Structural Basis of Nanomolar Inhibition of Tumor-Associated Carbonic Anhydrase IX: X-Ray Crystallographic and Inhibition Study of Lipophilic Inhibitors with Acetazolamide Backbone

Article abstract of DOI:10.1021/acs.jmedchem.0c01390

This study provides a structure-Activity relationship study of a series of lipophilic carbonic anhydrase (CA) inhibitors with an acetazolamide backbone. The inhibitors were tested against the tumor-expressed CA isozyme IX (CA IX), and the cytosolic CA I, CA II, and membrane-bound CA IV. The study identified several low nanomolar potent inhibitors against CA IX, with lipophilicities spanning two log units. Very potent pan-inhibitors with nanomolar potency against CA IX and sub-nanomolar potency against CA II and CA IV, and with potency against CA I one order of magnitude better than the parent acetazolamide 1 were also identified in this study, together with compounds that displayed selectivity against membrane-bound CA IV. A comprehensive X-ray crystallographic study (12 crystal structures), involving both CA II and a soluble CA IX mimetic (CA IX-mimic), revealed the structural basis of this particular inhibition profile and laid the foundation for further developments toward more potent and selective inhibitors for the tumor-expressed CA IX.

Full text of DOI:10.1021/acs.jmedchem.0c01390

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Article
Structural Basis of Nanomolar Inhibition of Tumor-Associated
Carbonic Anhydrase IX: X‑Ray Crystallographic and Inhibition Study
of Lipophilic Inhibitors with Acetazolamide Backbone
Jacob T. Andring, Mallorie Fouch, Suleyman Akocak, Andrea Angeli, Claudiu T. Supuran,*
Marc A. Ilies,* and Robert McKenna*
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ABSTRACT: This study provides a structure−activity relationship study of a series of lipophilic carbonic anhydrase (CA) inhibitors
with an acetazolamide backbone. The inhibitors were tested against the tumor-expressed CA isozyme IX (CA IX), and the cytosolic
CA I, CA II, and membrane-bound CA IV. The study identified several low nanomolar potent inhibitors against CA IX, with
lipophilicities spanning two log units. Very potent pan-inhibitors with nanomolar potency against CA IX and sub-nanomolar potency
against CA II and CA IV, and with potency against CA I one order of magnitude better than the parent acetazolamide 1 were also
identified in this study, together with compounds that displayed selectivity against membrane-bound CA IV. A comprehensive X-ray
crystallographic study (12 crystal structures), involving both CA II and a soluble CA IX mimetic (CA IX-mimic), revealed the
structural basis of this particular inhibition profile and laid the foundation for further developments toward more potent and selective
inhibitors for the tumor-expressed CA IX.
cellular and extracellular CO2/HCO3 pools.3,5 The distribu-
−
INTRODUCTION
■
A common feature of many aggressive tumors is hypoxia.1 Fast
tion of CA isozymes in humans differs from one isozyme to
another, with CA isozymes ubiquitously present in most cell
tumor growth places many tumor cells sufficiently far from
and tissue types (e.g., CA I, CA II), in specific organs (e.g., CA
IV in lungs, kidneys, ciliary processes of the eye), while others
have a relatively restricted tissue distribution (e.g., CA IX only
expressed in the epithelium lining the stomach and the small
intestine in normal physiology).3,11 However, CA IX as well as
CA XII have been identified as potential cancer targets. As
mentioned previously, the CA IX isozyme is upregulated in
tumors,5,12−14 where it functions to maintain the extracellular
blood vessels resulting in an inadequate amount of oxygen. As
a consequence, the hypoxia-inducible factor 1 (HIF-1) is
upregulated in hypoxic tumor cells, relocates to the nucleus,
and triggers the expression of a group of proteins needed for
the adaptation of cancer cells to survive the hypoxic
environment.2 Among them, carbonic anhydrase isozyme IX
(CA IX) plays a key role in maintaining tumor cell pH
homeostasis.3−10
Carbonic anhydrases (CAs) are a family of zinc metal-
loenzyme that catalyzes the reversible hydration of carbon
dioxide to bicarbonate ion and a proton (CO2 + H2O ⇌
Received: August 18, 2020
−
HCO3 + H+). These isozymes have different subcellular
localizations, such as cytosolic (CA I-III, VII, XIII),
mitochondrial (CA VA, VB), membrane-bound (CA IV, IX,
XII, XIV, and XV), or secreted (CA VI). Through each of
these CA isozymes, cells can quickly equilibrate the intra-
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Figure 1. Biochemistry of hypoxic tumor cells: ATP is produced in the absence of oxygen through the upregulation of glycolysis, which also
produces pyruvate and protons. Pyruvate is converted to lactate, regenerating NAD+ needed for glycolysis, and then lactate is excreted through
monocarboxylate transporter (MCT). Intracellular pH homeostasis is maintained without ATP consumption via the action of CA isozymes:
ubiquitously expressed cytosolic CA I, CA II, and the extracellular membrane-bound CA IX, which is overexpressed in hypoxic tumors. Efficient
inhibition of CA IX has thus the potential to kill tumor cells.
Chart 1. CA Inhibitors Used in the Clinics, Together with Some Representative CAIs with High Potency against Membrane-
Bound CA Isozymes, Including CA IX
pH homeostasis within normal limits, despite significant
upregulation of glycolysis with increased H+ production.
