Chemical structures of compounds used in this study and designed to investigate the covalent bindin capability to CA proteins. Compounds on the left of the vertical dashed line do not form the covalent bond, while the ones on the right form the covalent bond with the protein molecule. Moieties shown in red are important for structural comparison to visualize the chemical groups that are responsible for covalent interaction, high affinity, or high selectivity for CAIX.

Proposed rearrangement mechanism of compounds bearing the -SO2CH2CH2OCOR fragment to vinyl- sulfone and the formation of covalent bond with the histidine (His64) amino acid side chain of the CA protein.

Transformation of pre-drug to the active vinyl sulfone and a three-way recognition of CA isozymes. First, the negatively charged sulfonamide forms coordination bond with the Zn(II). Second, hydrophobic cyclooctyl rin fits to the hydrophobic pocket of CAIX isozyme and provides substantial selectivity over other CA isozymes. Third, covalent bond forms with the histidine providing irreversible inhibition of CAIX enzymatic activity.

Covalent interaction shown by X-ray crystallography. A. X-ray crystal structure of CAI with covalently bound compound 21. B. X-ray crystal structure of CAII with covalently bound compound 20. C. X-ray crystal structure of CAIX with covalently bound compound 23. 2Fo-Fc map is shown only for the ligand and the histidine residue with which it forms a covalent bond and is contoured at 1σ.

Covalent interaction shown by HRMS, and enzymatic activity recovery assay. A. MS spectra of CAXIII in th absence of compound (top panel), the presence of 1:1 molar ratio of compound 12 after 3-minute incubation (middle panel) and 2-hour incubation (bottom panel). B. MS spectra of CAIX in the absence of compound (top panel) and after the incubation of 1:1 molar ratio of compound 22 (carbamate) for 4 hours. C. Enzymatic activity of CAIX before dialysis, while D – after dialysis (32 hours, 4-times buffer change, black solid line – fully active CAIX, grey solid line – spontaneous CO2 hydration reaction (coincides with fully inhibited CAIX), blue solid line – CAIX with non-covalent 5, dotted red line – CAIX with covalent 20, and dashed green line – CAIX with covalent carbamate 22. The recombinant CAIX recovered almost full activity after dialyzing out the non-covalent compound, while th activity remained fully inhibited with the covalent compounds.

Dosing curves of covalent compounds applied to hypoxic live cell culture expressing CAIX: 22 – green; 18 - orange; 24 – cyan; 20 – red and 12 – black. The compounds competed with the fluorescein-labeled GZ19-32, added at 10 nM concentration to all samples. A competitive binding model applied to obtain the affinities of teste compounds for cell-surface CAIX (Table 1).

Affinities (apparent dissociation constants Kd,app) of covalent compound binding to cell-expressed CAIX determined by applying a competitive binding model to data in Figure 6 as previously described (45). Parameters used in the competitive model were: the CAIX protein concentration was 5 nM (Pt = 5 nM), the dissociatio constant of GZ19-32 was 150 pM (Kd_B = 150 pM), and the concentration of GZ19-32 was 10 nM (Lt_B = 10 nM).

Apparent affinity determination of compound 22 by the thermal shift assay. A. Raw FTSA data of compound 22 binding to CAIX (pH 7.0 for 37 °C). B. Enzyme melting temperature dependence on compound 22 concentration. Datapoints saturated due to the covalent nature of interaction and therefore did not fully fit to th model line.

The apparent dissociation constants Kd,app (in nM units) for compound interaction with human recombinant CA isozymes as determined by fluorescence-based thermal shift assay (FTSA) at pH 7.0 for 37 °C. The values ar logarithmic averages of several independent FTSA experiments.

A correlation map between chemical structures and binding affinities showing apparent dissociation constants of compounds for CAI, CAII, CAIX, and CAXII, in nM units. Apparent affinities are listed next to compound structures and the ratios of Kd,app – above or below the arrows connecting compounds that are compared. The two left compounds, both upper and lower, located to the left of the vertical dashed line do not form covalent bonds with the proteins, while the rest of the compounds form the covalent bond.