Inhibition of carbonic anhydrase-II by sulfamate and sulfamide groups: An investigation involving direct thermodynamic binding measurements

Article abstract of DOI:10.1021/jm058279n

This paper examines the relative effectiveness of bioisosteric sulfamate and sulfamide derivatives for inhibition of human carbonic anhydrase-II (CA-II) by using a direct binding assay based on the ThermoFluor method (Matulis et al. Biochemistry 2005, 44, 5258). Compounds 1-10, which represent five cognate sulfamate/ sulfamide pairs, were studied by ThermoFluor to obtain binding affinities (K_a values). The corresponding dissociation constants, K_d, provide an independent measure of CA-II activity relative to commonly used K_i values from enzyme kinetics studies. There was a sizable difference in potency between the sulfamates and sulfamides, with the sulfamides being much less potent, by factors ranging from 25 (7/8) to 1200 (3/4), These results are consistent with our recent report that sulfamides tend to be much weaker inhibitors of CA-II than their corresponding sulfamates (Maryanoff et al. J. Med. Chem. 2005, 48, 1941). Additionally, for arylsulfamides 10-12 the K_d values determined by ThermoFluor and the K_i values determined from enzyme kinetics are consistent. It appears that the sulfamide group is less suitable than the sulfamate group for obtaining potent inhibition of CA-II.

Full text of DOI:10.1021/jm058279n

3496
J. Med. Chem. 2006, 49, 3496-3500
Inhibition of Carbonic Anhydrase-II by Sulfamate and Sulfamide Groups: An Investigation
Involving Direct Thermodynamic Binding Measurements
Alexandra L. Klinger, David F. McComsey, Virginia Smith-Swintosky, Richard P. Shank, and Bruce E. Maryanoff*
Research & Early DeVelopment, Johnson & Johnson Pharmaceutical Research & DeVelopment, Spring House, PennsylVania 19477-0776
ReceiVed NoVember 8, 2005
This paper examines the relative effectiveness of bioisosteric sulfamate and sulfamide derivatives for inhibition
of human carbonic anhydrase-II (CA-II) by using a direct binding assay based on the ThermoFluor method
(Matulis et al. Biochemistry 2005, 44, 5258). Compounds 1-10, which represent five cognate sulfamate/
sulfamide pairs, were studied by ThermoFluor to obtain binding affinities (Ka values). The corresponding
dissociation constants, Kd, provide an independent measure of CA-II activity relative to commonly used Ki
values from enzyme kinetics studies. There was a sizable difference in potency between the sulfamates and
sulfamides, with the sulfamides being much less potent, by factors ranging from 25 (7/8) to 1200 (3/4).
These results are consistent with our recent report that sulfamides tend to be much weaker inhibitors of
CA-II than their corresponding sulfamates (Maryanoff et al. J. Med. Chem. 2005, 48, 1941). Additionally,
for arylsulfamides 10-12 the Kd values determined by ThermoFluor and the Ki values determined from
enzyme kinetics are consistent. It appears that the sulfamide group is less suitable than the sulfamate group
for obtaining potent inhibition of CA-II.
