Article

pubs.acs.org/jmc

StructureActivity Relationship for Sulfonamide Inhibition of

Helicobacter pylori Carbonic Anhydrase
Joyanta K. Modak,, Yu C. Liu, Claudiu T. Supuran,, and Anna Roujeinikova*,,,

Department of Microbiology, Monash University, Clayton, Victoria 3800, Australia

Infection and Immunity Program, Monash Biomedicine Discovery Institute, Monash University, Clayton, Victoria 3800, Australia

Laboratorio di Chimica Bioinorganica, Polo Scientico, Universita degli Studi di Firenze, Via della Lastruccia 3, Sesto Fiorentino,
Florence 50019, Italy

Neurofarba Department, Sezione di Scienze Farmaceutiche, Universita degli Studi di Firenze, Via U. Schi 6, Sesto Fiorentino,
Florence 50019, Italy

Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3800, Australia

S Supporting Information
*

ABSTRACT: -Carbonic anhydrase of Helicobacter pylori

(HpCA) plays an important role in the acclimation of this
oncobacterium to the acidic pH of the stomach. Sulfonamide
inhibitors of HpCA possess anti-H. pylori activity. The crystal
structures of complexes of HpCA with a family of
acetazolamide-related sulfonamides have been determined.
Analysis of the structures revealed that the mode of
sulfonamide binding correlates well with their inhibitory
activities. In addition, comparisons with the corresponding
inhibitor complexes of human carbonic anhydrase II (HCAII)
indicated that HpCA possesses an additional, alternative
binding site for sulfonamides that is not present in HCAII.
Furthermore, the hydrophobic pocket in HCAII that stabilizes the apolar moiety of sulfonamide inhibitors is replaced with a
more open, hydrophilic pocket in HpCA. Thus, our analysis identied major structural features can be exploited in the design of
selective and more potent inhibitors of HpCA that may lead to novel antimicrobials.

INTRODUCTION
Helicobacter pylori is a Gram negative, neutralophilic bacterium
that colonizes human gastric mucosa.1 Around half of the total
worlds population is infected with this bacteria, with the
prevalence reaching 80% of the population in some developing
countries.2,3 H. pylori infections are associated with diseases of
the upper gastrointestinal tract such as chronic gastritis, peptic
ulcer, gastric mucosa-associated lymphoid tissue (MALT),
lymphoma, and gastric cancer.48 The current standard therapy
for anti-H. pylori treatment consists of two broad-spectrum
antibiotics (clarithromycin and either metronidazole or
amoxicillin) and a proton pump inhibitor. However, the
success rate of this regimen has declined over time, falling
below 80% globally, mainly due to the spread of resistance to
clarithromycin and metronidazole.911 Therefore, there is a
clear need for identication of novel targets that can be used in
the development of alternative treatment strategies for H. pylori
infections.
Carbonic anhydrases (CAs) are zinc metalloenzymes that
catalyze a physiologically important process of reversible
hydration of CO2 to bicarbonate and protons.12,13 Bacterial
CAs provide substrates for many metabolic pathways including
pH regulation, CO2 xation, and cyanate degradation and play
2016 American Chemical Society

an important role in the survival of intracellular pathogens

within the host.1417 H. pylori possesses two dierent CAs,
periplasmic -carbonic anhydrase (HpCA) and cytoplasmic carbonic anhydrase (HpCA). Through the activities of
HpCA, HpCA, and urease, H. pylori buers its periplasm
at pH close to 6.1 as a means of acclimation to the harsh acidic
environment of the stomach.14,18 Because of their crucial role in
H. pylori survival in the host, HpCA and HpCA have now
gained interest as potential drug targets. Their activities are
inhibited by sulfonamides (RSO2NH2), including the clinically
used drugs acetazolamide (AAZ), ethoxzolamide (EZA),
methazolamide (MZA), topiramate, and sulpiride.19,20 Importantly, AAZ and MZA showed anti-H. pylori activity in in vitro
assays.21 Thus, the inhibition of HpCAs could be a novel
alternative approach for the treatment of H. pylori infections.
Because the sulfonamides in current clinical use were
designed to target human CA isoforms, their activity against
bacterial CAs, including HpCA and HpCA, is lower than
against human CAs.21 Understanding the structural basis of
inhibition and the dierences between the inhibitor-binding
Received: September 8, 2016
Published: November 21, 2016
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Figure 1. Structures of the two dierent classes of sulfonamide compounds discussed in this study. A representation of the generic structure is also
shown. (A) Class 1 is based on a 5-amino-1,3,4-thiadiazole-2-sulfonamide or 5-imino-4,5-dihydro-1,3,4-thiadiazole-2-sulfonamide scaold with
substituents R1 and R2. Oxygen atoms are colored red, nitrogen blue, sulfur yellow, carbon gray, and R1 and R2 groups cyan. (B) Class 2 has a
thiophene-2-sulfonamide scaold fused with group X.

sites in human CAs and their H. pylori orthologues would be

crucial for the design of inhibitors specic to H. pylori.
Crystallographic studies of HpCAs in complex with dierent
inhibitors would be an important step toward this goal.
The periplasmic location of HpCA makes it an easier target,
as its inhibitors do not need to cross the cytoplasmic
membrane. We have previously reported the crystal structures
of HpCA in complex with AAZ and MZA.22 We showed that
HpCA forms a dimer in solution, which is very similar to
other bacterial CAs, but distinct from the mammalian
monomeric form of CA. Despite the dierence in the

oligomeric state, the HpCA overall fold is very similar to

that of human carbonic anhydrase II (HCAII), which shares a
28% sequence identity with HpCA.22 The dierences between
the structures of the two enzymes are mainly in the length of
the surface loops. The active site of HpCA is located in the
cone-shaped cavity and contains a catalytic zinc ion that is
tetrahedrally coordinated by three histidine residues and a
water molecule/hydroxide ion. Notably, the entrance into the
HpCA active site cavity is wider than in HCAII due to a
shorter surface loop. However, in both HpCA and HCAII,
residues that are important for the recognition and correct
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Figure 2. Stereoview of the structural superposition of the active site residues of chains G and C, showing the two dierent binding modes of
compound 1. For clarity, the protein atoms and molecular surface of chain C are not shown. Binding mode A is shown in stick representation with
carbon atoms colored cyan; binding mode B is shown as thin lines with carbons in green. The main chain and/or side chains of the protein residues
that only form interactions with compound 1 in binding mode A are shown with carbons colored cyan, and those that only interact with compound
1 in binding mode B are shown with carbons colored green. The residues that form interactions with compound 1 in both binding modes are shown
with carbons colored gray. The coordinating bond between the inhibitor and the catalytic zinc ion is shown as a black dashed line; hydrogen bonds
are shown as blue dashed lines. Nitrogen, oxygen, and sulfur atoms are colored blue, red, and yellow, respectively.

