Fluorescent Silica Nanoparticles with Multivalent Inhibitory Effects towards Carbonic Anhydrases

Article abstract of DOI:10.1002/chem.201501037

Multifunctional silica nanoparticles decorated with fluorescent and sulfonamide carbonic anhydrase (CA) inhibitors were prepared and investigated as multivalent enzyme inhibitors against the cytosolic isoforms hCA I and II and the transmembrane tumor-asso

Full text of DOI:10.1002/chem.201501037

DOI: 10.1002/chem.201501037
Communication
&
Inhibitors |Hot Paper|
Fluorescent Silica Nanoparticles with Multivalent Inhibitory Effects
towards Carbonic Anhydrases
Nadia Touisni,[a] Nasreddine Kanfar,[a] SØbastien Ulrich,[a] Pascal Dumy,[a] Claudiu T. Supuran,[b]
Ahmad Mehdi,[c] and Jean-Yves Winum*[a]
design of more selective enzyme inhibitors, the nanoparticles
Abstract: Multifunctional silica nanoparticles decorated
with fluorescent and sulfonamide carbonic anhydrase (CA)
approach was applied by our group to carbonic anhydrases
and we demonstrated the possibility of activity modulation of
inhibitors were prepared and investigated as multivalent
carbonic anhydrases using nano-objects.[5,6] Due to their size,
enzyme inhibitors against the cytosolic isoforms hCA I and
ease of synthesis, and their potential applications, silica-based
II and the transmembrane tumor-associated ones hCA IX
nanoparticles received great attention for their biomedical ap-
and XII. Excellent inhibitory effects were observed with
plications both for the site-specific delivery of drugs or for
imaging purposes.[7–9] Moreover it constitutes a good platform
these nanoparticles, with KI values in the low nanomolar
range (6.2–0.67 nm) against all tested isozymes. A signifi-
cant multivalency effect was seen for the inhibition of the
for developing multivalent interactions, which is a promising
option for the specific treatment of diseases.
monomeric enzymes hCA I and II compared to the dimeric
Multivalent enzyme inhibitors that combine multiple copies
hCA IX and hCA XII isoforms, where no multivalent effect
of an inhibitor conjugated to a single scaffold may lead to an
was observed, suggesting that the multivalent binding is
overall increase of the binding affinity, avidity, and/or
occurring through enzyme clustering.
specificity.[10] These changes that are associated with the multi-
valent presentation of inhibitors depend on the nature of the
biological target and may arise from different mechanisms:
Carbonic anhydrases (CAs, EC 4.2.1.1) are well known zinc met-
alloproteins which catalyze a simple but essential physiological
reaction: carbon dioxide hydration to bicarbonate and
proton.[1] These enzymes are of clinical relevance in cancer
therapy as, among the 15 isoforms known in humans, some
catalytically active isozymes are critical in various fundamental
physiological processes and have been clinically exploited for
the treatment or prevention of various pathologies such as
glaucoma and neurological disorders. The lack of selectivity of
conventional inhibitors against the different CA isozymes have
led researchers to investigate new approaches for achieving
such a goal.[1] For example, different strategies were reported
in recent years for obtaining compounds that specifically
target the tumor-associated CA IX,[2] especially the sugar ap-
proaches developed by our group.[3,4] Beside the rational drug
1) a statistical rebinding effect, 2) a chelate effect, 3) a subsite
binding effect, or 4) a clustering effect.[11] Though the multiva-
lency approach has been extensively studied in the context of
lectins that contain multiple binding sites,[12] its application in
the field of enzyme inhibition has received less attention until
recently. For instance, the multivalency strategy has been suc-
cessfully applied in the case of glycosidase inhibitors.[13–14] In
the case of carbonic anhydrase, only studies on bivalent inhibi-
tors were recently reported. Indeed, Whiteside’s research
group described in 2012 the binding of monovalent and biva-
lent benzene sulfonamide ligands to a synthetic dimer of car-
bonic anhydrase.[15] The group of Neri reported the use of biva-
lent small molecule ligand–drug conjugates against carbonic
anhydrase IX.[16] Besides the classical small-molecule approach,
which has been applied with some success on the in vitro and
in vivo inhibition of human CA (hCA), the use of multivalent
nanoplatforms could therefore be of great interest and a
promising strategy in this field.