Carbonic anhydrase (CA) enzymes (EC 4.2.1.1), which are
involved in catalyzing the hydration of carbon dioxide and the
dehydration of bicarbonate [CO2 + 2 H2O / HCO3- + H3O+],
are biochemically well characterized and clinically relevant.1-4
CA-II, the most commonly studied isoform, is also the most
prevalent isoform in many different organs and cell types.2,4
For several decades, it has been widely recognized that potent
inhibitors of CA-II can be derived by having a primary
sulfonamide group (SO2NH2) on a suitable organic scaffold,
such as benzenoid or heterocyclic structures.3,5,6 This situation
is not too surprising since CA-II contains a functional Zn(II)
atom in its active site that is normally bound to the imidazole
nitrogen atoms of three histidine residues (His-94, His-96, and
His-119 in human CA-II).6,7 Other Zn(II)-coordinating groups
may also provide compounds that inhibit CA-II. However, it is
important to note that inhibitors based on such Zn(II) ligands
do not always operate with a high level of effectiveness (i.e.,
they can exhibit just moderate-to-weak potency).3
ality (-OSO2NH2).9 Coincident with our drug discovery efforts,
we were prompted to investigate the inhibition of CA-II, as a
possible mechanism of action and a potential source of additional
pharmacology.8,9a-c,10 In a recent article we reinforced the
viewpoint that topiramate is a moderate inhibitor of CA-II (Ki
value of ca. 500 nM),9c as opposed to being a potent inhibitor
(Ki value <50 nM).11 Interestingly, we also found that com-
pounds containing a primary sulfamide group (-NHSO2NH2)
can possess substantially weaker CA-II inhibitory potency than
their corresponding sulfamates.9c To be more specific, a direct
comparison of sulfamate/sulfamide bioisosteric pairs, such as
1/2, 3/4, and 5/6, revealed dramatically different CA-II inhibi-
tion, with CA-II Ki values of 500/650000 nM for 1/2 and 12/
20000 nM for 3/4, and CA-II IC50 values of 130/71000 nM for
5/6 (by the pH-shift method).9c However, this divergence in CA-
II inhibitory behavior between cognate sulfamates and sul-
famides has not met with universal acceptance.12 In 2003, Casini
et al. reported that several sulfamides of relatively simple
structure (e.g., PhNHSO2NH2) possess CA-II Ki values below
50 nM,13 and other papers have also mentioned sulfamides as
relatively potent CA-II inhibitors.11a,12,14 Since the degree of
CA-II inhibition attainable with sulfamate and sulfamide groups
is an important issue with respect to future drug design, we
sought to identify a means to reconcile these disparate views.
Are corresponding pairs of sulfamates and sulfamides nearly
the same or very different in CA-II inhibitory potency, and are
the sulfamides much weaker? Almost all CA-II inhibition data
in this field have emanated from enzyme kinetics studies, often
based on two standard assay protocols (“pH-shift” and “esterase”
methods).3b,9c,11-14 Despite the fact that we have obtained
consistent results with both of these methods,9c it is not unheard
of for enzyme kinetics studies in different laboratories to
experience discrepancies in outcomes because of differences in
experimental techniques and/or reaction conditions. To resolve
this conundrum, we decided to employ an independent meth-
odology that does not rely at all on enzyme kinetics, but rather
relies on the direct determination of binding affinity, in terms
of thermodynamic dissociation constants (Kd values). Thus, we
Since our discovery of the antiepileptic drug topiramate (1),8
we have developed an abiding interest in the pharmacological
actions of compounds containing a primary sulfamate function-
* To whom correspondence should be addressed. E-mail: bmaryano@
prdus.jnj.com. Fax: 215-628-4985. Phone: 215-628-5530.
10.1021/jm058279n CCC: $33.50 © 2006 American Chemical Society
Published on Web 05/19/2006

Inhibition of Carbonic Anhydrase-II
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12 3497
Table 1. CA-II Binding Affinity and Inhibition Data for Compounds
1-10
values at 37 °C) for the sulfamate/sulfamide pairs were
established as 290/25000 nM for 1/2, 10/12500 nM for 3/4, 140/
10000 nM for 5/6, 2000/50000 nM for 7/8, and 110/4600 nM
for 9/10. The ratio of Kd values for the five sulfamate/sulfamide
pairs ranged from 25 to 1250, with the sulfamide always having
the lower affinity (Table 1). It should be especially noted that
the Kd values for 1/2 and 3/4 are reasonably consistent with the
Ki values that we determined by using enzyme kinetics: 500/
650000 nM for 1/2 and 12/20000 nM for 3/4 (pH-shift method,
0 °C); 430/340000 nM for 1/2 and 38/25000 nM for 3/4 (esterase
method, 23 °C).9c This trend also was applicable to the three
other sulfamate/sulfamide pairs in Table 1: 5/6, 7/8, and 9/10.
