# Carborane-Based Carbonic Anhydrase Inhibitors: Insight into CAII/CAIX Specificity from a High-Resolution Crystal Structure, Modeling, and Quantum Chemical Calculations

Authors: Pavel Mader; Adam Pecina; Petr Cígler; Martin Lepšík; Václav Šícha; Pavel Hobza; Bohumír Grüner; Jindřich Fanfrlík; Jiří Brynda; Pavlína Řezáčová
Journal: BioMed Research International
 (2014)

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## Abstract

Carborane-based compounds are promising lead structures for development of inhibitors of carbonic anhydrases (CAs). Here, we report structural and computational analysis applicable to structure-based design of carborane compounds with selectivity toward the cancer-specific CAIX isoenzyme. We determined the crystal structure of CAII in complex with 1-methylenesulfamide-1,2-dicarba-closo-dodecaborane at 1.0 Å resolution and used this structure to model the 1-methylenesulfamide-1,2-dicarba-closo-dodecaborane interactions with CAIX. A virtual glycine scan revealed the contributions of individual residues to the energy of binding of 1-methylenesulfamide-1,2-dicarba-closo-dodecaborane to CAII and CAIX, respectively.


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## Body

## 1. Introduction

Carbonic anhydrases (CAs) play important roles in many physiological and pathophysiological processes. For example, extracellular CAs participate in tumor growth and progression [1]. CAIX, which is selectively expressed in a range of hypoxic tumors, is a validated diagnostic and therapeutic target (recently reviewed in [2–4]). There are 15 human CA isoenzymes, and due to the ubiquity of these enzymes in human tissues, selective inhibition is a very important aspect of drug design.Three main classes of CA inhibitors have been described to date (reviewed in [5]): (i) metal ion binders (sulfonamides, sulfamides, sulfamates, dithiocarbamates, thiols, and hydroxamates); (ii) compounds that anchor the zinc-coordinated water molecule/hydroxide ion (phenols, carboxylates, polyamines, esters, and sulfocoumarins); and (iii) coumarins and related compounds that bind further away from the metal ion.CA inhibitors from the first class (metal ion binders) contain specific functional groups that interact with the catalyticZn
2
+ ion in the CA active site. These metal-binding functionalities are typically joined to a “ring” structure. This moiety is not necessarily aromatic; however, it is usually consisting of a 5- or 6-membered hydrocarbon ring or conjugated ring system containing nitrogen, oxygen, and/or sulfur. Numerous functional groups have been added to the ring structure scaffold to modify inhibitor properties such as specificity toward a particular CA isoenzyme, pKa, or solubility (reviewed in [6]). Recently, we reported design of CA inhibitors containing space-filling carborane clusters in place of the typical ring structure [7]. We showed that various carborane clusters act as CA inhibitors and that modifying these clusters with an appropriately attached sulfamide group and other substituents leads to compounds with selectivity toward the cancer-specific CAIX isoenzyme.Boron is one of few chemical elements that can form binary hydrides composed of more than two atoms, which leads to formation of boron cluster compounds (boron hydrides or boranes). Their basic structural feature is formation of a polyhedron with triangular facets held together by 3-center 2-electron bonds with an extensive electron delocalization [8]. A typical structural archetype is represented by the divalentcloso-B
12
H
12
2
- anion, an extremely stable compound with a symmetrical 12-vertex icosahedron structure [9]. Replacement of one or more {
BH
-
} in borane cage with {
CH
} leads to series of carboranes and removal of BH vertices leads to various open-cage (nido-) species. Carboranes thus offer a large variety of structural archetypes that provide interesting counterparts to organic compounds [10].Many features of icosahedral 12-vertex carboranes are useful in the design of biologically active compounds. Carboranes have high thermal and chemical stability; therefore, they generally do not undergo catabolism and are nontoxic to the host organism [11, 12]. The basiccloso-C2B10H12 carborane cluster is highly hydrophobic [13]; however, its controlled deboronation can generate water soluble 11-vertexnido-C
2
B
9
H
12
-. These anions represent important intermediates in the synthesis of a family of mainly anionic metal bis(dicarbollides) accessible via metal insertion. Incorporation of carborane cages into the structures of certain substances of medicinal interest can enhance hydrophobic interactions between the boron cluster-coupled pharmaceuticals and their protein targets, increasein vivo stability, and facilitate uptake through cellular membranes [14, 15]. The successful use of boron clusters as hydrophobic pharmacophores has recently been increasing [16, 17]. Examples of carborane pharmacophores include boron-containing antifolates [18], HIV protease inhibitors [19, 20], and estrogen receptor agonists and antagonists [21], among others [16, 22, 23].Drug design efforts benefit greatly from knowledge of the 3D structures of protein-ligand complexes. X-ray crystallography has contributed considerably to the development of CA inhibitors; more than 500 structures of human CA isoenzymes (wild-type and mutant forms) in complex with various inhibitors have offered unprecedented insight into inhibitor binding modes (reviewed in [24]). Structural information coupled with experimental inhibition data can be used to validate various computational approaches to assess inhibitor binding strength. Once a particular theoretical approach reproduces the known data well, it can be used for prospective design. For studies involving metal ions and unusual compounds such as boranes, the use of quantum chemistry (QM) is warranted [25, 26]. Indeed, we recently used a quantum mechanics/molecular mechanics (QM/MM) methodology to quantitatively describe the binding of two carborane-based sulfamides to CAII [7] and to explain fundamental differences in the binding modes ofcloso- andnido-cages [27].Here, we report the X-ray structure of CAII with bound 1-methylenesulfamide-1,2-dicarba-closo-dodecaborane (compound1, Figure 1(a)) determined at 1.0 Å resolution. This atomic-level resolution allowed us to assess in detail the positions of carbon and boron atoms in the carborane cage of1. Additionally, we modeled the complex of1 with CAIX. We employed a virtual glycine scan to analyze the differences between the interactions of1 with CAII and CAIX.(a) Structural formula of1 with atom numbers used in the crystal structure coordinate file. The vertices in carborane cluster represent BH groups. (b) Crystal structure of CAII in complex with1. The CAII active site is shown in cartoon representation; residues involved in interactions with the Zn2+ ion (purple sphere) and1 are shown in stick representation with carbon atoms colored green. Boron atoms are colored pink, and other heteroatoms are colored according to standard color coding: oxygen, red; nitrogen, blue; sulfur, yellow. The 2Fo-Fc electron density map for1 is contoured at 1 σ.
(a)
(b)


