Two-prong inhibitors for human carbonic anhydrase II

Article abstract of DOI:10.1021/ja047271k

The enzyme inhibitors are usually designed by taking into consideration the overall dimensions of the enzyme's active site pockets. This conventional approach often fails to produce desirable affinities of inhibitors for their cognate enzymes. To circumve

Full text of DOI:10.1021/ja047271k

Published on Web 09/25/2004
Two-Prong Inhibitors for Human Carbonic Anhydrase II
Bidhan C. Roy, Abir L. Banerjee, Michael Swanson, Xiao G. Jia, Manas K. Haldar,
Sanku Mallik,* and D. K. Srivastava*
Department of Chemistry, Biochemistry & Molecular Biology, North Dakota State UniVersity,
Fargo, North Dakota 58105
Received May 10, 2004; E-mail: sanku.mallik@ndsu.nodak.edu; dk.srivastava@ndsu.nodak.edu
Carbonic anhydrases (EC 4.2.1.1) are ubiquitously distributed
Zn(II)-containing metalloenzymes1 that catalyze the reversible
hydration of CO2 to bicarbonate.2 In humans, there are 14 isozymes
of the R-type, some of which have been implicated in pathological
conditions.2 Thus, inhibitors of carbonic anhydrases have been
employed in the treatment of glaucoma, epilepsy, and cancer.2
Usually, these inhibitors possess a sulfonamide group, which
interacts with the active site Zn2+.2 While some inhibitors have
been obtained by structure-based design, capitalizing on this
interaction,3,4 there are limitations to this approach. Since the active
site pocket has defined spatial dimensions, variations in inhibitor
structure are not readily accommodated.5 Also, the intrinsic
flexibility in the protein allows for the binding of structurally
unrelated compounds, and it is difficult to predict the nature and
magnitude of such structural flexibility.3
Herein, we report a general strategy of inhibitor design which
overcomes the limitations of the active-site based design alone. The
proof-of-concept is demonstrated by converting a weak inhibitor
of human carbonic anhydrase II (hCAII) into very potent inhibitors.6
Our approach is based on attaching a metal chelating tether group
(containing bound Cu2+), which extends beyond the actiVe site
pocket of the enzyme and interacts with the surface exposed
histidine residues. In this two-prong approach, the binding energy
of the inhibitors is derived not only from the interactions at the
active site region but also from the interactions of IDA-Cu2+ to
the surface-exposed histidine residues.
There are some reports in the literature on linking two weak
binding ligands of an enzyme to generate potent inhibitors.7,8 In
these reports, the ligands interact with the enzymes via hydrophobic,
hydrogen bonding, and ion-pair interactions. There is one report
on enhancing the binding affinities of carbonic anhydrase inhibitors
by coordinating to His-64.7d This residue is situated close to the
enzyme’s active site pocket and is involved in catalysis.7d To the
best of our knowledge, there are no reports of using the metal-
ligand interactions involving surface amino acid residues to enhance
the binding affinities of “two-prong” inhibitors.
Benzenesulfonamide (1, Figure 1) and p-aminoethylbenzene-
sulfonamide (2, Figure 1) are weak inhibitors for hCAII (Ki ) 1.5
µM for 1 and 8.1 µM for 2; Table 1).9 These two compounds were
selected as the controls in the present studies. For chelating to Cu2+
ions, iminodiacetate (IDA) was selected as the ligand. IDA has a
very high affinity for Cu2+ (K ) 1012 M-1).10 In addition, CA binds
to the complex IDA-Cu2+ via the surface-exposed histidine
residues of the enzyme.11 For the studies described herein, the weak
inhibitors 1 and 2 were conjugated to the complex IDA-Cu2+ with
a variety of spacers to generate the potent inhibitors (similar
conjugates of IDA-Zn2+ precipitated above pH ) 5.5). The
structures of the resultant conjugates (5-12) are shown in Figure
1. The IDA-Cu2+ complexes 3 and 4 were also used as controls
Figure 1. Structures of the conjugates synthesized (5-12) and the controls
(1-4) used in this study.
Table 1. Inhibition Parameters for the Synthesized Conjugates
with HCAII
conjugate
Ki (nM)
K
d (1/Ka) (ITC), nM
1
2
3
4
5
6
7
8
1500 ( 140
8165 ( 312
no inhibition
no inhibition
131 ( 21
397 ( 30
27 ( 6
625 ( 110
4750 ( 1400
5500 ( 610
2130 ( 200
37( 3
196 ( 25
24( 3
35 ( 10
55( 2
9
104 ( 17
11 ( 3
100 ( 8
10
11
12
1.0 ( 0.05
77( 5
65 ( 9
52 ( 9
13 ( 0.8
during these studies. The syntheses of these compounds are included
in the Supporting Information.
The inhibitory potencies of the synthesized conjugates for the
recombinant human carbonic anhydrase II (hCAII)-catalyzed reac-
tion were determined by measuring the esterase activity of the
enzyme, using p-nitrophenyl acetate as a substrate.12 The inhibition
constants of the enzyme-inhibitor complexes (Ki values), deter-
mined by the best fit of the experimental data, are summarized in
Table 1. To ensure that the Ki values determined by the steady-
state kinetic method were true measures of the binding affinities
of the enzyme-inhibitor complexes, and not due to some unfore-
seen kinetic complexity, we performed the isothermal titration
microcalorimetry (ITC) experiments (Supporting Information). The
