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

Anyaporn Sangkaew,Jerapan Krungkrai,Chulee Yompakdee

doi:10.1186/s13568-018-0653-9

Abstract

Carbonic anhydrase (CA; EC 4.2.1.1) catalyzes the reversible hydration of carbon dioxide (CO2) 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 (Δnce103 strain) can grow under a high CO2 condition (5% (v/v) CO2) but not at an ambient level. We constructed Δnce103 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 CO2 condition. Using a multidrug-sensitive derivative of the Δnce103 strain expressing a low level of hCAII, we finally established a high throughput in vivo assay for hCAII inhibitors under a low CO2 condition. Cytotoxicity of the candidates obtained could be simultaneously determined under a high CO2 condition. However, their inhibitory activities against other CA isozymes remains to be established by further investigation.

Highlights

Carbonic anhydrases (CAs, EC 4.2.1.1) belong to the metalloenzymes family of proteins
Construction of a drug‐sensitive derivative of the nce103 null mutant of S. cerevisiae expressing various levels of human CA isozyme II (hCAII) from modified GAL1‐promoter‐based expression cassettes The yeast S. cerevisiae is generally highly tolerant to various drugs, which poses a serious obstacle for their use in a yeast-based in vivo drug assay.The yeast strain BY25929, was modified to attenuate the general permeability barriers for drugs by disruption of the ERG3 gene, which is involved in the biosynthesis of ergosterol, a major component of the cell membrane (Hemmi et al 1995)
In this study, a novel yeast-based screening system for the detection of compounds that could inhibit the function of hCAII in vivo was successfully established using a high drug sensitive nce103 null yeast mutant expressing hCAII as indicator cells

Summary

Introduction

Carbonic anhydrases (CAs, EC 4.2.1.1) belong to the metalloenzymes family of proteins They are a class of enzymes catalyzing the simple but physiologically essential process of carbon dioxide (CO2) hydration to bicarbonate and proton (CO2 + H2O ↔ HCO3− + H+). Sulfonamide compounds, which are classical CAI, have been used as commercial drugs for the treatment of glaucoma, epilepsy, edema and altitude sickness (Supuran 2008) They can inhibit all CA isoforms nonspecifically, diluting the drug effectiveness and causing undesired side effects from the off-target inhibition. CAIs were screened for by in vitro methods using a biochemical strategy, but this approach has several disadvantages It provides no information about drug uptake into cells, drug stability and, in particular, the cytotoxicity of the compounds (Bilsland et al 2013). Such disadvantages could be improved by using an in vivo assay

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