Exploring carbonic anhydrase inhibition with multimeric coumarins displayed on a fullerene scaffold

Article abstract of DOI:10.1039/c5ob01005e

Carbonic anhydrases (CAs) are ubiquitous Zn metallo-enzymes that catalyze the reversible hydration/dehydration of CO₂/HCO₃^-. CAs are involved in many key biological processes, therefore their inhibition has become an attractive research field. Distinct families of CA inhibitors (CAIs) have been reported, most of them interacting with the Zn(ii) at the active site. Some compounds such as the coumarins are hydrolyzed before binding the entrance of the active site cavity, and thus behave as "suicide" inhibitors. This study reports the first synthesis of multimeric suicide inhibitors, designed to address the selectivity and the potency of CA multivalent inhibition. Twelve coumarin units have been grafted to a central fullerene scaffold thanks to a CuAAC reaction and the final dodecamers were assayed against 4 relevant CAs. The multimers were always stronger inhibitors than the monomeric species but no strong "multivalent effect" was found. However, our study showed that the multimeric presentation of the coumarin around the C₆₀, indeed affected the selectivity of the relative inhibition among the 4 CAs assayed.

Full text of DOI:10.1039/c5ob01005e

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Exploring carbonic anhydrase inhibition
with multimeric coumarins displayed on
Cite this: DOI: 10.1039/c5ob01005e
a fullerene scaffold†
Marta Abellán-Flos,a Muhammet Tanç,b Claudiu T. Supuran*b and
Stéphane P. Vincent*a
Carbonic anhydrases (CAs) are ubiquitous Zn metallo-enzymes that catalyze the reversible hydration/
dehydration of CO2/HCO3−. CAs are involved in many key biological processes, therefore their inhibition
has become an attractive research field. Distinct families of CA inhibitors (CAIs) have been reported, most
of them interacting with the Zn(II) at the active site. Some compounds such as the coumarins are hydro-
lyzed before binding the entrance of the active site cavity, and thus behave as “suicide” inhibitors. This
study reports the first synthesis of multimeric suicide inhibitors, designed to address the selectivity and
the potency of CA multivalent inhibition. Twelve coumarin units have been grafted to a central fullerene
scaffold thanks to a CuAAC reaction and the final dodecamers were assayed against 4 relevant CAs. The
multimers were always stronger inhibitors than the monomeric species but no strong “multivalent effect”
Received 18th May 2015,
Accepted 4th June 2015
DOI: 10.1039/c5ob01005e
was found. However, our study showed that the multimeric presentation of the coumarin around the C60
,
www.rsc.org/obc
indeed affected the selectivity of the relative inhibition among the 4 CAs assayed.
recognition domain or are structurally multimeric, thus favour-
ing the interaction with multimeric presentation of their
Introduction
Multivalency has received much attention in biomedicine and carbohydrate ligand. Therefore, a vast research in the lectin-
bioorganic synthesis in the last few years because it plays a carbohydrate field has been carried out to design potent
critical role in major biological phenomena.1 Natural pro- inhibitors of biologically relevant lectins involved in pathologi-
cesses, especially at the cell interface, take advantage of the cal processes.3g–i,4 A typical example is the inhibition of the
multimeric presentation of biological entities to enhance the adhesion of pathogenic bacteria to their host tissues in order
efficiency of binding events. This is particularly true for carbo- to prevent bacterial infections.5
hydrate–protein interactions, which are notoriously weak at
On the other hand, the multivalent inhibition of enzymes
the monomer level, but can be dramatically strengthened if has not been substantially explored until 2009, when a clear
the ligand is presented in a multivalent manner.2 Inspired by multivalent effect was demonstrated and quantified in α-man-
these natural phenomena, scientists have dedicated tremen- nosidase inhibition by iminosugar clusters.6 Shortly after, a
dous effort to understanding the underlying mechanisms of fullerene bearing twelve copies of an iminosugar proved to be
multivalency but also to create artificial multivalent systems.3 a very potent inhibitor of jack bean mannosidase.7 In this
The most studied systems are probably lectin-carbohydrate study, a very strong binding enhancement was observed rela-
dyads. Lectins, which bind carbohydrate epitopes without cata- tive to the corresponding monomer. The term “multivalent
lyzing reactions, often display more than one carbohydrate effect”, which was only employed with carbohydrate-binding
proteins, could then be applied to the field of carbohydrate-
processing enzymes. Importantly, an increase of the inhibition
selectivity was also noticed thanks to the multivalent presen-
tation of glycosidase inhibitors. One year later, our group dis-
covered that glycofullerenes were also potent inhibitors of a
bacterial glycosyltransferase.8
This research field being very recent, many key questions
remain to be addressed and clarified. The first one is the ques-
tion of the inhibition mechanism: why and how multivalent
species do inhibit monomeric enzymes? The second important
aUniversity of Namur (UNamur), Académie Louvain, Département de Chimie,
Laboratoire de Chimie Bio-Organique, rue de Bruxelles 61, B-5000 Namur, Belgium.
