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    • To identify structurally novel autophagy inhibitors a medium

    2022-01-27

    To identify structurally novel autophagy inhibitors, a medium throughput screen of our in-house library of approximately 160,000 compounds was performed. In the screening assay, MCF7 Sitagliptin phosphate synthesis stably transfected with eGFP-tagged light chain 3 (LC3), were employed, which can be detected by automated fluorescence microscopy. Upon initiation of autophagy, cytosolic LC3-I is conjugated to phosphatidylethanolamine to produce LC3-II which localises to the autophagosomal membrane. The autophagosomes are represented as green punctae, while LC3-I fluorescence is visible as a diffuse signal throughout the cytosol. Autophagy inhibitors should reverse the phenotype of autophagosome formation, while autophagy enhancement results in an increase. Based on this screen, thienopyrimidines were identified as potential autophagy inhibitors (see the ). The most potent of these compounds was Authipyrin ( A–C, entry 1). Authipyrin inhibits starvation-induced autophagy with an IC of 0.02 ± 0.01 µM and rapamycin-induced autophagy with an IC of 0.18 ± 0.07 µM. Furthermore, the influence of Authipyrin on the autophagy marker LC3 was determined in different cell lines (D). An autophagy inhibitor should prevent LC3-II formation. Authipyrin significantly reduced LC3-II lipidation under starvation in the presence of chloroquine. This confirms the inhibitory effect of Authipyrin on autophagy. Furthermore, selective toxicity of Authipyrin under nutrient deprivation was investigated. While Authipyrin had no influence on cell viability under normal conditions, it had a pronounced dose-dependent toxicity upon glucose starvation (see the ; ). Several thienopyrimidine derived kinase inhibitors were reported., , A subset of these kinase inhibitors are also active in autophagy., Therefore, a single point full kinase panel with Authipyrin was performed at a concentration of 1 µM (see the , ). This resulted in the identification of four out of 485 kinases that featured a reduction in activity by >50% (). The affected kinases were PEAK1 (83%), the inactive version of the cyclin-dependent kinase CDK11 (70%), as well as haspin (51%) and the mutant DDR2 (N456S) protein (58%). Subsequently, the respective IC values for these kinases were determined. PEAK1 possessed the lowest IC of 0.65 µM. The cell-based IC of Authipyrin for autophagy inhibition upon starvation was 0.02 ± 0.01 µM. The IC for the purified target protein, which causes the inhibitory effect on autophagic flux should be comparable or even lower. Thus, it can be assumed that autophagy modulation by Authipyrin is not dependent on the inhibition of a kinase. Autophagy is an essential mechanism to maintain cellular homeostasis, by clearance of damaged organelles, including mitochondria. Furthermore, alteration of metabolic processes, such as oxidative phosphorylation, results in modulation of autophagic processes., , Therefore, the influence of Authipyrin on mitochondrial respiration was investigated by means of a Mito Stress Test assay employing the Seahorse XF analyzer. The readout is based on two different fluorophores. One fluorophore is sensitive to changes in the pH, which represents the extracellular acidification rate (ECAR). The ECAR is influenced by lactate excretion due to anaerobic glycolysis. The second fluorophore detects the cellular oxygen consumption rate (OCR). Initially, the basal respiration under resting conditions was determined (A and B). Subsequently, the inhibitor of interest was added to the cells, to determine the influence on mitochondrial respiration (point a). Injection of the known complex V inhibitor oligomycin reflects the amount of oxygen required for ATP production (point B). Subsequent addition of carbonyl cyanide-4-(trifluoro-methoxy)phenylhydrazone (FCCP) disrupts the proton gradient and causes a rise of mitochondrial respiration to maximal capacity (point c). This represents the cellular respiration under stress conditions. The range between basal and maximal OCR is the cellular spare capacity available to respond to increased energy requirement. The known complex I and III inhibitors rotenone and antimycin A are finally employed for complete inhibition of mitochondrial respiration (point c). The Mito Stress Test showed that Authipyrin inhibits oxidative phosphorylation in a dose-dependent manner. At a concentration of 0.1 µM and 1 µM cell treatment with Authipyrin resulted in a decrease of the OCR by 25% and 75% respectively (A). Simultaneously the ECAR showed a strong increase. (B). However, downregulation of mitochondrial respiration could be a general characteristic of this scaffold. Therefore, two structurally similar thienopyrimidines (, entry 15 and 27) were analyzed by means of MitoStress Test assay. These compounds were inactive in the initial autophagy screen. Both compounds did not display any effect on the OCR. (see ) Therefore, downregulation of mitochondrial respiration by Authipyrinis connected to the inhibition of autophagy.