The ratio of Ki values for the five sulfamate/sulfamide pairs
ranged from 220 to 1670, with the sulfamide always having a
much lower affinity. For topiramate (1), since the Kd value of
290 nM essentially recapitulates the Ki value of 500 nM,9c we
indicate again that topiramate is just a moderate inhibitor of
CA-II. It should be noted, however, that our Kd values for 1, 9,
and 10 are not consistent with the Ki values reported by other
researchers (esterase method): 5 nM for 1,11 1.3 nM for 9,11b
and 12 nM for 10.13
compd Kd (nM)a fold diffb
Ki (nM)c
fold diffb
1
2
3
4
5
6
7
8
9
10
290
25000
10
12500
140
10000
2000
50000
110
- -
88
- -
1250
- -
70
- -
25
- -
40
500d
650000d
12d
- -
1300
- -
1670
- -
550
- -
400
- -
20000d
130d
71000d
1020 (650-1590)e
408000 (255000-654000)e
36 (26-50)e
4550
7960 (4880-13000)e
220
a The binding affinity to human CA-II was determined by the Thermo-
Fluor method. b Fold difference in comparing the result for the sulfamide
with that for its corresponding sulfamate. c Inhibition of human CA-II by
measuring hydration of carbon dioxide via the shift in pH. d Data taken
from ref 9c. e New data. The 95% confidence intervals are given in
parentheses (N ) 2 for 7 and 8; N ) 3 for 9; N ) 4 for 10).
applied the specific thermochemical technique known as Ther-
moFluor,15 which was used effectively to evaluate the affinity
of six reference sulfonamide-based inhibitors of CA-II.16 Herein,
we report ThermoFluor results for five pairs of cognate
sulfamates and sulfamides. The Kd values observed for these
bioisosteric pairs are consistent with our Ki values from enzyme
kinetics experiments and lend further support to the fact that
the sulfamide group is not very effective for generating potent
CA-II inhibitors.
Results and Discussion
Given our result with 10, which departs markedly from the
potent Ki value reported,13 we decided to examine two analogous
arylsulfamides, 11 and 12. The interactions of 10-12 with CA-
II were assessed by using the ThermoFluor method and two
enzyme kinetics assays, hydration of CO2 and hydrolysis of
4-nitrophenyl acetate (4-NPA) (Table 2). The ThermoFluor
experiments were performed in duplicate under two different
conditions, A and B (see Experimental Section). Compound 10
had Kd values in the range of 2000-5000 nM, 11 had Kd values
in the range of 2000-5000 nM, and 12 had Kd values in the
range of 1000-1700 nM (all at 37 °C). The Ki values for 10-
12 from enzyme kinetics with the CO2 hydration assay (at 0
°C) were 7960, 14400, and 892 nM, respectively, and with the
4-NPA hydrolysis assay (at 23 °C) were 8020, 18800, and 1740
nM, respectively. For sulfamate 9, the ThermoFluor and kinetics
data are equally consistent and serve as a reference for a more
potent inhibitor of CA-II. In contrast to our observations, the
Ki values reported by other researchers for 9-12 in the 4-NPA
hydrolysis assay were 1.3, 12, 11, and 13 nM, respectively.11b,13
It is not clear what factors are behind the observed discrep-
ancy between the Ki values for 1, 9, 10, 11, and 12 measured
by the two different research groups. However, we have obtained
fairly consistent results for enzyme kinetics-derived Ki values
CA-II Inhibition and Binding. To address the apparent
inconsistencies in results for the inhibition of CA-II by sulfamide
derivatives on the basis of enzyme kinetics studies,9c,12-14 we
conducted a thermodynamically oriented investigation based on
the ThermoFluor method.15,16 This method provides a thermal
melting curve, derived from the change in fluorescence intensity
for an environmentally sensitive dye, such as 1-anilino-8-
naphthalenesulfonic acid (ANS), as a function of temperature.
