Multiple Binding Modes of Inhibitors to Carbonic Anhydrases: How
to Design Specific Drugs Targeting 15 Different Isoforms?
Vincenzo Alterio,
†
Anna Di Fiore,
†
Katia D’Ambrosio,
†
Claudiu T. Supuran,*
,‡
and Giuseppina De Simone*
,†
†
Istituto di Biostrutture e Bioimmagini-CNR, via Mezzocannone 16, 80134 Napoli, Italy
‡
Universita
degli Studi di Firenze, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3, I-50019 Sesto Fiorentino,
Firenze, Italy
CONTENTS
1. Introduction 4421
2. Structural Features of α-CAs 4423
3. Insights into CA Catalytic Mechanism: CO
2
and
HCO
3
−
Binding to hCA II 4423
4. Insights into CA Inhibition: Structural Features of
Zinc Binding Inhibitors 4424
4.1. Binding of Ureates and Hydroxamates 4425
4.2. Thiol Derivatives 4426
4.3. Metal-Complexing Anions 4428
4.4. Sulfonamides 4428
4.4.1. Benzenesulfonamides 4428
4.4.2. Thiophene, Thiadiazole, and Thiadiazo-
line Derivatives 4440
4.4.3. Sulfonamides Containin g Other Ri ng
Systems 4443
4.4.4. Thiazide Diuretics 4445
4.4.5. Aliphatic Sulfonamides 4446
4.5. Sulfamates and Sulfamides 4447
4.5.1. Aliphatic Sulfamates 4448
4.5.2. Sulfamate CAIs Also Acting as Steroid
Sulfatase and Aromatase Inhibitors 4450
4.6. Sulfonamides/Sulfamates/Sulfamides Con-
taining Sugar Moieties 4452
5. Insights into CA Inhibition: Structural Features of
Non-Zinc-Binding Inhibitors 4455
5.1. Compounds Anchoring to the Zinc Bound
Water Molecule 4455
5.1.1. Phenols 4455
5.1.2. Spermine and Related Polyamines 4456
5.2. Compounds Located at the Entrance of the
Active Site: Coumarins and Lacosamide 4457
6. Conclusions 4459
Author Information 4461
Corresponding Author 4461
Notes 4461
Biographies 4461
Acknowledgments 4462
Abbreviations 4462
References 4462
1. INTRODUCTION
Carbonic anhydrases (CAs, EC 4.2.1.1) are ubiquitous metallo-
enz ymes, pre sent throughout most living or ganisms and
encoded by five evolutionarily unrelated gene families: the α-,
β-, γ-, δ-, and ζ-CAs.
1−6
The α- β- and δ-CAs contain a Zn(II)
ion at the active site, the γ-CAs are probably Fe(II) enzymes
(but they are active also with Zn(II) or Co(II) ions), while the
metal ion is usually replaced by cadmium in the ζ-CAs.
5,7−9
All these enzymes catalyze the reversible hydration of carbon
dioxide to bicarbonate ion and proton (CO
2
+H
2
O ⇆ HCO
3
−
+H
+
), following a two step catalytic mechanism.
2
In the
hydration direction, the first step is the nucleophilic attack of a
Zn
2+
-bound hydroxide ion on CO
2
with consequent formation
of HCO
3
−
, which is then displaced from the active site by a
water molecule (eq 1). The second step, which is rate limiting,
regenerates the catalytically active Zn
2+
-bound hydroxide ion
through a proton transfer reaction from the Zn
2+
-bound water
molecule to an exogenous proton acceptor or to an active site
residue, generically represented by B in eq 2.
+
⇆
⇆+
+−
+−
+
−
E
Zn OH CO
EZn HCO
EZn H O HCO
2
2
2
3
2
23
(1)
+⇆ +
++−
+
E
Zn H O B EZn OH BH
2
2
2
(2)
All human CAs (hCAs) belong to the α-class; up to now,
fifteen isozymes have been identified, which differ by molecular
features, oligomeric arrangement, cellular localization, distribu-
tion in organs and tissues, expression levels, kinetic properties
and response to different classes of inhibitors (Table 1).