Thus, in the cytoplasm of hypoxic tumor cells, CA I and CA
II catalyze the conversion of glycolytic protons with
with poor prognosis in colorectal,14,19,20 breast,12,21,22
ovarian,23,24 pancreatic,25 head and neck,26 cervical,27,28
brain,29 and bladder cancers.30 Inhibition of CA IX has proved
to be a viable strategy to fight these aggressive metastatic
tumors.3,5,31,32
−
cytoplasmic HCO3 to yield CO2 and H2O. CO2 passes
through the cell membrane and is rehydrated in the
extracellular environment of the tumor by the overexpressed
However, clinically used CA inhibitors (Chart 1) such as
aromatic and heterocyclic primary sulfonamide acetazolamide
(1, AAZ), methazolamide (2, MZA), ethoxzolamide (3, EZA),
benzolamide (4, BZA), or dichlorphenamide (5, DCP),
although relatively potent against CA IX,3 have suboptimal
pharmacokinetic and biodistribution profiles due to their
hydrophilic nature, being essentially designed to inhibit CA
isozymes in kidneys (CA II, CA IV) and to act primarily as
diuretics.33 It was shown by our team that one can conjugate
the benzenesulfonamide and 1,3,4-thiadiazole-2-sulfonamide
zinc-binding motifs with different hydrophilic, amphiphilic, or
hydrophobic moieties34−38 to increase the potency and
selectivity of the resulting CA inhibitors (CAIs) by exploiting
CA IX, reforming the HCO3 and H+ ions. The bicarbonate
−
ion is subsequently imported into the cytoplasm via the anion
−
exchanger AE2, which exports Cl− in exchange for HCO3 . As
a net result of the action of CA IX, in tandem with CA I, CA II,
and AE2, hypoxic tumor cells can ensure pH homeostasis
despite the upregulated glycolysis, efficiently exporting the
glycolytic protons in the extracellular milieu without ATP
consumption (Figure 1).15 This key biochemical mechanism
allows malignant cells to secure the energy needed for
continuous growth and proliferation under hypoxic con-
ditions.16−18 Thus, CA IX expression has been associated
B
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Figure 2. Ribbon diagram (A) and active site details (B) of the 7/CAII (PDB 2HOC,58 rendered in light yellow), 8/CA II (PDB 3N4B,54 rendered
in light blue), 9/CA II (PDB 1ZE8,62 rendered in gray) adducts (superimposed), revealing the orientation of the tail of the inhibitor either toward
P1 (defined by residues Phe131, Val135, Leu198) for CAIs 7, 8 or P2 (defined by residues Ile91, Gln92, Phe131) for CAI 9.
Scheme 1. Synthetic Strategy for the Generation of Lipophilic CA Inhibitors 13−18, Starting from Acetazolamide
the additional interactions of these moieties with key residues
in the active site of CA isozymes in the so-called “tail
approach” to potent and selective CAIs.39 We applied this
strategy to CA IX isozyme by synthesizing the halogenated
aromatic and heterocyclic sulfonamides 6, 7 and congeners, in
the first CA IX inhibition study with synthetic inhibitors
(Chart 1).32 The same tail approach also yielded the ureido
and thioureido sulfonamides as potent CAIs,36 and subsequent
modulation of lipophilicity through halogen decoration yielded
powerful CA IX inhibitors such as 8.40 Other strategies involve
conjugation of CAIs with charged groups (as in CAIs 9 and
10)34,35,37,41,42 with sugar43−47 or with polymeric moieties (as
11, Chart 1).48 These lead compounds demonstrated the
ability to inhibit CA IX and control the growth of various
carcinomas in vitro and in vivo.45,49−51 Compound 8 (SLC-
0111) was proved to be efficient against metastatic breast
cancer40,52−54 and is currently in advanced clinical trials for the
treatment of hypoxic tumors that overexpress CA IX (Chart
1).3−5,49,55 The strategy was successfully used for the
generation of many potent CA IX inhibitors.3,5,46,51,55−57
In parallel with these synthetic efforts, we also conducted X-
ray crystallographic studies with these lead compounds to
reveal their binding in the active site of CAs and to elucidate
the structural basis of their potency and selectivity.5,58−61
Thus, crystal structures of the adducts of 7/CAII (PDB
2HOC58), 8/CA II (PDB 3N4B54), 9/CA II (PDB 1ZE862),
and related5,58−61 reveal that the tail approach is exploiting the
binding of the elongated CAIs in two major binding pockets,
denominated P1 and P2 (Figure 2).5,58,62−64 The first binding
pocket, denominated P1, is defined by residues Phe131,
Val135, and Leu198, while the second pocket, denominated
P2, is defined by residues Phe131, Ile91, and Gln92 (Figure
2).5,58,62−64 These crystallographic studies helped toward the
design of more potent CA IX inhibitors57,65−69 and benefitted
from the crystallization of the catalytic domain of CA IX7 and
by the creation of a CA IX mimic by replacing the key amino
acid residues in the active site of CA II with the corresponding
ones found in the CA IX structure.70 The abovementioned
crystallographic studies evidenced that there are major
structural differences between ubiquitous CA II and tumor-
overexpressed CA IX at the level of P2 site. CA II has a Phe in
position 131, which restricts the P2 site in this isozyme, while
CA IX has a Val, which makes the P2 site in this isozyme much
larger.
