A. Akdemir et al. / Bioorg. Med. Chem. 21 (2013) 5228–5232
5231
commonly observed binding poses within the hCA IX and hCA XII
active sites, respectively.
licate experiments were done for each inhibitor concentration, and
the values reported throughout the paper are the mean of such
results. The inhibition constants were obtained by non-linear
least-squares methods using PRISM 3, as reported earlier,10,11 and
represent the mean from at least three different determinations.
Human CA isozymes were prepared in recombinant form as
reported earlier by our groups.12,13
Trp5 and His64 are conserved in all four isoforms investigated
here, whereas Asn62 is conserved in hCA II, hCA IX and hCA XII
(Table 2). However, our docking studies suggest that only Trp5
and Gln92 are involved in binding interactions in both enzymes
(hCA IX aand XII). In general, the binding interactions with His64
and Gln67 of hCA IX and Asn62, Lys67 and Thr91 of hCA XII were
less frequently observed.
4.3. Ligand preparation
The docking studies also indicated that Phe131 in hCA II forms
hydrophobic stackings with the halophenyl or methoxyphenyl tails
of compounds 3 and 5 (Fig. 3). This residue is not conserved
amongst the other three enzymes (Table 2). In addition, the ligands
can form hydrogen bonding interactions with Gln92. In hCA I, a
bulky Leu131 is positioned at the Phe131 (hCA II) position. In addi-
tion, Phe91 is present close to Leu131 in hCA I. Phe91 is not cor-
rectly positioned with respect to the ligands to form hydrophobic
stackings with the ligands and it is expected to sterically hinder
the ligands to adopt similar poses as observed for hCA II, hCA IX
and hCA XII.
Compounds 3, 5 and the reference ligand AZ were built and
converted to three-dimensional structures using the MOE software
package (version 2011.10, Chemical Computing Group, Montreal,
Canada). The sulfonamide substituent of the phenyl group was as-
signed a negative charge (–SO2NHꢀ) because it was expected to
interact with the Zn2+ ion of the CA active sites. Subsequently, par-
tial atomic charges were calculated and the molecules were en-
ergy-minimized according to a steepest-descent protocol using
the MMFF94x force field in MOE. The ligands were saved as a mul-
ti-mol2 file.
In summary, the docking studies indicate that various hydrogen
bonds with the active site residues of hCA IX and hCA XII and
hydrophobic stacking with Phe131 of hCA II allow favorable bind-
ing interactions with these isoforms. Probably, the absence of
Phe131 and the presence of Phe91 and Leu131 residues in hCA I
(see Table 2) restrict the ligands to form similar binding interac-
tions with hCA I.
4.4. Template preparation
Crystal structures of hCA I (PDB: 3LXE; 1.90 Å),14 hCA II (PDB:
3B4F; 1.89 Å),13 hCA IX (PDB: 3IAI; 2.20 Å)15 and hCA XII (PDB:
1JD0; 1.50 Å)16 were obtained from the protein databank. Only 1
protein chain (chain A) and its corresponding Zn2+ ion and ligand
(CA inhibitor) was retained per PDB file and all other protein
chains, ions, buffer molecules and water molecules were deleted.
Hydrogen atoms and charges were added to the protein using
the protonate 3D tool of the MOE software package (version
2011.10, CCG, Montreal, Canada) and a steepest descent energy
minimization was applied (MMFF94x forcefield). All proteins were
3. Conclusions
The tested sulfonamides show very potent inhibition of four
physiologically relevant CA isoforms, hCA I, II, IX and XII, with KI
values in the nanomolar range, for the tumor-associated hCA IX
and hCA XII. Docking studies have revealed details regarding the
very favorable interactions between the scaffolds of this new class
of inhibitors and the active sites of the CA isoforms investigated
here. As there are reported cases of tumors overexpressing both
CA II and IX,7 such potent inhibitors for the two isoforms may have
clinical importance.
superposed on the hCA I structure using their Ca-atoms (RMSD:
1.561 Å) and the proteins were saved as mol2-files.
4.5. Docking studies
Compounds 3, 5 and the reference ligand AZ were docked into
the protein models of hCA I, hCA II, hCA IX and hCA XII using the
GOLD Suite docking package (version 5.1, CCDC, Cambridge,
UK)17 and the ChemScore scoring function (25 docking per ligand).
The binding pocket was defined as all residues within 12 Å of the
central carbon atom of the CA inhibitor topiramate (atom CAL of
topiramate in complex with hCA I; PDB: 3LXE). No restrictions
were applied for the ligand conformations and binding poses dur-
ing the docking procedure except for the requirement to form
hydrogen bonding with Thr199 (or its counterparts in other CA
isozymes).
4. Materials and methods
4.1. Chemistry
Compounds 3–5 investigated here as CAIs were recently de-
scribed by our group as Trypanosoma cruzi CA inhibitors.18
4.2. CA inhibition assay
An SX.18MV-R Applied Photophysics (Oxford, UK) stopped-flow
instrument has been used to assay the catalytic/inhibition of vari-
ous CA isozymes as reported by Khalifah.8 Phenol red (at a concen-
tration of 0.2 mM) has been used as indicator, working at the
absorbance maximum of 557 nM, with 10 mM Hepes (pH 7.4) as
buffer, 0.1 M Na2SO4 or NaClO4 (for maintaining constant the ionic
strength; these anions are not inhibitory in the used concentra-
tion),9 following the CA-catalyzed CO2 hydration reaction for a per-
iod of 5–10 s. Saturated CO2 solutions in water at 25 °C were used
as substrate. Stock solutions of inhibitors were prepared at a con-
centration of 10 mM (in DMSO–water 1:1, v/v) and dilutions up to
0.01 nM done with the assay buffer mentioned above. At least 7
different inhibitor concentrations have been used for measuring
the inhibition constant. Inhibitor and enzyme solutions were
preincubated together for 10 min at room temperature prior to
assay, in order to allow for the formation of the E–I complex. Trip-
Acknowledgments
This project was in part financed by the Istanbul University Sci-
entific Research Projects Department under project number UDP-
22409 and by an FP7 EU project (Metoxia).
References and notes
Akdemir, Atilla