dissociation constants (Kd ) 1/Ka) values of the individual enzyme-
inhibitor complexes have been compared with their corresponding
Ki values in Table 1. Note a marked similarity between the Ki and
Kd values for each inhibitor. To the best of our knowledge, there is
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C O M M U N I C A T I O N S
slow. The only way to release conjugate 11 from hCAII was to
add EDTA to the hCAII.11 complex. EDTA removes the cupric
ion from the conjugate 11, and thus the resultant metal-free
conjugate exhibits the off rate similar to that of control 2.
In conclusion, we have demonstrated that weak inhibitors of
hCAII can be converted to excellent inhibitors by conjugating with
surface binding groups. Conjugation of IDA-Cu2+ (using a spacer)
to benzenesulfonamides led to 800-fold improvement in the
inhibition constants. It should be noted that this method can be
used with other enzymes, which harbor histidine residues on their
surfaces, and in close proximity to their active sites (e.g., tyrosine
kinase, adenylate kinase, aldose reductase etc.). In addition, other
amino acid residues (i.e., besides histidines) present on the surface
of enzymes can be targeted by using other transition metal ion
conjugates.14
Figure 2. Dissociation “off-rates” of p-aminoethylbenzenesulfonamide and
conjugate 11 from hCAII. The main figure shows the stopped-flow trace
for the increase in the fluorescence intensity (λex ) 330 nm; cutoff filter )
335 nm) upon mixing of the enzyme-p-aminoethylbenzenesulfonamide
complex with a high concentration of dansylamide. The after-mixing
concentrations of the enzyme, inhibitor, and dansylamide were 2, 40, and
100 µM, respectively. The inset shows the spectrofluorimetric trace (λex
)
Acknowledgment. This research was supported by the National
Institutes of Health grants 1R01 GM 63404-01A1 to S.M. and 1R15
DK56681-01A1 to D.K.S..
330 nm, λem ) 448 nm) upon mixing 2 µM enzyme 2.5 µM conjugate 5
and 100 µM dansylamide, respectively.
only one previous report13a of ITC studies on inhibitor binding to
hCAII.
Supporting Information Available: A figure showing fluorescence
spectra of dansylamide and text and schemes giving synthetic details
for the conjugates 5-12 and experimental details for the ITC experi-
ments. This material is available free of charge via the Internet at
http://pubs.acs.org.
As apparent from the data of Table 1, all of the synthesized
conjugates are potent inhibitors for the enzyme. The conjugates
with triethylene glycol spacer show better inhibition as compared
to the others. The conjugate 10 showed the best inhibition, 800-
fold more pronounced than control 2. It was noted that the increase
in the inhibitory potencies of two Cu2+ ion complexes were modest
as compared to the corresponding one Cu2+ conjugates. This feature
indicates that only one of the cupric ions is binding to a histidine
residue on the surface of the enzyme. Currently, we are investigating
as to which histidine residue is specifically involved in the above
interaction by performing the site-directed mutagenesis. However,
when all the surface-exposed histidines residues are chemically
modified with diethyl pyrocarbonate, the inhibition potency of con-
jugate 8 (Ki ) 606 ( 59 nM) decreased by 17-fold compared to
theunmodifiedenzyme(Ki )35(10nM).Thisdemonstratestherole
of the surface-exposed histidine residues in the binding process.
To demonstrate the role of the cupric ions in the binding process,
two different types of experiments were conducted (described in
detail for the conjugate 8). The metal free ligand of complex 8
showed a weak inhibition (Ki ) 1.5 µM), comparable to that of
the controls 1 and 2. Similar results were obtained by the ITC
titration method. In addition, conjugate 8 was titrated by the enzyme
hCAII and the changes in the absorption maximum of the Cu2+
complex was monitored by the UV-vis spectrometry. The absorp-
tion maximum was found to shift from 730 to 665 nm, indicating
the coordination of histidines to the cupric ions of 8 (data not
shown).11
We ascertained the kinetic feasibility of dissociation of the
enzyme-bound inhibitors via the competitive displacement by an
active site directed ligand, dansylamide.13 The fluorescence emission
spectra of free and the enzyme-bound dansylamide (λex ) 330 nm)
indicated a marked difference in the spectral profile (around 448
nm) upon binding with hCAII (Supporting Information). As an
example, the dissociation off rates of the conjugate 11 and its parent
compound, p-aminoethylbenzenesulfonamide (compound 2), were
measured via the stopped-flow assembly, configured to detect the
time dependent fluorescence changes of the competitive inhibitor
dansylamide (λex ) 330 nm; “cutoff” filter ) 335 nm; Figure 2).
The data are best fitted by the single-exponential rate equation with
a rate constant of 0.094 s-1. When we attempted to perform the
above experiment involving conjugate 11, practically no change
in the fluorescence signal was noted up to 120 min of the reaction
time, suggesting that the dissociation off rate of 11 was extremely
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