From this melting curve the midpoint transition temperature Tm,
a measure of protein stability, can be determined. The binding
affinity (Ka ) 1/Kd) is directly related to the increase in protein
stability, and Tm, in the presence of the test compound (L).15
The magnitude of the change in Tm is proportional to both the
ligand concentration, log[L], and the binding affinity. Since
ThermoFluor is a plate-based technology, precise Tm values can
be established rapidly under diverse experimental conditions,
and with as many as 32 different inhibitor candidates in the
same experiment. The binding affinity at the Tm, namely Ka,Tm
,
is calculated with the aid of calorimetrically measured param-
eters for protein stability, and is then extrapolated to 37 °C.16
The ThermoFluor method was thoroughly validated for CA-II,
as well as for CA-I, with six sulfonamide inhibitor ligands.16
By using ThermoFluor, we have determined the behavior of
human carbonic anhydrase-II in the presence of compounds
1-10, which represent five cognate sulfamate/sulfamide pairs,
to derive Kd values (Table 1). The ThermoFluor-generated
curves for 1-10 with CA-II, which depict the response of Tm
relative to inhibitor concentration, are presented in Figure 1.
The Kd values were derived from these concentration-response
curves by using well-understood models of ligand-induced
perturbations on the thermal stability of proteins.16,17 To enable
this process, we measured two, key protein-specific thermody-
namic parameters for human CA-II by differential scanning
calorimetry (DSC): the Gibbs free energy [∆UG(T)], which is a
function of the calorimetric enthalpy [∆UH(T)], and the heat
capacity of unfolding (∆UCp). Thus, the binding affinities (Kd
Table 2. CA-II Inhibition and Binding Data for Arylsulfamides 10-12
CO2 hydration Ki,
4-NPA hydrol Ki,
Kd
Kd
compd
nMa
nMb
(nM)c (nM)d
10
11
12
7960 (4880-13000)
8020 (5760-11200)
2000 3750
14400 (8790-23600) 18800 (13100-26900) 2650 5000
892 (546-1430)
1740 (1140-2660)
1000 1500
a Inhibition of human CA-II by measuring hydration of carbon dioxide
via the shift in pH. The 95% confidence intervals are given in parentheses
(N ) 4 for 10; N ) 3 for 11 and 12). b Inhibition of human CA-II by
measuring the rate of hydrolysis of 4-nitrophenyl acetate (4-NPA) by
absorption of light at 400 nm. The 95% confidence intervals are given in
parentheses (N ) 5 for 10-12). c Binding affinity to human CA-II
determined by ThermoFluor under the “A” conditions. d Binding affinity
to human CA-II determined by ThermoFluor under the “B” conditions.

3498 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12
Klinger et al.
Figure 1. ThermoFluor concentration-response curves (Tm vs log[L]) for 1-10 (L ) ligand). The data points for sulfamates are filled circles; the
data points or sulfamides are filled squares; each set of data in Panels A-E is represented by different colors (see below). Experiments were
performed in quadruplicate. Error bars represent the standard deviation of measured Tm values for each log[L]. Inhibitor dissociation constants (Kd)
were derived from the concentration effect of ligand on Tm for each test compound. The solid lines are simulated (see Experimental Section16) by
using ∆UH ) 197 kcal/mol, ∆UCp ) 2.5 kcal mol-1K-1, and Tm ) 55 °C with the determined Kd values. Legends for plots: Panel A, red circle 1
(290 nM), green square 2 (25000 nM); Panel B, yellow circle 3 (12 nM), blue square 4 (12500 nM); Panel C, purple circle 5 (100 nM), light green
square 6 (10000 nM); Panel D, yellow circle 7 (2000 nM), light blue square 8 (50000 nM); Panel E, pink circle 9 (100 nM), violet square 10 (4500
nM).
with two assay systems,9c and there is agreement with the
ThermoFluor-derived, direct binding parameters (Kd values).
Under the circumstances, we must conclude that our measured
levels of CA-II inhibition (Ki values) are the more accurate ones.