asymmetric unit. The structures of the protein subunits from

the four complexes could be superposed with an average
pairwise C root-mean-square deviation (rmsd) value of 0.5 ,
showing no signicant dierences. The analysis and discussion
therefore focuses on the similarities and dierences in the
relative positions of the respective inhibitors in the enzymes
active site.
Two Dierent Binding Modes of Compound 1 and
Comparison with AAZ. The chemical structure of compound
1 (R1 = R2 = H) is a truncated version of that of AAZ (Figure
1). Dierence Fourier maps calculated for the HpCA/
compound 1 complex, based on the molecular replacement
(MR) solution obtained with the coordinates of the free
enzyme, clearly showed the presence of one inhibitor molecule
bound in the active site of each protein subunit. Interestingly,
structural alignment of the eight protein subunits in the
asymmetric unit revealed that compound 1 binds to HpCA in
two dierent modes (A and B) (Figure 2 and Supporting
Information, Figure S1), in both of which the N atom of the
sulfonamide moiety of the inhibitor coordinates the catalytic
zinc ion in the active site. Binding mode A of compound 1,
observed in four out of eight subunits, is very close to that of
the AAZ molecule previously observed in the crystal structure
of the HpCA/AAZ complex (Supporting Information, Figure
S1A).22 In this mode, the N atom of sulfonamide moiety forms
a hydrogen bond with the O atom of Thr191 (2.9 ), while
one of the two sulfonamide oxygen atoms is within hydrogen
bonding distance from the backbone NH of Thr191 (2.9 )
(Figure 2 and Supporting Information, Figure S1A). The
sulfonamide group is further stabilized by van der Waals
contacts with the side chains of Val141, Thr191, and Trp201.
The thiadiazole ring is located in the hydrophobic side of the
active site and makes van der Waals contacts with Val131,
Leu190, and Ala192. The 5-amino group of the inhibitor points
out of the pocket and is exposed to the solvent.
In binding mode B, the 5-amino-thiadiazole ring of
compound 1 is bound in a small, partially hydrophobic cavity
formed by residues Trp23, Thr83, His84, Thr86, His110, and
Ala192, and the inhibitor is oriented at an approximate angle of
60 to binding mode A (Figure 2 and Supporting Information,

orientation of the substrate molecule (CO2) are conserved and,

hence, HpCA is likely to follow the same reaction mechanism
as HCAII, where the substrate is converted into HCO3 via a
nucleophilic attack of the reactive zinc-bound H2O/OH on
CO 2. 22 Our structural analysis revealed that the two
sulfonamide oxygens of the inhibitors occupy positions similar
to those of the oxygen atoms of the CO2 substrate, whereas its
Zn-coordinating sulfonamide nitrogen atom is bound at the site
for the catalytic H2O molecule. Sulfonamides AAZ (KI, 21 nM)
and MZA (KI, 225 nM) therefore act as site-directed inhibitors
of HpCA by mimicking the catalytic transition state of the
CO2 hydration reaction.
In this study, we present the analysis of the structureactivity
relationship for a family of AAZ- and MZA-related sulfonamide
inhibitors of HpCA, whose structures, IUPAC names, and
inhibitory constants (KI) are presented in Figure 1 and
Supporting Information, Table S1. We identify the features that
dene their inhibitory activity against HpCA and pinpoint the
dierences with HCAII that can be exploited in the design of
specic and more potent inhibitors of HpCA.

RESULTS AND DISCUSSION

To investigate the structureactivity relationship for sulfonamide inhibition of HpCA, we determined the crystal
structures of HpCA in complex with four dierent
sulfonamide inhibitors representing two dierent classes of
sulfonamides, modeled the complexes with three other
sulfonamides from this family and integrated this data with
the previously determined structures of the complexes with
acetazolamide (AAZ) and methazolamide (MZA).22 The
inhibitors discussed in this study include clinical drugs AAZ,
MZA, EZA, dorzolamide (DZA), and brinzolamide (BRZ) and
related compounds 1, 2, 3, and benzolamide (BZA) (Figure 1).
The chemical structure of all inhibitors has a ve-membered
ring linked to a sulfonamide moiety and either decorated with
R1 and N-R2 substituents at positions 4 and 5 (class 1),
respectively, or fused with a six-membered ring (class 2) as
illustrated in Figure 1. The crystals of the HpCA complexes
with BZA, EZA, compound 1, and compound 3 belonged to
the P21 space group, with eight protein subunits in the
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Figure 3. BZA binding in the active site of HpCA and comparison to HCAII. Residue labels in black refer to HpCA. (A) The simulated-annealing
omit |2Fo Fc| electron density map for BZA bound to HpCA calculated at 2.4 resolution and contoured at 1.0 level. The BZA molecule is
shown in all-atom ball-and-stick representation with carbon atoms colored green. Amino acid residues that interact with BZA are shown in stick
representation with carbon atoms colored wheat. The catalytic zinc ion that is coordinated tetrahedrally is shown as a black sphere. Hydrogen bonds
are shown as blue dashed lines. (B) Superposition of all chains (except chain F) present in the asymmetric unit of the crystal complex HpCA/BZA
showing variation in the position of BZA. (C) Two dierent BZA binding modes observed in the active site of chain E of HpCA. The amino acid
residues that interact with the phenyl ring only in the rst binding mode are shown with carbon atoms colored cyan, those interacting only with the
second mode green, and the residues that form hydrogen bonds with the 5-sulfonamido group of benzolamide in both conrmations are shown with
carbon atoms colored white. The molecular surface was calculated with the inhibitor molecule excluded. (D) Superposition of the active site residues
of the HpCA/BZA complex (wheat) with those of the HCAII/BZA complex (green) showing the similar binding mode of BZA. The catalytic zinc
ion and protein residues that form interactions with BZA are shown. The helix 130136 in HCAII is drawn as a ribbon. The HCAII residues that
form additional stabilizing interactions with BZA are shown using ball-and-stick representation and marked with boxed labels in green.

