Fluorinated benzenesulfonamide anticancer inhibitors of carbonic anhydrase IX exhibit lower toxic effects on zebrafish embryonic development than ethoxzolamide

Abstract

The toxic effects of two recently discovered inhibitors (VD12-09 and VD11-4-2) that selectively and with extraordinary strong, picomolar binding affinity to human carbonic anhydrase (CA) isoform IX were investigated on zebrafish embryonic development. CA IX has been recently introduced as an anticancer target since it is highly overexpressed in numerous human cancers but nearly absent in normal tissues. Morphological changes in zebrafish treated by the compounds were studied by light-field microscopy and histological analysis. Homology models of zebrafish CA II and CA IX were built to identify the conserved amino acid residues in the active site of zebrafish CAs. The toxicity studies here showed that the LC₅₀ values at 120 hours post-fertilization (hpf) were 13  μM for VD12-09, 120  μM for VD11-4-2, and 9  μM for ethoxzolamide (EZA), a non-selective CA inhibitor commonly used as a drug in clinics. Thus, EZA was the most toxic of the three compounds. The zebrafish embryos exposed to LC₅₀ doses of VD12-09 and VD11-4-2 showed fewer phenotypic abnormalities compared with the embryos exposed to the corresponding dose of EZA. Histochemical studies did not show any gross morphological changes in the embryos treated with VD12-09 and VD11-4-2 unlike EZA. The results of our study indicate that the compounds exhibited 10-fold lower toxicity and induced fewer side effects in zebrafish than EZA. Therefore, the exposure to VD11-4-2 and VD12-09 at concentrations below LC₅₀ did not lead to deleterious effects on the zebrafish embryonic development and thus both inhibitors may be further developed as drugs.

• Carbonic anhydrase IX
• zebrafish toxicity
• benzenesulfonamides

Acknowledgements

Authors thank Aulikki Lehmus and Marrianne Kuuslahti for the skillful technical assistance with the experiments. The authors thank Alma Yrjänäinen and Linda Urbański for their help with histochemical analysis experiments. Thanks are also due to Leena Mäkinen, Hannaleena Piippo for their help with zebrafish embryos. The authors thank Prof. Saulius Šumanas for his valuable comments and discussion.

Declaration of interest

The authors have no competing interests. This research was partially funded by the Research Council of Lithuania (No. TAP LLT-1/2016).