## 2. Materials and Methods

### 2.1. Protein Crystallization and Diffraction Data Collection

For crystallization of human CAII (Sigma, catalogue number C6165) in complex with 1-methylenesulfamide-1,2-dicarba-closo-dodecaborane (compound1), we adapted a previously described procedure [28]. CAII (at a concentration of 4 mg·mL−1, dissolved in water) was incubated in aqueous solution containing a 2-fold molar excess ofp-hydroxymercuribenzoate (Sigma, catalogue number 55540). The protein was concentrated to 10 mg·mL−1 and unboundp-hydroxymercuribenzoate was removed with Amicon Ultra-4 concentrators (Merck-Millipore MWCO 10 kDa).The complex of CAII with1 was prepared by adding a 1.1-fold molar excess of1(in DMSO) to the 10 mg·mL−1 solution of CAII in water without pH adjustment (the final DMSO concentration did not exceed 5% v/v).The best diffracting crystals were obtained using the hanging-drop vapor diffusion method under the following conditions: 2μL protein-inhibitor complex solution was mixed with 2 μL precipitant solution [2.5 M (NH4)2SO4, 0.3 M NaCl, and 100 mM Tris-HCl, pH 8.2] and equilibrated over a reservoir containing 1 mL of precipitant solution at 18°C. Crystals with dimensions of 0.3 mm × 0.1 mm × 0.1 mm grew within 7 days.For cryoprotection, the crystals were incubated in mother liquor supplemented with 25% glycerol for approximately 30 s, flash-frozen, and stored in liquid nitrogen. Diffraction data for the CAII complex were collected at 100 K at the X14.2 BESSY beamline in Berlin, Germany [29]. Data were collected in two passes: the high-resolution range (11.75–1.00 Å) and the low-resolution range (21.08–1.20 Å). The two datasets were integrated with iMOSFLM [30] and merged and scaled with SCALA [31]. Data collection and refinement statistics are summarized in Table 1.Table 1
Data collection and refinement statistics.
Data collection statistics
Space group
P
2
1
Cell parameters (Å; °)
42.20, 41.73, 72.16; 90.0, 104.4, 90.0
Wavelength (Å)
0.9184
Resolution (Å)
21.08–1.00 (1.05–1.00)
Number of unique reflections
108,781 (15,490)
Multiplicity
3.5 (2.5)
Completeness (%)
83.1 (81.4)
R
merge
a
0.056 (0.375)
AverageI
/
σ(I)
10.8 (2.3)
Wilson B (Å2)
6.5
Refinement statistics
Resolution range (Å)
69.90–1.00 (1.03–1.00)
No. of reflections in working set
97,856 (7,831)
No. of reflections in test set
5,426 (412)
R value (%)b
17.5 (24.4)
R
free value (%)c
20.0 (26.2)
RMSD bond length (Å)
0.011
RMSD angle (°)
1.53
Number of atoms in AU
2297
Number of protein atoms in AU
2081
Number of water molecules in AU
176
Mean ADP value protein/inhibitor (Å2)
12.0/17.6
Ramachandran plot statisticsd
Residues in favored regions (%)
96.56
Residues in allowed regions (%)
3.44
The data in parentheses refer to the highest-resolution shell.
a
R
merge
=
∑
h
k
l
∑
i
I
i
(
h
k
l
)
-
〈
I
(
h
k
l
)
〉
∣
/
∑
h
k
l
∑
i
I
i
(
h
k
l
), where I
i
(
h
k
l
) is the individual intensity of the ith observation of reflection h
k
l and 〈
I
(
h
k
l
)
〉 is the average intensity of reflection h
k
l with summation over all data.
b
R value = |
|
F
o
|
-
|
F
c
|
|
/
|
F
o
|, where F
o and F
c are the observed and calculated structure factors, respectively.
c
R
free is equivalent to R value but is calculated for 5% of reflections chosen at random and omitted from the refinement process [32].
das determined by Molprobity [33].


### 2.2. Structure Determination, Refinement, and Analysis

Crystal structures were solved by difference Fourier method using the CAII structure (PDB code 3IGP [34]) as a starting model. The model was refined using REFMAC5 [35], part of the CCP4 program suite [36]. The model was initially refined with isotropic atomic displacement parameters (ADPs); hydrogen atoms in riding positions were added later. For the final rounds of refinement, we used a mixed isotropic-anisotropic model of ADPs: anisotropic ADPs were used for all atoms, and only atoms in alternative conformations were refined isotropically. Atomic coordinates for the structure of1 were generated by quantum mechanics computation with DFT-D methodology [37] using the B-LYP functional and SVP basis set [38] in the Turbomole program [39]. A geometric library for1 was generated using the Libcheck program from the CCP4 suite. Coot [40] was used for rebuilding. The quality of the refined model was assessed using MolProbity [33]. The coordinates and structure factors were deposited in the PDB under accession code 4Q78. Final refinement statistics are summarized in Table 1. All structural figures were prepared using PyMOL 1.4.1 [41].