E-mail: stephane.vincent@unamur.be; Fax: +32 81 72 45 17
bUniversità degli Studi di Firenze, Polo Scientifico, Laboratorio di Chimica
Bioinorganica, Rm. 188, Via della Lastruccia 3, 50019 Sesto Fiorentino, Florence,
Italy. E-mail: claudiu.supuran@unifi.it; Fax: +39055 457 3385;
Tel: +39055 457 3005
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
c5ob01005e
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question is related to the type of enzymes that could possibly possibility of designing selective inhibitors, therefore with less
be inhibited in a multivalent manner: Presently, there is not side effects.19 Moreover, coumarins can be readily synthesized
enough data available to be able to predict whether a Multi- and derived through distinct substitutions patterns without
valent Enzyme Inhibition (MEI) may occur or not. Strikingly, a much difficulty. The interesting properties of coumarins
very recent study demonstrated that polymeric iminosugars prompted us to design a set of multivalent derivatives in order
could improve the enzymatic activity of some glycosyl-hydro- to evaluate their activity against several biologically relevant
lases, and thus act as effectors, not inhibitors.9
hCAs. The targeted molecules for this study are depicted in
To date, the glycosidase family is the enzyme class for Fig. 1. The two fullerenes C60(A)12 and C60(B)12 bear twelve
which the largest amount of data on MEI is available.6,7,9,10 coumarins and differ in their structures only by the length of
First, it can be concluded that, among all glycosidases assayed, the spacer group linking the ligands and the C60 core. The
a minority could be inhibited with a significant multivalent four monomeric ligands A1, A2, B1 and B2 were designed to
effect. Second, MEI seems to be extremely sensitive to the determine whether a multivalent effect occurs with the tar-
structure of the multimeric inhibitor: the topology, the geted enzymes by comparison with the dodecavalent fuller-
valency, the structure of the central scaffold and the length/ enes. In addition, having two kinds of monomeric coumarins
rigidity of the tether to which each inhibitor is grafted to the with or without a triazole ring, allowed assessing how this
scaffold are parameters that need to be fine-tuned in order to heterocycle affects the inhibition activity.
observe and optimize a multivalent inhibition.
The fullerene (C60) was the core of choice due to its exclu-
Since the fullerene scaffold gave excellent multivalent inhi- sive physical, electrochemical and chemical properties, which
bition of glycosidases, we decided to explore further this class makes this carbon nanomaterial appealing for biotech and
of molecules to another biologically and therapeutically rele- biomedical applications.20 Several examples of clinical drugs
vant class of enzymes. Therefore, we decided to study the which contain fullerene moieties can be already found and
multivalent binding of carbonic anhydrases since this family plenty of functionalized fullerenes are under intense research
of enzymes, besides its essential biological functions, offers a for a great variety of applications such as drug delivery, MRI
large panel of macromolecular structures, and thus various contrast agents or gene delivery.21 Previous research has been
binding features.
done in the field of coumarin-fullerene dyads,22 though the
Carbonic anhydrases (CAs, EC 4.2.1.1) are ubiquitous fullerene derivatives synthesized for these studies displayed
Zn based metallo-enzymes which catalyze the reversible only one coumarin moiety. The objective of the latter studies
hydration/dehydration of carbon dioxide/bicarbonate. All the was to investigate the photophysical properties and energy-
human CAs (hCAs) belong to the α-class and differ mainly in transfers between the coumarin and the fullerene within the
their location within the cell and tissues. To date fifteen molecules.
