Conjugation of poor inhibitors with surface binding groups: A strategy to improve inhibition

Article abstract of DOI:10.1039/b305179j

Conjugation of surface binding groups with inhibitors for carbonic anhydrase leads to the conversion of weak inhibitors to strong inhibitors.

Full text of DOI:10.1039/b305179j

Conjugation of poor inhibitors with surface binding groups: a strategy to
improve inhibition†
Bidhan C. Roy, Ryan Hegge, Theresa Rosendahl, Xiao Jia, Rachael Lareau, Sanku Mallik* and D. K.
Srivastava
Department of Chemistry and Molecular Biology, North Dakota State University, Fargo, North Dakota,
USA. E-mail: Sanku.Mallik@ndsu.nodak.edu; Fax: 701-231-8831; Tel: 701-231-8829
Received (in Corvallis, OR, USA) 8th May 2003, Accepted 25th July 2003
First published as an Advance Article on the web 4th August 2003
Conjugation of surface binding groups with inhibitors for
carbonic anhydrase leads to the conversion of weak in-
hibitors to strong inhibitors.
amylase) compared to the flexible counterparts. However, in
these reported examples, the enhancement of biological proper-
ties is due to the rigidity of the structures induced by the metal
ions.
Recognition of protein surfaces by synthetic molecules has been
successfully used to disrupt protein–protein interactions,1 alter
electron transport properties,2 inhibit receptor–ligand inter-
actions,3 etc. These ligands recognize their targets by several,
simultaneous, weak interactions (e.g., hydrogen bonding, ion
pairing, etc.). Recently, we have demonstrated a protein surface
recognition strategy employing strong and directional metal–
ligand interactions4 for the enzyme carbonic anhydrase (bovine
erythrocyte).
Inhibition of carbonic anhydrase is important for the
treatment of glaucoma and cancer.5,6 Usually, the clinically-
approved inhibitors are the sulfonamide class of compounds.
Conjugation of the high-affinity sulfonamides with bile acids,7
short peptides,8 amino-polycarboxylate ligands and their metal
complexes9 further enhances inhibition efficiency.
Herein, we report a strategy to convert a poor inhibitor to a
good inhibitor by attaching a surface-histidine recognition
group to the inhibitor. Benzene sulfonamide, a rather weak
inhibitor for carbonic anhydrase (Kd = 120 mM, Table 1), was
converted to a very good inhibitor for the enzyme (Kd = 130
nM, Table 1) as a result of this conjugation.
For the studies reported herein, the ligand iminodiacetic acid
(IDA) was used to chelate the cupric ions (K = 1012 M21).11
The length of the spacer separating the benzene sulfonamide
group from IDA was varied in these complexes. Benzene
sulfonamide (6) and the di-Cu2+ complex 7 (lacking the benzene
sulfonamide moiety) were used as controls for these studies.
The syntheses of sulfonamide-based metal complexes are
depicted in Scheme 1. The reported Na-salt of IDA (7)12 was
coupled with the sulfonamides (6) using BOP reagent (benzo-
triazol-1-yloxytris(dimethylamino)phosphonium hexafluoro-
phosphate). The synthesis of complex 5 was carried out by
reacting cyanuric chloride and 2 equiv. of amine-IDA ester
(ESI†).13 It was then combined with 4-(aminoethyl)benzene
sulfonamide (experimental details are included in ESI†).
Complexes 2, 3 and 4 have two cupric ions ~ 8 Å apart.14
Complex 5 is flexible and the distances between the Cu2+ ions
was estimated to be 8–12 Å (employing BioMed CAChe,
version 6.0).
The binding constants of these complexes with carbonic
anhydrase (bovine erythrocyte, Sigma Chemical Company,
mixture of isozymes) were determined employing isothermal
titration calorimetry (25 mM HEPES buffer, pH = 7.0, Table
1). The two controls, benzene sulfonamide (control 6) and the
di-IDA-Cu2+ complex 7 (lacking the benzene sulfonamide
group) showed weak affinity for the enzyme. Affinities of the
conjugates were considerably higher compared to the controls.
Complex 3 showed the highest affinity for the enzyme, three
orders of magnitude higher compared to the controls. The
similarity of binding constants for complexes 1 (one Cu2+ ion)
and 3 (two Cu2+ ions) possibly indicates that one cupric ion is
binding to one histidine on the surface of the protein. Since the
enzyme preparation included a mixture of isozymes, it is
To demonstrate the proof-of-concept, five Cu2+-complexes
(Fig. 1) were designed and synthesized. In these complexes, the
benzene sulfonamide binds to the active-site Zn2+ ion of the
enzyme. It was estimated by molecular modelling (BioMed
CAChe 6.0, Fujitsu America, Beaverton, OR) that the Cu2+ ions
of the complexes are then capable of binding to His-4 or His-17
on the surface of carbonic anhydrase (bovine erythrocyte,
protein data bank file: 1g6v.pdb). The targeted histidine
residues are close to the N-terminus of the enzyme. The protein
backbone in this region is flexible and has a random coil
structure, facilitating the binding of the cupric ions to the
histidines when the benzene sulfonamide is bound to the Zn2+
ion in the active site.
There are literature reports of flexible peptides converted to
rigid structures by coordination to transition metal ions (Cu2+,
Zn2+).10 These rigid peptides demonstrated enhanced biological
properties (including improved inhibition of the enzyme a-
Table 1 Binding parameters of the complexes with carbonic anhydrase
Compound
Binding constant
Enthalpy/kcal mol21
Complex 1
Complex 2
Complex 3
Complex 4
Complex 5
Control 6
(4.6 ± 0.07) 3 106
(1.9 ± 0.03) 3 105
(7.5 ± 0.1) 3 106
(5.4 ± 0.02) 3 105
(4.3 ± 0.03) 3 105
(9.0 ± 0.1) 3 103
(22.8 ± 1.3) 3 103
226.4 ± 0.8
251.7 ± 5.8
236.9 ± 4.2
230.3 ± 2.6
245.5 ± 2.2
231.2 ± 1.6
2129.0 ± 3.2
Control 7
† Electronic supplementary information (ESI) available: experimental
details and UV-Vis titration data. See http://www.rsc.org/suppdata/cc/b3/
b305179j/
Fig. 1 The structures of the metal complexes.
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CHEM. COMMUN., 2003, 2328–2329
This journal is © The Royal Society of Chemistry 2003

