Carbonic anhydrase inhibitor coated gold nanoparticles selectively inhibit the tumor-associated lsoform IX over the cytosolic isozymes I and II

Article abstract of DOI:10.1021/ja805558k

An approach for the synthesis of carbonic anhydrase (CA, EC 4.2.1.1) inhibitor coated gold nanoparticles is reported. This nanomaterial selectively inhibited the tumor-associated isoform CA IX overexpressed in hypoxic cancers over the ubiquitous, cytosoli

Full text of DOI:10.1021/ja805558k

Published on Web 11/07/2008
Carbonic Anhydrase Inhibitor Coated Gold Nanoparticles Selectively Inhibit
the Tumor-Associated Isoform IX over the Cytosolic Isozymes I and II
Maamar Stiti,†,‡ Alessandro Cecchi,§ Marouan Rami,† Mohamed Abdaoui,‡
Ve´ronique Barragan-Montero,† Andrea Scozzafava,§ Yannick Guari,| Jean-Yves Winum,*,† and
Claudiu T. Supuran*,§
Institut des Biomole´cules Max Mousseron (IBMM) UMR 5247 CNRS-UM1-UM2 Baˆtiment de Recherche Max
Mousseron, Ecole Nationale Supe´rieure de Chimie de Montpellier, Montpellier Cedex, France, Laboratoire de
Chimie Applique´e, UniVersite´ de Guelma, Guelma, Algeria, UniVersita` degli Studi di Firenze, Polo Scientifico,
Laboratorio di Chimica Bioinorganica, Florence, Italy, and Institut Charles Gerhardt, UMR 5253, CMOS,
UniVersite´ Montpellier II, Montpellier Cedex, France
Received July 25, 2008; E-mail: winumj@univ-montp2.fr; claudiu.supuran@unifi.it
Scheme 1. Synthesis of Au NPs Coated with Sulfonamide CAI, of
Type GNP-1 and GNP-2
The carbonic anhydrases (CAs, EC 4.2.1.1) are ubiquitous metallo-
enzymes with five independently evolved (R,ꢀ,γ,δ,ς) classes reported
up to date.1-3 These enzymes catalyze the reversible hydration of
carbon dioxide to bicarbonate and protons by means of a metal-
hydroxide (Lig3M2+(OH)-) mechanism.2
In addition to the established role of the carbonic anhydrase
inhibitors (CAIs) as diuretics and antiglaucoma drugs, it has recently
emerged that CAIs could have potential as novel antiobesity, anticancer,
and anti-infective drugs.1,2 A critical problem in the design of CAIs
is related to the high number of isoforms in mammals (15), their diffuse
localization in many tissues/organs, and the lack of isozyme selectivity
of the presently available inhibitors.1-4
Recently, nanoparticles received great attention for their bio-
medical applications both for the site-specific delivery of drugs7
or for imaging purposes.7,8 Several recent such examples include
HIV inhibition with a CCR5 antagonist attached to multivalent gold
nanoparticles (Au NPs), tumor targeting with NPs loaded with
hydroxycamptothecin, paclitaxel, or fumagillin, and magnetic
resonance imaging (MRI) techniques of tumors based on integrins
targeting with Fe3O4 NPs, computer tomography (CT) or MRI
imaging with gadolinium chelate Au NPs, etc.7,8
Human CA IX (hCA IX) is an extracellular, transmembrane isoform
which was recently shown to constitute a novel and interesting target
for the anticancer therapy due to its overexpression in many cancer
tissues and not in their normal counterparts.1b,9 Its expression is
strongly induced by hypoxia present in many tumor tissues and
correlated with a bad response to classical chemo- and radiotherapies.1b,9
CA IX was shown to acidify the extratumoral medium leading both