### 2.3. Model of CAIX-1 Complex

The complex of CAIX and1 was modeled by aligning the existing crystal structures of the CAIX catalytic domain (PDB code 3IAI [42]) with the CAII-1 complex (PDB code 4MDG [7]) using PyMOL version 1.2 [43]. Preparation of structure coordinate files for further calculations was performed as described before for CAII [27].The complex was fully optimized using a QM/MM procedure. We used ONIOM-like subtractive scheme [44] with link atoms and mechanical embedding to be consistent with our previous studies [27, 45–48]. The QM part is described at the DFT-D TPSS/TZVP//BLYP/SVP level of theory [39] and comprises 218 atoms including the atoms present in1 and 8 amino acids (Trp5, Asn62, His64, Gln67, Gln92, Val131, Leu135, and Pro202). The MM part constituted the remainder of the protein, and the surrounding solvent was approximated by a generalized Born (GB) implicit model. Detailed description of the procedure was published in [27]. One crystal water molecule (Wat272) bridging the inhibitors and CAII residues Thr199, Glu106, and Tyr7 was retained to maintain the integrity of the active site. Other water molecules present in the crystal structures were omitted.The positions of the added hydrogen atoms,1, and 15 amino acids surrounding the ligand (Trp5, Asn62, Gly63, His64, Gln67, Leu91, Gln92, Leu123, Val131, Leu135, Leu141, Thr200, Pro201, Pro202, and Ala204) were relaxed in a GB implicit solvent model using the FIRE algorithm followed by 10 ps annealing from 100 K or 150 K to 0 K using the Berendsen thermostat [49] in the SANDER module of the AMBER 10 package [50].


### 2.4. Virtual Glycine Scan

The contribution of the active site amino acids to inhibitor binding was examined by virtual glycine scanning. Individual amino acids in contact with1 in the CAIX-1 model and CAII-1 crystal structure were substituted with glycine. The energy contributions (Δ
Δ
G
int
⁡
′) were calculated as the difference between the original Δ
G
int
⁡
′ at the QM/MM level with the wild-type amino acid and the new Δ
G
int
⁡
′ with the mutated glycine residue [27].


## 2.1. Protein Crystallization and Diffraction Data Collection

For crystallization of human CAII (Sigma, catalogue number C6165) in complex with 1-methylenesulfamide-1,2-dicarba-closo-dodecaborane (compound1), we adapted a previously described procedure [28]. CAII (at a concentration of 4 mg·mL−1, dissolved in water) was incubated in aqueous solution containing a 2-fold molar excess ofp-hydroxymercuribenzoate (Sigma, catalogue number 55540). The protein was concentrated to 10 mg·mL−1 and unboundp-hydroxymercuribenzoate was removed with Amicon Ultra-4 concentrators (Merck-Millipore MWCO 10 kDa).The complex of CAII with1 was prepared by adding a 1.1-fold molar excess of1(in DMSO) to the 10 mg·mL−1 solution of CAII in water without pH adjustment (the final DMSO concentration did not exceed 5% v/v).The best diffracting crystals were obtained using the hanging-drop vapor diffusion method under the following conditions: 2μL protein-inhibitor complex solution was mixed with 2 μL precipitant solution [2.5 M (NH4)2SO4, 0.3 M NaCl, and 100 mM Tris-HCl, pH 8.2] and equilibrated over a reservoir containing 1 mL of precipitant solution at 18°C. Crystals with dimensions of 0.3 mm × 0.1 mm × 0.1 mm grew within 7 days.For cryoprotection, the crystals were incubated in mother liquor supplemented with 25% glycerol for approximately 30 s, flash-frozen, and stored in liquid nitrogen. Diffraction data for the CAII complex were collected at 100 K at the X14.2 BESSY beamline in Berlin, Germany [29]. Data were collected in two passes: the high-resolution range (11.75–1.00 Å) and the low-resolution range (21.08–1.20 Å). The two datasets were integrated with iMOSFLM [30] and merged and scaled with SCALA [31]. Data collection and refinement statistics are summarized in Table 1.Table 1
Data collection and refinement statistics.
Data collection statistics
Space group
P
2
1
Cell parameters (Å; °)
42.20, 41.73, 72.16; 90.0, 104.4, 90.0
Wavelength (Å)
0.9184
Resolution (Å)
21.08–1.00 (1.05–1.00)
Number of unique reflections
108,781 (15,490)
Multiplicity
3.5 (2.5)
Completeness (%)
83.1 (81.4)
R
merge
a
0.056 (0.375)
AverageI
/
σ(I)
10.8 (2.3)
Wilson B (Å2)
6.5
Refinement statistics
Resolution range (Å)
69.90–1.00 (1.03–1.00)
No. of reflections in working set
97,856 (7,831)
No. of reflections in test set
5,426 (412)
R value (%)b
17.5 (24.4)
R
free value (%)c
20.0 (26.2)
RMSD bond length (Å)
0.011
RMSD angle (°)
1.53
Number of atoms in AU
2297
Number of protein atoms in AU
2081
Number of water molecules in AU
176
Mean ADP value protein/inhibitor (Å2)
12.0/17.6
Ramachandran plot statisticsd
Residues in favored regions (%)
96.56
Residues in allowed regions (%)
3.44
The data in parentheses refer to the highest-resolution shell.
a
R
merge
=
∑
h
k
l
∑
i
I
i
(
h
k
l
)
-
〈
I
(
h
k
l
)
〉
∣
/
∑
h
k
l
∑
i
I
i
(
h
k
l
), where I
i
(
h
k
l
) is the individual intensity of the ith observation of reflection h
k
l and 〈
I
(
h
k
l
)
〉 is the average intensity of reflection h
k
l with summation over all data.
b
R value = |
|
F
o
|
-
|
F
c
|
|
/
|
F
o
|, where F
o and F
c are the observed and calculated structure factors, respectively.
c
R
free is equivalent to R value but is calculated for 5% of reflections chosen at random and omitted from the refinement process [32].
das determined by Molprobity [33].


