Hydroxamate represents a versatile zinc binding group for the development of new carbonic anhydrase inhibitors

Article abstract of DOI:10.1039/c2cc34275h

Hydroxamates (R-CONHOH) have been scarcely investigated as carbonic anhydrase (CA, EC 4.2.1.1) inhibitors (CAIs). An inhibition/structural study of PhCONHOH is reported against all human isoforms. Comparing aliphatic (R = Me and CF₃) and aromatic (R = Ph) hydroxamates as CAIs, we prove that CONHOH is a versatile zinc binding group. Depending on the nature of the R moiety, it can adopt different coordination modes to the catalytic ion within the CA active site.

Full text of DOI:10.1039/c2cc34275h

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Hydroxamate represents a versatile zinc binding group for the
development of new carbonic anhydrase inhibitors†
Anna Di Fiore,a Alfonso Maresca,b Claudiu T. Supuran,b* and Giuseppina De Simonea*
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
5
DOI: 10.1039/b000000x
Thr199, which is conserved among all isoforms. Additional
hydrophobic and/or hydrophilic interactions can be established by
50 the R moiety with active site residues (Figure 1).
Hydroxamates (R-CONHOH) were scarcely investigated as
carbonic anhydrase (CA, EC 4.2.1.1) inhibitors (CAIs). An
inhibition/structural study of PhCONHOH is reported
against all human isoforms. Comparing aliphatic (R=Me and
10 CF3) and aromatic (R=Ph) hydroxamates as CAIs, we prove
that CONHOH is a versatile zinc binding group. Depending
on the nature of the R moiety, it can adopt different
coordination modes to the catalytic ion within the CA active
site.
15 Human αꢀcarbonic anhydrases (CA, EC 4.2.1.1) occur as 15
isoforms, which differ by their tissue distribution, cellular
localization and kinetic properties.1,2 All these enzymes catalyze
the reversible hydration of carbon dioxide to bicarbonate and a
proton, a very simple reaction, which is however critically
20 important for many physiological processes. Indeed, defective
activities or expression levels of several human (h) CAs have
been associated with various human diseases, such as glaucoma,
obesity, cancer, epilepsy, etc.1,3 For this reason, last years have
seen an increasing interest in the utilization of αꢀCA isozymes as
25 targets for the design of inhibitors and activators with biomedical
applications.1,4,5 αꢀCA inhibitors (CAIs) found their first
applications as diuretics, antiglaucoma agents, and antiepileptics,
while recently they are becoming to be considered for the
development of antiobesity drugs, and diagnostic and therapeutic
30 tools for cancer treatment.6,7 However, none of the currently
clinically used CAIs shows selectivity for a specific isozyme,
thus there is a constant need of improving the selectivity profile
of such molecules, in order to avoid side effects due to the
simultaneous inhibition of different isoforms.1,8
35 Derivatized sulfonamides of type RꢀSO2NH2 represent the class
of CAIs mostly utilized and best characterized both from a
structural and functional viewpoint. Indeed, a large number of
crystallographic studies are available on the adducts that such
molecules form with several CA isozymes, such as CA I, II, IV,
40 VII, IX and XIII among others.2 Such studies have clarified the
main factors responsible for the binding of the sulfonamide
moiety to the CA active site and have provided an explanation for
the characteristic properties of this metalꢀanchoring group. In
particular, in all the studied adducts the binding of the
45 sulfonamide derivatives is principally driven by the coordination
of the deprotonated sulfonamide nitrogen to the catalytic Zn2+
ion, and by two Hꢀbonds of the sulfonamide moiety with residue
Figure 1 Schematic illustration of the key interactions between
a
generic sulfonamide inhibitor and the CA active site.
