Switching the Inhibitor-Enzyme Recognition Profile via Chimeric Carbonic Anhydrase XII

Abstract

A key part of the optimization of small molecules in pharmaceutical inhibitor development is to vary the molecular design to enhance complementarity of chemical features of the compound with the positioning of amino acids in the active site of a target enzyme. Typically this involves iterations of synthesis, to modify the compound, and biophysical assay, to assess the outcomes. Selective targeting of the anti-cancer carbonic anhydrase isoform XII (CA XII), this process is challenging because the overall fold is very similar across the twelve CA isoforms. To enhance drug development for CA XII we used a reverse engineering approach where mutation of the key six amino acids in the active site of human CA XII into the CA II isoform was performed to provide a protein chimera (chCA XII) which is amenable to structure-based compound optimization. Through determination of structural detail and affinity measurement of the interaction with over 60 compounds we observed that the compounds that bound CA XII more strongly than CA II, switched their preference and bound more strongly to the engineered chimera, chCA XII, based on CA II, but containing the 6 key amino acids from CA XII, behaved as CA XII in its compound recognition profile. The structures of the compounds in the chimeric active site also resembled those determined for complexes with CA XII, hence validating this protein engineering approach in the development of new inhibitors.

Keywords: X-ray diffraction; drug design; enzyme models; fluorescence spectroscopy; sulfonamides.

Figure 1

The general scheme of engineering the chimeric CA XII (chCA XII). The CA XII‐selective inhibitor is shown in gray. The hypothetical inhibitor is unable to bind to CA II (violet) but is supposed to bind well to chCA XII (violet‐green) and CA XII (green). To obtain chCA XII, six residues in the active site of CA II were replaced by the ones present in CA XII at the same locations of the active site (N67→K67, A65→S65, I91→T91, F130→A130, V134→S134, and L203→N203).

Figure 2

Experimental data of stability, catalytic activity and inhibitor binding of CA II (violet squares), CA XII (dark green circles) and chCA XII (light green squares) by FTSA (panels A, C, E, H), ITC (panels D, F) and SFA (panels G, I) techniques. (A) Thermal stability profiles of CA's at pH 4.5–10.0. (B) Catalytic constant of CA dependence on pH in the range 6.0–9.0. (C) Unfolding curves of the proteins at 0, 25 and 200 μM inhibitor 24 concentrations. (D) Raw curves of CA titrations with inhibitor 13. (E) Thermal shifts of CA melting temperature at increasing inhibitor 24 concentrations up to 200 μM. (F) Integrated titration curves for inhibitor 13 binding to CA. (G) Acidification curves of spontaneous and catalyzed CO₂ hydration reaction by CA at several inhibitor 34 concentrations. (H) Correlations of observed pK _d’s between chCA XII and CA XII (R²=0.90) or CA II (R²=0.24). The theoretically perfect positive correlation is represented as a black dashed line and linear regression models are shown as green (y=1.03x–0.31) and violet (y=0.52x+3.20) lines. (I) Dose‐response curves of inhibition of CA enzymatic activity by 34. Overall, all shown figures here confirm that the chCA XII protein is recognized by compounds similarly to CA XII, but different from CA II.

Figure 3

Chemical structures of compounds 1–34 used in this study arranged according to their selectivity. Benzenesulfonamides 1–22 bound more than 10‐fold stronger to CA II than to CA XII, while compounds 23–34 were more than 10‐fold stronger binders of CA XII.

Figure 4

X‐ray crystallographic structures of CA II‐selective compounds bound to CA II, chCA XII and CA XII. Zn(II) is shown as magenta sphere. Black line denotes the shape of the active site, whereas blue transparent ellipse denotes the hydrophilic part of active site which contains water molecules. Protein side chains and ligands bound to CA II are colored magenta, chCA XII‐light green, CA XII‐dark green. (A) Compound 6 bound in the active site of CA II (PDB: 4KNJ), chCA XII (PDB: 6YH5) and CA XII (PDB: 4KP5). (B) Compound 9 bound in the active site of CA II (PDB: 4WW6), chCA XII (PDB: 6YHA) and CA XII (PDB: 5MSA). (C) Compound 11 bound in the active site of CA II (PDB: 4PZH), chCA XII (PDB: 6YH4) and CA XII (PDB: 5MSB). (D) Compound 13 bound in the active site of CA II (PDB: 4HT0), chCA XII (PDB: 6YHB) and CA XII (PDB: 4HT2).

Figure 5

CA XII‐selective inhibitors: 26 (EA12‐3), 29 (EA12‐7) and 30 (EA11‐7) and their affinities for all twelve human CA isoforms determined by fluorescent thermal shift assay. The white line on the bars represents the limit of affinity determination (K _d 200 μM). Substitution of sulfur atom to nitrogen atom in the para position decreased the binding affinity for all 12 CAs, but increased inhibitor selectivity for CA XII over CA II from 33‐fold to 155‐fold and up to 10000‐fold over remaining 10 CA isoforms. However, additional methylation of carboxy group at meta substituent increased the binding affinity for all CAs up to 25‐fold, but reduced selectivity towards CA XII.

Figure 6

Chemical structures of compounds 35–63, that bound to CA II and CA XII with similar binding affinities (<10‐fold differences). Their binding affinities were also close for chCA XII and thus were uninformative about the switch of affinities in the chimeric protein.

Figure 7

X‐ray crystallographic structures of several nonselective compounds bound to CA II, chCA XII and CA XII. Zn(II) is shown as magenta sphere. Black line denotes the shape of active site, whereas blue transparent area marked the hydrophilic part of active site which contains water molecules. Protein side chains and ligands bound to CA II are colored magenta, chCA XII‐light green, CA XII‐dark green. (A) Compounds 43 (PDB: 6YH8) and 44 (magenta, PDB: 6YH6) bound in chCA XII. (B) Compound 48 bound in CA II (PDB: 4QIY), chCA XII (PDB: 6YH9) and CA XII (PDB: 4QJ0, main orientation (from 4 protein subunits: 3 vs 1)). (C) Compound 50 bound in CA II (PDB: 5DRS), chCA XII (PDB: 6YH7) and CA XII (PDB: 5LLO). (D) Compound 52 bound in CA II (PDB: 5EHE), chCA XII (PDB: 6YHC) and CA XII (PDB: 4QJW).