Table 2 Kinetic parameters of the carbonic anhydrase catalyzed reaction in
the absence and presence of selected conjugates
possible that different histidine residues from different iso-
zymes contribute to this binding.
In order to demonstrate the binding of histidine residues to
the cupric ions, the free ligands for these complexes (i.e., 9a, 9b
and 9c) were titrated with the enzyme. The affinities were found
to be much lower, similar to those of the controls 6 and 7 (data
not shown). In addition, the Cu2+ complexes were titrated with
the enzyme employing UV-Vis spectrometry.4 The absorbance
maxima for the cupric complexes were found to shift from 735
nm to 666 nm upon sequential addition of carbonic anhydrase,
indicating the coordination of histidines to the cupric ions4 (data
for complex 3 are included in ESI†).
The kinetic parameters (Km, Vmax, and Ki) of the carbonic
anhydrase catalyzed reactions were determined by measuring
the hydrolysis of p-nitrophenyl acetate at 450 nm (Table 2). The
substrate concentration dependent kinetic data in the absence
and presence of inhibitors were analyzed by the non-linear
regression analysis program, Grafit 4.0, and presented in the
form of the double reciprocal plots (Fig. 3S, ESI†). The analyses
of the kinetic data conformed to the competitive inhibition
Inhibitor
Km/mM
Vmax (DA450)/min21Ki/mM
No inhibitor
Complex 2
Complex 3
Complex 4
15.70
28.30
36.10
29.20
0.31
0.25
0.33
0.31
0.74
0.124
0.814
model, and excluded other (viz., non-competitive and un-
competitive) models. It should be noted that the Ki values
determined by the kinetic method are similar to the dissociation
constants (Kd = 1/Ka) of the corresponding enzyme–inhibitor
complexes (Table 1, also see ESI†), determined via the
isothermal titration microcalorimetric method.
In conclusion, we have demonstrated that the conjugation of
a poor inhibitor (for the enzyme carbonic anhydrase) with a
surface-binding functionality enhances the inhibitor efficiency
by three orders of magnitude.
This research was supported by the National Institutes of
Health grants 1R01 GM 63404-01A1 and 1P20 RR15566-01 to
SM.
Notes and references
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Scheme 1 Syntheses of the copper complexes 2, 3 and 4. The syntheses of
1, 4 and 6 are included in ESI.†
14 N. Shirai, Y. Watanabe and Y. Sato, J. Org. Chem., 1990, 55, 2767.
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