to the acquisition of metastasic phenotypes and to chemoresistance
with many anticancer drugs, these processes being reverted by
inhibition of the enzyme catalytic activity with sulfonamide inhibitors.1b,9
The development of selective CA IX inhibitors might provide useful
tools for highlighting the exact role of CA IX in hypoxic cancers, to
control the pH imbalance of tumor cells and lead to novel diagnostic
or therapeutic applications for the management of such tumors.1b,9
Although many sulfonamide/sulfamate/sulfamide potent CA IX inhibi-
tors were reported, few of them show an acceptable level of selectivity
for inhibiting the transmembrane-tumor associated target isoform IX
over the cytosolic, ubiquitous isozymes hCA I and II.1b,9 Considering
the extracellular localization of the target CA isoform and the fact
that nanomaterials generally show membrane impermeability,10 we
report here the synthesis of CAI coated Au NPs which show excellent
CA IX inhibitory properties and selectivity for the inhibition of the
tumor-associated isoform over hCA I and II.
The key intermediate 3a was synthesized by coupling lipoic acid 1
with 4-aminoethylbenzene sulfonamide 2a in the presence of EDCI/
DMAP, as outlined in Scheme 1. The CAI coated Au NPs (GNP-1)
were then prepared in a single step by reduction of chloroaurate with
NaBH4 in the presence of the lipoic acid tailed sulfonamide 3a.11,12
The same strategy has been used to prepare a sulfathiazole-lipoic acid
Au NPs conjugate (GNP-2) which has been used as a control, since
substituted sulfonamides do not act as CAIs.1 The NPs were character-
ized by transmission electron microscopy (TEM) (Supporting Informa-
tion, Figure 1) being observed that they are roughly spherical in shape.
These particles are monodispersed with an average particle size of
3.3 nm which corresponds to ∼720-724 Au atoms. Energy dispersive
X-ray analysis (EDX) and elemental analysis allowed us to estimate
the number of sulfonamide ligands attached to the NP as being 144
for GNP-1 and 135 for GNP-2. The average surface area occupied
by one ligand unit 3a is ∼0.24 nm2. GNP-1 has the empirical formula
[Au720(C16H24O3N2S3)144], and GNP-2 [Au724(C17H21O3N3S4)135], which
were confirmed by elemental analysis data. The powdered materials
GNP-1/2 showed an intense plasmon band absorbance at 540 nm
characteristic of Au(0) NPs.12 The powder X-ray diffraction (XRD)
patterns of the Au NPs GPN-1/2 in the range 2θ (20°-140°)
(Supporting Information Figure 2) presented seven of the main
diffractions characteristic of the Au cubic phase at 38.6°, 44.9°, 64.9°,
78.1°, 82.2°, 111.2°, and 135.5°. The peak broadness may be explained
by the small size of the crystalline domains. The peak of the d111
reflection has been deconvoluted to the Lorentzian curve for determin-
ing the full width at half-maximum (FMWH) value. The crystalline
domain has been calculated from the Debye-Scherrer formula using
the FWMH value of the corresponding index peaks giving an average
† Ecole Nationale Supe´rieure de Chimie de Montpellier.
‡ Universite´ de Guelma.
§ Universita` degli Studi di Firenze.
| Universite´ Montpellier II.
9
16130 J. AM. CHEM. SOC. 2008, 130, 16130–16131
10.1021/ja805558k CCC: $40.75
2008 American Chemical Society

