0256: A screening strategy to identify modulators of the mitochondrial ADP/ATP translocase (ANT) for cardioprotection

In many pathological conditions, a sudden lack of energy, accompanied by an increased reactive oxygen (ROS) level and calcium overload, accelerates cell death with necrotic features. As the mitochondrion is the main source of cellular ATP and ROS, it has become an attractive therapeutic target for human pathology such as cardiac, neuronal and hepatic ischemia-reperfusion injury. In this context, we and others have shown that pharmacological modulation of ANT can prevent three acute pathologies in mice1 and recently, we hypothesized that activation of ANT might be an innovative strategy for cardioprotection. Here, we present the design and the implementation of enzymatic, cellular and subcellular assays and their optimization for the screening of modulators of ANT (i.e. inhibitors and/or activators). We miniaturized the ADP/ATP exchange measurement in cardiac interfibrillar and subsarcolemmal isolated mitochondria from rat heart, evaluated its robustness (Z-factor) in 96 well-microtiter plates and screened an in silico-generated library of ANT-potential ligands and small molecules, i.e. about 100 molecules. Cytotoxicity was evaluated in rat H9c2 and human HT29 cell lines and non-cytotoxic compounds (i.e. with LD50 >200 μM) were selected for further studies. To confirm potential hits activity directly on ANT, we purified the native protein from rat hearts in the presence of Triton X-100, confirmed its purity by western-blot and incorporated it into small unilamellar vesicles (100 nm of diameter) as described.2 Validation of a novel ANT-containing proteoliposome bioluminescent assay is currently on-going to develop a method to quantify the ability of molecules to modulate ADP/ATP exchange efficacy in dose-response and calculate EC50. If robust, this assay will be adapted to our high throughput screening platform. For a therapeutic perspective, identified molecules will be evaluated for their toxicity, druggability, and for their ability to influence the disease outcome in animal models as a prerequisite for future clinical studies.