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

• Published in: ChemistryOpen (Weinheim) 2021-05, Vol.10 (5), p.567-580
• Main Authors: Smirnovienė, Joana, Smirnov, Alexey, Zakšauskas, Audrius, Zubrienė, Asta, Petrauskas, Vytautas, Mickevičiūtė, Aurelija, Michailovienė, Vilma, Čapkauskaitė, Edita, Manakova, Elena, Gražulis, Saulius, Baranauskienė, Lina, Chen, Wen‐Yih, Ladbury, John E., Matulis, Daumantas
• Format: Article
• Language: English
• Subjects: Amides - chemistry; Amino Acid Sequence; Amino acids; Binding sites; Cancer; Carbonic anhydrase; Carbonic Anhydrase Inhibitors - chemistry; Carbonic Anhydrase Inhibitors - metabolism; Carbonic Anhydrases - metabolism; Catalytic Domain; Chimera - metabolism; Crystallization; Drug Design; enzyme models; Enzymes; fluorescence spectroscopy; Glaucoma; Humans; Hydration; Hydrophobic and Hydrophilic Interactions; Inhibitors; Ligands; Medical prognosis; Models, Molecular; Mutant Proteins; Mutation; Optimization; Physiology; Protein Binding; Protein Conformation; Protein Isoforms; Proteins; Recognition; Reverse engineering; Structure-Activity Relationship; sulfonamides; Sulfonamides - chemistry; Sulfonamides - pharmacology; X-ray diffraction
• ISSN: 2191-1363; 2191-1363
• DOI: 10.1002/open.202100042

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. Chimeric carbonic anhydrase XII (chCA XII), based on CA II with six key amino acids in the active site mutated according to CA XII, recognized ligands as CA XII and not CA II. The compounds that bound CA XII more strongly than CA II switched their preference and interacted with higher affinity with chCA XII.