Figure S1B). In common with mode A, the N atom of the

sulfonamide group forms a hydrogen bond with the O atom of
Thr191 (2.6 ), and the O1 atom of the sulfonamide group
forms a hydrogen bond with the backbone NH moiety of
Thr191 (3.1 ). An additional hydrogen bond that is present in
mode B, but absent in mode A, is that between the O1 atom of
the sulfonamide group and the backbone NH group of Ala192
(2.9 ). The thiadiazole ring in binding mode B forms van der
Waals interactions with Trp23, His110, and Ala192. The 5amino group, which is completely exposed to the solvent in
mode A, is stabilized by van der Waals contacts with the
carbonyl oxygen atoms of Thr83 and His84 and the side chain
of Thr86 in mode B. Structural superposition revealed that the
pocket accommodating the 5-amino-thiadiazole ring bound in
mode B is occupied by a molecule of cryoprotectant glycerol in
the HpCA/AAZ complex. Modeling the AAZ molecule based
on the coordinates of compound 1 bound to HpCA in mode
B resulted in a clash between the acetamido group of AAZ and
protein residues Thr83, His84, and Thr86, which explains why
binding mode B was not observed for AAZ.22

Compound 1 (KI = 323 nM) is a less potent inhibitor of

HpCA than AAZ (KI = 21 nM). Superposition of the
structure of the HpCA complex with AAZ with that of the
corresponding complex with compound 1 in binding mode A
shows that the acetamido moiety of AAZ contributes additional
favorable interactions between the inhibitor molecule and the
enzyme in the form of a hydrogen bond between the acetamido
group and the N atom of Asn108 and a van der Waals contact
with the side chain of Lys88 (Supporting Information, Figure
S1).
Comparison of Compound 1 Binding Sites in HpCA
and HCAII. Superposition of the structures of HpCA and its
human orthologue HCAII revealed that 206 of 258 C atoms
could be overlapped with an rmsd of 1.6 , showing the overall
similarity of the two enzymes despite only 28% amino acid
sequence identity between them.22 Inspection of their
respective superposed complexes with compound 1 (PDB
2HNC)23 revealed that the binding mode of the inhibitor in
HCAII is remarkably close to mode A in HpCA (Supporting
Information, Figure S2), with a large number of conserved
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dierences between the BZA binding sites of the two enzymes

are the presence of a short helix 130136 in HCAII that is
absent in the H. pylori enzyme and an Ala Thr substitution
in HCAII at a position corresponding to Ala192 in HpCA.
These dierences result in additional interactions that stabilize
BZA bound to HCAII: hydrophobic interactions between the
apolar side chains of the helix residues Phe131 and Val135 and
the phenyl moiety of BZA and a hydrogen bond between the
side chain of Thr200 and a nitrogen atom of the ve-membered
heterocyclic ring of BZA. These additional stabilizing
interactions are consistent with a signicantly higher potency
of BZA toward HCAII (KI = 9 nM) than HpCA (KI = 315
nM).
tert-Butyl Substituent on Phenyl Ring of Compound 3
Forms Stabilizing Interactions with HpCA. Despite the
lower resolution (2.9 ) of the data for the crystal complex of
HpCA with compound 3, dierence Fourier maps unambiguously indicated the position of the inhibitor molecule (Figure
4). The electron density around this molecule was somewhat

residues involved in stabilizing the inhibitor in HpCA and

HCAII (in parentheses) as follows: Val131 (Val121), Val141
(Val143), Leu190 (Leu198), Thr191 (Thr199), and Trp201
(Trp209). Modeling binding mode B for compound 1 in the
active site of HCAII based on this superposition resulted in a
steric clash between the thiadiazole ring and the C atom of
Thr200 (Ala192 in HpCA) (Supporting Information, Figure
S2), consistent with the HCAIIs preference for mode A.
Furthermore, the presence of a hydroxyl group on Thr200 in
HCAII, as compared to Ala192 in HpCA, allows formation of
an additional hydrogen bond with the nitrogen atom of the
compound 1s heterocyclic ring, resulting in a tighter binding to
the human orthologue (KI (HpCA) = 323 nM, KI (HCAII) =
60 nM, Supporting Information, Table S1).
Evidence of Positional Disorder of Benzolamide (BZA)
Bound to HpCA. Electron density maps calculated for the
HpCA crystals soaked in a solution containing BZA showed
the location of BZA molecules in each subunit of HpCA,
except chain F, where the occupancy of the binding site was
very low. The 2-sulfonamide moiety of BZA forms interactions
with the active-site Zn2+ ion and the main chain of Thr191
(Figure 3A) that are very similar to those observed in the
crystal complexes with compound 1, AAZ, and MZA.22 The
thiadiazole ring forms van der Waals contacts with His110,
Val131, Val141, Leu190, Thr191, Ala192, and Pro193. The O1
and O2 oxygen atoms of the 5-sulfonamido group are within
hydrogen bonding distance from the side chains of Asn108 and
Lys88, respectively. The apolar phenyl ring lies in an
energetically unfavorable environment, with one side of it
exposed to the solvent and the other side packing against the
protein surface area that contains both polar (Asp107, Asn108,
Lys133) and apolar (Val131 and Leu139) residues (Figure 3A).
Superposition of the structures of all subunits in the
asymmetric unit revealed a signicant degree of positional
disorder of this inhibitor in the crystal (Figure 3B). The BZA
molecule has been trapped in several slightly dierent
orientations in dierent protein subunits, which is a
manifestation of the fact that, in solution, the BZA molecule
bound to HpCA has some degree of thermal disorder and
performs 8 pivotal movement about the sulfur atom of the 2sulfonamide moiety and 25 rotation about the SCNS
torsion angle, scanning through dierent possible binding
modes. Furthermore, the electron density for the inhibitor in
subunit E indicates the presence of two distinctly dierent
binding modes, in one of which the phenylsulfonamido moiety
ips by 180 (Figure 3C) and binds in the cavity lined with
polar residues Thr83, His84, Thr86, and His110, likely at high
energetic cost. The positional disorder of BZA observed in the
crystal is likely to be the consequence of multiple unfavorable
interactions between the phenyl ring of the inhibitor and the
protein atoms, consistent with a low inhibitory potency of BZA
toward HpCA (KI = 315 nM).
Comparison of BZA Binding Sites in HpCA and
HCAII. Superposition of the structure of the HpCA/BZA
complex with the corresponding complex of HCAII (PDB
3D8W)24 (also produced by crystal soaking in the inhibitor
solution) revealed that the mode of BZA binding is similar
(Figure 3D), which is in agreement with the fact that a large
number of residues involved in BZA binding in HpCA are
strongly conserved in its human orthologue (in parentheses)
Asn108 (Gln92), His110 (His94), Val131 (Val121), Leu139
(Leu141), Val141 (Val143), Leu190 (Leu198), Thr191
(Thr199), and Pro193 (Pro201). The most signicant

