Out of the active site binding pocket for carbonic anhydrase inhibitors

Article abstract of DOI:10.1039/c4cc07320g

A structural study of the adduct which 2-benzylsulfinylbenzoic acid forms with human carbonic anhydrase II is reported, showing a binding mode completely different from any other class of carbonic anhydrase inhibitors investigated so far; this carboxylate binds in a pocket situated out of the enzyme active site. This journal is

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Out of the active site binding pocket for Carbonic
Anhydrase inhibitors†
Cite this: DOI: 10.1039/x0xx00000x
Katia D’Ambrosio,a,* Simone Carradori,b Simona M. Monti,a Martina
Buonanno,a Daniela Secci,b Daniela Vullo,c Claudiu T. Supuranc and
Giuseppina De Simone.a,*
Received 00th January 2012,
Accepted 00th January 2012
DOI: 10.1039/x0xx00000x
www.rsc.org/
and out.8-10 In the in conformation the histidine is orientated toward
A
structural
study
of
the
adduct
which
2-
the interior of the active site, probably to accept a proton from a
relay of water molecules, which connect it to the zinc-bound water
molecule, whereas in the out conformation this residue is orientated
toward the external of the active site to deliver the proton to the bulk
solvent.8-10
benzylsulfinylbenzoic acid forms with human carbonic
anhydrase II is reported, showing a binding mode completely
different from any other class of carbonic anhydrase
inhibitors investigated so far: this carboxylate binds in a
pocket situated out of the enzyme active site.
Although being a very simple reaction, the hydration of carbon
dioxide is of fundamental importance to many processes based on
gas exchange, ion transport, and pH balance, thus connecting hCAs
to a variety of physiological processes. Since abnormal levels and/or
activities of these enzymes have been associated with several human
diseases, such as glaucoma, obesity, cancer, epilepsy, etc,2 they
represent interesting targets for pharmaceutical research. Indeed,
several CA inhibitors (CAIs) are currently clinically used for the
treatment or prevention of diseases such as glaucoma and epilepsy.1,2
The main drawback associated with the use of CAIs as drugs is
related to their lack of selectivity caused by the high sequence and
structural similarity of the different hCA isoforms. Thus, the last
years saw a large number of efforts devoted to the development of
isozyme-selective inhibitors.
At least two major classes of CAIs have been so far characterized:
compounds which bind to the enzyme active site coordinating the
catalytic zinc ion,1,2 and compounds which bind in the active site but
do not interact with the metal ion.11-16 Among the zinc binders the
most important CAIs are the primary sulfonamides and their
isosteres, such as sulfamates and sulfamides.1,17,18 They bind as
deprotonated anions to the Zn(II) from the CA active site in
Human carbonic anhydrases (hCAs) are zinc-requiring
metalloenzymes, which catalyze the reversible hydration of carbon
dioxide to bicarbonate and proton.1,2 15 different isoforms have been
so far identified, among which 12 are catalytically active (CAs I–IV,
VA–VB, VI–VII, IX, and XII–XIV), whereas the CA-related
proteins (CARPs) VIII, X, and XI are devoid of catalytic activity.1,2
The hCAs catalytic mechanism is a well understood process, which
takes place in two separate steps described by equations (1) and
(2).3,4
-
EZn2+-OH- + CO2 ⇆ EZn2+-HCO3- ⇆ EZn2+-H2O + HCO3 (1)
EZn2+-H2O + B ⇆ EZn2+-OH- + BH+
(2)
The first catalytic step is the nucleophilic attack of a zinc-bound
hydroxide ion on a carbon dioxide molecule entrapped within a
hydrophobic pocket of the active site, which leads to the formation
of a bicarbonate ion coordinated to the metal ion. The binding of
bicarbonate to zinc is rather weak and thus this ion is readily
substituted by a water molecule, generating the acidic form of CA,
which is catalytically unproductive (equation 1). In the second step
of the reaction, which is the rate limiting one, the zinc-bound
hydroxide is regenerated through a proton transfer reaction from the
zinc-coordinated water molecule to the external buffer.4 This step is
generally facilitated by a residue placed in the middle of the active
site, which is able to assist the proton transfer reaction, acting thus as
a proton shuttle. In the majority of human CA isoforms this residue
is a histidine (e.g., His64 in hCA II, IV, VII, IX, etc).5,6 In some
other isoforms, the absence of this histidine has been related to the
low catalytic efficiency of the enzyme. For example in hCA III,
which has the lowest catalytic efficiency among the active isoforms,
this histidine is substituted by a lysine.7 Further support to the role of
His64 as proton shuttle comes from the observation that in a large
number of studies, conducted mainly on the ubiquitous isoform hCA
II, this residue is observed in two different conformations, termed in
tetrahedral
geometries.
