Intrinsic binding of 4‐substituted‐2,3,5,6‐tetrafluorobenezenesulfonamides to native and recombinant human carbonic anhydrase VI

Kazokaitė, Justina; Milinavičiūtė, Goda; Smirnovienė, Joana; Matulienė, Jurgita; Matulis, Daumantas

doi:10.1111/febs.13196

Cited by 20 publications

(20 citation statements)

References 48 publications

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“…Therefore, it is essential to evaluate inhibitor affinity to all human CAs, including CA VI. Since our previous study 40 indicated a low yield of recombinant CA VI from E . coli , we generated CA II mutant as a CA VI model protein (CA VI-mimic) for inhibitor screening.…”

Section: Resultsmentioning

confidence: 99%

Engineered Carbonic Anhydrase VI-Mimic Enzyme Switched the Structure and Affinities of Inhibitors

Kazokaitė

Kairys

Smirnovienė

et al. 2019

Sci Rep

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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 CO 2 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.

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Section: Resultsmentioning

confidence: 99%

Engineered Carbonic Anhydrase VI-Mimic Enzyme Switched the Structure and Affinities of Inhibitors

Kazokaitė

Kairys

Smirnovienė

et al. 2019

Sci Rep

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“…CA VI is a potential drug target for cariogenesis and cancer of the salivary glands. It is the only secreted human CA isozyme that is found in saliva and milk [Kazokaitė et al, 2015]. The relevance of CA VI in oral health is still underinvestigated.…”

Section: Discussionmentioning

confidence: 99%

Children with a Higher Activity of Carbonic Anhydrase VI in Saliva Are More Likely to Develop Dental Caries

Picco¹,

Lopes²,

Marques³

et al. 2017

Caries Res

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Objective: This study aimed to analyze the concentration and activity of carbonic anhydrase (CA) VI in the saliva of school children. We investigated the relationship among caries, CA VI concentration/activity, flow rate, pH, and buffering capacity. Materials and Methods: Seventy-four school children were divided into a caries-free group and a caries group. Clinical examinations were conducted by one examiner according to World Health Organization criteria + early caries lesions. Salivary flow rate, pH, and buffering capacity were analyzed. Salivary CA VI concentration and activity were evaluated by ELISA and zymography, respectively. The data were analyzed using Student's t test and the Mann-Whitney test, and Pearson and Spearman correlation analyses were also done. In multivariate modeling, associations between variables were expressed as odds ratios. Results: The results showed that salivary flow rate, salivary pH, and BC were significantly higher in the saliva of caries-free children. Also, the salivary CA VI concentration was significantly higher in the saliva of caries-free children. The salivary CA VI activity was higher in children with caries. We found a negative correlation between BC and dental caries. Also, in the caries group we found a positive correlation between the concentration and the activity of CA VI and a negative correlation between BC and CA VI activity. A negative correlation between salivary pH and CA VI concentration was observed in the caries-free group. A high activity of CA and a low salivary flow rate were associated with dental caries. Conclusion: These results support the conclusion that dental caries is highly affected by the activity of CA VI in saliva as well as by the salivary flow rate.

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“…The standard error of the K d measurements is approximately 1.69 of the K d value isoforms [50][51][52][53][54] and are explained by the binding-linked protonation equilibria. First, a sulfonamide compound has to undergo deprotonation in order to bind to the Zn in the active center of the enzyme.…”

Section: Inhibitor Binding To Ca Vb By Itcmentioning

confidence: 94%

Thermodynamic characterization of human carbonic anhydrase VB stability and intrinsic binding of compounds

Kasiliauskaitė

Časaitė

Juozapaitienė

et al. 2015

J Therm Anal Calorim

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The thermodynamics of low molecular weight synthetic sulfonamide inhibitor binding to carbonic anhydrase (CA) VB was determined by the isothermal titration calorimetry (ITC) and the fluorescent thermal shift assay (FTSA). ITC provided the enthalpic and entropic contributions to the binding affinity of ethoxzolamide to CA VB. FTSA is a high-throughput assay that measures protein thermal stabilization by added ligands. FTSA enabled determination of extremely high affinity of several compounds binding to CA VB. CA VB is one of two isoforms that are expressed in mitochondria, participate in carbon metabolism and pH homeostasis and are implicated in diseases such as obesity. Therefore CA VB is a drug target. Here a series of para-substituted tetrafluoro benzenesulfonamides were investigated as high affinity inhibitors of CA VB. Thermodynamic equilibrium binding measurements such as ITC and FTSA provide only the observed parameters. Dissection of binding-linked reactions is necessary to obtain the intrinsic parameters that in turn could be correlated with the chemical structure of the inhibitors. Intrinsic dissociation constants of the inhibitors were estimated and they reached 1 pM, one of the strongest binding reactions observed between any protein-ligand binding.

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Intrinsic binding of 4‐substituted‐2,3,5,6‐tetrafluorobenezenesulfonamides to native and recombinant human carbonic anhydrase VI

Cited by 20 publications

References 48 publications

Engineered Carbonic Anhydrase VI-Mimic Enzyme Switched the Structure and Affinities of Inhibitors

Engineered Carbonic Anhydrase VI-Mimic Enzyme Switched the Structure and Affinities of Inhibitors

Children with a Higher Activity of Carbonic Anhydrase VI in Saliva Are More Likely to Develop Dental Caries

Thermodynamic characterization of human carbonic anhydrase VB stability and intrinsic binding of compounds

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