## 2.2. Structure Determination, Refinement, and Analysis

Crystal structures were solved by difference Fourier method using the CAII structure (PDB code 3IGP [34]) as a starting model. The model was refined using REFMAC5 [35], part of the CCP4 program suite [36]. The model was initially refined with isotropic atomic displacement parameters (ADPs); hydrogen atoms in riding positions were added later. For the final rounds of refinement, we used a mixed isotropic-anisotropic model of ADPs: anisotropic ADPs were used for all atoms, and only atoms in alternative conformations were refined isotropically. Atomic coordinates for the structure of1 were generated by quantum mechanics computation with DFT-D methodology [37] using the B-LYP functional and SVP basis set [38] in the Turbomole program [39]. A geometric library for1 was generated using the Libcheck program from the CCP4 suite. Coot [40] was used for rebuilding. The quality of the refined model was assessed using MolProbity [33]. The coordinates and structure factors were deposited in the PDB under accession code 4Q78. Final refinement statistics are summarized in Table 1. All structural figures were prepared using PyMOL 1.4.1 [41].


## 2.3. Model of CAIX-1 Complex

The complex of CAIX and1 was modeled by aligning the existing crystal structures of the CAIX catalytic domain (PDB code 3IAI [42]) with the CAII-1 complex (PDB code 4MDG [7]) using PyMOL version 1.2 [43]. Preparation of structure coordinate files for further calculations was performed as described before for CAII [27].The complex was fully optimized using a QM/MM procedure. We used ONIOM-like subtractive scheme [44] with link atoms and mechanical embedding to be consistent with our previous studies [27, 45–48]. The QM part is described at the DFT-D TPSS/TZVP//BLYP/SVP level of theory [39] and comprises 218 atoms including the atoms present in1 and 8 amino acids (Trp5, Asn62, His64, Gln67, Gln92, Val131, Leu135, and Pro202). The MM part constituted the remainder of the protein, and the surrounding solvent was approximated by a generalized Born (GB) implicit model. Detailed description of the procedure was published in [27]. One crystal water molecule (Wat272) bridging the inhibitors and CAII residues Thr199, Glu106, and Tyr7 was retained to maintain the integrity of the active site. Other water molecules present in the crystal structures were omitted.The positions of the added hydrogen atoms,1, and 15 amino acids surrounding the ligand (Trp5, Asn62, Gly63, His64, Gln67, Leu91, Gln92, Leu123, Val131, Leu135, Leu141, Thr200, Pro201, Pro202, and Ala204) were relaxed in a GB implicit solvent model using the FIRE algorithm followed by 10 ps annealing from 100 K or 150 K to 0 K using the Berendsen thermostat [49] in the SANDER module of the AMBER 10 package [50].


## 2.4. Virtual Glycine Scan

The contribution of the active site amino acids to inhibitor binding was examined by virtual glycine scanning. Individual amino acids in contact with1 in the CAIX-1 model and CAII-1 crystal structure were substituted with glycine. The energy contributions (Δ
Δ
G
int
⁡
′) were calculated as the difference between the original Δ
G
int
⁡
′ at the QM/MM level with the wild-type amino acid and the new Δ
G
int
⁡
′ with the mutated glycine residue [27].


## 3. Results and Discussion

### 3.1. Crystal Structure of CAII in Complex with1 at Atomic Resolution

The overall structure of CAII in complex with1 was refined to 1.0 Å resolution. This high resolution allowed us to observe details that could not be fully resolved in the complex structure determined previously at lower resolution. Atomic resolution was achieved by derivatization of CAII using the 4-(hydroxymercury)benzoic acid (abbreviated MBO in the cif library of small molecules) method described by [28]. The mercury atom of MBO covalently binds to Sγ of Cys206. This modification allows formation of a hydrogen bond between the OZ1 oxygen of the MBO carboxyl group and the main-chain amino group of Tyr40 in the neighboring protein molecule, reinforcing the crystal lattice and increasing the diffraction quality of the crystal. In our structure, MBO is modeled in two alternative conformations with occupancies of 0.6 and 0.2.When our atomic resolution structure is compared with the structure of the CAII-1 complex determined at 1.35 Å resolution (PDB code 4MDG [7]), the RMSD value for superposition of the Cα atoms of residues 4–261 is 0.142 Å, a value typical for superposition of identical structures [51]. The N-terminal residue His3 is traced differently in the two structures; double conformations of numerous side chains (e.g., Glu14, His64, and Gln74) are resolved in the atomic resolution structure. We found an additional difference in the loop formed by amino acid residues 124–139, with a maximum difference of 0.738 Å for the position of Gln136 Cα. Gln136 forms van der Waals contacts with the MBO covalently attached to Cys206. The positions of Phe131 and Val135, which form a hydrophobic rim at the active site, are also influenced by MBO binding. This results in a subtle positional shift of the inhibitor, with an RMSD of 0.145 Å for superposition of 12 atoms in the carborane cage of1 bound to CAII and CAII derivatized by MBO. This value is below the value observed for superposition of identical structures [51].Atomic-level resolution allowed us to resolve the carbon and boron atom positions in the symmetrical carborane cage of1. When analyzing the values of the electron density map at positions of atoms bonded to the C1 atom, we can assume that positions with higher density levels are more likely to be carbon than boron atoms. Similar analysis was done by others for boron-containing inhibitor of human dihydrofolate reductase [18]. The C2 atom of the carborane cage (Figure 1(a)) was modeled into the position with an electron density value of 1.16 e/Å3, which was approximately 0.15 e/Å3 higher than those for the B3, B4, B5, and B6 atoms. To exclude the possibility that higher density is caused by model bias, we altered the composition of the cage by replacing the C2 atom with a boron atom. Electron density values did not change significantly after several rounds of refinement cycles.Thus, we can conclude that the most probable position of the second carbon atom in the carborane cage of1 is the position assigned to the C2 atom in our crystal structure. This is in good agreement with the recently published QM/MM modeling study [27].


