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Pharmaceuticals are found in the environment but the impact of this contamination on human and animal health is poorly known. The liver could be particularly targeted since a significant number of these drugs are hepatotoxic, in particular via oxidative stress and mitochondrial dysfunction. Notably, the latter events can also be observed in liver diseases linked to obesity, so that the obese liver might be more sensitive to drug toxicity. In this study, we determined the effects of a chronic exposure to low doses of pharmaceuticals in wild-type and obese mice, with a particular focus on mitochondrial function. To this end, wild-type and ob/ob mice were exposed for 4 months to a cocktail of 11 pharmaceuticals provided in drinking water containing 0.01, 0.1, or 1 mg/L of each drug. At the end of the treatment, liver mitochondria were isolated and different parameters were measured. Chronic exposure to the pharmaceuticals reduced mitochondrial respiration driven by succinate and palmitoyl-l-carnitine in wild-type mice and increased antimycin-induced ROS production in ob/ob mice. Hyperglycemia and hepatic histological abnormalities were also observed in treated ob/ob mice. Investigations were also carried out in isolated liver mitochondria incubated with the mixture, or with each individual drug. The mitochondrial effects of the mixture were different from those observed in treated mice and could not be predicted from the results obtained with each drug. Because some of the 11 drugs included in our cocktail can be found in water at relatively high concentrations, our data could be relevant in environmental toxicology. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1375-1389, 2017.

Mitochondria deserve special attention as sensors of cellular energy homeostasis and metabolic state. Moreover, mitochondria integrate intra- and extra-cellular signals to determine appropriate cellular responses that range from proliferation to cell death. In autoimmunity, as in other inflammatory chronic disorders, the metabolism of immune cells may be extensively remodeled, perturbing sensitive tolerogenic mechanisms. Here, we examine the distribution and effects of the therapeutic 21-mer peptide called P140, which shows remarkable efficacy in modulating immune responses in inflammatory settings. We measured P140 and control peptide effects on isolated mitochondria, the distribution of peptides in live cells, and their influence on the levels of key autophagy regulators. Our data indicate that while P140 targets macro- and chaperone-mediated autophagy processes, it has little effect, if any, on mitochondrial autophagy. Remarkably, however, it suppresses NET release from neutrophils exposed to immobilized NET-anti-DNA IgG complexes. Together, our results suggest that in the mitochondrion-lysosome axis, a likely driver of NETosis and inflammation, the P140 peptide does not operate by affecting mitochondria directly.

International audience; Mitochondrial dysfunction is a major mechanism whereby different drugs can induce liver injury such as cytolytic hepatitis, microvesicular and/or macrovacuolar steatosis, steatohepatitis, and possibly cholestasis. In the most severe cases, drug-induced mitochondrial dysfunction and liver injury can require liver transplantation or lead to the death of the patient. Moreover, these adverse effects can lead to the withdrawal of drugs from the market, or earlier during clinical trials. Drugs can induce mitochondrial dysfunction by different mechanisms including inhibition of fatty acid oxidation, impairment of oxidative phosphorylation and respiratory chain activity, and alteration of the integrity of the mitochondrial membranes. The present chapter focuses on different drugs for which enough clinical and experimental evidence indicates the potential role of mitochondrial dysfunction in the pathogenesis of liver injury acetaminophen, amiodarone, fialuridine, linezolid, nucleoside reverse transcriptase inhibitors (e.g., stavudine, zidovudine, and didanosine), tamoxifen, tetracycline, troglitazone, and valproic acid. Because drug-induced mitochondrial dysfunction and liver injury are major issues for public health and pharmaceutical companies, mitochondrial liability should be systematically investigated during preclinical safety studies. © 2018 John Wiley and Sons, Inc. All rights reserved.

International audience; Mitochondrial dysfunction can be a major mechanism whereby different drugs can induce adverse effects affecting different tissues such as liver, heart and skeletal muscle. In the most severe cases, drug-induced mitochondrial dysfunction can require a hospitalization, or lead to the death of the patient. Moreover, these adverse effects can lead to the withdrawal of drugs from the market, or earlier during clinical trials. Drugs can induce mitochondrial dysfunction by different mechanisms including inhibition of fatty acid oxidation, impairment of oxidative phosphorylation and respiratory chain activity as well as alteration of the integrity of the mitochondrial membranes. Some drugs also impair mitochondrial function via the production of reactive oxygen species and the generation of reactive metabolites, which can covalently bind to key mitochondrial proteins. The present chapter focuses on different drugs for which enough clinical and experimental evidence indicates the potential role of mitochondrial dysfunction in the pathogenesis of adverse effects such as liver injury, myopathy and cardiotoxicity acetaminophen, amiodarone, doxorubicin, nucleoside reverse transcriptase inhibitors (e.g. stavudine, zidovudine, didanosine), statins (e.g. atorvastatin, cerivastatin, simvastatin) and valproic acid. Notably, these drugs epitomize the diversity of the mechanisms whereby xenobiotics can induce mitochondrial dysfunction and also the variety of the targeted tissues. Other drugs affecting mitochondrial function by similar mechanisms are discussed more briefly in the present chapter. Because drug-induced mitochondrial dysfunction and related adverse events are major issues for public health and pharmaceutical companies, mitochondrial liability should be systematically investigated during preclinical safety studies. © Springer International Publishing AG, part of Springer Nature 2018.