One can notice that this site is also hydrophobic, pointing to
the fact that one can increase simultaneously the potency and
the selectivity of a CAI toward CA IX through the use of
lipophilic moieties, which can locate either in the P1 or in the
P2 site. Therefore, we have decided to systematically
investigate the effect of conjugating the 2-amino-5-sulfonami-
do-1,3,4-thiadiazole pharmacophore of acetazolamide with
lipophilic moieties of increased size toward inhibition of CA
IX, in a comparative manner with ubiquitously spread CA II
and to double this synthetic and inhibition study with a
comprehensive X-ray crystallographic study on CA II and CA
IX.
RESULTS AND DISCUSSION
■
The new CAIs were synthesized starting from the same key
intermediate 2-amino-5-sulfonamido-1,3,4-thiadiazole 12 via
acylation with the corresponding acid chlorides in the presence
of pyridine as base, in acetonitrile (method A), or with the
corresponding acid anhydrides in acetonitrile, at reflux
(method B). The key intermediate 12 was synthesized from
C
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acetazolamide (AAZ) 1 via acid deprotection, followed by
neutralization of the corresponding hydrochloride (Scheme 1).
CAIs 13−18 were purified by flash chromatography, crystal-
lized, and characterized by standard analytical methods (see
the Materials section).
The lipophilic CAIs 13−18 were tested for their ability to
inhibit the tumor-overexpressed CA IX isozyme, the
ubiquitous cytosolic CA I and CA II isozymes, and the
membrane-bound isozyme CA IV that can be reached in vivo
by compounds 13−18. The parent Acetazolamide 1 was also
included for comparison (Table 1). Data from Table 1
To provide a solid structural basis of the inhibitory profile of
CAIs 13−18, we undertook an extensive X-ray crystallographic
study using the cytosolic CA II and the CA IX mimic.70 The
results are summarized in Figures 3−5. The 12 crystal
structures were solved with high resolutions, ranging from
1.3 to 1.7 Å (Supporting Information Tables 1 and 2). The
inhibitors bound as expected within active sites of both CA II
and CA IX, anchoring their sulfonamide moiety through the
catalytic zinc. All compounds displayed full electron density
including tail atoms as seen in Figure 3. While examining these
figures, one can observe that the tail of inhibitors 13−18
resides only in the hydrophobic pocket P1 in the case of the
CA IX.
As seen in Figure 4B,I, compound 13 binds in a similar
fashion in both CA isoforms with the exception of the final
methyl group on the tail portion. In CAII, the methyl tail is
positioned away from the bulky F131 and resides in pocket P2.
In contrast, in CA IX, the methyl tail of compound 13 binds in
pocket P1 adjacent to residue 131. Mention must be made that
P1 pocket is larger in CA IX as compared with CA II, having a
Val instead of a Phe in position 131, and therefore can
accommodate easily hydrophobic tails of increased steric bulk,
from Et (congener 13) to adamantyl (congener 18). The
location of the hydrophobic tail of compounds 13−18, in the
large, hydrophobic pocket P1 of CA IX explains the constant
nanomolar potency of these compounds against this target
despite the significant increase in steric bulk and lipophilicity
(log P’s between 0.8 and 2.28, Table 1). Even the bulkiest
adamantyl derivative 18 can be fully accommodated in the P1
of CA IX, which explains why this derivative is most potent
against CA IX. The pattern displayed by compound 13 binding
in CA IX is repeated with the rest of the compounds, as seen in
Figure 4C−G.
Table 1. Physicochemical Properties and Inhibition Data
against Human CA Isoforms CA I, II, IV, and IX with
Derivatives 13−18 Reported Here and with the Standard
Inhibitor Acetazolamide (AAZ, 1) by a Stopped-Flow CO2
Hydrase Assay
a
KI (nM)
comp
R
log P
CA I
CA II
CA IV
CA IX
AAZ 1
13
14
15
16
Me
Et
nPr
iPr
tBu
cHex
Ad
0.14
0.8
250
12.1
2.7
74
25.8
12.8
2.1
2.4
8.2
66.8
53.8
75.1
63.4
62.8
883
0.78
0.37
0.44
0.43
0.36
349
1.21
1.36
2.07
2.12
2.28
0.52
0.59
0.66
0.53
11.0
17
18
15.7
6.4
a
Mean from three different assays, by a stopped-flow technique
(errors were in the range of 5−10% of the reported values).
Monomeric (recombinant) human enzymes used in all cases.
revealed an excellent inhibitory profile of compounds 13−18
against CA II, CA IV, and CA IX, with nanomolar and even
sub-nanomolar potency of some representatives against these
targets. The CA I isozyme was the least susceptible to be
inhibited with CAIs 13−18. The inhibitory potency of the new
compounds increased while elongating the tail length from Me
(AAZ 1) to Et (13) and nPr (14). Tail branching to iPr
(compound 15), tBu (compound 16), or cHexyl (compound
17) does not significantly increase the potency of the CAI
toward CA I. However, the substitution of cHexyl moiety from
17 with much bulkier adamantly (Ad in Scheme 1) in 18
lowers the inhibitory potency with 1 order of magnitude
(Table 1). The same trend is valid for the other three
isozymes: the initial elongation of the tail of the inhibitor
increases the potency of the inhibitor, with nPr congener 14
being the most potent representative against CA II, IV, and IX,
and reaching low nanomolar potency for CA IX and sub-
nanomolar potency for CA II and CA IV. Branching of the tail
in iPr (compound 15), tBu (compound 16), and cHexyl
(compound 17) does not alter much the inhibitory potency
against CA II and CA IV, which remain sub-nanomolar.