Sulfamates vs Sulfamides as CA-II Inhibitors. We have
commented on the comparison of sulfamate and sulfamide
molecules, which are strict bioisosteres, as inhibitors of CA-
II.9c Compound 3 is a very potent inhibitor of human CA-II
with a Ki value of 12 nM and a Kd value of 10 nM and thus
serves as a benchmark for sulfamates. Its sulfamide isostere, 4,
is markedly less potent, with a Ki value of 20000 nM (1600-
fold less potent) and a Kd value of 12500 nM (1200-fold less
potent). A similar disparity exists for topiramate (1), with a Ki
value of 500 nM and Kd value of 290 nM, and its sulfamide
congener (2), with a Ki value of 650000 nM and a Kd value of
25000 nM. This pattern of substantially diminished CA-II
inhibition/affinity for a sulfamide vs a sulfamate occurred for
the five pairs of compounds that we studied, including the simple
compounds 9 and 10 (Kd ) 110 and 4600 nM, respectively).
Considering the clear-cut potency difference for the sulfamate/
sulfamide bioisosteric pairs, it would appear that the sulfamide
moiety is not particularly desirable for obtaining potent inhibi-
tion of, or affinity to, CA-II.
The most straightforward explanation for the weaker potency
of sulfamides relative to sulfamates could be their difference
in pKa. For sulfamates 1 and 3, we had determined pKa values
of 8.66 and 8.51, respectively; whereas for sulfamide 2, we had
determined a pKa value of 10.7.9c This difference of 2 orders
of magnitude in pKa is very significant. Since 2 is much less

Inhibition of Carbonic Anhydrase-II
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12 3499
evaporation. Measurements were made in quadruplicate on each
plate. The medium was comprised of 10 mM HEPES (pH 7.5),
100 µM Na2SO4, 100 µM ANS, and 1.5% DMSO (“A” conditions).
For 10-12, measurements were also made by using 25 mM HEPES
and no salt at pH 7.4 (“B” conditions). Plates were heated at a rate
of 1 °C/min on a thermal block and illuminated with band-passed
filtered UV light (380-400 nm). Fluorescence was measured by
using an overhead CCD (charge-coupled device) camera that was
filtered to detect wavelengths of 500 ( 25 nm.
To determine the Kd values from the melting curves, expressions
were used that describe the total ligand concentration needed to
raise the protein Tm values to a given value. Since these transcen-
dental expressions cannot be solved explicitly, we simulated 12-
point Tm concentration-response curves by calculating the con-
centration of total added ligand required to produce the experimentally
observed Tm values. This simulation process was based on key
protein-specific thermodynamic parameters for human CA-II that
determine the temperature dependence of the Gibbs free energy of
unfolding [∆UG(T)], which were measured by differential scanning
calorimetry (DSC; with a VP-DSC calorimeter; Microcal, Inc.,
North Hampton, MA) according to reported methodology.16 The
calorimetric enthalpy [∆UH(T)] was 197 kcal mol-1 and the heat
capacity of unfolding (∆UCp) was 2.5 kcal mol-1 K-1. Since the
dissociation binding constants determined by ThermoFluor are
affinities at the melting temperatures, the Kd values were extrapo-
lated to 37 °C by using established methods and previously
measured binding enthalpy and heat capacity values. Under the
standard ThermoFluor conditions (“A”) with these computational
methods, acetazolamide afforded a Kd value of 50 nM.21
acidic than 1, it will have a much lower population of the anionic
form that is required for binding to Zn(II) in the active site of
CA-II.6,7,18
Conclusion
We have examined compounds 1-10, which represent five
sulfamate/sulfamide bioisosteric pairs, for their binding to human
carbonic anhydrase-II by using the ThermoFluor method.15,16
The resultant thermodynamically based binding parameters (Kd
values) provide an independent measure of the effectiveness of
the sulfamate and sulfamide groups compared with the inhibition
of CA-II through enzyme kinetics studies (Ki values). The
sulfamides were much less potent than the sulfamates by factors
ranging from 25 (7/8) to 1200 (3/4). Additionally, we determined
Ki values by enzyme kinetics and Kd values by ThermoFluor
for 9-12 and obtained consistent results between each approach.