2,10
Twelve isoforms (CA I−IV, VA−VB, VI−VII, IX, and XII−
XIV) show a variable degree of enzymatic activity, whereas
three isoforms (VIII, X, and XI), the so-called CA-related
proteins (CARPs), are devoid of any catalytic activity (Table
Received: May 18, 2011
Published: May 18, 2012
Review
pubs.acs.org/CR
© 2012 American Chemical Society 4421 dx.doi.org/10.1021/cr200176r | Chem. Rev. 2012, 112, 4421−4468
1).
2,5,11
Several studies demonstrated important roles of CAs in
a variety of physiological processes, and showed that abnormal
levels or activities of these enzymes have been often associated
with different human diseases.
2,3
Consequently, in recent years
CA isozymes have become an interesting target for the design
of inhibitors or activators with biomedical applications.
2,12−18
CA activators may have pharmacological applications in
pathologies in which learning and memory are impaired, such
as Alzheimer’s disease or aging.
19
On the other hand, CA
inhibitors (CAIs) have been originally used as diuretics,
antiglaucoma agents, antiepileptics, and in the management of
altitude sickness,
2
while novel generation compounds are
undergoing clinical investigation as antiobesity, and antitumor
drugs/diagnostic tools.
2,20−26
Table 2 shows a brief presentation of the various hCA
isoforms as drug targets/off-targets. Indeed, some of them
(such as CA I and II) are ubiquitous and may be both targets
for some diseases and off-targets, and in this case their
inhibition should be avoided (e.g., CA IX and XII in tumors
should be inhibited by compounds, which do not affect the
activity of CA I, II, VA, and VB).
27
Specifically CA I is found in
many tissues, but a seminal study from Feeener’s group
28
demonstrated that this enzyme is involved in retinal and
cerebral edema, and its inhibition may be a valuable tool for
fighting these conditions. CA II is involved in several diseases,
such as glaucoma, edema, epilepsy, and probably altitude
sickness.
26,29−33
CA III is involved in the oxidative stress,
characterizing a lot of inflammatory diseases. It is not yet
understood whether this feature is due to the CO
2
hydration
activity of CA III (which is quite low), or to other enzyme
properties, such as a different enzymatic activity or the presence
of Cys residues on its surface, responsible for the antioxidant
effects of this protein.
34−36
CA IV is surely a drug target for
several pathologies, including glaucoma (together with CA II
and XII), retinitis pigmentosa and stroke.
37−40
The mitochon-
drial isoforms CA VA and VB are targets for obtaining
antiobesity agents,
20−22,41
whereas CA VI is implicated in
cariogenesis.
42,43
CA VII has been noted for its contributions to
epileptiform activity together with CA II and XIV.
26,31,44
CA
VIII was demonstrated to be involved in neurodegenerative
diseases, as the CA8 gene has been associated with ataxia, mild
mental retardation and quadrupedal gait in humans and with
lifelong gait disorder in mice, suggesting an important role for
CA VIII in the brain.
45
It was also found to be involved in the
development of colorectal and lung cancers in humans, and its
overexpression has been observed in several other cancers.
45,46
However, nothing is known regarding the involvement of the
remaining two acatalytic isoforms, CA X and XI, in human
pathologies or what is the physiological function of these two
proteins conserved all over the phylogenetic tree in
vertebrates.
45
CA IX and XII are well-established anticancer
Table 1. Organ/Tissue Distribution, Subcellular Localization, CO
2
Hydrase Activity, and Affinity for Sulfonamides of the 15
Human α-CA Isozymes
2,10
organ/tissue distribution subcellular localization
catalytic activity (CO
2
hydration)
affinity for
sulfonamides
CA I erythrocytes, gastrointestinal tract, eye cytosol low medium
CA II erythrocytes, eye, gastrointestinal tract, bone osteoclasts, kidney, lung,
testis, brain
cytosol high very high
CA III skeletal muscle, adipocytes cytosol very low very low
CA IV kidney, lung, pancreas, brain capillaries, colon, heart muscle, eye membrane-bound medium high
CA VA liver mitochondria low high
CA VB heart and skeletal muscle, pancreas, kidney, spinal cord, gastrointestinal
tract
mitochondria high high
CA VI salivary and mammary glands secreted into saliva and
milk
low very high
CA VII central nervous system cytosol high very high
CA VIII central nervous system cytosol acatalytic ND
a
CA IX tumours, gastrointestinal mucosa transmembrane high high
CA X central nervous system cytosol acatalytic ND
a
CA XI central nervous system cytosol acatalytic ND
a
CA XII kidney, intestine, reproductive epithelia, eye, tumors transmembrane low very high
CA XIII kidney, brain, lung, gut, reproductive tract cytosol low high
CA XIV kidney, brain, liver, eye transmembrane low high
a
ND = not determined.