[a] Dr. N. Touisni, N. Kanfar, Dr. S. Ulrich, Prof. Dr. P. Dumy, Dr. J.-Y. Winum
Institut des BiomolØcules Max Mousseron (IBMM)
UMR 5247 CNRS-ENSCM-UniversitØ de Montpellier, ENSCM
8 rue de l’Ecole Normale, 34296 Montpellier Cedex (France)
E-mail: jean-yves.winum@univ-montp2.fr
In this paper, we describe the synthesis and multivalent
inhibition of sulfonamide carbonic anhydrase inhibitor-coated
silica nanoparticles against cytosolic/transmembrane CA
isoforms.
[b] Prof. Dr. C. T. Supuran
Neurofarba Department
Section of Pharmaceutical and Nutriceutical Sciences
Università degli Studi di Firenze
CA inhibitor-coated silica nanoparticles were prepared
following the synthetic strategy depicted in Scheme 1. First,
two organosilylated ligands 3 and 6 were synthesized, the first
one by coupling 3-aminopropyltriisopropoxylsilane 1 with
fluorescein isothiocyanate 2, the latter by coupling of 3-amino-
propyltriisopropylsilane 1 with 4-isothiocyanato(ethyl) benzene
sulfonamide 5. Previously, 6 has been prepared by reacting 4-
aminoethylbenzene sulfonamide 4 with dicyclohexylcarbodi-
Via Ugo Schiff 6, 50019 Sesto Fiorentino, Florence (Italy)
[c] Prof. Dr. A. Mehdi
Institut Charles Gerhardt
UMR 5253, Equipe Chimie MolØculaire et Organisation du Solide
UniversitØ de Montpellier
Place Eug›ne Bataillon, 34095 Montpellier Cedex 05 (France)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201501037.
Chem. Eur. J. 2015, 21, 10306 – 10309
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particles. The results showed
that the particles 9 were mono-
disperse with
a diameter of
about (3.5Æ0.8) nm (Figure 1).
The nitrogen sorption analy-
ses showed that the NFHS parti-
cles are not porous, and the
Brunauer–Emmett–Teller (BET)
specific surface areas are re-
spectively 330 m2gÀ1 for NFHS
particle 7 and 260 m2gÀ1 for
NFHS particle 9. This diminution
shows clearly the surface modi-
fication by the ligand 6. With
the help of elemental analyses,
a silica/ligand molar ratio of
around 250 was obtained for
nanoparticle 9. Based on NFHS
particle size and this ratio, the
number of sulfonamide ligands
per nanoparticle was estimated
to be between 12 and 14 (see
the Supporting Information).
The NFHS particles 8 and 9
have been assayed for their in-
hibitory potency against the
physiologically relevant cytosol-
ic hCA I and II, as well as against
the two tumour-associated iso-
forms hCA IX and XII. It can be
observed from the data in
Table 1 that no inhibition or
a weak inhibition was observed
Scheme 1. Schematic representation of NFHS particle synthesis with carbonic anhydrase inhibitors immobilization.
imide (DCC) and carbon disulfide.[17,18] Isothiocyanate 5 was re-
acted with n-butylamine in acetone to afford the thiourea 10,
which was used as a monovalent reference in the enzymatic
inhibition assays. The nanometer-sized fluorescent hybrid silica
(NFHS) particles 7 were then prepared by a hydrolysis and
polycondensation approach, using the water-in-oil microemul-
sion method.[19–21] Co-hydrolysis and polycondensation of a mix-
ture of the fluorescent silylated precursor 3 and tetramethoxy-
silane in the presence of a base as catalyst in an ultrasound
bath[22] led to fluorescein-labelled nanoparticle 7, which was
isolated after centrifugation and several treatments with etha-
nol and water. The silica surface was then modified, on the
one hand, by treatment with methanol overnight under reflux.
The capping of SiÀOH functions was performed by treatment
with hexamethyldisilazane (HMDS) for four hours to yield the
nanoparticle 8.[23] On the other hand, the bifunctional (fluores-
cent+inhibitor) system was prepared by reacting nanoparticle
7 with silylated inhibitor ligand 6 in methanol under reflux
overnight. The same treatment with HMDS was subsequently
performed to afford nanoparticle 9 (Scheme 1).
The size of the composite NFHS particles was investigated
by transmission electron microscopy (TEM) and the mean
diameter and standard derivation of the particles were
determined from TEM pictures by averaging the size of 150
Figure 1. TEM picture of NFHS particle 9.