However, CA IX seems to be more sensitive to this branching
effect, with KI’s values increasing from nPr congener 14 to iPr
congener 15, then to tBu congener 16 and cHexyl congener
17. The combined effect of tail branching and bulkiness in
adamantyl congener 18 decreases the potency of the CAI
relative to cHexyl congener 17 1 order of magnitude for CA II
(similarly to CA I) and 2 orders of magnitude for CA IV.
Interestingly, adamantane derivative 18 retained nanomolar
potency for CA IX, although about three times weaker than
nPr congener 14.
In contrast, in the case of CA II isozyme, the hydrophobic
tail of compounds 13−18 was found to be located mostly in
P2, opposite with the case of parent acetazolamide AAZ 1 that
has the methyl located in the P1 pocket,59,71 thus revealing the
importance of the tail approach also used in this study (Figure
4J−N). Interestingly, the only CAI that had the tail located
outside the P2 pocket of CA II was the tBu derivative 16. In
this case, the tBu moiety is in fact located in between P1 and
P2 pockets, displacing gently the Phe131 residue that normally
separates the P1 and P2 pockets in CA II from its normal
position (Figures 3−5). This translates into a slight decrease of
potency relative to congeners 15 and 17 (Table 1).
Compounds 13−17 each had higher specificity toward CA II
over CA IX. With their smaller, nonbulky tails, these inhibitors
were able to bind in the P2 pocket of CA II, resulting in stable
and high-affinity binding. On the contrary, in CA IX, these
inhibitors’ tails went to the less sterically hindered P1 pocket
binding near Val131 (Figure 3). However, with the addition of
a bulky adamantyl group to the tail, steric clashes start to
interfere with P2 pocket binding, thus specificity toward CA II
drops. On the contrary, in CA IX, the adamantyl group can still
bind with relatively high affinity to the less bulky P1 pocket of
CA IX (Figure 3).
CONCLUSIONS
■
A detailed structure−activity relationship study was carried out
within a series of lipophilic CAI with acetazolamide backbone
13−18. The CAIs were profiled against the tumor-overex-
pressed CA IX, against ubiquitous cytosolic CA I and CA II
and against membrane-bound CA IV. We identified several low
D
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Figure 3. continued
E
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Figure 3. CA Inhibitors 13−18 electron density. Panels (A−F) are CA II and (G−L) are CA IX mimic complexes. CA II complex with (A) CAI 13
green, (B) CAI 14 blue, (C) CAI 15 pink, (D) CAI 16 yellow, (E) CAI 17 peach, and (F) CAI 18 purple. CA IX complex with (G) CAI 13 green,
(H) CAI 14 blue, (I) CAI 15 pink, (J) CAI 16 yellow, (K) CAI 17 peach, and (L) CAI 18 purple. The 2FO-FC electron density maps (blue mesh)
for each of the inhibitors are contoured to 1.0σ. Active site zinc is depicted as a magenta sphere.
Synthesis of 5-Amino-1,3,4-thiadiazole-2-sulfonamide (2).
5-Amino-1,3,4-thiadiazole-2-sulfonamide 2 was synthesized by de-
protection of acetazolamide as previously described.34,72
nanomolar potent inhibitors against CA IX, with lipophilicities
spanning two log units. We also identified very potent pan
inhibitors such as 14, with nanomolar potency against CA IX
and sub-nanomolar potency against CA II and CA IV, and a
good inhibition profile (1 order of magnitude) better than
parent acetazolamide 1 against CA I. The adamantane
derivative 18 displayed high potency against CA IX and CA
II, involved in tumor pH homeostasis (Figure 1) and selectivity
against membrane-bound CA IV, features that recommend this
compound for translation to in vivo studies for the treatment of
hypoxic solid tumors. We revealed the structural basis of these
particular inhibition profiles through a comprehensive X-ray
crystallographic study involving both CA II and CA IX
comparatively, thus laying the basis for further developments
toward more potent and selective inhibitors for the tumor-
overexpressed CA IX isozyme.
General Procedure for the Synthesis of Acyl Derivatives
13−16 (Method A). In a 100 mL round-bottom flask, 5-amino-1,3,4-
thiadiazole-2-sulfonamide 2 (1.8 g, 10 mmol) was suspended in dry
acetonitrile (30 mL) and cooled to 0−5 °C using an ice bath. The
corresponding anhydride (12 mmol) was added dropwise to the cold
suspension, under stirring. The ice bath was removed and the reaction
mixture was stirred at room temperature for another 30 min, followed
by heating it to reflux (TLC control, MeOH/CH2Cl2 20/80 v/v).
After all amine was consumed (1−3 h reflux), the reaction mixture
was cooled and the solvent was evaporated to a small volume using a
rotary evaporator when the product precipitates. The crude product
was filtered out, washed with cold acetonitrile, and dried under
vacuum. Recrystallization from alcohols (MeOH, EtOH) usually
yielded the pure product, as evidenced by LC-MS (>96%). When
purity was not satisfactory, flash chromatography was performed using
MeOH/CH2Cl2 gradients. The pure fractions (by TLC) were
grouped, evaporated to dryness, and crystallized from MeOH or
EtOH. Reaction advancement was checked by TLC, and its
completion was confirmed by LC-MS.