International audience; Drug-induced liver injury (DILI) in humans is difficult to predict using classical in vitro cytotoxicity screening and regulatory animal studies. This explains why numerous compounds are stopped during clinical trials or withdrawn from the market due to hepatotoxicity. Thus, it is important to improve early prediction of DILI in human. In the present study, we hypothesized that this goal could be achieved by investigating drug-induced mitochondrial dysfunction as this toxic effect is a major mechanism of DILI. To this end, we developed a high-throughput screening platform using isolated mouse liver mitochondria. Our broad spectrum multiparametric assay was designed to detect the global mitochondrial membrane permeabilization (swelling), inner membrane permeabilization (transmembrane potential), outer membrane permeabilization (cytochrome c release) and alteration of mitochondrial respiration driven by succinate or malate/glutamate. A pool of 124 chemicals (mainly drugs) was selected, including 87 with documented DILI and 37 without reported clinical hepatotoxicity. Our screening assay revealed an excellent sensitivity for clinical outcome of DILI (94 or 92% depending on cut-off) and a high positive predictive value (89 or 82%). A highly significant relationship between drug-induced mitochondrial toxicity and DILI occurrence in patients was calculated (P

The adenine nucleotide translocator (ANT) is a mitochondrial bi-functional protein, which catalyzes the exchange of ADP and ATP between cytosol and mitochondria and participates in many models of mitochondrial apoptosis. The human adenine nucleotide translocator sub-family is composed of four isoforms, namely ANT1-4, encoded by four nuclear genes, whose expression is highly regulated. Previous studies have revealed that ANTI and 3 induce mitochondrial apoptosis, whereas ANT2 is anti-apoptotic. However, the role of the recently identified isoform ANT4 in the apoptotic pathway has not yet been elucidated. Here, we investigated the effects of stable heterologous expression of the ANT4 on proliferation, mitochondrial respiration and cell death in human cancer cells, using ANT3 as a control of pro-apoptotic isoform. As expected, ANT3 enhanced mitochondria-mediated apoptosis in response to lonidamine, a mitochondriotoxic chemotherapeutic drug, and staurosporine, a protein kinase inhibitor. Our results also indicate that the pro-apoptotic effect of ANT3 was accompanied by decreased rate of cell proliferation, alteration in the mitochondrial network topology, and decreased reactive oxygen species production. Of note, we demonstrate for the first time that ANT4 enhanced cell growth without impacting mitochondria] network or respiration. Moreover, ANT4 differentially regulated the intracellular levels of hydrogen peroxide without affecting superoxide anion levels. Finally, stable ANT4 overexpression protected cancer cells from lonidamine and staurosporine apoptosis in a manner independent of Bcl-2 expression. These data highlight a hitherto undefined cytoprotective activity of ANT4, and provide a novel dichotomy in the human ANT isoform sub-family with ANT1 and 3 isoforms functioning as pro-apoptotic while ANT2 and 4 isoforms render cells resistant to death inducing stimuli. (C) 2010 Elsevier Ltd. All rights reserved.

Increased production of amyloid-β (Aβ)42 peptide, derived from the amyloid-β protein precursor, and its subsequent aggregation into oligomers and plaques constitutes a hallmark of Alzheimer's disease (AD). We here report on a family of low molecular weight molecules, the Aftins (Amyloid-β Forty-Two Inducers), which, in cultured cells, dramatically affect the production of extracellular/secreted amyloid peptides. Aftins trigger β-secretase inhibitor and γ-secretase inhibitors (GSIs) sensitive, robust upregulation of Aβ42, and parallel down-regulation of Aβ38, while Aβ40 levels remain stable. In contrast, intracellular levels of these amyloids appear to remain stable. In terms of their effects on Aβ38/Aβ40/Aβ42 relative abundance, Aftins act opposite to γ-secretase modulators (GSMs). Aβ42 upregulation induced by Aftin-5 is unlikely to originate from reduced proteolytic degradation or diminished autophagy. Aftin-5 has little effects on mitochondrial functional parameters (swelling, transmembrane potential loss, cytochrome c release, oxygen consumption) but reversibly alters the ultrastructure of mitochondria. Aftins thus alter the Aβ levels in a fashion similar to that described in the brain of AD patients. Aftins therefore constitute new pharmacological tools to investigate this essential aspect of AD, in cell cultures, allowing (1) the detection of inhibitors of Aftin induced action (potential 'anti-AD compounds', including GSIs and GSMs) but also (2) the identification, in the human chemical exposome, of compounds that, like Aftins, might trigger sustained Aβ42 production and Aβ38 down-regulation (potential 'pro-AD compounds').

