Development of a high throughput yeast-based screening assay for human carbonic anhydrase isozyme II inhibitors

• Published in: AMB Express Vol. 8; no. 1; pp. 124 - 12
• Main Authors: Sangkaew, Anyaporn, Krungkrai, Jerapan, Yompakdee, Chulee
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer Berlin Heidelberg 04.08.2018; Springer Nature B.V; SpringerOpen
• Subjects: Assaying; Baking yeast; Bicarbonates; Biocompatibility; Biomedical and Life Sciences; Biotechnology; Carbon dioxide; Carbonic anhydrase; Carbonic anhydrase inhibitor; Carbonic anhydrases; Cytotoxicity; Human carbonic anhydrase isozyme II; In vitro methods and tests; Inhibitors; Isoenzymes; Life Sciences; Localization; Low level; Microbial Genetics and Genomics; Microbiology; NCE103; Original; Original Article; Pharmacology; Reaction kinetics; Resazurin; Saccharomyces cerevisiae; Screening; Toxicity; Yeast; Yeast-based assay; Resazurin; Yeast-based assay; Human carbonic anhydrase isozyme II; Carbonic anhydrase inhibitor; Saccharomyces cerevisiae; NCE103
• ISSN: 2191-0855; 2191-0855
• DOI: 10.1186/s13568-018-0653-9

Carbonic anhydrase (CA; EC 4.2.1.1) catalyzes the reversible hydration of carbon dioxide (CO 2 ) to bicarbonate and proton. There are 16 known isozymes of α-CA in humans, which differ widely in their kinetics, subcellular localization and tissue-specific distribution. Several disorders are associated with abnormal levels of CA, and so the inhibition of CA has pharmacological application in the treatment of many diseases. Currently, searching for novel CA inhibitors (CAI) has been performed using in vitro methods, and so their toxicity remains unknown at the time of screening. To obtain potentially safer CAIs, a screening procedure using an in vivo assay seems to have more advantages. Here, we developed a yeast-based in vivo assay for the detection of inhibitors of the human CA isozyme II (hCAII). The yeast Saccharomyces cerevisiae mutant deprived of its own CA (Δ nce103 strain) can grow under a high CO 2 condition (5% (v/v) CO 2 ) but not at an ambient level. We constructed Δ nce103 strains expressing various levels of hCAII from a plasmid harboring the hCAII gene arranged under the control of variously modified GAL1 promoter and relying on the expression of hCAII for growth under low CO 2 condition. Using a multidrug-sensitive derivative of the Δ nce103 strain expressing a low level of hCAII, we finally established a high throughput in vivo assay for hCAII inhibitors under a low CO 2 condition. Cytotoxicity of the candidates obtained could be simultaneously determined under a high CO 2 condition. However, their inhibitory activities against other CA isozymes remains to be established by further investigation.