N-(5-Sulfamoyl-1,3,4-thiadiazol-2-yl)propionamide (13). Mp
249−252 °C (lit33 mp 147−148 °C, lit73 mp 247−248 °C); yield
85.2%; 1H NMR (DMSO-d6, δ, ppm): 12.96 (s, 1H, −CONH), 8.32
(s, 2H, −SO2NH2), 2.54 (q, J = 7.5 Hz, 2H, CH2CONH), and 1.13
(t, J = 7.5 Hz, 3H, CH3CH2CONH); 13C NMR (DMSO-d6, δ, ppm):
172.9 (−CONH), 164.2 (C5 TDA), 161.1 (C2, TDA), 28.2
(−CH2CONH), and 8.7 (CH3CH2CONH); LC-MS: C5H8N4O3S2,
exact mass: 236.0; found: 237.0 (MH+).
EXPERIMENTAL SECTION
■
Materials. The following materials were used as received:
propionic anhydride, butyric anhydride isobutyric anhydride (TCI
America, Portland, OR), acetazolamide, pivaloyl chloride, cyclohexane
carboxylic acid, adamantane carboxylic acid (Sigma, St Louis, MO);
other salts, acids, and solvents (HPLC quality) were purchased from
Fisher Scientific (Pittsburgh, PA), EMD (Gibbstown, NJ), and VWR
International (West Chester, PA), respectively.
Techniques. The purity and the structure identity of the
intermediary and the final products were assessed by a combination
of techniques that included thin-layer chromatography (TLC),
1
HPLC-MS, H NMR, COSY, and 13C NMR, high-resolution mass
N-(5-Sulfamoyl-1,3,4-thiadiazol-2-yl)butyramide (14). Mp 276−
280 °C (lit33 mp 260−262 °C); yield 81.5%; 1H NMR (DMSO-d6, δ,
ppm): 12.99 (s, 1H, −CONH), 8.32 (s, 2H, −SO2NH2), 2.53 (m,
spectrometry (HR-MS). TLC was carried out on SiO2-precoated
aluminum plates (silica gel with F254 indicator); layer thickness 200
μm; pore size 60 Å, from Sigma-Aldrich. Melting points were
determined using a Thermolyne heating stage microscope (Dubuque,
IA), equipped with an Olympus 5X objective, at heating/cooling rate
of ∼4 °C/min and were uncorrected. The purity of compounds was
assessed via liquid chromatography-mass spectrometry (LC-MS)
using an Agilent 1200 HPLC-DAD-MS system equipped with a
G1315A DAD and a 6130 Quadrupole MS using a ZORBAX SB-C18
column, eluted with H2O (0.1% HCOOH)/MeCN (0.1% HCOOH)
95/5 to 0/100 linear gradient. NMR spectra were recorded at ≈300 K
with a Bruker Avance III 400 Plus spectrometer equipped with a 5
2H, CH2CONH), and 1.65 (sext,
C H 3 C H 2 CH 2 CO NH ); 0. 90 (t,
J
=
7.4 Hz, 3H,
J
= 7. 4 Hz , 3 H,
CH3CH2CH2CONH); 13C NMR (DMSO-d6, δ, ppm): 172.1
(−CONH), 164.2 (C5 TDA), 161.0 (C2, TDA), 36.6
(−CH2 CONH), 17.8 (−CH2 CH2 CONH), and 13.4
(CH3CH2CH2CONH); LC-MS: C6H10N4O3S2, exact mass: 250.0;
found: 251.0 (MH+).
N-(5-Sulfamoyl-1,3,4-thiadiazol-2-yl)isobutyramide (15). Mp
281−283 °C (lit33 mp 280−283 °C); yield 79.2%; 1H NMR
(DMSO-d6, δ, ppm): 12.98 (s, 1H, −CONH), 8.32 (s, 2H,
−SO2NH2), 2.82 (hep, J = 6.8 Hz, 1H, CHCONH), and 1.15 (d, J
= 6.9 Hz, 6H, (CH3)2CHCONH); 13C NMR (DMSO-d6, δ, ppm):
176.0 (−CONH), 164.3 (C5 TDA), 161.2 (C2, TDA), 33.9
(−CHCONH), and 18.8 (CH3 )2 CHCONH; LC-MS:
C6H10N4O3S2, exact mass: 250.0; found: 251.0 (MH+).
1
mm indirect detection probe, operating at 400 MHz for H NMR, at
100 MHz for 13C NMR. Chemical shifts are reported as δ values,
using tetramethylsilane (TMS) as an internal standard for proton
spectra and the solvent resonance for carbon spectra. Assignments
were made based on chemical shifts, signal intensity, COSY, HMQC,
1
and HMBC sequences. For H NMR, data are reported as follows:
General Procedure for the Synthesis of Acyl Derivatives
17,18 (Method B). In a 100 mL round-bottom flask, 5-amino-1,3,4-
thiadiazole-2-sulfonamide 2 (1.8 g, 10 mmol) was suspended in dry
chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, sep =
septet, m = multiplet), coupling constants J (Hz), and integration.