International audience; Dengue viruses belong to the Flavivirus family and are responsible for hemorrhagic fever in Human. Dengue virus infection triggers apoptosis especially through the expression of the small membrane (M) protein. Using isolated mitochondria, we found that synthetic peptides containing the C-terminus part of the M ectodomain caused apoptosis-related mitochondrial membrane permeabilization (MMP) events. These events include matrix swelling and the dissipation of the mitochondrial transmembrane potential (DeltaPsi(m)). Protein M Flavivirus sequence alignments and helical wheel projections reveal a conserved distribution of charged residues. Moreover, when combined to the cell penetrating HIV-1 Tat peptide transduction domain (Tat-PTD), this sequence triggers a caspase-dependent cell death associated with DeltaPsi(m) loss and cytochrome c release. Mutational approaches coupled to functional screening on isolated mitochondria resulted in the selection of a protein M derived sequence containing nine residues with potent MMP-inducing properties on isolated mitochondria. A chimeric peptide composed of a Tat-PTD linked to the 9-mer entity triggers MMP and cell death. Finally, local administration of this chimeric peptide induces growth inhibition of xenograft prostate PC3 tumors in immuno-compromised mice, and significantly enhances animal survival. Together, these findings support the notion of using viral genomes as valuable sources to discover mitochondria-targeted sequences that may lead to the development of new anticancer compounds.

Mitochondria play a central role in the intrinsic pathway of apoptosis. In response to many pro-apoptotic stimuli, mitochondria undergo an irreversible process called mitochondrial membrane permeabilization (MMP). The detection of MMP in isolated mitochondria is most often based on assays that monitor either the loss of the inner transmembrane potential (DYm; classically with Rhodamine 123), permeability transition (PT, cyclosporin A-sensitive matrix swelling), or the release of critical pro-apoptotic intermembrane space effectors. To gain complementary information on MMP mechanisms, we have systematically used three additional assays optimized for the 96-well microplate format: (1) inner membrane permeability, (2) VDAC-associated NADH reductase activity, and (3) ATP/ADP translocase activity. We report that ad hoc combinations of ANT and VDAC ligands, carbonyl cyanide m-chlorophenylhydrazone (CCCP), mastoparan and Vpr52-96 peptide and PT inhibitors, permit to explore relationships between enzymatic functions of sessile mitochondrial proteins (i.e. ANT, VDAC) and MMP. These assays should be useful tools to investigate mitochondrial apoptosis, decipher the implication of inner and outer membrane permeabilization and provide a multi-parametric approach for drug discovery.

International audience; The HIV-1 encoded apoptogenic protein Vpr induces mitochondrial membrane permeabilization (MMP) via interactions with the voltage-dependent anion channel (VDAC) and the adenine nucleotide translocator (ANT). We have designed a peptide, TEAM-VP, composed of two functional domains, one a tumor blood vessel RGD-like 'homing' motif and the other an MMP-inducing sequence derived from Vpr. When added to isolated mitochondria, TEAM-VP interacts with ANT and VDAC, reduces oxygen consumption and overcomes Bcl-2 protection to cause inner and outer MMP. TEAM-VP specifically recognizes cell-surface expressed alpha(V)beta(3) integrins, internalizes, temporarily localizes to lysosomes and progressively co-distributes with the mitochondrial compartment with no sign of lysosomal membrane permeabilization. Finally TEAM-VP reaches mitochondria of angiogenic endothelial cells to induce mitochondrial fission, dissipation of the mitochondrial transmembrane potential (DeltaPsi(m)), cytochrome c release and apoptosis hallmarks. Hence, this chimeric peptide constitutes the first example of a virus-derived mitochondriotoxic compound as a candidate to kill selectively tumor neo-endothelia.