Other
zinc-binders
are
the
dithiocarbamates,19 xanthates20 as well as small inorganic anions
such as halides, pseudohalides, etc.21 The second class of inhibitors
is characterized by compounds that can either be anchored to the
zinc-coordinated water molecule/hydroxide ion, as observed for
phenols,22,23 and polyamines,12 or bind to the border of the active
cavity partially occluding its entrance, as reported for coumarins14,15
and lacosamide.24
Interestingly the carboxylic acids, a group of compounds containing
many milli- micro-molar CAIs,21 can show binding modes belonging
to both classes mentioned above. Indeed, some of them (formate and
acetate), coordinate to the Zn(II) ion25-28 while others, such as
phenolic acids 1 and 2 (Figure 1),29 interact with the enzyme
hydrogen bonding the zinc-bound water molecule. However the
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molecular determinants responsible of one or the other binding mode Crystallization experiments on the unbound hCA II were performed,
are not at all understood yet.
as previously described (see Supporting information for
experimental details),31,32 using the hanging-drop vapor-diffusion
method and ammonium sulfate as precipitant. Crystals of the hCA
II/3 adduct were then obtained by soaking the crystals of the enzyme
in a saturated solution of the inhibitor for 48 hours. The structure of
the enzyme-inhibitor complex was analyzed by difference Fourier
techniques using 1CA2 structure as starting model33 and refined to a
resolution of 1.50 Å with final crystallographic R-factor/R-free
values of 0.163/0.184. Statistics for data collection and refinement
are shown in Table S1.
Figure 1. Chemical structures of compounds 1-3
Table 1. hCA I, II, IX and XII inhibition data with compound 3 and
acetazolamide (AAZ, 5-acetamido-1,3,4-thiadiazole-2-sulfonamide) as
standard inhibitor, by CO2 hydration, stopped-flow technique.
KI (ꢀM)[a]
In a general research project aimed to investigate in more detail
these compounds, we have undertaken a kinetic and structural study
of
an
ortho-substituted
benzoic
acid,
namely
2-
benzylsulfinylbenzoic acid 3, showing that this molecule not only
possesses an interesting inhibition profile against several
physiologically relevant hCA isoforms, but it also presents a binding
mode completely different from any other class of CAIs investigated
so far: this carboxylate binds in a pocket which is not situated within
the enzyme active site, but OUT of it.
hCA I
hCA II
hCA IX
hCA XII
Compound
3
>10
0.15
1.29
>10
AAZ
0.25
0.012
0.025
0.006
[a]Mean from 3 different assays, errors were within ± 5-10% of the reported
values.
Compound 3 was synthesized by selectively oxidizing the sulfide 4
to the corresponding sulfoxide, as reported in the literature (Scheme
1).30 Its physico-chemical characterization was in full agreement
with the reported structure (see Supporting information).
Analysis of the electron density maps within the catalytic site of
the enzyme surprisingly did not show the binding of any inhibitor
molecule, neither to the catalytic zinc ion nor to the residues in its
immediate neighbourhood. On the contrary, an inhibitor molecule,
rather well defined in the electron density (Figure 2), was observed
in a cavity located on the protein surface, about 14 Å far from the
Zn2+ ion, and delimitated by residues Gly6, Tyr7, Gly8, Asn11,
His64, Phe231, Asn232 and Glu239 (Figure 3).