different CA isoforms and CA-related proteins (CARPs) have
Therefore, as far as we know, this study reports the first
been reported.11 CAs play an essential role in a great deal of synthesis of multimeric suicide inhibitors, designed to
physiological processes in animals such as respiration, pH address the selectivity and the potency of CA multivalent
regulation, biosynthesis, calcification and photosynthesis in inhibition.
plants. CA inhibitors (CAIs) are acknowledged as diuretics and
antiglaucoma drugs. Besides, they have recently been demon-
Synthesis
strated to be promising as anticonvulsant, antiobesity agents The synthesis of the monovalent coumarins (Scheme 1),
and in fighting pathological processes such as tumour for- started with the preparation of the linkers that would be later
mation, metastasis and the virulence of several pathogens.12 attached to 4-methylumbelliferone, a coumarin derivative
The Zn(II) cation is crucial for the catalytic activity of these already known as CA inhibitor.19 An azidation was first carried
enzymes.13 The active site of all the CA isoforms is highly con- out from 2-bromoethan-1-ol and 2-(2-chloroethoxy)ethan-1-ol
served14 and most CAIs act by binding either to the Zn(II) or to to provide 1 and 3, which were tosylated to give intermediates
the water molecule coordinated to it. Namely, sulfonamides 2 and 4, respectively. Williamson reactions of the latter mole-
and their isosteres, metal complexing anions, phenols, thio- cules with 4-methylumbelliferone yielded azides 5 and 7,
phenols and polyamines. In addition, it was recently discov- which were then engaged in both the synthesis of the mono-
ered that ortho-substituted benzoic acid derivatives can inhibit valent clicked control molecules and the final multivalent
CA without entering the active site.15 Moreover, coumarins16 compounds. The monovalent controls 6 and 8 containing the
and lacosamide17 inhibit the enzyme activity by occluding the 1,2,3-triazole moiety were carried out through the CuAAC click
entrance of the catalytic site. It has recently been proved that reaction by coupling the compounds 5 and 7 with 4-pentyn-1-
coumarins are suicide inhibitors which are hydrolyzed within ol. Therefore, molecules 6 and 8 are the exact monomeric
the CA active site with formation of 2-hydroxycinnamic acids, units that will be present onto fullerenes C60(A)12 and C60(B)12.
displaying a lengthened two-arm conformation that blocks the
The two targeted multimeric coumarins were prepared
entrance to the active site.18 This new class of CAIs is very from known fullerene hexakis-adduct 1023 that displays twelve
promising due to its mechanism of action. These inhibitors TMS-protected alkynes prone to cycloaddition reactions
bind to the entrance of the active site cavity, which is the most (Scheme 2). The azido-coumarins 5 and 7 were then grafted
differentiated region among the CA isoforms, offering the onto the fullerene scaffold through copper-catalyzed alkyne-
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Fig. 1 Targeted multivalent coumarin derivatives and the corresponding monovalent controls.
Scheme 1 Synthesis of the monovalent coumarin derivatives. Reagent and conditions: (a) NaN3, H2O, 18 h, 80 °C (1: from 2-bromoethan-1-ol, 75%;
3: from 2-(2-chloroethoxy)ethan-1-ol, 99%). (b) p-TsCl, Et3N, CH2Cl2, 16 h, r.t. (2: from 1, 90%; 4: from 3, 88%). (c) 2 or 4, K2CO3, KI, DMF, 6 h, 60 °C
(5: from 2, 84%; 7: from 4, 96%). (d) 5 or 7, 4-pentyn-1-ol, CuSO4/NaAsc, 1,4-dioxane/CH2Cl2/H2O, 5 h, 60 °C (6: from 5, 86%; 8: from 7, 60%).
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Scheme 2 Synthesis of the multivalent coumarin derivatives. Complete structures are shown in Fig. 1.
azide cycloaddition (CuAAC) based on the protocol developed cannot arise from an incomplete functionalization of the full-
by Nierengarten et al.24
erene scaffold.