Figure 4. HpCA complex with compound 3 superposed on the

HpCA complex with BZA. The compound 3 and BZA molecules are
shown with carbon atoms colored light-green and cyan, respectively.
For clarity, only the side chains lining the inhibitor-binding pocket in
the complex with compound 3 are shown (with carbon atoms colored
wheat). The molecular surface was calculated with the inhibitor
molecule excluded. The gure illustrates the similar mode of binding
of compound 3 and BZA and shows the detail of the additional
stabilizing interactions provided by the tert-butyl group. The catalytic
zinc ion that is coordinated tetrahedrally is shown as a black sphere;
hydrogen bonds are shown as blue dashed lines. The simulatedannealing omit |2Fo Fc| electron density map for compound 3 bound
to HpCA was calculated at 2.9 resolution and contoured at 1.0-
level.

better in subunit A. Therefore, the detailed analysis of

compound 3 interactions with HpCA is discussed with
reference to that subunit. Compared to the BZA structure,
compound 3 contains a CH3 substituent at position 4 of the
thiadiazole ring and a tert-butyl substituent at position 4 of the
phenyl ring (Figure 1). Similar to BZA and other sulfonamide
inhibitors of HpCA described above, the 2-sulfonamide N
atom of compound 3 coordinates directly to the Zn2+ ion, while
the 2-sulfonamide moiety and thiadiazole ring form interactions
with His110, Val131, Val141, Leu190, Thr191 Ala192, Pro193,
and Trp201 (Figure 4). In common with BZA, the oxygen
atoms of the 5-sulfonamido group of compound 3 form
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Figure 5. Binding site for EZA in its crystal complex with HpCA and comparison with the HpCA/AAZ and HCAII/EZA crystal complexes.
Residue labels in black refer to HpCA. (A) The simulated-annealing omit |2Fo Fc| electron density for EZA is shown in light-blue. The map was
calculated at 2.2 resolution and contoured at 1.0 level. The EZA molecule is shown in all-atom stick representation with carbon atoms colored
light-green. Amino acid residues that interact with EZA are shown in stick representation with carbon atoms colored wheat. The tetrahedrally
coordinated catalytic zinc ion is shown as a black sphere. Hydrogen bonds are shown as blue dashed lines. (B) Superposition of the EZA and AAZ
complexes of HpCA. The EZA and AAZ molecule are shown in all-atom stick representation with carbon atoms colored light-green and cyan,
respectively. For clarity, protein residues and protein surface are shown for the EZA complex only. The surface was calculated with the inhibitor
molecule excluded and colored according to the electrostatic potential. (C) Superposition of the active site residues of the HpCA/EZA complex
(wheat) with the corresponding complex of HCAII (green) showing the short helix 130136 that stabilizes the ethoxy group of EZA in the HCAII/
EZA complex. EZA molecule is shown using stick representation. The catalytic zinc ion and protein residues that form interactions with EZA are
shown. The HCAII residues that form additional stabilizing interactions with EZA are shown using ball-and-stick representation and marked with
boxed labels in green.

hydrogen bonds with Lys88 and Asn108. The CH3 substituent

at the thiadiazole ring (absent in BZA) forms a van der Waals
contact with Pro194. The tert-butyl phenyl ring of compound 3
binds in the same pocket as the phenyl group of BZA (Figure
4). The aliphatic tert-butyl substituent forms stabilizing
hydrophobic interactions with the side chain of Leu139 and
the aliphatic part of the side chain of Lys133, which
compensates for an unfavorable contact with the polar side
chain of Asp107 and accounts for the tighter binding of
compound 3 to HpCA (KI = 8.2 nM) in comparison to BZA.
Analysis of Ethoxzolamide Binding Mode and
Comparison to AAZ. The structure of the ethoxzolamide
(EZA) complex of HpCA was determined at 2.2 resolution.
The Fourier maps in the active site showed unambiguous
electron density for the inhibitor (Figure 5A) in all chains
except chain D. Comparisons with the crystal structures of the
dierent HpCA/sulfonamide inhibitor complexes discussed
above showed that the 2-sulfonamide moiety of EZA overlaps
well with that of other sulfonamides, and the thiazole ring
forms similar hydrophobic interactions with Val131, Leu190,
and Ala192. Inspection of the superposition of the structures of

the HpCA complexes with EZA and AAZ (Figure 5B) showed
that the bicyclic ring of the EZA molecule and the thidiazole
ring of AAZ partially overlap. In comparison to AAZ, the EZA
molecule is pivoted about the sulfur atom of the 2-sulfonamide
moiety in the plane of the ring toward the loop 189199. The
benzene ring fused to the thiazole moiety of EZA forms van der
Waals contacts with Pro193 and Pro194 (Figure 5). In
comparison to AAZ, the proximity of the benzene ring of the
EZA molecule to the partially negatively charged carbonyl
oxygen of Pro193 is energetically unfavorable. Furthermore,
EZA forms one less hydrogen bond with the protein than AAZ,
as it lacks the acetamido group which, in the crystal complex
with AAZ, is hydrogen-bonded to the side chain of Asn108
(Figure 5B). In addition, the apolar ethyl tail of the EZA
molecule is mostly solvent exposed. Cumulatively, these
energetically unfavorable factors result in a higher inhibitory
constant value of EZA for HpCA (KI = 193 nM) in
comparison to AAZ (KI = 21 nM).
Comparison of EZA Binding Sites in HpCA and
HCAII. Superposition of the crystal structures of the respective
EZA complexes of HpCA and HCAII (PDB 3CAJ)25 revealed
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positioned its methyl group close to the partially negatively