### 3.2. Detailed Analysis of Inhibitor Interactions with CAII

The crystal structure of human CAII in complex with1 determined at 1.0 Å resolution confirmed the key interactions that our group observed previously [7]. The compound fits very well into the CAII active site cavity and makes numerous polar and nonpolar interactions with the residues in the enzyme active site. The sulfamide moiety, which forms key polar interactions with the active site Zn2+ ion, also makes polar interactions with Thr199 typical of other sulfamide inhibitors of CAII (Figure 2). The linker NH group forms an additional polar interaction with Oγ of Thr200. The compound makes several van der Waals interactions with residues Gln92, His94, His96, His119, Val121, Phe131, Leu198, and Thr200 (Figure 2). All interactions between the inhibitor and protein are summarized in Table 2.Table 2
List of contacts between CAII and1.
CAII
1
Residue
Atom
Atoma
Distance [Å]b
Zn
ZN
N2
1.87c
Zn
ZN
S
3.04
Zn
ZN
O2
3.05
92
Gln
OE1
B6
3.47
92
Gln
OE1
B11
3.52
92
Gln
CD
B6
3.84
94
His
CE1
O2
2.97
94
His
NE2
N2
3.23
94
His
NE2
O2
3.31
94
His
CE1
C3
3.67
94
His
NE2
S
3.81
94
His
CE1
N2
3.82
94
His
CE1
S
3.84
94
His
NE2
C3
3.94
96
His
NE2
N2
3.14
96
His
CE1
N2
3.56
119
His
ND1
N2
3.39
119
His
ND1
O2
3.88
119
His
CE1
N2
3.96
121
Val
CG2
O2
3.82
131
Phe
CZ
B8
3.83
131
Phe
CZ
B7
3.97
198
Leu
CA
O1
3.09
198
Leu
C
O1
3.36
198
Leu
CB
O1
3.60
198
Leu
CD2
O1
3.63
198
Leu
CD1
B3
3.86
199
Thr
N
O1
2.70
199
Thr
OG1
N2
2.74
199
Thr
OG1
O1
3.58
199
Thr
OG1
S
3.78
199
Thr
N
S
3.83
199
Thr
CA
O1
3.83
199
Thr
CB
N2
3.98
200
Thr
OG1
N1
3.02
200
Thr
OG1
C3
3.14
200
Thr
OG1
B4
3.36
200
Thr
OG1
B3
3.56
200
Thr
OG1
C1
3.66
aAtom labels correspond to those shown in Figure 1(a).
bAll contacts with a distance between ligand and protein (or Zn) atoms less than or equal to 4 Å are listed.
cPolar interactions are highlighted in bold.Figure 2
Interactions of1 with CAII. The protein is shown in cartoon representation; residues involved in interactions with the Zn2+ ion (gray sphere) and1 are shown in stick representation. Polar interactions are represented by blue dashed lines; Zn2+ ion coordination is shown as black dashed lines.The idea of designing CA inhibitors containing a carborane cluster moiety originated from our previous structural studies of isoquinoline-containing sulfonamide inhibitors (Figure3(a)). Structural analysis of CAII in complex with 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinolin-2-ylsulfonamide (2, PDB code 3IGP, [34]) and 6,7-dimethoxy-1-methyl-1,2,3,4-tetrahydroisoquinolin-2-ylsulfonamide (3, PDB code 3PO6, [52]) revealed two distinct binding modes that engage two opposite sides of the enzyme active site cavity (Figure 3(b)). Following this analysis, we hypothesized that the binding space within the enzyme active site cavity could be effectively filled with a bulky hydrophobic molecule with a spherical structure. This led to design of1 which exhibited inhibitory property to CAII and CAIX with Ki values in submicromolar range. Structural analysis of CAII-1 indicates that our structure-based design was sound. We found that the carborane cluster interacts with both sides of the enzyme active site as predicted (Figure 3(c), Table 3) and that the position of1 in the CAII active site superposes well with the two binding modes observed for 2 and 3 (Figure 3(d)).Table 3
CAII or CAIX residues interacting with1, 2, and 3.
CAII
CAIX
1a
2b
3c
1d
Trp5
Asn62
Asn67e
Gln92
Gln92
Gln92
His94
His94
His94
His94
His96
His96
His96
His96
Glu106
His119
His119
His119
His119
Val121
Val121
Val121
Phe131
Phe131
Phe131
Val143
Val143
Leu198
Leu198
Leu198
Leu198
Thr199
Thr199
Thr199
Thr199
Thr200
Thr200
Thr200
Thr200
Pro201
Pro201
Pro202
Trp209
Trp209
Interacting residues were identified fromacrystal structure 4Q78 (this work); bcrystal structure 3IGP [34]; ccrystal structure 3PO6 [52]; dcomputational model (this work); eresidues making polar interactions are highlighted in bold.(a) Structural formulas of2 and3. (b) Interactions of2 and3 with the CAII active site. Compound2 is represented with golden carbon atoms, while the carbon atoms of3 are colored turquoise. Surface of residues making contacts with the isoquinoline moiety of2 and3 are highlighted in yellow and blue, respectively. Surface of residues colored orange make contacts with both compounds. Atoms involved in contacts with the sulfonamide groups are not highlighted. (c) Interactions of1 with the CAII active site. Surface of residues making contacts with the carborane and linker moiety of1 are highlighted in green. Atoms involved in contacts with the sulfonamide groups are not highlighted. (d) Superposition of binding poses of1,2, and3 in the CAII active site. Superposition of the complex structures was based on the best fit for Cα atoms of CAII residues 6–261.
(a)
(b)
(c)
(d)