55
These studies highlighted that the sulfonamide group is an ideal
ligand of the CA active site since it combines the negative charge
of the deprotonated nitrogen with the positively charged zinc ion,
and at the same time, it has no affinity for other metalloenzymes
60 possessing the same zinc coordination, such as for example the
matrix metalloproteinases (MMPs).9 However, unfortunately the
presence of such a good zinc binding group (ZBG) in these
derivatives presents an important drawback, since, although these
molecules generally behave as very potent CAIs, they do not
65 show selectivity for the different isoforms. Indeed, due to the
predominant role played by the sulfonamide moiety in the
interaction with the enzyme, any change in the thermodynamics
of binding, caused by the nature of the R substituent, has
generally a rather marginal effect on the enzymeꢀinhibitor
70 affinity.10 Consequently, much efforts were dedicated in last
years to the development of new ZBGs that, although presenting
lower affinity for the CA active site, would be able to be more
selective toward the different isoforms. In this context we
recently demonstrated that the introduction in the model
75 compound benzeneꢀsulfonamide 1 of hydroxyꢀ (2) and methoxyꢀ
moieties (3) (Figure 2) at the sulfonamide nitrogen generates
interesting lead compounds for the development of more selective
CAIs.10 In this paper, we are continuing such studies evaluating
the effect of the substitution in the model compound 1 of the
80 sulfonamide moiety with a hydroxamate group (see compound 4).
Hydroxamate derivatives are potent inhibitors of several zinc
enzymes among which the pharmaceutically significant MMPs;11ꢀ
13 nevertheless their interaction with the different αꢀCA isozymes
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and the threeꢀdimensional structure analyzed by difference
has been so far only poorly investigated,14ꢀ18 and a systematic
comparative study with sulfonamide CAIs is completely missing. 55 Fourier techniques, the crystals being isomorphous to those of the
Such a study has been undertaken in this paper where Nꢀ
(Hydroxy)ꢀbenzamide 4 has been assayed for the inhibition of all
catalytically active human CA isoforms (CA IꢀXIV) and
compared to compounds 1-3 containing the same R moiety.
Furthermore, the Xꢀray structure of the complex of 4 with hCA II
has been solved, allowing us to unravel interesting aspects related
to the inhibition mechanism of hydroxamates, as well as to the
native protein.19 The statistics for data collection and refinement
are summarized in Table S1.
5
Table 1: Inhibition of CA isozymes IꢀXIV (of human = h, and murine =
60 m origin) and MMPsꢀ2/8 with compounds 1-4.
1
2
3
4
KI (ꢀM)a
hCA I
hCA II
0.086b
0.101b
2.26b
2.73b
>1000b
8.96b
5.72b
39.5b
6.70b
10.7b
27.0b
9.56b
64.3b
8.32b
6.21b
1.57b
>1000
>1000
83.1
179
5.47b
4.42b
24.6b
5.28b
67.9b
86.2b
8.73b
60.3b
1.53b
4.97b
1.66b
70.0
10 design of more isoformꢀselective CAIs.
hCA III
hCA IV
hCA VA
hCA VB
hCA VI
hCA VII
hCA IXc
hCA XIIc
mCA XIII
hCA XIV
MMPꢀ2
MMPꢀ8
44.8
84.7
67.4
53.6
78.1
70.7
45.9
9.51
23.0
0.94
215
7.96b
1.68b
8.82b
0.097b
0.095b
0.097b
0.090b
0.100b
0.092b
>1000
>1000
Figure 2
Chemical structures of compounds 1- 4
74.14
326
Data of Table 1 show the inhibition of all catalytically active
15 hCA isoforms and two MMPs (MMPꢀ2 and MMPꢀ8) with
compounds 1-4. It may be noted that hydroxamate 4 inhibited all
12 CA isoforms, with inhibition constants in the range of 0.94 –
179 ꢁM, being thus less effective as CAI compared to the
sulfonamide 1, but showing an activity comparable to that of the
20 Nꢀsubstituted sulfonamides 2 and 3. However, there are several
notable features of the inhibition profile of compound 4 with
respect to the other three compounds. In particular, the dominant,
offtarget isoform hCA II was the least inhibited by 4 (KI of 179
ꢁM) whereas sulfonamide 1 potently inhibits this isoform, and
25 derivatives 2 and 3 also show good inhibition. The other highly
abundant cytosolic isoform hCA I was also relatively resistant to
inhibition by 4 but is highly inhibited by 1 and 2. The other
interesting aspect of the hydroxamate 4 was that it strongly
inhibited two transmembrane isoforms, hCA XII and XIV, with
30 inhibition constants in the range of 0.94 – 9.51 ꢁM. As many
transmembrane isoforms are important drug targets for the
development of antiglaucoma or anticancer therapies, this class of
underexplored CAIs may constitute an interesting starting point
for compounds with increased selectivities for such isoforms over
35 the cytosolic, highly abundant offtarget ones hCA I and II. It
should be also noted that many of the CA isoforms investigated
here were modestly inhibited by 4, with KIs in the range of 23.0 –
84.7 ꢁM. Overall, the inhibition profile of the hydroxamate 4 is
very different from those of the unsubstituted or Nꢀsubstituted
40 sulfonamides 1-3, which constitutes an important finding in the
search of isoformꢀselective CAIs. It should be also noted that
only Nꢀhydroxybenzenesulfonamide 2 and phenyl hydroxamate 4
were also MMP inhibitors, whereas 1 and 3 did not inhibit
significantly these enzymes. Interestingly, phenyl hydroxamate is
45 a rather poor MMP inhibitor (KIs in the range of 215ꢀ326 ꢁM),
aErrors in the range of ± 5 % of the reported data from three different
assays. bData from ref. 10. cCatalytic domain.
