Abstract 350: Study of A3 adenosine receptor interactions and identification of novel ant-/agonist using a specific fluorescent probe

Abstract Introduction: Adenosine receptors belong to G protein-coupled receptors (GPCRs). There are four types of adenosine receptors (A1, A2A, A2B, and A3). These receptors play roles in several pathological conditions. It has been proven that the A3 adenosine receptor (A3AR) is expressed at high densities in various tumor cells. Activation of this receptor leads, in some cases, to tumor growth and cell proliferation and, in other cases, to activation of apoptotic pathways and mediation of antiproliferative effects. Therefore A3AR is a promising therapeutic target in anticancer therapy. Here we introduce the microscopic technique for A3AR visualization and functional studies using a novel A3AR-specific fluorescent probe. Our method could be used in living cells and competitive binding assays to identify and validate novel A3AR agonists and antagonists. Material and methods: The reporter cell line overexpressing A3AR was treated by the A3AR-specific fluorescent probe (fluorescently labeled A3AR antagonist - CELT-228; Celtarys, Spain), the fluorescent images were taken by spinning disc confocal microscopy (Yokogawa CV8000) at different timepoints and concentrations. Then the localization and intensity of the signal were analyzed by Columbus HCA software. The competitive assay based on the CELT-228 fluorescent probe was developed to study interactions of the potential A3AR agonists or antagonists and used for the High Content Screening of newly synthesized nucleoside-based compounds. The competition assay was used to test the interaction of newly identified A3AR ant-/agonists on cancer cell lines derived from tumors of various histogenetic origins with A3AR expression. The results were correlated with cytotoxicity and molecular pathway alterations induced by the studied compounds. Results: The fluorescent signal of the bound probe was localized on the cell membrane of the reporter cell line and the majority of the cancer cell lines. Interestingly in some cell lines, such as MIA PaCa-2, the signal was also observed in the cytoplasm, which will be further studied. Our data from competition assays indicated that the newly synthesized potential anticancer compound binds to the same (orthosteric) binding site on the A3 receptor and competes for it with the fluorescent-labeled probe. Conclusions: We introduced the technique suitable for observing A3R expression, small molecule competition monitoring, and confirmation of binding to A3AR of novel compounds even in native conditions. This work was supported by European Union - Programme EXCELES, ID Project No. LX22NPO5102, the Czech Ministry of Education, Youth and Sports (CZ-OPENSCREEN - LM2018130, EATRIS-CZ - LM2018133), and by the internal grant of Palacky University Olomouc (IGA_LF_2022_033) and European Regional Development Fund - Project ENOCH (No. CZ.02.1.01/0.0/0.0/16_019/0000868). Citation Format: Katerina Jecmenova, Jana Kotulova, Sona Gurska, Maria Majellaro, Marian Hajduch, Eddy Sotelo, Claudia Gioe-Gallo, Petr Dzubak. Study of A3 adenosine receptor interactions and identification of novel ant-/agonist using a specific fluorescent probe [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 350.