### 3.3. Model of the CAIX-1 Complex

The CAII-1 crystal structure was used to model binding of compound1into the CAIX active site using QM/MM methods (Figure 4).Figure 4
Interactions of1 with the CAIX active site. Atoms making contacts with the carborane and linker moiety of1 are highlighted in magenta. Atoms involved in contacts with the sulfonamide groups are not highlighted. Superposition of the binding pose of1 in CAII is shown as black lines. Superposition is based on the best fit for Cα atoms of all residues of CAII onto CAIX.The substrate binding sites of CAII and CAIX differ by only six amino acids: Asn67 of CAII is replaced by Gln in CAIX, Ile91 by Leu, Trp123 by Leu, Phe131 by Val, Val135 by Leu, and Leu204 by Ala. These variations result in a differently shaped active site cavity, which accommodated1 in a slightly different pose (Figure 4). While the position of the sulfamide anchor remained unchanged, the carborane cluster shifted by 2.1 Å (expressed as a difference in the position of B12) away from the central β-sheet. In CAIX-1, the carborane interacts more with the opposite site of the active site, specifically with amino acid residues His94, His96, Glu106, Leu198, Thr199, Thr200, and Pro201 (Figure 4, Table 3). All polar and van der Waals interactions between CAIX and1 are summarized in Table 4.Table 4
Interactions between CAIX and1.
CAIX
1
Residue
Atom
Atoma
Distance [Å]b
Zn
ZN
N2
2.1c
Zn
ZN
S
3.3
Zn
ZN
O2
3.5
5
Trp
CZ2
B5
3.74
5
Trp
CZ2
B10
3.81
94
His
CE1
O2
3.15
94
His
CE1
C3
3.74
94
His
NE2
N2
3.36
94
His
NE2
S
3.88
94
His
NE2
O2
3.45
94
His
NE2
C3
3.76
96
His
CE1
N2
3.99
96
His
NE2
N2
3.49
106
Glu
OE2
N2
3.71
119
His
ND1
N2
3.37
119
His
CE1
N2
3.83
121
Val
CG2
O2
3.58
198
Leu
CA
O1
3.04
198
Leu
CB
O1
3.4
198
Leu
CD2
O1
3.43
198
Leu
C
O1
3.38
199
Thr
N
S
3.88
199
Thr
N
O1
2.79
199
Thr
CA
O1
3.96
199
Thr
CB
N2
3.85
199
Thr
OG1
N2
2.63
199
Thr
OG1
S
3.69
199
Thr
OG1
O1
3.65
200
Thr
OG1
C1
3.77
200
Thr
OG1
B5
3.56
200
Thr
OG1
N1
3.13
200
Thr
OG1
C3
3.31
200
Thr
OG1
B4
3.64
201
Pro
O
B4
3.6
201
Pro
O
B10
3.49
201
Pro
O
B8
3.96
209
Trp
CZ2
O1
3.74
aAtom labels correspond to those shown in Figure 1(a).
bAll contacts with a distance less than or equal to 4 Å between ligand and protein (and Zn) atoms are listed.
cPolar interactions are highlighted in bold.We used a virtual glycine scan to study the roles of individual amino acid side chains in the active sites of CAII and CAIX in binding of1. The changes in free energy of interaction (Δ
Δ
G
int
⁡
′) upon mutation of a given amino acid residue to glycine are shown in Figure 5.Figure 5
Results of virtual glycine scan showing contributions of individual residues to the energy of binding of1 to CAII and CAIX, respectively.The largest energy change (2.6 kcal/mol) occurred for Trp5, which is positioned closer to1 in CAIX-1 than in CAII-1. The side chain of Trp5 forms several dihydrogen bonds with the carborane cage of1. The shortest one has a H
⋯
H distance of 2.3 Å. The other major contributor to strong CAIX-1 binding was Asn62; the energy of binding exceeded that in CAII-1 by nearly 1 kcal/mol. These contributions were cancelled out by differences in binding energy contributions of amino acid residues 131 (Phe/Val) and 135 (Val/Leu), which were lower in CAIX by 0.7 and 0.9 kcal/mol, respectively. The energy changes of other residues were small.When we compared binding of1 to CAII and CAIX, we noted that the favorable energy changes in CAIX-1 due to the binding of residues Trp5, Asn62, and His64 were slightly larger than the unfavorable changes in binding caused by the different amino acids at residues 131 and 135. This is in qualitative agreement with the experimental K
i values, which are 700
±
141 nm for inhibition of CAII and 380
±
111 nM for inhibition of CAIX [7].


## 3.1. Crystal Structure of CAII in Complex with1 at Atomic Resolution

The overall structure of CAII in complex with1 was refined to 1.0 Å resolution. This high resolution allowed us to observe details that could not be fully resolved in the complex structure determined previously at lower resolution. Atomic resolution was achieved by derivatization of CAII using the 4-(hydroxymercury)benzoic acid (abbreviated MBO in the cif library of small molecules) method described by [28]. The mercury atom of MBO covalently binds to Sγ of Cys206. This modification allows formation of a hydrogen bond between the OZ1 oxygen of the MBO carboxyl group and the main-chain amino group of Tyr40 in the neighboring protein molecule, reinforcing the crystal lattice and increasing the diffraction quality of the crystal. In our structure, MBO is modeled in two alternative conformations with occupancies of 0.6 and 0.2.When our atomic resolution structure is compared with the structure of the CAII-1 complex determined at 1.35 Å resolution (PDB code 4MDG [7]), the RMSD value for superposition of the Cα atoms of residues 4–261 is 0.142 Å, a value typical for superposition of identical structures [51]. The N-terminal residue His3 is traced differently in the two structures; double conformations of numerous side chains (e.g., Glu14, His64, and Gln74) are resolved in the atomic resolution structure. We found an additional difference in the loop formed by amino acid residues 124–139, with a maximum difference of 0.738 Å for the position of Gln136 Cα. Gln136 forms van der Waals contacts with the MBO covalently attached to Cys206. The positions of Phe131 and Val135, which form a hydrophobic rim at the active site, are also influenced by MBO binding. This results in a subtle positional shift of the inhibitor, with an RMSD of 0.145 Å for superposition of 12 atoms in the carborane cage of1 bound to CAII and CAII derivatized by MBO. This value is below the value observed for superposition of identical structures [51].Atomic-level resolution allowed us to resolve the carbon and boron atom positions in the symmetrical carborane cage of1. When analyzing the values of the electron density map at positions of atoms bonded to the C1 atom, we can assume that positions with higher density levels are more likely to be carbon than boron atoms. Similar analysis was done by others for boron-containing inhibitor of human dihydrofolate reductase [18]. The C2 atom of the carborane cage (Figure 1(a)) was modeled into the position with an electron density value of 1.16 e/Å3, which was approximately 0.15 e/Å3 higher than those for the B3, B4, B5, and B6 atoms. To exclude the possibility that higher density is caused by model bias, we altered the composition of the cage by replacing the C2 atom with a boron atom. Electron density values did not change significantly after several rounds of refinement cycles.Thus, we can conclude that the most probable position of the second carbon atom in the carborane cage of1 is the position assigned to the C2 atom in our crystal structure. This is in good agreement with the recently published QM/MM modeling study [27].


