Abstract
Secretory human carbonic anhydrase VI (CA VI) has emerged as a potential drug target due to its role in pathological states, such as excess acidity-caused dental caries and injuries of gastric epithelium. Currently, there are no available CA VI-selective inhibitors or crystallographic structures of inhibitors bound to CA VI. The present study focuses on the site-directed CA II mutant mimicking the active site of CA VI for inhibitor screening. The interactions between CA VI-mimic and a series of benzenesulfonamides were evaluated by fluorescent thermal shift assay, stopped-flow CO2 hydration assay, isothermal titration calorimetry, and X-ray crystallography. Kinetic parameters showed that A65T, N67Q, F130Y, V134Q, L203T mutations did not influence catalytic properties of CA II, but inhibitor affinities resembled CA VI, exhibiting up to 0.16 nM intrinsic affinity for CA VI-mimic. Structurally, binding site of CA VI-mimic was found to be similar to CA VI. The ligand interactions with mutated side chains observed in three crystallographic structures allowed to rationalize observed variation of binding modes and experimental binding affinities to CA VI. This integrative set of kinetic, thermodynamic, and structural data revealed CA VI-mimic as a useful model to design CA VI-specific inhibitors which could be beneficial for novel therapeutic applications.
Highlights
carbonic anhydrase VI (CA VI) is the only secreted human CA isoform found in saliva[10], serum[11], milk[12], respiratory airways[13], and alimentary canal[14]
Inhibitor affinities towards CA II and CA VI-mimic were expected to differ in the way imitating inhibitor binding to CA VI, but not CA II (Fig. 1A)
Nowadays enzymes encompass over one-third of drug targets investigated by large pharmaceutical companies[43], thereby emphasizing the relevance of target-based drug approach
Summary
CA VI is the only secreted human CA isoform found in saliva[10], serum[11], milk[12], respiratory airways[13], and alimentary canal[14]. Www.nature.com/scientificreports limitations, such as the largely unknown CO2 concentration and unfeasibility to measure inhibition constant below several nM26 Biophysical techniques, such as the fluorescent thermal shift assay (FTSA) and isothermal titration calorimetry (ITC), are promising alternatives to screen CA-targeting derivatives. Most studies on the development of CA inhibitors usually provide only observed binding parameters, which are dependent on experimental conditions and might be misleading. Both the CA and inhibitor exist in different protonation states in the solution compared with ones in the complex. Due to the recent advances in the structural and in silico biology, production of target recombinant proteins, including CAs, in large quantities is of high demand for in vitro inhibitor screening of drug-candidates during preclinical research. Different, more efficient strategies to obtain functionally active recombinant proteins in high yield are required for screens of chemical compounds with the aim to identify hits in the initial stages of drug discovery
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