F
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Figure 4. continued
G
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Figure 4. Interactions of inhibitors 13−18 with CA II and CA IX. Panels (A) and (H) are the unbound active sites of CA II (PDB 3KS3) and CA
IX mimic (PDB 4ZAO), respectively. Panels (B−G) are CA II and panels (I−N) are CA IX mimic complexes. CA II complex with (B) CAI 13
green, (C) CAI 14 blue, (D) CAI 15 pink, (E) CAI 16 yellow, (F) CAI 17 peach, and (G) CAI 18 purple. CA IX complex with (I) CAI 13, (J)
CAI 14 blue, (K) CAI 15 pink, (L) CAI 16 yellow, (M) CAI 17 peach, and (N) CAI 18 purple. Active site zinc is depicted as a magenta sphere,
and active site histidines are represented by sticks. Ordered active site waters are represented by red spheres.
Figure 5. Superposition of CAIs 13−18 binding. Surface representation of (A) CA II and (B) CA IX mimic in complex with CAIs 13−18. CAI 13
green, CAI 14 blue, CAI 15 pink, CAI 16 yellow, CAI 17 peach, and CAI 18 purple. Note that residue 131 has a key role in the binding modes of
the tails of the inhibitors in CA II and CA IX.
H
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acetonitrile (20 mL), under stirring. Pyridine (0.95 g, 12 mmol) was
added via a syringe and the reaction mixture was cooled to 0−5 °C
using an ice bath. The corresponding acyl chloride (12 mmol, made
from the corresponding acid with thionyl chloride), dissolved in 5−10
mL of dry acetonitrile, was added dropwise to the cold suspension,
under stirring. Stirring was continued for 1 h when a white precipitate
of pyridinium chloride was formed. The ice bath was removed and the
reaction mixture was stirred at room temperature until completion
(TLC control, MeOH/CH2Cl2 20/80 v/v). The solvent was
evaporated to dryness using a rotary evaporator and the residue was
treated with 10 mL of DI water, stirred for 5 min, then filtered, and
washed with cold DI water. Recrystallization from alcohols (MeOH,
EtOH) usually yielded the pure product, as evidenced by LC-MS
(>96%). When purity was not satisfactory, flash chromatography was
performed using MeOH/CH2Cl2 gradients. The pure fractions (by
TLC) were grouped, evaporated to dryness, and crystallized from
MeOH or EtOH. Reaction advancement was checked by TLC and its
completion was confirmed by LC-MS.
were transferred to an overnight growth in nutrient-rich Luria Broth.
Cultures were then allowed to grow to an optical density of 0.6 at 600
nm in a 2 L culture flask in the presence of selecting antibiotic. The E.
coli was then induced by the addition of 0.5 mM isopropyl β-D-1-
thiogalactoside (IPTG) and 1 mM zinc sulfate and incubated for an
additional 3 h. Cells were then lysed using a microfluidizer set to
18 000 psi and purified using affinity chromatography with a
benzenesulfonamide resin. Purity was determined with SDS-Page
and concentration was determined by UV−vis spectroscopy at 280
nm.
X-ray Crystallography. CAII and CA IX mimic were cocrystal-
lized with a series of 6 CA inhibitors 3−8. Each crystallization drop
was prepared by adding 4.5 μL of protein in a 1:1 ratio with 4.5 μL of
the precipitant solution of 1.6 M sodium citrate and 50 mM Tris at a
pH of 7.8 to a final volume of 9 μL. Inhibitor stock solutions of
varying concentrations were made in dimethyl sulfoxide (DMSO) to
determine solubility. Approximately, 1 μL of 200 mM of each
inhibitor was added to the crystallization drop to a final volume of 10
μL. Crystal trays were incubated at room temperature and
undisturbed for 2 weeks until crystals formed. The complexes of
CAI 8 could not be obtained via cocrystallization due to the high
log P value of the inhibitor. Instead, the preformed crystals of CA II
and CA IX mimic were soaked with ∼80 mM CAI 8 in 10% DMSO.
Data was collected at cryogenic temperatures at Cornell High Energy
Synchrotron Source (CHESS) and Stanford Synchrotron Radiation
Lightsource (SSRL), using a Pilatus 6M detector. The diffraction
images were indexed, integrated, merged, and scaled to the P21 space
group using XDS via the CCP4 program suite78 The diffraction data
was phased with standard Molecular replacement methods using the
software package PHENIX with the PDB entry 3KS3 as the search
model.79,80 Coordinate refinements and inhibitor restraints were
calculated using PHENIX.79 The program Coot was utilized in
between refinements to add a solvent and inhibitor molecules and
make individual real space refinements of each residue when
needed.81 Figures were generated in the molecular graphical software
PyMol (Schrodinger LLC), and protein−ligand interactions and bond
lengths were determined using LigPlot Plus.82 The inhibitor surface
interactions were determined using online server PDB Pisa.83
N-(5-Sulfamoyl-1,3,4-thiadiazol-2-yl)pivalamide (16). Mp 256−
258 °C (lit74 mp 252−254 °C); yield 82.1%; 1H NMR (DMSO-d6, δ,
ppm): 12.73 (s, 1H, −CONH), 8.32 (s, 2H, −SO2NH2), and 1.28 (s,
9H, (CH3)3CCONH); 13C NMR (DMSO-d6, δ, ppm): 177.4
(−CONH), 164.4 (C5 TDA), 161.9 (C2, TDA), 30.6
(−CCONH), and 26.3 (CH3)3CCONH; LC-MS: C7H12N4O3S2,
exact mass: 264.0; found: 265.0 (MH+).
