Abstract

The HTS (high-throughtput screening) is widely used in the field of pharmaceuticals and academic institutes as a primary tool for early-stage drug discovery. This technique accelerates the screening of a large number of potential biological modulators against selected and specific targets. The aim of this screening compaign was to identify new inhibitors of human carbonic anhydrase IX (CA IX). This enzyme plays a crucial role in cancer cell proliferation and metastases. Its overexpression in hypoxic tumors is associated with malignant progression and poor treatment outcome. Therefore the inhibition of CA IX activity could be a promising approach to novel anticancer therapies. To identify small molecule inhibitors of CA IX in HTS conditions a new fluorescence-based assay was designed. In this assay, pyranine, as a fluorescent indicator of pH change, was used. The assay was validated on the Prestwick Chemical Library. Then in the primary screen, the inhibition activity of over 10.000 unique compounds from IMTM Proprietary Library was analyzed at one concentration (10 µM). The PI (percentage of inhibition) values were calculated for all tested compounds and those with highest inhibition activity were selected as hits. To quantify the suitability of the assay in HTS, the Z-factor was determined for each plate. Data were analyzed by Dotmatics software. Obtained results of validation and primary screen as well as selected hits will be presented and discussed. This work was supported by European Union – Programme EXCELES, ID Project No. LX22NPO5102, the Czech Ministry of Education, Youth and Sports (CZ-OPENSCREEN - LM2023052, EATRIS-CZ - LM2023053), and by the Internal Grant of Palacky University Olomouc (IGA_LF_2023_025) and European Regional Development Fund - Project ENOCH (No. CZ.02.1.01/0.0/0.0/16_019/0000868).