Table 2. hCA Isoforms as Drug Targets/Offtargets in
Various Diseases
a
isoform
disease in which is
involved
possible offtargets among other
hCAs
CA I retinal/cerebral edema
28
unknown
CA II glaucoma
29
hCA I
edema
30
unknown
epilepsy
26,31
unknown
altitude sickness
32,33
unknown
CA III oxidative stress
34−36
unknown
CA IV glaucoma
37
hCA I
retinitis pigmentosa
38,39
unknown
stroke
40
unknown
CA VA/VB obesity
20−22,41
hCA I, hCA II
CA VI cariogenesis
42,43
hCA II
CA VII epilepsy
26,31,44
unknown
CA VIII neurodegeneration
45
unknown
cancer
45
unknown
CA IX cancer
24,27,47
hCA I, hCA II
CA XII cancer
24,47,48
hCA I, hCA II
glaucoma
49
unknown
CA XIII sterility
50
unknown
CA XIV epilepsy
26,51
unknown
retinopathy
52
unknown
a
No data are available in the literature on CA X and XI involvement in
diseases.
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drug targets. CA IX is a marker of disease progression in many
types of hypoxic tumors, and recently its inhibition has been
shown to be associated with a significant inhibition of the
growth of both primary tumors and metastases. Furthermore,
CAIs targeting this isoform can also be used for imaging of
hypoxic tumors.
24,27,47
CA XII is less investigated but basically
it is also an antitumor target.
24,47−49
There are a few reports
regarding CA XIII showing that it is involved in the sperm
motility processes
50
(probably together with CA XIV) and the
fact that its inhibition may be used in obtaining contraceptive
agents. CA XIV is involved in epileptogenesis and, similarly to
CA IV, in some retinopathies, and may be a drug target for
innovative agents useful in the management of such
disorders.
26,51,52
It should be stressed that none of the currently clinically used
CAIs show selectivity for a specificisozyme.
2,5
Thus,
developing isozyme-specific CAIs should be highly beneficial
in obtaining novel classes of drugs devoid of various undesired
side-effects. Recently, a large number of structural studies has
provided a scienti fic basis for the rational drug design of more
selective enzyme inhibitors.
53
However, although X-ray crystal
structures are already available for the majority of the twelve
catalytically active members of the human CA family,
54−65
most
of the reported complexes with inhibitors regards just isozyme
II, the most thoroughly characterized CA isoform.
The aim of this review is to provide an exhaustive description
of the structural studies on α-CAs so far reported. In particular,
the main structural features of the catalytically active α-CA
isozymes characterized to date will be described and the current
state of the art on complexes of hCA II with the principal
classes of inhibitors will be summarized. A comparison with the
corresponding adducts of other isoforms, when available, will
be also performed, in order to find for each isoform the key
residues responsible for enzyme/inhibitor interaction. Finally,
recent advances in the field of drug design of selective CAIs will
be illustrated.
2. STRUCTURAL FEATURES OF α-CAs
As mentioned above, human catalytically active α-CAs differ for
subcellular localizaton: CA I, II, III, VII and XIII exist in
cytosol, CA IV, IX, XII, and XIV are membrane-associated, CA
VA and VB reside in mitochondria, whereas CA VI is secreted
in saliva and milk (Figure 1).
2,5
To date the three-dimensional
structures of all isoforms except CA VB have been
determined.
54−65
The analysis of these structures shows that
independently on their subcellular localization and, as expected
on the basis of their high sequence homology (Figure 2), these
enzymes present a similar structure, characterized by a central
twiste d β-sheet surrounded by helical connections and
additional β-strands (Figure 3). The active site is located in a
large, conical cavity, approximately wide 12 Å and deep 13 Å,
which spans from the protein surface to the center of the
molecule. The catalytic zinc ion is located at the bottom of this
cavity, exhibiting a tetrahedral coordination with three
conserved His residues and a water molecule/hydroxide ion
as ligands (Figure 3).