Scheme 1. Synthesis of compound 3
Sulfoxides have rarely been studied as ligands for transition metal
ions in metalloenzymes, despite their zwitterionic form endowed
with chelating properties. Furthermore, the sulfoxide moiety displays
good synthetic versatility towards steric and electronic
modifications, and induces a chiral center important in recognition
by enzyme active sites. Thus, our initial working hypothesis was that
the concurrent presence of two functional groups (COOH and S=O)
could infer an ambidentate character to this derivative. The
inhibition profile of four physiologically dominant isoforms hCAs I,
II, IX and XII with compound 3 and acetazolamide (AAZ), as
standard sulfonamide inhibitor, was investigated (Table 1) (see
Supporting information for experimental details). These isoforms
have been used in the assay due to the fact that the cytosolic hCAs I
and II are widespread house-keeping enzymes, involved in a host of
physiologic processes, whereas the transmembrane hCAs IX and XII
are tumor-associated enzymes, abundant in hypoxic tumors and
recently validated as antitumor targets.1 Interestingly, hCA I was not
Figure 2. σA-weighted |2Fo−Fc| map (at 1.0 σ), relative to the inhibitor
molecule 3. Residues involved in inhibitor recognition are also shown.
The inhibitor was stabilized in this cavity by several polar
interactions. In particular, one oxygen of the carboxylate moiety
accepted a hydrogen bond from the backbone nitrogen atom of Tyr7,
whereas the other oxygen interacted with the ND2 atom of Asn11
(Figure 2). Moreover, both oxygen atoms of the carboxylate group
were involved in two strong hydrogen bonds with water molecules,
which in turn were bound to the backbone oxygen of Trp5 and to the
ND1 atom of His64. Interestingly, this latter water-mediated
hydrogen bond froze the His64 residue in its out-conformation.
Several other interactions stabilized the organic scaffold of the
inhibitor within the cavity. In particular, the phenyl ring established
a number of strong van der Waals contacts (distance < 4.0 Å) with
residues Gly6, Tyr7, Gly8, Phe231 and Glu239, while the
benzysulfinyl moiety interacted with Phe231 and Asn232 (Figure 2).
Binding of inhibitors in this region of the enzyme has already been
observed in the complexes which hCA II forms with two other
benzoic acids, namely 1 and 2 (Figure 1). Indeed, the structural
superposition of these latter two complexes with the one under
significantly inhibited by benzoic acid
3 up until 10 ꢀM
concentration of inhibitor, whereas the other cytosolic isoform, hCA
II, was inhibited in the submicromolar range (KI of 0.15 ꢀM).
Moreover 3 was a micromolar inhibitor of hCA IX but did not
significantly inhibit hCA XII. It is also interesting to note that the
sulfide 4 did not significantly inhibit any of these CA isoforms (KIs
> 10 ꢀM, data not shown). The inhibition data of 3 are rather
atypical, prompting us to hypothesize a different mechanism of
action with respect to all known inhibitors. For this reason we
investigated in detail the interaction of the benzoic acid 3 with hCAs
by means of X-ray crystallography, choosing hCA II for the study
since it is easily to crystallize and the best studied isoform.1
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investigation showed that the phenyl rings of the three inhibitors are shows a restoration of the proton shuttling activity blocked by 3,
perfectly superimposable and their interactions with water molecules
and both enzyme residues Tyr7 and Asn11 are all retained (Figure
4).
even at low concentrations of imidazole (2 ꢀM), with an almost total
regain of the initial catalytic activity at 15-20 ꢀM of imidazole in the
assay system. It should be mentioned that imidazole itself acts as a
CA activator (CAA),38 binding in a site at the entrance of the active
site cavity,5 and providing an alternative route for proton release. We
also tried to rescue the catalytic activity of hCA II complexed to 3
with a buffer which does not bind within the CA active site, such as
HEPES or TRIS (in concentrations of 2-20 ꢀM). In these cases no
rescue of activity was observed, which is another confirmation of the
proposed inhibition mechanism.