charged main chain carbonyl oxygen of His84. This structural
analysis explains the reduced inhibitory potency (KI = 549 nM)
of compound 2 compared to compound 1 (KI = 310 nM).
BRZ and DZA were modeled based on the crystal structure
of the complex with the structurally similar EZA, the 6-ethoxy1,3-benzo group of which was replaced with the substituted
thiazine ring for BRZ and substituted thiopyran ring for DZA
(Figure 1). The sulfonamide and thiophene moieties of BRZ
and DZA were placed in the same position as the respective
sulfonamide and thiazole moieties of EZA, and their
heterocyclic six-membered rings were overlapped with the
benzene ring of EZA (Figure 7). The modeling placed the two

a remarkably similar binding mode of EZA in the two enzymes

(Figure 5C). The most noticeable dierences in the EZA/
protein interactions concern the -helix 130136 in HCAII
(absent in HpCA), and residues Thr200 and Gln92 in HCAII
that occupy positions corresponding to Ala191 and Asn108 in
HpCA, respectively. The 130136 -helix of HCAII forms a
wall of the hydrophobic pocket accommodating the 6-ethoxy
tail of EZA, with the apolar side chain of Phe131 forming
stabilizing van der Waals interactions with the inhibitor
molecule. The side chain of Thr200 forms a hydrogen bond
with the nitrogen atom of the inhibitors thiazole ring, while the
side chain of Gln92 is within the van der Waals contact distance
from the benzene ring of EZA. These additional multiple
stabilizing interactions present in the HCAII/EZA complex are
consistent with a much higher potency (KI = 8 nM) of EZA
toward the human CAII as compared to HpCA (KI = 193
nM).
Docking Studies of Compound 2, Brinzolamide (BRZ),
and Dorzolamide (DZA). To further investigate structure
activity relationships in sulfonamides, three related sulfonamide
derivatives (compound 2, BRZ and DZA) were modeled into
the HpCA active site. The chemical structure of compound 2
is very similar to that of compound 1, with the main dierence
being an additional R1 methyl group at position 4 of the
thiadiazole ring (Figure 1). The crystal structure of the
HpCA/compound 1 complex was therefore used for
modeling the binding mode of compound 2 by placing its
sulfonamide and thiadiazole moiety in the same position as
those of compound 1 in its respective crystal complex.
Modeling compound 2 in binding mode A observed for
compound 1 (Figure 6) positioned its apolar 4-methyl group
within energetically unfavorable distance from the partially
negatively charged main chain carbonyl oxygens of Ala192 and
Pro193, similar to the situation observed for the structurally
similar MZA in its respective crystal complex with HpCA.22
Modeling compound 2 in binding mode B (Figure 6)

Figure 7. BRZ and DZA inhibitor molecules modeled into the active
site of HpCA using the crystal structure of the HpCA/EZA
complex as a template. The EZA, BRZ, and DZA molecules are shown
in all-atom stick representation with carbon atoms colored light-gray,
magenta, and light-green, respectively. Possible additional hydrogen
bonds between BRZ/DZA and protein (not observed in the EZA
complex) are shown.

oxygen atoms of the sulfodioxide moiety of both BRZ and DZA

within a hydrogen bonding distance from the side chain amines
of Lys88 and Lys133. In addition, the N atom of the ethylamine
group of BRZ and DZA was within a hydrogen bonding
distance from the main chain carbonyl oxygen of Pro193.
Furthermore, the ethyl moiety of BRZ and DZA was within van
der Waals contact distance from Trp23, His84, and Ala192
(Figure 7). However, superposition of the modeled BRZ and
DZA complexes of HpCA onto the crystal structures of the
respective complexes of HCAII26 did not produce a good
overlap of the inhibitor molecules despite a signicant degree of
conservation between the active sites of the two enzymes
(Figure 8). Thus, on the basis of our modeling study, we
cannot exclude the possibility that the actual binding modes of
BRZ and DZA in HpCA may be somewhat dierent to that of
EZA. Detailed analysis of these superpositions revealed that
some of the key stabilizing interactions between the protein and
BRZ/DZA in HCAII cannot be formed in the H. pylori enzyme.
One of the dierences concerns the 130136 helix in HCAII
that has no structural equivalent in HpCA. This helix harbors
apolar residues Phe131 and Val135 that contribute to the
hydrophobic environment for the apolar methoxypropyl/
methyl substituent of the six-membered ring of BRZ/DZA
(Figure 8). In addition, the polar side chain of Thr196 in
HpCA is substituted with leucyl (Leu204) in human CAII,
which enhances the hydrophobicity of the binding pocket for

Figure 6. Compound 2 modeled in the active site of HpCA using

coordinates for compound 1 in the respective complex as a template.
Binding modes A and B are shown with carbon atoms colored cyan
and light-green, respectively. In mode A, the weaker binding of
compound 2 in comparison to compound 1 is likely due to
energetically unfavorable interaction between the additional aliphatic
methyl group of compound 2 and partially negatively charged carbonyl
oxygen atoms of the main chain peptides of Ala192 and Pro193. In
mode B, binding of compound 2 would be less tight than compound 1
due to an unfavorable proximity between its methyl group and the
carbonyl oxygen of His84.
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Figure 8. Superposition of the HpCA/BRZ (A) and HpCA/DZA (B) complexes, modeled using the crystal structure of the HpCA/EZA
complex as a template, with the crystal structures of the respective complexes of HCAII.25,26 The HpCA and HCAII complexes are colored wheat
and green, respectively. The catalytic zinc ion and protein residues that form interactions with BRZ and DZA are shown. The BRZ and DZA
molecules are drawn using stick representation. Residue labels in black refer to HpCA. The HCAII residues that form additional stabilizing
interactions with BRZ and DZA are shown using ball-and-stick representation and marked with boxed labels in green.