For many years, medical drug discovery has extensively exploited peptides as lead compounds. Currently, novel structures of therapeutic peptides are derived from active pre-existing peptides or from high-throughput screening, and optimized following a rational drug design approach. Molecules of interest may prove their ability to influence the disease outcome in animal models and must respond to a set of criteria based on toxicity studies, ease of administration, the cost of their synthesis, and logistic for clinical use to validate it as a good candidate in a therapeutic perspective. This applies to the potential use of peptides to target one central intracellular organelle, the mitochondrion, to modulate (i.e. activate or prevent) apoptosis. Putative mitochondrial protein targets and the strategies already elaborated to correct the defects linked to these proteins (overexpression, inactivation, mutation…, etc.) are described, and recent advances that led or may lead to the conception of therapeutic peptides via a specific action on these mitochondrial targets in the future are discussed.

During development as well as in pathological situations, neurons that fail to find appropriate targets or neurotrophic factors undergo cell death. Using primary cortical neurons subjected to acute serum-deprivation (SD), we have examined caspases activation, mitochondrial dysfunction and cell death parameters. Among a panel of metabolic, signaling and caspases inhibitors only those able to interfere with caspase-2 like activity protect primary neurons against SD-induced cell death. In situ detection and subcellular fractionation demonstrate a very early activation of cytosolic caspase-2, which controls Bax cleavage, relocalization and mitochondrial membrane permeabilization (MMP). Both z-VDVAD-fmk and a siRNA specific for caspase-2 abolish Bax changes, mitochondrial membranes permeabilization, as well as cytochrome c release-dependent activation of caspase-9/caspase-3, nuclear alterations, phosphatidylserine exposure, neurites dismantling and neuronal death. Hence, caspase-2 is an early checkpoint for apoptosis initiation in primary neurons subjected to serum deprivation.

International audience; The Trans-activator protein (Tat) of human immunodeficiency virus (HIV) is a pleiotropic protein involved in different aspects of AIDS pathogenesis. As a number of viral proteins Tat is suspected to disturb mitochondrial function. We prepared pure synthetic full-length Tat by native chemical ligation (NCL), and Tat peptides, to evaluate their direct effects on isolated mitochondria. Submicromolar doses of synthetic Tat cause a rapid dissipation of the mitochondrial transmembrane potential (ΔΨ(m)) as well as cytochrome c release in mitochondria isolated from mouse liver, heart, and brain. Accordingly, Tat decreases substrate oxidation by mitochondria isolated from these tissues, with oxygen uptake being initially restored by adding cytochrome c. The anion-channel inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) protects isolated mitochondria against Tat-induced mitochondrial membrane permeabilization (MMP), whereas ruthenium red, a ryanodine receptor blocker, does not. Pharmacologic inhibitors of the permeability transition pore, Bax/Bak inhibitors, and recombinant Bcl-2 and Bcl-XL proteins do not reduce Tat-induced MMP. We finally observed that Tat inhibits cytochrome c oxidase (COX) activity in disrupted mitochondria isolated from liver, heart, and brain of both mouse and human samples, making it the first described viral protein to be a potential COX inhibitor.

Since fifteen years ago, the mitochondrion has been progressively recognized as an integrator-coordinator of apoptosis. In this context recent anti-cancer drugs in development are targeting mitochondrial apoptotic machinery to induce tumor cell death. By this strategy, drugs can interact with apoptosis-regulating proteins of Bcl-2 family to trigger Bax/Bak oligomerization and mitochondrial membrane permeabilization. This event conducts to the release of pro-apoptotic factors such as cytochrome c into the cytosol that are required for caspases activation and apoptosis process. In this study, we selected another strategy and investigated the effect of ANT-targeting small molecules to induce cancer cells apoptosis. The adenine nucleotide translocator (ANT) is a mitochondrial protein of the inner membrane involved in the exchange of ADP and ATP between the mitochondrial matrix and the cytosol. Medicinal chemistry approach coupled with in silico studies yield to several small organic compounds, which proved to be specific for ANT and fulfill druggability criteria (good cell penetration and biodisponibility). We identified a family of compounds that inhibited ATP translocation and induced apoptosis by a pathway involving Bax-dependent cytochrome c release and caspases activation. One of the hit compound was optimized to obtain molecules able to induce apoptosis of HT-29 and BxPC3 cells at 100 nM. We are currently investigating which ANT isoform(s) is (are) targeted in cells. In vivo tumor regression experiments will be performed to achieve the proof of concept of ANT-ligands small molecules as an interesting anti-cancer strategy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4078. doi:10.1158/1538-7445.AM2011-4078