## 3.2. Detailed Analysis of Inhibitor Interactions with CAII

The crystal structure of human CAII in complex with1 determined at 1.0 Å resolution confirmed the key interactions that our group observed previously [7]. The compound fits very well into the CAII active site cavity and makes numerous polar and nonpolar interactions with the residues in the enzyme active site. The sulfamide moiety, which forms key polar interactions with the active site Zn2+ ion, also makes polar interactions with Thr199 typical of other sulfamide inhibitors of CAII (Figure 2). The linker NH group forms an additional polar interaction with Oγ of Thr200. The compound makes several van der Waals interactions with residues Gln92, His94, His96, His119, Val121, Phe131, Leu198, and Thr200 (Figure 2). All interactions between the inhibitor and protein are summarized in Table 2.Table 2
List of contacts between CAII and1.
CAII
1
Residue
Atom
Atoma
Distance [Å]b
Zn
ZN
N2
1.87c
Zn
ZN
S
3.04
Zn
ZN
O2
3.05
92
Gln
OE1
B6
3.47
92
Gln
OE1
B11
3.52
92
Gln
CD
B6
3.84
94
His
CE1
O2
2.97
94
His
NE2
N2
3.23
94
His
NE2
O2
3.31
94
His
CE1
C3
3.67
94
His
NE2
S
3.81
94
His
CE1
N2
3.82
94
His
CE1
S
3.84
94
His
NE2
C3
3.94
96
His
NE2
N2
3.14
96
His
CE1
N2
3.56
119
His
ND1
N2
3.39
119
His
ND1
O2
3.88
119
His
CE1
N2
3.96
121
Val
CG2
O2
3.82
131
Phe
CZ
B8
3.83
131
Phe
CZ
B7
3.97
198
Leu
CA
O1
3.09
198
Leu
C
O1
3.36
198
Leu
CB
O1
3.60
198
Leu
CD2
O1
3.63
198
Leu
CD1
B3
3.86
199
Thr
N
O1
2.70
199
Thr
OG1
N2
2.74
199
Thr
OG1
O1
3.58
199
Thr
OG1
S
3.78
199
Thr
N
S
3.83
199
Thr
CA
O1
3.83
199
Thr
CB
N2
3.98
200
Thr
OG1
N1
3.02
200
Thr
OG1
C3
3.14
200
Thr
OG1
B4
3.36
200
Thr
OG1
B3
3.56
200
Thr
OG1
C1
3.66
aAtom labels correspond to those shown in Figure 1(a).
bAll contacts with a distance between ligand and protein (or Zn) atoms less than or equal to 4 Å are listed.
cPolar interactions are highlighted in bold.Figure 2
Interactions of1 with CAII. The protein is shown in cartoon representation; residues involved in interactions with the Zn2+ ion (gray sphere) and1 are shown in stick representation. Polar interactions are represented by blue dashed lines; Zn2+ ion coordination is shown as black dashed lines.The idea of designing CA inhibitors containing a carborane cluster moiety originated from our previous structural studies of isoquinoline-containing sulfonamide inhibitors (Figure3(a)). Structural analysis of CAII in complex with 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinolin-2-ylsulfonamide (2, PDB code 3IGP, [34]) and 6,7-dimethoxy-1-methyl-1,2,3,4-tetrahydroisoquinolin-2-ylsulfonamide (3, PDB code 3PO6, [52]) revealed two distinct binding modes that engage two opposite sides of the enzyme active site cavity (Figure 3(b)). Following this analysis, we hypothesized that the binding space within the enzyme active site cavity could be effectively filled with a bulky hydrophobic molecule with a spherical structure. This led to design of1 which exhibited inhibitory property to CAII and CAIX with Ki values in submicromolar range. Structural analysis of CAII-1 indicates that our structure-based design was sound. We found that the carborane cluster interacts with both sides of the enzyme active site as predicted (Figure 3(c), Table 3) and that the position of1 in the CAII active site superposes well with the two binding modes observed for 2 and 3 (Figure 3(d)).Table 3
CAII or CAIX residues interacting with1, 2, and 3.
CAII
CAIX
1a
2b
3c
1d
Trp5
Asn62
Asn67e
Gln92
Gln92
Gln92
His94
His94
His94
His94
His96
His96
His96
His96
Glu106
His119
His119
His119
His119
Val121
Val121
Val121
Phe131
Phe131
Phe131
Val143
Val143
Leu198
Leu198
Leu198
Leu198
Thr199
Thr199
Thr199
Thr199
Thr200
Thr200
Thr200
Thr200
Pro201
Pro201
Pro202
Trp209
Trp209
Interacting residues were identified fromacrystal structure 4Q78 (this work); bcrystal structure 3IGP [34]; ccrystal structure 3PO6 [52]; dcomputational model (this work); eresidues making polar interactions are highlighted in bold.(a) Structural formulas of2 and3. (b) Interactions of2 and3 with the CAII active site. Compound2 is represented with golden carbon atoms, while the carbon atoms of3 are colored turquoise. Surface of residues making contacts with the isoquinoline moiety of2 and3 are highlighted in yellow and blue, respectively. Surface of residues colored orange make contacts with both compounds. Atoms involved in contacts with the sulfonamide groups are not highlighted. (c) Interactions of1 with the CAII active site. Surface of residues making contacts with the carborane and linker moiety of1 are highlighted in green. Atoms involved in contacts with the sulfonamide groups are not highlighted. (d) Superposition of binding poses of1,2, and3 in the CAII active site. Superposition of the complex structures was based on the best fit for Cα atoms of CAII residues 6–261.
(a)
(b)
(c)
(d)