Figure 9. Classication of the HpCA residues forming interactions with dierent classes of sulfonamide inhibitors discussed in this study and
analysis of the dierences with the respective sulfonamide binding site in HCAII. (A) The superposition of the crystal structures of HpCA in
complex with compound 1 (magenta), compound 3 (blue), AAZ (red), BZA (black), and EZA (orange) showing dierent pockets in and around the
active site that accommodate these inhibitors. Only the protein moiety of the respective AAZ complex is shown for clarity. The HpCA protein
surface that contributes to binding of all inhibitors is colored green and comprises residues Val131, Val141, Leu190, Thr191, Ala192, and Trp201.
The residues that form interactions with some sulfonamides but not others are shown in stick representation and colored according to the pocket
they are in. The residues colored red form the pocket that accommodates the alternative binding mode (mode B) of compound 1 and BZA. The
residues colored cyan form the walls of the pocket that accommodates the R2 substituent of AAZ, BZA in the binding mode A, and compound 3.
Residues Pro193 and Pro194 that interact with the R1 substituent (methyl group) of compound 3 and MZA, and with the fused bicyclic ring of EZA,
are colored orange. (B) Superposition of the crystal structures of HpCA (wheat) in complex with compound 3 (thin black lines) and HCAII (gray)
highlighting dierences between the sulfonamide binding sites in the two enzymes. The additional helix 130136 in HCAII is shown in green. Its
structural equivalent in HpCA is a short loop colored blue. The residues involved in inhibitor binding that are not conserved between the two
enzymes are shown using ball-and-stick representation and labeled in black and green in HpCA and HCAII, respectively. The catalytic zinc ion is
shown only in HpCA as a light-blue sphere.

the methoxypropyl tail. These structural dierences between

HpCA and HCAII are consistent with the respective
dierences in potency of BRZ/DZA (KI = 210 nM/4360 nM
for HpCA compared to 3 nM/9 nM for HCAII).
Overview of the Pockets in the HpCA Active Site
That Accommodate Dierent Classes of Sulfonamide
Inhibitors. In this study, we presented analysis of the
complexes between HpCA and a series of sulfonamide
inhibitors containing a common structural core consisting of
a ve-membered ring linked to a sulfonamide moiety. On the
basis of this analysis, we classied HpCA residues forming
interactions with dierent classes of sulfonamide inhibitors

discussed in this study, according to the binding pocket they

belong to. Residues Val131, Val141, Leu190, Thr191, Ala192,
and Trp201 (colored green in Figure 9A) interact with the
common structural core and therefore contribute to binding of
all inhibitors. Residues Trp23, Thr83, His84, Thr86, and
His110 (colored red) form a hydrophilic pocket that
accommodates the alternative binding mode (mode B) of
compound 1 and BZA. Residues Pro193 and Pro194 (orange)
form a surface that interacts with the R1 substituent (methyl
group) of compound 3 and MZA and with the fused bicyclic
ring of EZA (Figure 9A). The R2 substituents of AAZ, BZA in
the binding mode A, and compound 3 are bound within the
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hydrophobic (Figure 9B). In contrast, this helix is absent in the

HpCA structure, the pocket is more open to the solvent and is
lined on this side with polar residues Asp107 (corresponding to
Ile91 in HCAII), Lys133, and Thr196 (corresponding to
Leu204 in HCAII). This suggests that sulfonamides with polar
or amphipathic R2/X group are likely to have preference
toward HpCA over HCAII. On the other side, this pocket is
lined with mainly polar residues in both HpCA and its human
orthologue (Figure 9B). Although the shape and hydrophilicity
of this surface is similar in the two proteins, the residues that
form it are not conserved between the two enzymes (Thr86,
Thr83, Lys88, and Asn108 in HpCA corresponding to Ala65,
Asn62, Asn67, and Gln92 in HCAII), and these additional,
subtle dierences may therefore also be exploited in the design
of more selective inhibitors targeting HpCA.

pocket lined with mostly polar residues Lys88, Asp107, Asn108,

Lys133, and Leu139 (colored cyan in Figure 9A). We noted
that the most potent inhibitor in the analyzed series, compound
3 (KI = 8 nM), forms extensive interactions with this pocket via
its bulky apolar R2 tert-butyl phenyl moiety. While the
hydrophobic nature of this moiety is matched by the apolar
side of the pocket formed by the side chain of Leu139 and the
aliphatic part of the side chain of Lys133, its proximity to the
polar side chains of Lys88, Asp107 and Asn108 is a
destabilizing factor. Our structural analysis therefore suggests
that sulfonamides with a bulky, polar, or amphipathic R2
substituent group may have higher potency toward HpCA
than compound 3.
Summative Analysis of the Dierences between the
Sulfonamide Binding Sites in HpCA and HCAII. To
identify the structural dierences between HpCA and its
human orthologue CAII that can be exploited in the rational
design of H. pylori-specic inhibitors, we have summarized the
analysis of the superpositions of the corresponding sulfonamide
complexes of HpCA and HCAII. The results are presented in
Figure 9B by mapping these dierences on the representative
structure of the HpCA/sulfonamide crystal complex (with
compound 3) and the crystal structure of HCAII (PDB
2VVA).27
We note that the residues that interact with the common
structural core of all sulfonamides discussed here, and therefore
play an important role in the binding of all inhibitors, are
strongly conserved in HpCA and HCAII (residue IDs for
HCAII in parentheses): Val131 (Val121), Val141 (Val143),
Leu190 (Leu198), Thr191 (Thr199), Ala192 (Thr200), and
Trp201 (Trp209). This observation is consistent with the fact
that the two enzymes showed a remarkably similar mode of
binding to compound 1, the structure of which represents the
common motif present in all inhibitors in this study. The only
dierence between the residues surrounding this common
structural core in the two enzymes is Thr200 in HCAII in place
of Ala192 in HpCA. Comparisons between the HpCA
complex structures and the crystal structures of sulfonamide
complexes of HCAII2325 showed that this substitution results
in one additional hydrogen bond between the side chain of
Thr200 and the nitrogen atom of the ve-membered
heterocyclic ring of the inhibitor. All of the sulfonamides in
this study that contain a nitrogen atom in the respective
position of the ve-membered heterocyclic ring showed higher
potency toward HCAII than HpCA (not including compound
3, for which KI for HCAII is not available) (Supporting
Information, Table S1), which indicates that this additional
hydrogen bond between HCAII and the common structural
core of the inhibitor plays an important role in stabilizing the
bound inhibitor in the HCAII active site.
Further analysis of this superposition revealed that the small
hydrophilic pocket that accommodates the alternative binding
mode (mode B) of compound 1 and BZA in HpCA
(highlighted in red in Figure 9A) is obstructed by the C and
O atoms of Thr200 in HCAII (Figures 3D and 9B, and
Supporting Information, Figure S2). Inhibitors targeting this
pocket would therefore be selective to HpCA over HCAII.
The most signicant structural dierences between the two
enzymes are in the pocket that accommodates the R2/X group
of the sulfonamide inhibitors (highlighted in cyan in Figure
9A). In HCAII, this pocket is anked on one side by the helix
harboring apolar residues Phe131 and Val135 and by the side
chains of Leu204 and Ile91, which render this surface strongly