The inspection of the initially calculated electron density maps in
65 the activeꢀsite region showed clear evidence of one inhibitor
molecule bound in the hCA II active site cavity (Figure 3A).
Figure 3 (A) Active site region in the hCA II–4 complex. The simulated
70
annealing omit 2|Fo|ꢀ|Fc| electron density map, contoured at 1.0 σ,
associated to the inhibitor molecule is also shown. (B) Zn2+ coordination
geometry of Nꢀ(Hydroxy)ꢀbenzamide 4. (C) Zn2+ coordination geometry
of acetohydroxamic acid (PDB code 1AM6).18
The binding of the inhibitor molecule did not cause any
75 significant change in the overall protein structure as judged by a
r.m.s. deviation of only 0.25 Å for the superposition of the Cα
atoms of native protein with those of the complexed one. Nꢀ
(Hydroxy)ꢀbenzamide 4 binds to the hCA II active site with the
CO and OH groups which simultaneously coordinate to the zinc
80 ion to form an energetically favorable 5ꢀmembered chelate
complex (Figure 3A, and 3B). Inhibitor binding is also stabilized
by several other interactions with enzyme active site residues; in
particular, the nitrogen atom forms a hydrogen bond with the
Thr200OG1 atom (NꢀꢀꢀꢀThr200OG1=3.12 Å), while the phenyl
85 ring, whose position is rather well superimposable to that of the
phenyl ring in the hCA II/2 adduct (Figure 4), is involved in a
number of van der Waals interactions with the side chains of
residues Gln92, Val121, Phe131, Leu141, Val143, Leu198 and
Thr200 (Figure 3A). Although, several controversial data have
90 been reported on the putative deprotonation site of hydroxamates
being much more effective as
a
CAI. NꢀHydroxy
benzenesulfonamide 2 is a more efficient MMP inhibitor
compared to 4 (Table 1).
The binding mode of Nꢀ(Hydroxy)ꢀbenzamide 4 to the CA active
50 site was investigated by means of Xꢀray crystallographic studies
on the adduct that this molecule forms which the best
characterized and easily crystallizable CA isoform, namely hCA
II. Crystals of the complex were obtained as described in the ESI
2
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of type RꢀCONHOH, highlighting that the solvent and the 50 suggest that the enzymeꢀinhibitor interaction of this new CAI
environment can play a key role in favoring the Nꢀdeprotonation
or the Oꢀdeprotonation,20ꢀ22 the observation that compound 4
coordinates to the catalytic zinc ion through its CO and OH
groups suggests that in this case the Oꢀdeprotonated form is the
most probable. For this reason the deprotonated oxygen atom
cannot form a hydrogen bond with the Thr199OG1 atom,
although being at a distance of only 2.74 Å from it. Indeed, the
Thr199OG1 atom is known to be involved in the classical Hꢀbond
class can be largely modulated by exploring different substitution
patterns at the R group, thus providing interesting hints for the
development of new CAIs of the nonꢀsulfonamide type with
pharmaceutical applications in the treatment of various diseases.
5
55 Notes and references
1. C.T. Supuran, Nat. Rev. Drug Discov. 2008, 7, 168.
2. V. Alterio, A. Di Fiore, K. D’Ambrosio, C.T. Supuran, G. De Simone.
Chem. Rev. 2012, DOI: 10.1021/cr200176r.