As mentioned above, compound 3 is a good hCA II and hCA IX
inhibitor, but does not inhibit hCA I and hCA XII (Table 1). It is
difficult to identify the reasons of this behaviour; however, an
hypothesis can be done superimposing the residues defining the
cavity where 3 is bound in hCA II, to the corresponding residues in
the other isoforms (data not shown). From this superposition it can
be observed that the substitution of Glu239 with an arginine in CA
XII and of Gly8 with an aspartate in hCA I can cause a steric
hyndrance for the positioning of the inhibitor. More generally, it can
be seen that this cavity is delimited by different residues in the
diverse isoforms, thus it can be considered an interesting target to be
explored in drug design studies of selective CAIs.
Figure 3. Solvent accessible surface of hCA II in its complex with 3. The
cavity where the inhibitor is bound is highlighted in magenta. The catalytic
zinc ion is represented as a cyan sphere at the bottom of the active site cavity.
Figure 4. Structural superposition of the hCA II/1 (magenta) (PDB code
4E3D), hCA II/2 (cyan) (PDB code 4E3F) and hCA II/3 (green) complexes
in the protein region where compound 3 is bound.
Figure 5. A detail of the active site of the hCA II/1 adduct with inhibitor 3
superimposed to 1 in two different orientations (A) and (B), demonstrating
that it is not possible its accommodation in the active site without clashing
with enzyme active site residues.
However, it is worth noting that in hCA II/1 and hCA II/2 adducts
another inhibitor molecule has been observed anchored to the zinc-
bound water molecule.29 In order to understand why 3, although
being a substituted benzoic acid like 1 and 2, does not bind to the
zinc-bound water molecule in the same manner of its analogues, we
superimposed it to the molecules 1 and 2 bound in the active site.
From this superposition it is clear that compound 3 in this position
would strongly clash by means of its ortho substituent with residues
which delimit the active site cavity (Figure 5).
It is important to highlight that this is the first time that the binding
in the aforementioned cavity on the protein surface is not associated
to a contemporary binding within the active site, thus indicating that
it alone can cause the inhibition of the enzyme catalytic activity.
Table 2. hCA II inhibition by 10 ꢀM of benzoic acid 3, varying
concentrations of imidazole (Im), by a CO2 hydration, stopped-flow
technique.
Inhibitor
No inhibitor
3 10 ꢀM
3 10 ꢀM + Im 2 ꢀM
3 10 ꢀM + Im 5 ꢀM
3 10 ꢀM + Im 10 ꢀM
3 10 ꢀM + Im 15 ꢀM
% CA activity[a]
100%
58%
61%
73%
84%
96%
100%
147%
Considering the catalytic mechanism, described above, which needs 3 10 ꢀM + Im 20 ꢀM
Im[b] 20 ꢀM
a flexible His64 to restore the active form of the enzyme, it is
a]Errors in the range of 2-4 % from three different measurements; [b]Im alone
is a known CA activator.
reasonable to hypothesize that this inhibition is due to the inability of
His64 to change its conformation and thus participate in the proton
shuttling needed to regenerate the nucleophilic species of the
enzyme. Previously reported studies, showing that the mutation of
His64 to Ala in hCA II caused a decrease in the rate of reaction by
about 20 fold, are in agreement with this idea.6 In these studies it
was also shown that the decrease in catalysis could be rescued by the
addition of exogenous proton acceptors in solution such as
histamine, small imidazoles, pyridines and their derivatives.6,34-37
Thus we decided to test our hypothesis verifying if the addition of
imidazole could restore the enzyme catalytic activity (inhibited by
3). The inhibition of hCA II by 3 with or without imidazole was then
measured and the data are reported in Table 2. Analysis of these data
In conclusion, these findings, together with previously reported
studies, indicate that carboxylic acids can present different
inhibition mechanisms against CAs. Indeed, they can
coordinate directly to the zinc ion, as observed for hCA
II/formate27 and hCA II/acetate,28 form a hydrogen bond with
the zinc-bound water molecule, as observed for hCA II/
hCA II/ , and freeze the out conformation of His64, as
observed for hCA II/ , even though it is difficult to predict
1 and
2
3
a
priori the binding mode adopted by a given carboxylate.
Moreover, it cannot be excluded that the same molecule could
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