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Communication
lency effects for inhibiting the tumor-associated CA IX (but
were much less effective as CA I and II inhibitors).[5] For the
nanoparticles reported here, based on silica nanoparticles, the
opposite effect was observed: for the cytosolic isoforms hCA I
and II, the inhibition constants decreased from 142 to 4.4 nm
for hCA I, and from 48 to 0.67 nm for hCA II, corresponding to
an inhibitory potency improvement of 32 and 72 times, respec-
tively, when compared to the monovalent system. However,
multivalency effects were not observed for the transmembrane
isoforms hCA IX and XII. The main difference between the cy-
tosolic isoforms hCA I and II on the one hand, and hCA IX and
XII on the other hand (apart from the different cellular localiza-
tion) is that the two transmembrane isoforms are dimers,
whereas the cytosolic CA I and II are monomers.[28,29] Although
the active sites of CA I, II, IX and XII are rather similar, the fact
that the two transmembrane isoforms have two different
active sites in their molecule may have consequences for the
access of a multivalent inhibitor in both of them concomitant-
ly. In fact, the sulfonamide 10 is a better inhibitor of hCA IX
and XII compared to hCA I and II. However, this effect is not
preserved when 10 was attached to the NFHS nanoparticles.
However, a significant multivalency effect was seen for the
inhibition of hCA I and II (monomeric enzymes) probably
because there are no steric hindrance effects related to the
access of the derivatized nanoparticle to the enzyme active
site. This may suggest that multivalent binding occurs in this
case through enzyme clustering.[11] As hCA I and II are impor-
tant drug targets for obtaining diuretics,[30] antiglaucoma,[31]
and anticonvulsant agents,[32] we estimate that this type of
NFHS nanoparticle may have interesting biomedical therag-
nostic applications, combining therapy through carbonic
anhydrase inhibition and fluorescence imaging.
Table 1. hCA I, II, IX, XII inhibition data for monovalent inhibitor 10 and
with NFHS particles 8 and 9 determined by a stopped-flow, CO2 hydration
assay method[24] at pH 8.4.
KI [nm][a]
hCA I
hCA II
hCA IX
hCA XII
10
8
9
rp
rp/n
142
38800
4.4
32.3
2.7
48.1
268
0.67
71.8
6
38.3
560
2.7
14.2
1
29.8
92650
6.2
4.8
0.4
[a] Mean from three different assays, errors in the range of Æ10% of the
reported values (data not shown). rp: relative potency=KI(10)/KI(9).
rp/n: relative potency/number of sulfonamide units.
for all isoforms with the inhibitor-free nanoparticle 8. However,
nanoparticle 9 exhibited excellent inhibitory effects, in low
nanomolar range, with KI values in the range 6.2–0.67 nm
against all tested isozymes.
Multivalent effects were clearly seen for nanoparticle 9 when
compared with monovalent inhibitor 10. With hCA I and II, the
inhibition constants KI decrease from 142 to 4.4 nm for hCA I
and from 48.1 nm to 670 pM for hCA II, which corresponds to
an inhibitory potency improvement of 32 and 72 times, respec-
tively (Table 1). Interestingly, no multivalent effect was charac-
terized in the case of hCA IX, where a linear inhibitory effect
was observed in reference to the ligand 10, the relative poten-
cy normalized to the sulfonamide units (rp/n) being equal to 1.
For the isoform XII, an increase of the overall inhibitory poten-
cy from 29.8 to 6.2 nm was observed between the monovalent
compound 10 and the multivalent system 9, but amounted to
a negative multivalent effect; the relative potency of a sulfon-
amide inhibitor being weaker in the nanoplatform compared
to the single compound reference. These results indicate that
the multivalency approach can be a promising strategy for en-
hancing not only the binding affinity of CA inhibitors but also
the selectivity between different isoforms. For instance, the
selectivity was modified when shifting from a monovalent
(KI(hCA II)/KI(hCA IX)=1.25) to a multivalent system (KI(hCA II)/
KI(hCA IX)=0.24), the monovalent inhibitor becoming a hCA II-
selective multivalent inhibitor compared to hCA IX and hCA XII.
Note also the inhibition data observed with inhibitor-free
nanoparticles 8. Even if inhibitory activity is relatively weak,
a significant selectivity was noticed with hCA II and hCA IX
compared to other isoforms tested. The inhibitory mechanism
could be explained by adsorption phenomena of silica nano-
particles on the border of the active site cavity.[25]
Acknowledgements
This work was supported by the French Ministry of Research
(MESR) with a Ph.D fellowship for N.K. We thank the CNRS and
LabEx CheMISyst (ANR-10-LABX-05-01) and La Ligue contre le
Cancer (ComitØ des PyrØnØes-Orientales) for funding. Authors
thank Simon Cassegrain for technical assistance designing the
cover page.
Keywords: carbonic anhydrase · inhibitors · multivalency ·
nanoparticles · sulfonamide
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Received: March 17, 2015
Published online on May 12, 2015
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