Deprotection of the trimethylsilyl-protected alkynes was
accomplished in situ by addition of TBAF 1 M in THF into the
CA Inhibition assays
reaction mixture. To assure the complete functionalization of The monovalent compounds 5–8 and the two fullerenes conju-
the fullerene core, 15 equivalents of the azido-coumarins were gated with coumarins C60(A)12 and C60(B)12 were assayed
required. Dichloromethane was used as solvent due to the in vitro for inhibition against the four physiologically relevant
high solubility of all the compounds and CuBr·SMe2 was CA isoforms, the cytosolic hCA I and II as well as the transmem-
found to be the catalyst of choice compared to CuSO4, which brane, tumor-associated hCA IX and XII (Table 1). Acetazol-
requires heterogeneous solvent systems.24 After 20 h at room amide (5-acetamido-1,3,4-thiadiazole-2-sulfonamide) was used
temperature, the reaction mixtures were diluted with dichloro- as a standard in the assay.25
methane and extracted with water. The purifications of the
As many coumarin derivatives, the investigated compounds
reaction mixtures were performed by silica gel chromatography from the present paper also showed low inhibitory properties
followed by size exclusion chromatography on lipophilic against the cytosolic isoforms hCA I and II, with most of them
Sephadex-LH20. The two final compounds were obtained possessing inhibition constants >50 µM. The exceptions are
in moderate to very good yields, bearing in mind that twelve the low molecular weight coumarin 7 and C60(B)12 against hCA
reactions had taken place. Importantly, the possibility of puri- I, which were medium potency inhibitors with KIs in the range
fication of the final molecules by two successive chromato- of 7.0–8.1 µM. Against hCA II the simple coumarins 5–8 were
graphies after the click reaction facilitates the total removal of ineffective inhibitors whereas the two fullerene conjugates
Cu salts, as confirmed by ICP.
C60(A)12 and C60(B)12 were medium efficacy inhibitors with KIs
The structures of the final compounds were determined by in the range of 9.9–10.0 µM.
1H and 13C NMR, IR and mass spectrometry (see ESI†). The
The tumor-associated hCA IX was better inhibited both by
1H NMR clearly indicated the presence of the newly formed tri- the coumarins 5–8 (KIs in the range of 0.06–0.18 µM) as well as
azoles (around 7.5 ppm in CDCl3 at 20 °C) and the disappear- by the fullerene conjugates (KIs of 0.04 µM for both deriva-
ance of the TMS-alkynes and azides functionalities. Moreover, tives). A similar behavior was observed for the inhibition of
13C NMR spectra were instrumental for the demonstration of the second transmembrane isoform investigated here, hCA XII;
the final structures. The presence of only two sp2 carbon against which coumarins 5–8 showed KIs of 1.08–1.92 µM,
atoms belonging to the fullerene core confirms the Th-sym- whereas the two fullerene-coumarin conjugates of 0.16–0.19 µM.
metry of the core and the monodispersity of the final com- It is clear that the multimers are always more potent than
pounds. A comparison of the spectra of the precursors and the their corresponding monomers, an effect likely due to the
final molecule C60(B)12 is shown in Fig. 2. Furthermore, mass local ligand concentration around the fullerene core. It is also
spectrometry provided a definitive proof of structures (see worth to note that a small positive triazole effect was observed
ESI†). All the peaks correlate with the expected molecule and for the extracellular isoforms hCA IX and hCA XII in every
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Fig. 2 Comparison of 13C NMR spectra of molecules 10, 7 and C60(B)12 in CDCl3 at 20 °C.