N-(5-Sulfamoyl-1,3,4-thiadiazol-2-yl)cyclohexanecarboxamide
1
(17). Mp 255−259 °C; yield 78.6%; H NMR (DMSO-d6, δ, ppm):
12.96 (s, 1H, −CONH), 8.32 (s, 2H, −SO2NH2), 2.55 (m, 1H,
CHCONH), and 1.10−1.90 (m, 10H, cyclohexyl); 13C NMR
(DMSO-d6, δ, ppm): 175.0 (−CONH), 164.3 (C5 TDA), 161.2
(C2, TDA), 42.3 (CαCONH), 28.5 (2C, CβCONH), 25.1
(CδCONH), and 24.9 (2C, CγCONH); LC-MS: C9H14N4O3S2,
exact mass: 290.0; found: 291.0 (MH+); anal (C9H14N4O3S2) C, H,
N. Requires: C 37.23, H 4.86, N 19.30; found: C 37.20, H 4.95, N
19.36.
N-(5-Sulfamoyl-1,3,4-thiadiazol-2-yl)adamantanecarboxamide
(18). Mp 246−248 °C (lit75 mp 246−248 °C); yield 75.2%; 1H NMR
(DMSO-d6, δ, ppm): 12.68 (s, 1H, −CONH), 8.31 (s, 2H,
−SO2NH2), 1.82−2.10 (m, 9H, Ad), and 1.60−1.80 (m, 6H, Ad);
13C NMR (DMSO-d6, δ, ppm): 176.7 (−CONH), 164.4 (C5 TDA),
161.9 (C2, TDA), 40.8 (CαCONH Ad), 37.3 (3C, CβCONH Ad),
35.6 (3C, CγCONH Ad), and 27.3 (3C, CδCONH Ad); LC-MS:
C13H16N4O3S2, exact mass: 340.0; found: 341.0 (MH+).
ASSOCIATED CONTENT
* Supporting Information
■
sı
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01390.
CA Inhibition Assay. An applied photophysics stopped-flow
instrument has been used for assaying the CA-catalyzed CO2
hydration activity.76 Phenol red (at a concentration of 0.2 mM) has
been used as an indicator, working at the absorbance maximum of 557
nm, with 20 mM Hepes (pH 7.5) as buffer, and 20 mM Na2SO4 (for
maintaining constant the ionic strength), following the initial rates of
the CA-catalyzed CO2 hydration reaction for a period of 10−100 s.
The CO2 concentrations ranged from 1.7 to 17 mM for the
determination of the kinetic parameters and inhibition constants. 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 (0.1 mM) were
prepared in distilled-deionized water and dilutions up to 0.01 nM
were done thereafter with the assay buffer. Inhibitor and enzyme
solutions were preincubated together for 15 min 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 the Cheng−Prusoff equation, as
reported earlier,38 and represent the mean from at least three different
determinations. All CA isoforms were recombinant ones obtained in-
house as reported earlier.21,32,75,77
Crystallographic CA II complex structure statistics; and
crystallographic CA IXmimic-complex structure statistics
(PDF)
HPLC traces of investigated compounds (PDF)
Smiles (CSV)
AUTHOR INFORMATION
Corresponding Authors
■
Claudiu T. Supuran − NEUROFARBA Department,
Pharmaceutical Sciences Section, Universita degli Studi di
Firenze, 50019 Sesto Fiorentino, Florence, Italy; orcid.org/
0000-0003-4262-0323; Phone: 39-055-4573005;
Email: claudiu.supuran@unifi.it; Fax: 39-055-4573385
Marc A. Ilies − Department of Pharmaceutical Sciences and
Moulder Center for Drug Discovery Research, Temple University
School of Pharmacy, Philadelphia, Pennsylvania 19140, United
States; orcid.org/0000-0003-0694-411X; Phone: 215-
707-1749; Email: mailies@temple.edu; Fax: 215-707-5620
Robert McKenna − Department of Biochemistry and Molecular
Biology, College of Medicine, University of Florida, Gainesville,
Florida 32610, United States; Phone: (352) 294-8395;
Email: rmckenna@ufl.edu
Protein Expression and Purification. CA II and CA IX mimic
were expressed and purified according to previously published
protocols.70 Briefly, the gene-containing plasmid was transformed
into competent BL21 Escherichia coli cells via a standard BL21
transformation protocol. After growth in SOC media, the cultures
I
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Article
(6) Pastorek, J.; Pastorekova, S.; Callebaut, I.; Mornon, J. P.; Zelnik,
V.; Opavsky, R.; Zat’ovicova, M.; Liao, S.; Portetelle, D.; Stanbridge,
E. J. Cloning and characterization of MN, a human tumor-associated
protein with a domain homologous to carbonic anhydrase and a
putative helix-loop-helix DNA binding segment. Oncogene 1994, 9,
2877−2888.
