3.22
Thr202
2.41
1.80
Zn
2.17
Zn
2.26
2.11
2.45
Gln94
Gln92
Gln69
Asn67
His64
His66
Figure 6. Representative snapshots of CA II and CA VII complexes with compound 11 showing its interaction with water molecules and Thr residues. Representative residues
of the active site are highlighted in green and the Zn atom is represented as a nonbonded gray sphere. The Carbon atoms of compound 11 are highlighted in purple. The
distances (in Å) correspond to average values.
important for this isozyme: Aromatic and aliphatic compounds
show similar action (compare for example compounds 7 and 9).
In the same line, and contrasting with CA I and CA II, the N,N0-
disubsitution favors the binding in some cases (compare for exam-
ple 5 and 6 to 3 and 4). In fact, the most active compound of the set
is 11, which bears two cyclopropyl rings as substituents of the sulf-
amide function. Moreover, a tetra-substitued ligand shows selec-
tivity towards this isoform, which has to be associated with a
different coordination mode than the one analyzed here. In order
to better understand the effects that can contribute to this different
trend, we run 12 ns molecular dynamic simulations of the CA VII-
11 complex, and CA II-11 complex, for which the ligand exerts no
The interactions with Thr residues previously described re-
mained stable for both isozymes (Fig. 6). Regarding the solvation
pattern, water molecules are positioned close to the active site dur-
ing both MD simulations. The water H atom acts as donor for the
H-bond interaction with the sulfamide N atom that is involved in
the Zn coordination (Fig. 6). This negative effect has been reported
before for mono-substituted sulfamides,7 and it might be one of
the factor that significantly diminish the activity in both isozymes.
Figure 7 shows a superposition of representative snapshots for
both enzymes. The surface representation shows that the cleft that
ends with the active site is more elongated in CA VII (yellow) than
in CA II (red), especially in the hydrophilic region delimited by
Asn64, His66, Gln69, Lys93 and Gln94 (Figure 7). Gln69 and
Lys93 are distinctive residues for CA VII, and they are replaced
by Asn67 and Ile91 in CA II. The larger volume available in the
activity (Ki >200 lM). We built the initial structure of the com-
plexes using, as a template, the conformations obtained from the
docking procedure.

L. Gavernet et al. / Bioorg. Med. Chem. 21 (2013) 1410–1418
1417
Compound 11
Lys93
Zn
Ile91
His66
Gln94
Asn67
Asn64
Gln69
Figure 7. Surface representation of the superimposition of the active sites of CA VII (yellow) and CA II (red). Representative residues of the lipophilic cleft are highlighted in
yellow for CA VII. For CA II the residues Asn67 and Ile91 are highlighted in red. The Carbon atoms of compound 11 in complex with CAVII are highlighted in purple and the Zn
Atom is represented as a nonbonded gray sphere.
active site entrance in CA VII allows it to accommodate bulkier sul-
famides interacting with the Zn center. This provides a possible
explanation for the origin of the activity of disubsituted ligands,
but does not justify the activity of compound 13. We did not fur-
ther analyzed the interaction of this compound using computa-
tional techniques as we are comparing the trends for ligands that
are modeled as anionic binders.
Consejo Nacional de Investigaciones Científicas y Técnicas de la
República Argentina (CONICET) and P. Palestro is a fellowship
holder of CONICET. The work described herein was done in part
during a Postdoctoral leave of Dr. L. Gavernet of the University of
Notre Dame to whom she gratefully acknowledges. This research
was supported in part by the National Science Foundation (TG-
CHE090124) through TeraGrid resources provided by NICS and
LONI and by the Agencia de Promoción Científica y Tecnológica
(PICT 01774/2010), CONICET, the Universidad Nacional de La Plata,
Argentina, and the Walther Cancer Research Center, University of
Notre Dame. The computations were performed on Kraken (a Cray
XT5) at the National Institute for Computational Sciences (http://
www.nics.tennessee.edu/) and on Queen Bee at the Louisiana Opti-
cal Network Initiative. Generous allocation of computing resources
by the Center for Research Computing at the University of Notre
Dame is also acknowledged. Work from CTS lab has been financed
by an European Union grant of the seventh FP programme (Metox-
ia project).
4. Conclusions
Sulfamides and sulfamates are the most common bioisosteres of
sulfonamides, a classical zinc binding function that interacts di-
rectly with the metal Zn in the active site of CAs. Compounds bear-
ing these functionalities has been proved to be potent inhibitors of
several isozymes.1,53 In this investigation we provide an explana-
tion for the different activity of sulfamides in different isozymes.
To this end we applied molecular modeling techniques supported
by experimental data.
We analyzed the influence of the nature of the residues close to
the active site, especially the ones that are distinctive for each iso-
zyme. We also included in the models the different orientations
that can adopt the His64 residue, providing a new conformation
of the CA II and CA VII active sites for further docking. We found
that not all the compounds interact with the same conformation
of CA II. The most active compounds interact better with the ‘in’
conformation of CA II whereas the ‘out’ conformation was pre-
ferred for other ligands. We also found that disubstitution not al-
ways impairs the inhibitory activity. The effect depends on the
capability of the isozymes to accommodate the extra hydrocarbon
chain (as it was shown for CA VII). This information will allow us to
design more potent and isoform-selective inhibitors, with the re-
lated impact that it has in the treatment of several diseases in
which the inhibition of CA is involved.
Supplementary data
Supplementary data (additional figures that describe the de-
fined ‘in’ and ‘out’ conformation for CAII and CA VII, the interaction
of compounds 7 and 8 in the CA VII active site and the interactions
of compound 5 with CA II are given) associated with this article can
be found, in the online version, at http://dx.doi.org/10.1016/
j.bmc.2012.10.048.
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