Table 1 Inhibition constants (KIs) by a stopped flow CO2 hydrase assay,
with coumarins and fullerene-coumarin conjugates, and acetazolamide
AAZ as standard drug25
Discussion and conclusions
This work addressed the question of multivalent enzyme inhi-
bition applied to a novel class of enzymes for this topic. The
family of carbonic anhydrases was selected as enzymatic target
because they play critical roles in numerous biological pro-
cesses. For instance CAIs are already recognized as antiglau-
coma and diuretic drugs while they are being investigated as
anticancer and anti-virulence drugs, among other biomedical
applications. In 2008, Supuran et al. synthesized nanoparticles
functionalized by sulphonamides.26 Although no multivalent
effect was observed, the nanoparticles were potent inhibitors
of CA and were shown not to penetrate into cells, contrarily to
the corresponding monovalent species. This property could be
extremely advantageous in case an extracellular enzymatic
activity is targeted for a therapeutic purpose, which is the case
for the tumour associated CA IX activity. In the present study,
the synthesis of multimeric suicide CAIs is reported for the
KI (µM)
Compound
hCA I
hCA II
hCA IX
hCA XII
5
6
7
8
>50
>50
8.1
>50
>50
7.0
>50
>50
>50
>50
9.9
10.0
0.012
0.14
0.06
0.18
0.14
0.04
0.04
0.025
1.92
1.44
1.66
1.08
0.16
0.19
0.006
C60(A)12
C60(B)12
AAZa
0.25
a Acetazolamide (AAZ) was used as a standard inhibitor for all CAs
investigated here. The data represent the mean of three different
determinations. Errors were in the range of 10% of the reported
values (data not shown).
case. Indeed, the inhibitors 6 and 8 containing a 1,2,3-triazole first time, and their activity against biologically relevant hCAs
moiety are slightly better than their corresponding azides 5 has been studied. Our main findings are (i) the modest inhi-
and 7. However, in the particular case of the cytosolic hCA I, bition power of coumarins against cytosolic hCAs are generally
the monovalent azido-inhibitor 7, showed a higher inhibitory improved by displaying the inhibitors in
a multivalent
power than the rest of monovalent molecules (KIs of 8.1 µM, manner, (ii) the multimeric suicide inhibitors always showed a
while for the others KIs > 50 µM). Therefore, for this enzyme, better activity against the extracellular CAs isoforms, (iii) a
there is a substantial effect of the linker length and the pres- selective inhibition among cytosolic hCAs is possible by fine-
ence of triazole unit in the binding potency.
tuning the length and structures of the linker bridging the C60
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and the ligand without modifying the coumarin itself (iv) the
effect of the triazole moiety can be either positive or negative
depending on the hCA isoform. Although a clear multivalent
effect could not be observed (if calculated on a “per-coumarin”
basis), the multivalent compounds were always better inhibi-
tors than the monovalent controls. Therefore, we believe that
the multivalent inhibition of CAs is possible and that, as for
the well-studied glycosidase family, the level of inhibition
potency can be improved by optimizing the length and rigidity
of the linker, the core scaffold itself and the substitution
pattern on the coumarin derivative. We also believe that the
field of multienzyme inhibition should indeed find biomedical
applications especially if the enzymes are extracellular, as for
the tumor-associated carbonic anhydrases, or if they are mem-
brane-anchored and exposed to the external cellular medium
such as the viral neuraminidases.27
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Experimental
Synthetic procedures and analytical data: see ESI†
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CA inhibition. A stopped-flow instrument (SX.18MV-R
Applied Photophysics model) was used for assaying the CA-
catalyzed CO2 hydration activity.25 Inhibitor and enzyme were
preincubated for 15 min for allowing the complete formation
of the enzyme-inhibitor adduct. IC50 values were obtained
from dose response curves working at eight different concen-
trations of test compound (from 0.01 nM to 50 μM), by fitting
the curves using PRISM (http://www.graphpad.com) and non-
linear least squares methods, the obtained values representing
the mean of at least three different determinations. The inhi-
bition constants (KI) were derived from the IC50 values by
using the Cheng–Prusoff equation, as follows: KI = IC50/(1 +
[S]/Km) where [S] represents the CO2 concentration at which
the measurement was carried out, and Km the concentration of
substrate at which the enzyme activity is at half maximal. All
enzymes used were recombinant, produced in E. coli as
reported earlier.28 The concentrations of enzymes used in the
assay varied between 8.4 nM and 12.8 nM.
5 (a) A. Imberty, Y. M. Chabre and R. Roy, Chem. – Eur. J.,
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Acknowledgements
We thank the People Programme (Marie Curie Actions) of the
European Union’s Seventh Framework Programme “DyNano”
FP7/2007-2013/under REA grant agreement no. 289033 for the
financial support.
6 J. Diot, M. I. Garcia-Moreno, S. G. Gouin, C. Ortiz Mellet,
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