C O M M U N I C A T I O N S
Table 1. CA Inhibition Data Against Isoforms CA I, II and IX With
the Standard Sulfonamide in Clinical Use Acetazolamide AZA, the
New Sulfonamides 3a,b, and GNP-1/2a
ments were performed by incubation of RBCs with millimolar
concentrations of sulfonamide inhibitors (AZA, 3a, and GNP-1).
Incubation with AZA and sulfonamide 3a led to saturation with the
inhibitor of the two isozymes present in erythrocytes (CA I and II),
after 30-60 min (Supporting Information, Table 2).15,16 This is due
to the high diffusibility through membranes of these inhibitors. On
the contrary, GNP-1 was only detected in negligible amounts within
the RBCs (even after 2-24 h incubation time), proving that the CAI
coated Au NPs are unable to penetrate through biological membranes.
These experiments show that the CAI coated Au NPs are totally
membrane-impermeant, which is a highly desirable feature for a
compound that should inhibit selectively only CA IX which possesses
and extracellular active site. Thus, GNP-1 constitutes an interesting
candidate to be investigated for both imaging and treatment purposes
of tumors overexpressing CA IX.
Ki (nM)
compound
hCA I
hCA II
hCA IX
AZA
3a
GNP-1
3b
GNP-2
Au@
250 ( 12
214 ( 9
581 ( 18 (128)
>50 000
28 550
12 ( 1
25 ( 1
230 ( 10
451 ( 21 (116)
>50 000
30 400
41 ( 2
32 ( 2 (2.4)
>50 000
31 050
32 000
31 600
29 560
a Data in parentheses show the inhibition constants when enzyme and
inhibitor were incubated for 2 h.14
value of ∼4.0 nm which is concomitant with the value 3.3 ( 1.4 nm
obtained from TEM images.12
Inhibition data of 3a,b, GNP-1/2, the standard, clinically used CAI
acetazolamide AZA (5-acetamido-1,3,4-thiadiazole-2-sulfonamide),
and Au NPs (Au@) as control, against isoforms hCA I and II (cytosolic
isozymes) and the transmembrane, tumor-associated isozyme hCA IX
(a construct incorporating the catalytic domain and proteoglycan
regions of the enzyme),13 are shown in Table 1.14 A stopped-flow
method has been used for assaying the CA-catalyzed CO2 hydration
activity with Phenol red as indicator, working at the absorbance
maximum 557 nm, following the initial rates of the CA-catalyzed CO2
hydration reaction for 10-100 s. For each inhibitor at least six traces
of the initial 5-10% of the reaction have been used for determining
the initial velocity. The uncatalyzed rates were determined in the same
manner and subtracted from the total observed rates. Stock solutions
of inhibitor (0.01 µM) were prepared in distilled-deionized water with
5% DMSO, and dilutions up to 0.01 nM were done thereafter with
distilled-deionized water. The NPs were soluble in this solvent mixture.
Inhibitor (concentration range 0.01 µM-0.01 nM) and enzyme
solutions ([E] ) 10 nM) were preincubated together for 15 min-2 h
at room temperature prior to assay, to allow for the formation of the
E-I complex. The inhibition constants were obtained by nonlinear
least-squares methods using PRISM 3, and represent the mean from
at least three different determinations. In the standard conditions used
to determine the inhibition constants, i.e., incubation time 15 min, the
sulfonamides AZA, 3a and GNP-1 were modest hCA I inhibitors (KI’s
of 214-581nM), AZA was an effective CA II and IX inhibitor (KIs
of 12-25nM) whereas the new sulfonamides 3a and GNP-1 were
moderate-weak CA II inhibitors (KI’s of 230-451 nM) and effective
CA IX inhibitors (KI’s of 32-41 nM). However, when inhibitors and
enzymes were incubated for 2 h (or longer) GNP-1 (but not the other
sulfonamides) showed an enhanced inhibitory activity against all three
isozymes, with inhibition constants of 128 nM against hCA I, 116
nM against hCA II, and 2.4 nM against hCA IX, respectively (Table
1). In control experiments, the sulfathiazole lipoic acid conjugate 3b,
its Au NPs derivative GNP-2, and uncoated Au NPs were assayed
under the same conditions. It may be observed that 3b, GNP-2, and
Au@ show very weak, micromolar inhibition or no inhibition at all
against all CA isoforms. Thus, the inhibition observed with GNP-1 is
due to the interactions of its sulfonamide moiety with the enzyme active
site. Inhibitors 3a and GNP-1 also showed good selectivity for
inhibiting CA IX over CA I and II (Table 1), probably due to the fact
that some key residues for the binding of inhibitors1,2,4 (such as Phe131
and Gly132) are different in the cytosolic and transmembrane isozymes,
as rationalized earlier by us.9
Acknowledgment. This research was supported in part by a
Franco-Algerian Intergouvernemental Program and by two EU
grants of the 6th Framework Programme (EUROXY and DeZnIT
projects).
Supporting Information Available: The synthesis, characterization,
and in Vitro/ex ViVo enzymatic studies are described in detail. This
material is available free of charge Via the Internet at http://pubs.acs.org.
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