CONCLUSIONS
Analysis of the crystal structures of HpCA in complex with a
family of sulfonamide inhibitors, including the newly
synthesized compound 3, allowed us to investigate the
structural features of the enzymeinhibitor interactions that
modulate binding anity, and to identify similarities and
dierences of the inhibitor binding sites in HpCA and its
human orthologue HCAII. Our study revealed that the binding
mode of dierent sulfonamides correlates well with their
inhibitory activities against HpCA. Compound 3 displayed
nanomolar inhibition (KI = 8 nM), the lowest that has ever
been reported for any sulfonamide inhibitor of HpCA. The
structural comparisons showed that HpCA possesses an
additional small pocket in the active site (absent in HCAII) that
serves as an alternative binding site for sulfonamides.
Furthermore, the pocket that accommodates the R2/X group
of sulfonamide inhibitors in HpCA is more open and
hydrophilic in nature, as compared to the respective hydrophobic pocket in HCAII. The structural features of HpCA
sulfonamide interactions identied in our study can be
exploited in the design of selective and more potent inhibitors
of HpCA, which may lead to novel anti-H. pylori drugs.

EXPERIMENTAL SECTION

Synthesis of the Inhibitors. EZA was purchased from SigmaAldrich. Compounds 1 and 2 and BZA were synthesized as reported
earlier.28,29 To synthesize compound 3, 5 mmol of compound 2 was
dissolved in 15 mL of an aqueous 2.5 M solution of NaOH and cooled
to 05 C in a saltice bath. 4-tert-Butylphenylsulfonyl chloride (5
mmol) was added in small portions concomitantly with 10 mL of a
cold 2 M NaOH solution, with the temperature maintained below 10
C. The reaction mixture was stirred at room temperature for 510 h
with monitoring by thin-layer chromatography, then the pH was
adjusted to 2.0 with 5 N HCl, and the precipitated sulfonamides were
ltered and recrystallized from aqueous ethanol. The purity of all
compounds was >95% as determined by HPLC.
5-(4-tert-Butylphenylsulfonamido)-4-methyl-2-1,3,4-thiadiazoline-2-sulfonamide, 3. White crystals, mp 2501 C (EtOH).
IR (KBr) (cm1) 1133 and 1175 (SO2 sym), 1310 and 1332 (SO2 as).
1
H NMR (DMSO-d6) (, ppm; J, hertz) 0.96 (s, 9H, t-Bu), 3.42 (s,
3H, N-Me), 7.70 (d, 2H, AABB, 8.7), 7.84 (d, 2H, AABB, 8.7), 8.52
(s, 2H, SO2NH2), 13.657 (br s, 1H, SO2NH). 13C NMR (DMSO-d6)
(, ppm): 13.23 (Me), 46.81 (N-Me), 61.69 (C from t-Bu), 128.21
(C2/C3 of Ph), 131.50 (C3/C2 of Ph), 141.23 (C1/C4 of Ph), 144.78
(C4/C1 of Ph), 162.69 (C-thiadiazoline), 164.52 (C-thiadiazoline).
Anal. (C13H18N4O4S3) C, H, N.
Inhibition Studies. Inhibitory constant of AZA, MZA, BZA, EZA,
BRZ, DZA, compound 1, and compound 2 were reported earlier.19
The inhibitory activity of compound 3 was measured by following the
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Table 1. X-ray Data Collection Statistics

complex

BZA

EZA

compd 1

compd 3

space group
cell dimension (a,b,c (), (deg)
observed reections
unique reections
resolution range ()
Rmergea
average I/(I)
completeness (%)
redundancy

P21
42.2, 134.4, 165.9, 90.1
129205
55072
42.62.4 (2.52.4)
0.077 (0.319)
8 (2.7)
78.3 (81.1)
2.3 (2.2)

P21
42.9, 138.8, 168.2, 90.1
282181
96783
39.92.2 (2.32.2)
0.067 (0.491)
9.8 (2.4)
97.1 (98.5)
2.9 (2.9)

P21
41.8, 136.9, 166.3, 90.1
213336
64612
302.5 (2.62.5)
0.047 (0.315)
16.6 (3.7)
99.8 (99.7)
3.3 (3.1)

P21
41.6, 133.9, 166.6, 90.3
138343
39929
29.92.9 (3.02.9)
0.108 (0.265)
8.3 (4.0)
99.8 (99.7)
3.5 (3.5)

R merge =

(h i |Ihi Ih|)
h i |Ihi|

, where Ihi is the intensity of the ith observation of reection h.