10 with Glu1061,23 and thus it does not have further hydrogens to
donate to the hydroxamate functionality (Figure 3B).
3. Drug Design of Zinc-Enzyme Inhibitors: Functional, Structural, and
60 Disease Applications; Supuran, C. T., Winum, J.ꢀY., Eds.; Wiley:
Hoboken, New Jersey, 2009; Part II.
4. C.T. Supuran. Future Med. Chem. 2011, 3, 1165.
5. S. Pastorekova, S. Parkkila, J. Pastorek, C.T. Supuran, J. Enzyme
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65 6. S.M. Monti, C. T. Supuran, G. De Simone. Curr. Med. Chem. 2012,
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7. C.T. Supuran, A. Di Fiore, G. De Simone. Expert. Opin. Emerg.
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Figure 4 Superposition of compounds 4 (green) and 2 (magenta) (PDB
code 3T5U)10 when bound to the hCA II active site.
15 It is worth noting that the Zn2+ coordination observed in the
adduct here reported is identical to that described for the majority
of the MMP/hydroxamate complexes so far structurally
characterized,24 but is completely different from that observed in
other CA/aliphatic hydroxamate adducts studied earlier.18 Only
20 two such compounds have been so far structurally characterized
in their adducts with hCA II, namely acetohydroxamic and
trifluoroacetohydroxamic acids. In these studies it has
surprisingly been reported that the acetohydroxamic acid
coordinates to the Zn2+ ion in a tetrahedral coordination by means
25 of the nitrogen atom, thus suggesting in this case the occurrence
of an Nꢀdeprotonation (Figure 3C).18 The same coordination was
observed for the trifluoroacetohydroxamic acid.18 However, in
the last case a weakly polar CꢀF→Zn2+ interaction was also
present. The observation that hydroxamate derivatives of type Rꢀ
30 CONHOH can adopt such completely different coordination
modes to the CA catalytic zinc ion, depending on the nature of
the R substituent, strongly suggests that this ZBG is very versatile
and can represent an interesting alternative to the classical
sulfonamides for the development of more selective CAIs.
35 In conclusion, we prove here that the hydroxamates, an
underexplored class of CAIs, may constitute interesting leads for
the development of compounds with enhanced selectivity for
pharmaceutically relevant CA isoforms, such as the
transmembrane ones CA XII and XIV. The very simple model
40 compound phenyl hydroxamate was investigated as inhibitor of
12 catalytically active hCA isoforms. The compound, unlike the
unsubstituted or Nꢀsubstituted sulfonamides, selectively inhibited
the transmembrane isoforms being less effective against the
cytosolic or mitochondrial ones. The Xꢀray crystal structure of
45 the compound bound to hCA II also afforded interesting hints
regarding the versatility of the hydroxamate as a ZBG for
designing CAIs. Indeed, we showed that depending on the nature
of the R moiety, this ZBG can adopt different coordination modes
to the catalytic zinc ion within the CA active site. These findings
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Chem. Commun. 2011, 47, 11636.
75 11. J. F. Fisher, S. Mobashery. Cancer Metastasis Rev. 2006, 25, 115.
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Scozzafava, C.T. Supuran. Bioorg. Med. Chem. 2007, 15, 2298.
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May, T.R. Dean, D.W. Christianson. J. Am. Chem. Soc. 1997, 119, 850.
90 19. A.E. Eriksson, T.A. Jones, A. Liljas. Proteins. 1988, 4, 274.
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23. S. Lindskog, D. W. Silverman. In The Carbonic Anhydrase: New
Horizons; Chegwidden, W. R.; Edwards, Y. H.; Carter, N. D., Eds.;
Birkhäuser Verlag: Basel, Switzerland, 2000; pp 175ꢀ195.
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100 aIstituto di Biostrutture
e
Bioimmagini-CNR, Napoli, Italy Fax:
+390812536642; Tel: +390812534579; E-mail: gdesimon@unina.it
bUniversità degli Studi di Firenze, Sesto Fiorentino (Florence); Italy Fax:
+390554573385; Tel: +390554573005; E-mail: claudiu.supuran@unifi.it
† Electronic Supplementary Information (ESI) available: Experimental
105 details and Table of Crystallographic data. See DOI: 10.1039/b000000x/
Acknowledgments: This research was financed in part from an FP7 EU
project (Metoxia).
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