The phosphatidylinositol 3-kinase subunit PI3K is frequently mutated in human cancers and therefore represents an interesting therapeutic target. Cancer resistance to therapies is often related to expression of anti-apoptotic Bcl-2 family members which negatively regulate mitochondrial cell death. We investigated the influence of PI3K mutations on Bcl-2 family proteins both at the cellular and mitochondrial level. We used human breast epithelial cell lines with an endogenous “knock-in” PI3K mutation (HME-1 cells harbouring the H1047R activating mutation) from which we purified the mitochondria to homogeneity (95%). We first characterized these mitochondria for their sensitivity to reference compounds (Calcium, t-Bid) on 3 parameters: swelling, m loss and cytochrome c release. Mitochondrial preparations from wild-type and mutated HME-1 were analysed for their protein pattern in Bcl-2 family members. We observed accumulation of anti-apoptotic (Bcl-xl and Bcl-2) and pro-apoptotic (Bad) proteins in mitochondria isolated from PI3K mutated cells. We showed that the excess of anti-apoptotic proteins at the mitochondria does not change their sensitivity to the Bcl-2 family inhibitor, ABT-737. Investigations with PI3K-inhibitors are currently undergoing to determine the mitochondrial on-target biomarker for drug development. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-14.

Current limitations of chemotherapy include toxicity on healthy tissues and multidrug resistance on malignant cells. A number of recent anti-cancer strategies are targeting the mitochondrial apoptotic machinery to induce tumor cell death. In this study, we set up protocols to isolate mitochondria from various origins with high purity, quality and functionality. We analyzed the effect of putative Bcl-2 inhibitors on both healthy and tumor isolated mitochondria. We measured mitochondrial membrane permeabilization (swelling), mitochondrial transmembrane potential (ΔΨm) and mitochondrial outer membrane permeabilization (MOMP) in both isolated healthy and tumoral cell mitochondria. Like t-Bid and some BH3 peptides, the molecule ABT-737 was found to present a tumor-specific mitochondrio-toxicity, while compounds like HA-14.1, YC-137, Chelerythrine, Gossypol, TW-37 or EM20-25 did not. We thus demonstrated that ABT-737 can induce a relatively large and targeted MOMP triggering the release of apoptotic proteins from tumoral cell mitochondria (Cytochrome c, Smac/Diablo and Omi/HtrA2 but not AIF) however without any mitochondrial swelling. Furthermore, ABT-737 addition to isolated tumoral cell mitochondria induced specific Bax/Bak channel formation by oligomerizing monomeric Bax and/or Bak already inserted in the mitochondrial membrane. We also provided evidence that tumoral cell mitochondria can be either resistant or sensitive to ABT-737-induced MOMP, depending on their origin and interactions between pro- and anti-apoptotic proteins. Finally, this method based on MOMP is an interesting screening tool, tailored for identifying Bcl-2 antagonists with selective toxicity profile against tumoral cell mitochondria but devoid of toxicity against healthy mitochondria. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1021.

International audience; Current limitations of chemotherapy include toxicity on healthy tissues and multidrug resistance of malignant cells. A number of recent anti-cancer strategies aim at targeting the mitochondrial apoptotic machinery to induce tumor cell death. In this study, we set up protocols to purify functional mitochondria from various human cell lines to analyze the effect of peptidic and xenobiotic compounds described to harbour either Bcl-2 inhibition properties or toxic effects related to mitochondria. Mitochondrial inner and outer membrane permeabilization were systematically investigated in cancer cell mitochondria versus non-cancerous mitochondria. The truncated (t-) Bid protein, synthetic BH3 peptides from Bim and Bak, and the small molecule ABT-737 induced a tumor-specific and OMP-restricted mitochondrio-toxicity, while compounds like HA-14.1, YC-137, Chelerythrine, Gossypol, TW-37 or EM20-25 did not. We found that ABT-737 can induce the Bax-dependent release of apoptotic proteins (cytochrome c, Smac/Diablo and Omi/HtrA2 but not AIF) from various but not all cancer cell mitochondria. Furthermore, ABT-737 addition to isolated cancer cell mitochondria induced oligomerization of Bax and/or Bak monomers already inserted in the mitochondrial membrane. Finally immunoprecipatations indicated that ABT-737 induces Bax, Bak and Bim desequestration from Bcl-2 and Bcl-xL but not from Mcl-1L. This study investigates for the first time the mechanism of action of ABT-737 as a single agent on isolated cancer cell mitochondria. Hence, this method based on MOMP (mitochondrial outer membrane permeabilization) is an interesting screening tool, tailored for identifying Bcl-2 antagonists with selective toxicity profile against cancer cell mitochondria but devoid of toxicity against healthy mitochondria.

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.