Crystal structure of the human carbonic anhydrase II adduct with 1-(4-sulfamoylphenyl-ethyl)-2,4,6-triphenylpyridinium perchlorate, a membrane-impermeant, isoform selective inhibitor

Figures & data

Figure 1. Chemical structures of clinically used CAIs and pyridinium containing sulfonamides 1 and 2.

Table 1. Inhibition of isozymes hCA I, hCA II, hCA IV, hCA IX, and hCA XII with the pyridinium salts 1, 2, and the standard, clinically used sulfonamide CAIs.

	KI (nM)
Compound	hCA I	hCA II	hCA IV	hCA IX	hCA XII
1	4000^a	21^a	60^a	14^a	7.0^a
2	270,000^b	419^b	1830^b^,^d	95^b	12.5^c
Dorzolamide	50,000^a	9^a	8500^a	52^a	3.5^a
Brinzolamide	45,000^a	3^a	3950^a	37^a	3.0^a
Acetazolamide	250^a	12^a	74^a	25^a	5.7^a
Methazolamide	50^a	14^a	6200^a	27^a	3.4^a
Ethoxzolamide	25^a	8^a	93^a	34^a	22^a
Dichlorophenamide	1200^a	38^a	1500^a	50^a	50^a

^a From Ref. (1).

^b From Ref. (39).

^c This work.

^d This Ki value refers to bCA IV.

Table 2. Data collection and refinement statistics for the hCA II/2 complex.

Crystal parameters
Space group	P21
a (Å)	42.1
b (Å)	41.3
c (Å)	71.9
γ (°)	104.2
Number of independent molecules	1
Data collection
Resolution (Å)	25.3–1.65
Wavelength (Å)	1.54178
Temperature (K)	100
R-merge (%)^a	5.9 (26.6)
<I>/<σ(I)>	25.7 (3.8)
Total reflections	172066
Unique reflections	27539
Redundancy	6.2 (2.7)
Completeness (%)	94.5 (79.6)
Refinement
Resolution (Å)	25.3–1.65
R-work (%)^b	17.5
R-free (%)^b	21.0
RMSD from ideal geometry:
Bond lengths (Å)	0.010
Bond angles (°)	1.6
Number of protein atoms	2076
Number of water molecules	195
Number of inhibitor atoms	36
Average B factor (Å^2):
All atoms	15.0
Protein atoms	14.2
Inhibitor atoms	26.5
Water molecules	22.2
Ramachandran statistics (%):
Most favoured	88.2
Additionally allowed	11.4
Generously allowed	0.5
Disallowed	0

Values in parentheses are statistics for the highest resolution shell (1.68–1.65 Å).

^a R-merge = Σ_hklΣ_i|I_i(hkl) − <I(hkl)>|/ Σ_hklΣ_iI_i(hkl), where I_i(hkl) is the intensity of an observation and <I(hkl)> is the mean value for its unique reflection; summations are over all reflections.

^b R-work = Σ_hklǁF_o(hkl)| − |F_c(hkl)ǁ/Σ_hkl|F_o(hkl)| calculated for the working set of reflections. R-free is calculated as for R-work, but from 4.9% of the data that was not used for refinement.

Figure 2. Active site region of the hCA II/2 adduct, showing σA-weighted |2F_o − F_c| simulated annealing omit map (contoured at 1.0σ) relative to the inhibitor molecule. Active site Zn²⁺ coordination (red continuous lines) and hydrogen bonds (red dotted lines) are also reported.

Figure 3. Structural superposition between 1 (cyan, PDB code 1ZE8)⁴¹ and 2 (green) when bound to hCA II active site.

Figure 4. (A) Solvent accessible surface of hCA II/1 active site⁴¹. Residues delimiting the hydrophobic pocket, where the trimethyl-pyridinium ring is located, are highlighted in red (Ile91, Gln92, Phe131). Compound 1 and residues Ile91, Gln92, and Phe131 are represented as ball-and-stick. (B) Solvent accessible surface of hCA II active site. Colour code is as in (A). The hypothetical conformation that compound 2 would have had if its pyridinium ring was positioned in the same hydrophobic pocket of 1 is shown. Yellow arrows indicate protein residues which clashes with inhibitor (Ile91, Gln92, Val121, Phe131).

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