Table 2. Properties of the Final Models

resolution range ()
no. of reections
residues/atoms/waters
R/Rfree
average B (protein) (2)
average B (water) (2)
average B (Zn ions) (2)
average B (inhibitors) (2)
bond-length deviation from ideality ()
bond-angle deviation from ideality (deg)
Ramachandran space (%)
favored
allowed
outliers

HpCABZA

HpCAEZA

HpCA1

HpCA3

41.32.4
55054
1726/14479/155
0.19/0.23
43
29
40
38
0.01
1.6

39.92.2
96747
1751/15081/538
0.20/0.24
45
35
38
39
0.01
1.4

29.92.5
64588
1756/14565/103
0.22/0.27
52
36
39
47
0.01
1.3

29.92.9
39892
1785/14827/0
0.18/0.24
46
35
37
0.01
1.6

93
7
0

96
4
0

94
6
0

93
7
0

excluded. MR revealed that the asymmetric unit of all complexes

contains four dimers. Renements were carried out using PHENIX,37
with the twin law (h, -k, -l) and torsion noncrystallographic symmetry
restraints applied. COOT38 was used to inspect the electron density
maps and rebuild the models manually where necessary. The
dierence Fourier maps clearly revealed phase-unbiased electron
density for one zinc ion and respective bound inhibitors in each
subunit of all complexes except in chain F and chain D of the BZA and
EZA complexes, respectively. The topology and restraints les of the
inhibitors were generated using the COOT Ligand Builder interface
and PHENIX eLBOW,39 respectively. Renement of the structures
using PHENIX and iterative model building using COOT was
continued until the R and Rfree converged. The average B factors for
the Zn ions and the respective inhibitors molecules in the nal rened
model were close to those of the surrounding protein atoms,
suggesting that the ions and inhibitor molecules are bound at close
to full occupancy. An automatic water molecules search was performed
using PHENIX, but waters were only retained after renement if their
B-factor remained below 50 2. The validity of the nal models was
conrmed using MOLPROBITY.40 The nal model renement
statistics are summarized in Table 2. The structure gures were
produced using PYMOL.41
Molecular Modeling. Compound 2 was modeled in the active site
of HpCA using the coordinates of its complex with compound 1 as a
template. BRZ and DZA were modeled using the HpCA/EZA
complex coordinates as a template by superposing the sulfonamide
moiety and the ve-membered ring of BRZ and DZA onto those of
EZA and selecting a conformationally allowed rotamer of the
ethylamine and methoxypropyl groups that does not clash with
protein atoms. The models were generated and analyzed using COOT.

initial rates of the HpCA-catalyzed hydration of CO2 in buer

containing 20 mM Hepes (pH 7.5) and 20 mM Na2SO4 using an
Applied Photophysics stopped-ow instrument as previously
described.30 HpCA was preincubated with compound 3 for 15 min
at room temperature prior to the assay. The KI value was calculated by
nonlinear least-squares methods using PRISM 3.
Protein Purication, Crystallization, and X-ray Diraction
Data Collection. HpCA from strain 26695 was expressed in
Escherichia coli using the pET151/D-TOPO vector and puried by
following the previously described procedure.31 Crystals of the
HpCA complex with AAZ were obtained by hanging-drop vapor
diusion method from buered PEG 1.5 K solutions as described
earlier.22 To obtain HpCA crystals in complex with BZA, EZA,
compound 1, or compound 3, the crystals grown in the presence of
AAZ were soaked for 34 h in a stabilizing solution containing 30%
PEG 1.5 K, 240 mM dibasic ammonium citrate, and 1.2 mM ZnCl2
and then soaked overnight in a stabilizing solution containing 5 mM
BZA, EZA, compound 1, or compound 3. The crystals were then
cryoprotected in a stabilizing solution supplemented with 20% (v/v)
glycerol and 5 mM respective ligand and ash-cooled in liquid
nitrogen for data collection. X-ray diraction data were collected at
cryogenic temperatures using the MX1 and MX2 beamlines of the
Australian Synchrotron. All data processing and scaling were
performed using iMOSFLM32 and SCALA,33 respectively, from the
CCP4 software suite.34 A summary of the data processing statistics is
presented in Table 1. The crystals of all complexes were isomorphous,
belonged to space group P21, had the value close to 90, and
displayed pseudomerohedral twinning with the twin law (h, -k, -l)
detected by using PHENIX Xtriage.35
Structure Determination and Analysis. The crystal structures
of the HpCA complexes with BZA, EZA, compound 1, and
compound 3 were determined using MR (PHASER)36 with the
coordinates of the HpCA dimer in complex with AAZ (PDB
4YGF)22 as a search model, with ions, AAZ, and water molecules
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(8) Parsonnet, J.; Hansen, S.; Rodriguez, L.; Gelb, A. B.; Warnke, R.
A.; Jellum, E.; Orentreich, N.; Vogelman, J. H.; Friedman, G. D.
Helicobacter pylori infection and gastric lymphoma. N. Engl. J. Med.
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ASSOCIATED CONTENT

S Supporting Information
*

The Supporting Information is available free of charge on the

ACS Publications website at DOI: 10.1021/acs.jmedchem.6b01333.
HpCA and HCAII inhibition data for the sulfonamide
compounds discussed in this study, gures showing a
comparison of the compound 1 and AAZ binding modes
in HpCA, and the superposition with the respective
HCAII/compound 1 complex (PDF)
Molecular formula strings (CSV)
Accession Codes

The coordinates and structure factors for the HpCA

complexes with BZA, EZA, compound 1, and compound 3
have been deposited in the Protein Data Bank, www.rcsb.org,
under accession codes 5TT8, 5TT3, 5TUO, and 5TV3,
respectively. Authors will release the atomic coordinates an
experimental data upon article publication.

AUTHOR INFORMATION

Corresponding Author

*Phone: +61399029194. E-mail: Anna.Roujeinikova@monash.

edu.
ORCID

Claudiu T. Supuran: 0000-0003-4262-0323

Anna Roujeinikova: 0000-0001-8478-4751
Notes

The authors declare no competing nancial interest.

ACKNOWLEDGMENTS
J.K.M. is the recipient of the Postgraduate Publication Award
(PPA), 2016, Monash University.
ABBREVIATIONS USED
HpCA, -carbonic anhydrase of H. pylori; HCAII, human
carbonic anhydrase II; MALT, mucosa-associated lymphoid
tissue; CA, carbonic anhydrase; HpCA, -carbonic anhydrase
of H. pylori; AAZ, acetazolamide; EZA, ethoxzolamide; MZA,
methazolamide; DZA, dorzolamide; BRZ, brinzolamide; BZA,
benzolamide; MR, molecular replacement

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DOI: 10.1021/acs.jmedchem.6b01333
J. Med. Chem. 2016, 59, 1109811109