## 3.3. Model of the CAIX-1 Complex

The CAII-1 crystal structure was used to model binding of compound1into the CAIX active site using QM/MM methods (Figure 4).Figure 4
Interactions of1 with the CAIX active site. Atoms making contacts with the carborane and linker moiety of1 are highlighted in magenta. Atoms involved in contacts with the sulfonamide groups are not highlighted. Superposition of the binding pose of1 in CAII is shown as black lines. Superposition is based on the best fit for Cα atoms of all residues of CAII onto CAIX.The substrate binding sites of CAII and CAIX differ by only six amino acids: Asn67 of CAII is replaced by Gln in CAIX, Ile91 by Leu, Trp123 by Leu, Phe131 by Val, Val135 by Leu, and Leu204 by Ala. These variations result in a differently shaped active site cavity, which accommodated1 in a slightly different pose (Figure 4). While the position of the sulfamide anchor remained unchanged, the carborane cluster shifted by 2.1 Å (expressed as a difference in the position of B12) away from the central β-sheet. In CAIX-1, the carborane interacts more with the opposite site of the active site, specifically with amino acid residues His94, His96, Glu106, Leu198, Thr199, Thr200, and Pro201 (Figure 4, Table 3). All polar and van der Waals interactions between CAIX and1 are summarized in Table 4.Table 4
Interactions between CAIX and1.
CAIX
1
Residue
Atom
Atoma
Distance [Å]b
Zn
ZN
N2
2.1c
Zn
ZN
S
3.3
Zn
ZN
O2
3.5
5
Trp
CZ2
B5
3.74
5
Trp
CZ2
B10
3.81
94
His
CE1
O2
3.15
94
His
CE1
C3
3.74
94
His
NE2
N2
3.36
94
His
NE2
S
3.88
94
His
NE2
O2
3.45
94
His
NE2
C3
3.76
96
His
CE1
N2
3.99
96
His
NE2
N2
3.49
106
Glu
OE2
N2
3.71
119
His
ND1
N2
3.37
119
His
CE1
N2
3.83
121
Val
CG2
O2
3.58
198
Leu
CA
O1
3.04
198
Leu
CB
O1
3.4
198
Leu
CD2
O1
3.43
198
Leu
C
O1
3.38
199
Thr
N
S
3.88
199
Thr
N
O1
2.79
199
Thr
CA
O1
3.96
199
Thr
CB
N2
3.85
199
Thr
OG1
N2
2.63
199
Thr
OG1
S
3.69
199
Thr
OG1
O1
3.65
200
Thr
OG1
C1
3.77
200
Thr
OG1
B5
3.56
200
Thr
OG1
N1
3.13
200
Thr
OG1
C3
3.31
200
Thr
OG1
B4
3.64
201
Pro
O
B4
3.6
201
Pro
O
B10
3.49
201
Pro
O
B8
3.96
209
Trp
CZ2
O1
3.74
aAtom labels correspond to those shown in Figure 1(a).
bAll contacts with a distance less than or equal to 4 Å between ligand and protein (and Zn) atoms are listed.
cPolar interactions are highlighted in bold.We used a virtual glycine scan to study the roles of individual amino acid side chains in the active sites of CAII and CAIX in binding of1. The changes in free energy of interaction (Δ
Δ
G
int
⁡
′) upon mutation of a given amino acid residue to glycine are shown in Figure 5.Figure 5
Results of virtual glycine scan showing contributions of individual residues to the energy of binding of1 to CAII and CAIX, respectively.The largest energy change (2.6 kcal/mol) occurred for Trp5, which is positioned closer to1 in CAIX-1 than in CAII-1. The side chain of Trp5 forms several dihydrogen bonds with the carborane cage of1. The shortest one has a H
⋯
H distance of 2.3 Å. The other major contributor to strong CAIX-1 binding was Asn62; the energy of binding exceeded that in CAII-1 by nearly 1 kcal/mol. These contributions were cancelled out by differences in binding energy contributions of amino acid residues 131 (Phe/Val) and 135 (Val/Leu), which were lower in CAIX by 0.7 and 0.9 kcal/mol, respectively. The energy changes of other residues were small.When we compared binding of1 to CAII and CAIX, we noted that the favorable energy changes in CAIX-1 due to the binding of residues Trp5, Asn62, and His64 were slightly larger than the unfavorable changes in binding caused by the different amino acids at residues 131 and 135. This is in qualitative agreement with the experimental K
i values, which are 700
±
141 nm for inhibition of CAII and 380
±
111 nM for inhibition of CAIX [7].


## 4. Conclusions

We determined to atomic resolution the crystal structure of CAII in complex with 1-methylenesulfamide-1,2-dicarba-closo-dodecaborane (1), a parent compound of a recently reported series of CA inhibitors containing carborane cages [7]. Comparing this crystal structure with those of CAII complexes with conventional organic inhibitors showed that the three-dimensional cluster fills the enzyme active site cavity. Atomic-level resolution allowed us to distinguish the positions of carbon and boron atoms in the carborane cage. The crystal structure also served as a model for construction of the CAIX-1computational model. Virtual glycine scan enabled us to quantify the contributions of individual residues to the energy of binding of1 to CAII and CAIX and uncover differences of the enzyme active site cavities. Structural and computational analysis will be used in future structure-based design of carborane compounds with selectivity toward the cancer-specific CAIX isoenzyme.



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*Source: 389869-2014-09-18.xml*