Consequently, it would appear that the sulfamide group is not
particularly suitable for obtaining potent CA-II inhibition.
Experimental Section
General Procedures. Reactions were conducted under an
atmosphere of argon in solvents that were dried with molecular
sieves (4A). Melting points were determined on a Thomas-Hoover
apparatus calibrated with a set of melting point standards. 1H NMR
spectra were acquired on a Bruker Avance 300-MHz spectrometer
(abbreviations used: s, singlet; d, doublet; t, triplet; m, multiplet;
br, broad). Thin-layer chromatography was conducted on Whatman
silica gel GF plates (250 µm) with iodine staining. Column
chromatography was performed on silica gel 60 (40-63 µm; EM
Science). Unless otherwise specified, mass spectra were electrospray
(ES) and were run on a Micromass Platform LC single quadrupole
mass spectrometer in the positive or negative mode as indicated.
Chemical-ionization (CI) mass spectra were recorded on a Finnigan
3300 mass spectrometer with ammonia as the reagent gas. Elemental
analyses were obtained from Quantitative Technologies, Inc.,
Whitehouse, NJ; percentage of water was determined by the Karl
Fischer method.
Carbonic Anhydrase Inhibition. Carbon Dioxide Hydration
and 4-Nitrophenyl Acetate Hydrolysis Assays. Purified human
CA-II was used. Inhibition of CA-II was determined for both assays
according to the procedures that were described earlier in detail.9c
The temperatures for the CO2 hydration and 4-NPA hydrolysis
assays were 0-2 °C and 23 °C, respectively.9c
Acknowledgment. We thank James Kranz for assistance in
the differential scanning calorimetry work. We are grateful to
Matthew Todd for helpful suggestions and for developing the
methods to determine Kd values from Tm concentration-response
curves.
Materials. Compounds 1-7 were described previously by us.8a,9c
Sulfamides 10-12 were prepared by reacting the commercially
available amines with sulfamide, according to the method described
in our preceding paper.9c Sulfamate 9 was prepared in the usual
manner from phenol, sulfamoyl chloride, and NaH.8a Analytical
data for 9-12 are presented in the Supporting Information.19 Human
carbonic anhydrase-II (purified from erythrocytes) and 4-nitrophenyl
acetate (4-NPA) were purchased from Sigma Chemical Co. (St.
Louis, MO). 1-Anilino-8-naphthalenesulfonic acid (ANS) was
obtained from Molecular Probes at Invitrogen Corp. (Carlsbad, CA).
N-(2,2-Dimethyl-1,3-dioxolan-4-ylmethyl)sulfamide (8). 2,2-
Dimethyl-1,3-dioxolane-4-methanamine (5.24 g, 40 mmol; Aldrich
Chemical Co.) was dissolved in 1,4-dioxane (100 mL), sulfamide
(19.2 g, 200 mmol) was added, and the reaction was heated at reflux
for 2 h. After concentration on a rotary evaporator, the residue was
triturated with ethyl acetate, the solid was filtered off, and the filtrate
was evaporated to give crude product, which was purified by flash-
column chromatography (10% MeOH in CH2Cl2) to give an oil,
which was redissolved in CH2Cl2. The solution was dried (Na2-
SO4) and evaporated in vacuo to afford the titled compound as a
Supporting Information Available: Analytical data for com-
pounds 9-12. ThermoFluor graphs of the original data for 10-12
that is represented in Table 2. This material is available free of
charge via the Internet at http://pubs.acs.org.
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1
clear, colorless, viscous oil (5.46 g, 65%). H NMR (CDCl3) δ
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3500 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12
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(19) See the paragraph at the end of this paper regarding Supporting
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(20) ThermoFluor is a registered trademark in the United States and certain
other countries.
(21) For acetazolamide, we reported: CO2 hydration Ki ) 3 nM; 4-NPA
hydrolysis Ki ) 18 nM.9c
JM058279N