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Citation
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HERO ID
3603720
Reference Type
Journal Article
Title
Picomolar inhibitors of carbonic anhydrase: Importance of inhibition and binding assays
Author(s)
Smirnovienė, J; Smirnovas, V; Matulis, D
Year
2017
Is Peer Reviewed?
Yes
Journal
Analytical Biochemistry
ISSN:
0003-2697
EISSN:
1096-0309
Volume
522
Page Numbers
61-72
Language
English
PMID
28153585
DOI
10.1016/j.ab.2017.01.022
Web of Science Id
WOS:000395228100008
URL
http://
://WOS:000395228100008
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Abstract
The Ki of carbonic anhydrase (CA) inhibitors is often determined by the stopped- flow CO2 hydration assay, the method that directly follows the inhibition of CA enzymatic activity. However, the assay has limitations, such as largely unknown concentration of CO2 and the inability to determine the Ki below several nM. The widely used direct binding assay, isothermal titration calorimetry, also does not determine the Kd below several nM. In contrast, the thermal shift assay can accurately determine picomolar affinities. New equations estimating CO2 concentration were developed for the determination of kcat and KM of CA I and CA II. The inhibitor dose-response curves were analyzed using Hill and Morrison equations demonstrating that only the Morrison model is applicable for the determination of tight-binding inhibitor Ki. The measurements of interactions between ten inhibitors and seven CA isoforms showed the limitations and advantages of all three techniques. Inhibitor 6 exhibited the Kd of 50 pM and was highly selective towards human CA IX, an isoform which is nearly absent in healthy human, but highly overexpressed in numerous cancers. Combination of inhibition and binding techniques was necessary for precise determination of CA-high-affinity inhibitor interactions and future drug design.
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Naphthalene
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Naphthalene (2021 Evidence mapping publication)
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