11,66−68
The Zn
2+
-bound water molecule/
hydroxide ion is involved in a network of hydrogen bonds
which helps to enhance its nucleophilicity. In particular, it is
hydrogen bonded with the hydroxyl moiety of a conserved Thr
residue (Thr199) and with two water molecules, located on
two opposite sides: the first one, also called the “deep water”,is
located in a hydrophobic cavity delimited by conserved residues
in position 121, 143, 198, and 209, while the second one is in a
hydrophilic environment toward the entrance of the active site
(Figure 4).
69
In all the CA isoforms the active site cavity is
divided into two very different environments: the first one is
delimited by a cluster of hydrophobic amino acids, whereas the
other one is lined with hydrophilic residues (Figure 5). Several
studies suggested that these two peculiar active site environ-
ments are responsible of the rapid catalytic cycling of CO
2
to
bicarbonate;
70
indeed, the hydrophobic region is necessary to
sequester the CO
2
substrate and orient the carbon atom for
nucleophilic attack by the zinc-bound hydroxide,
70,71
while the
hydrophilic region creates a well ordered hydrogen-bonded
solvent network, which is necessary to allow the proton transfer
reaction from the zinc-bound water molecule to the bulk
solvent (eq 2 and Figure 4).
70,72−75
Despite the high sequence
and structu ral homology among all the isozymes, the
oligomeric state represents a discriminating factor at least for
three of them. Indeed, all the enzymes are monomeric, except
the two membrane-associated isoforms CA IX and CA XII and
the secreted CA VI, which in contrast are dimeric.
63,65
Intriguingly, the dimeric structures of these three isoforms are
different, and none influences the catalytic properties of the
enzyme. The observation of a dimeric arrangement in only
three α-CAs is quite surprising and probably suggests that the
oligomeric arrangement of these isoforms responds to the
necessity to perform specific functions in the tissues, where
they are generally present.
59,63
3. INSIGHTS INTO CA CATALYTIC MECHANISM: CO
2
AND HCO
3
−
BINDING TO hCA II
Even if in the past a high number of studies provided
hypotheses on the putative binding site of CO
2
in the CA active
Figure 1. Schematic illustration of domain composition and
subcellular localization of catalytically active human α-CAs: the
cytosolic CA I, II, III, VII, and XIII and the mitochondrial CA VA
and VB consist only of the CA domain; the membrane-associated CA
IV, IX, XII, and XIV have a transmembrane anchor and, except for CA
IV, also a cytoplasmic tail, while CA IX is the only isozyme with an N-
terminal proteoglycan-like domain; CA VI is secreted and contains a
short C-terminal extension.
Chemical Reviews Review
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site,
71,76−81
the first clear stru ctural evidence on the CO
2
binding region was obtaine d only recent ly with two
independent crystallographic studies showing the entrapment
of this substrate in the CA II active site.
69,70
Figure 6A shows
the binding interactions of CO
2
as determined by these
structural studies. In particular, the CO
2
molecule is bound in
the hydrophobic side of the active site, in the small pocket
delimited by residues Val121, Val143, Leu198 and Trp209 and
in an orientation that makes it suited for the nucleophilic attack
on the carbon atom by the zinc bound hydroxide ion. One of
the CO
2
oxygen atoms lies about in the same position formerly
occupied by the “deep water”, which is now shifted in a new
position, where it establishes novel hydrogen bond interactions
(see Figure 4 and Figure 6A). These structural data are in
agreement with previously reported mutagenesis studies which
demonstrated that substitutions of Val121, Val143, and Leu198
with bulkier residues, leading to the reduction of the volume of
the CO
2
binding cavity, caused significant decreases in the
activity.
80−83
Interestingly, the presence of the Zn
2+
ion seems
to be not important in the binding and orientation of CO
2
.
70,84
The structure of hCA II/HCO
3
−
complex was also recently
provided by Sjoblom and co-workers.
69
As shown in Figure 6B
the HCO
3
−
lies in the same plane defined by the CO
2
molecule
(Figure 6A) and the Zn
2+
-bound hydroxide ion and is
tetrahedrally coordinated to the catalytic metal ion through
one of its oxygen atoms. The availability of both hCA II/CO
2
and hCA II/HCO
3
−
complex structures allowed to obtain for
the first time a detailed description of the first step of the
enzymatic reaction providing new perspectives in the structural
studies of this exciting enzyme family.