(7) Alterio, V.; Hilvo, M.; Di Fiore, A.; Supuran, C. T.; Pan, P.;
Parkkila, S.; Scaloni, A.; Pastorek, J.; Pastorekova, S.; Pedone, C.;
Scozzafava, A.; Monti, S. M.; De Simone, G. Crystal structure of the
catalytic domain of the tumor-associated human carbonic anhydrase
IX. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 16233−16238.
(8) Tureci, O.; Sahin, U.; Vollmar, E.; Siemer, S.; Gottert, E.; Seitz,
G.; Parkkila, A. K.; Shah, G. N.; Grubb, J. H.; Pfreundschuh, M.; Sly,
W. S. Human carbonic anhydrase XII: cDNA cloning, expression, and
chromosomal localization of a carbonic anhydrase gene that is
overexpressed in some renal cell cancers. Proc. Natl. Acad. Sci. U.S.A.
1998, 95, 7608−7613.
Authors
Jacob T. Andring − Department of Biochemistry and Molecular
Biology, College of Medicine, University of Florida, Gainesville,
Florida 32610, United States; orcid.org/0000-0002-6632-
6391
Mallorie Fouch − Department of Pharmaceutical Sciences and
Moulder Center for Drug Discovery Research, Temple University
School of Pharmacy, Philadelphia, Pennsylvania 19140, United
States
Suleyman Akocak − Department of Pharmaceutical Sciences
and Moulder Center for Drug Discovery Research, Temple
University School of Pharmacy, Philadelphia, Pennsylvania
19140, United States
Andrea Angeli − NEUROFARBA Department, Pharmaceutical
Sciences Section, Universita degli Studi di Firenze, 50019 Sesto
Fiorentino, Florence, Italy; orcid.org/0000-0002-1470-
7192
(9) Whittington, D. A.; Waheed, A.; Ulmasov, B.; Shah, G. N.;
Grubb, J. H.; Sly, W. S.; Christianson, D. W. Crystal structure of the
dimeric extracellular domain of human carbonic anhydrase XII, a
bitopic membrane protein overexpressed in certain cancer tumor cells.
Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 9545−9550.
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.0c01390
(10) Shabana, A. M.; Ilies, M. A. Drug delivery to hypoxic tumors
targeting carbonic anhydrase ix, in targeted nanosystems for
therapeutic applications: new concepts, dynamic properties, efficiency,
and toxicity. Am. Chem. Soc. 2019, 223−252.
(11) Zamanova, S.; Shabana, A. M.; Mondal, U. K.; Ilies, M. A.
Carbonic anhydrases as disease markers. Expert Opin. Ther. Pat. 2019,
29, 509−533.
Author Contributions
The manuscript was written through the contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Notes
The authors declare no competing financial interest.
(12) Austin, R. P.; Barton, P.; Cockroft, S. L.; Wenlock, M. C.; Riley,
R. J. The influence of nonspecific microsomal binding on apparent
intrinsic clearance, and its prediction from physicochemical proper-
ties. Drug Metab. Dispos. 2002, 30, 1497−1503.
ACKNOWLEDGMENTS
■
We would like to thank the staff, operators, and scientists at
SSRL and CHESS for their assistance in X-ray crystallography
data acquisition. MAI acknowledges the financial support of
NIH (Grant R03EB026189) and of Edward N. & Della
Thome Memorial Foundation. Suleyman Akocak acknowl-
edges the financial support of the Turkish Minister of
Education for a PhD scholarship. The support of Temple
Undergraduate Research Program is also gratefully acknowl-
edged.
́
(13) Pastorekova, S.; Pastorek, J. Cancer-related carbonic anhydrase
isozymes and their inhibition. Carbonic Anhydrase 2004, 255−281.
(14) Saarnio, J.; Parkkila, S.; Parkkila, A. K.; Haukipuro, K.;
Pastorekova, S.; Pastorek, J.; Kairaluoma, M. I.; Karttunen, T. J.
Immunohistochemical study of colorectal tumors for expression of a
novel transmembrane carbonic anhydrase, MN/CA IX, with potential
value as a marker of cell proliferation. Am. J. Pathol. 1998, 153, 279−
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A. L.; Vaughan-Jones, R. D. Tumor-associated carbonic anhydrase 9
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Cancer Res. 2006, 66, 5216−5223.
ABBREVIATIONS
■
CA, carbonic anhydrase; CAI, carbonic anhydrase inhibitor;
HIF-1, hypoxia-inducible factor 1; ATP, adenosine triphos-
phate; AAZ, acetazolamide; MZA, methazolamide; EZA,
ethoxzolamide; BZA, benzolamide; DCP, dichlorphenamide;
IPTG, isopropyl β-D-1-thiogalactoside; CHESS, Cornell High
Energy Synchrotron Source; SSRL, Stanford Synchrotron
Radiation Lightsource
(17) Gatenby, R. A.; Gillies, R. J. Why do cancers have high aerobic
glycolysis? Nat. Rev. Cancer 2004, 4, 891−899.
̌
́
(18) Svastova, E.; Hulikova, A.; Rafajova, M.; Zat’ovicova, M.;
Gibadulinova, A.; Casini, A.; Cecchi, A.; Scozzafava, A.; Supuran, C.
T.; Pastorek, J.; Pastorekova, S. Hypoxia activates the capacity of
tumor-associated carbonic anhydrase IX to acidify extracellular pH.
FEBS Lett. 2004, 577, 439−445.
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