Fig 1.
The interactions between CA II and the 2-chlorobenzenesulfonamide-bearing compound.
Panel (a): The negatively charged amino group of the sulfonamide forms a coordination bond with the Zn(II) in the active site and prevents the CO2 substrate binding. Additional R substituents form hydrophobic and other contacts with the protein amino acids. Panel (b): The chlorine (green) of the 2-chloro-benzenesulfonamide (orange) occupies the binding site cavity and orients the compound in the active site of CA II as compared to the benzenesulfonamide (grey) (PDB IDs 2WEH and 2WEJ [10]).
Fig 2.
Chemical structures of compounds used in this study.
Table 1.
Dissociation constants (Kd, nM) of the compounds for 12 human CA isoforms.
Fig 3.
Binding isotherms of chlorothiazide 3 interaction with CA II determined by three assays.
FTSA—panels (a) and (b)), SFA—panels (c) and (d), and ITC—panels (e) and (f). The left panels show the raw data, while the right panels—the dosing curves.
Fig 4.
Comparison of compound 3, 4 and 16 binding affinities for CA II as determined by SFA (panel a) and FTSA (panel b).
Panel (a) shows the SFA data: CA II binding to hydrochlorothiazide 4, CA IV–chlorothiazide 3 and CA XII–chlorthalidone 16. Panel (b) shows the FTSA data: CA II– 4, CA IV– 3 and CA XII– 16. Panel (c) compares the values and shows that both techniques yielded the same values (within the 2x error margin) for a wide range of affinities (micromolar to nanomolar compounds). The data points lie close to the solid diagonal line showing would-be-perfect agreement.
Fig 5.
Selectivities of compound binding to CA isoforms are shown as affinity bars expressed in pKd.
Panel (a) shows the binding of compounds to CA II. Affinities differed up to three orders of magnitude for various compounds. Panel (b) shows hydrochlorothiazide 4 binding to the twelve catalytically active human CA isoforms. Affinities varied by up to approximately one order of magnitude among CA isoforms. Most isoforms exhibited affinity of single-digit micromolar order of magnitude. The white horizontal line shows the lower limit of binding detection of 200 μM by FTSA.
Fig 6.
A comparison of two structurally related compound affinities.
The addition of chemical groups that are shown in red may enhance or diminish compound affinity for a particular CA isoform. This effect is shown on the left axis as the difference in the standard Gibbs energies of binding of both compounds corresponding to the ratio of the dissociation constants of both compounds on the right axis. The addition of some groups led to a nearly universal increase in binding affinity (a, f), or affected only a narrow subset of CA isoforms (d).