4. INSIGHTS INTO CA INHIBITION: STRUCTURAL
FEATURES OF ZINC BINDING INHIBITORS
Analysis of all available structural studies for CA/inhibitor
complexes allowed to divide CA inhibitors into two main
classes: those that bind to the enzyme active site anchoring
themselves to the catalytic zinc ion, and those that are bound to
the active site but do not interact directly with the metal ion.
Four different groups of zinc binding inhibitors have been
studied crystallographically: the ureates/hydroxamates, the
mercaptophenols, the metal-complexing anions and the
sulfonamides with their bioisosteres, such as sulfamates and
sulfamides.
53
In this chapter the main structural features of the
binding of these molecules to the CA II active site will be
schematically summarized.
Figure 2. Structure-based sequence alignment of α-CAs with known three-dimensional structure. hCA II secondary structure elements are shown
schematically: helices are represented by solid cylinders and β-strands as arrows. Helix and β-strand regions for the various isozymes are highlighted
in red and yellow, respectively. Conserved residues are underlined, catalytic triad, Thr199 and Glu106 are indicated with black triangles, while
residues delimiting the active site cavity are marked with asterisks. β-strand and helix regions are named as reported by Eriksson et al.
55
The
following PDB entries were used in the alignment: 1CA2 (hCA II);
55
2CAB (hCA I);
54
1Z93 (hCA III);
61
1ZNC (hCA IV);
58
1DMX (mCA VA);
57
3FE4 (hCA VI);
65
3ML5 (hCA VII);
64
3IAI (hCA IX);
63
1JCZ (hCA XII);
59
3D0N (hCA XIII);
62
1RJ5 (mCA XIV).
60
Chemical Reviews Review
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4.1. Binding of Ureates and Hydroxamates
hCA II is not only a very effective catalyst for the
interconversion between carbon dioxide and bicarbonate, but
shows some catalytic versatility, participating in several other
hydrolytic processes which presumably involve nonphysiolog-
ical substrates (see Scheme 1). These reactions include the
hydration of cyanate to carbamic acid (eq 3),
85
or of cyanamide
to urea (eq 4),
86,87
the aldehyde hydration to gem-diols (eq
5),
88,89
the hydrolysis of some carboxylic (eq 6)
90−92
or
sulfonic acid esters (eq 7),
92
as well as other less investigated
hydrolytic processes (eq 8−11). Among these reactions, the
hydration of cyanamide to urea has been carefully characterized
by means of spectroscopic, kinetic, and crystallographic
techniques.
86,87
These studies demonstrated that cyanamide
acts as a weak inhibitor of the esterase activity of hCA II,
interacting with the zinc ion within the enzyme active site.
Interestingly, when hCA II crystals were soaked with cyanamide
for 90 min two different adducts with different occupancy were
observed in the active site. In the high occupancy form the
cyanamide replaces the hydroxide ion coordinating to the zinc
ion with a regular tetrahedral geometry. In the low occupancy
Figure 3. Ribbon diagram of hCA II structure (PDB code 1CA2),
which has been chosen as representative CA isoform. The active site
Zn
2+
coordination is also shown. Helix and β-strand regions are
colored in red and yellow, respectively.
Figure 4. The active site of hCA II, which has been chosen as
representative CA isoform. The Zn
2+
is tetrahedrally coordinated by
the three catalytic histidines and a water molecule/hydroxide ion,
which is engaged in a well-defined network of hydrogen bonds. Water
molecules are indicated as red circles. The side chain of His64 is
shown in both the in and out conformations.
Figure 5. Solvent accessible surface of hCA II. Residues delimiting the
hydrophobic half of the active site cleft are shown in red (Ile91,
Phe131, Val121, Val135, Leu141, Val143, Leu198, Pro202, Leu204
Val207 and Trp209), while residues delimiting the hydrophilic one are
shown in blue (Asn62, His64, Asn67 and Gln92).
Figure 6. Active site of hCA II showing: (A) the position of CO
2
molecule (PDB code 2VVA), (B) the binding of the bicarbonate ion
(PDB code 2VVB). The Zn
2+
coordination and polar interactions are
also reported.
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