acknowledges financial support from Italian Ministry for Education and Research (Fondo di Finanziamento per le Attivit Base di Ricerca, FFABR 2017)

acknowledges financial support from Italian Ministry for Education and Research (Fondo di Finanziamento per le Attivit Base di Ricerca, FFABR 2017). Conflicts of Interest The authors declare no conflict of interest. Footnotes Sample Availability: Samples of the compounds are not available from the authors.. B and acetylcholinesterase, cytoprotection from oxidative insult, and activation of carnitine/acylcarnitine carrier, as concurring biological activities responsible for neuroprotection. 0.05 and ** 0.01. The reducing properties of DAN were studied on the recombinant WT CAC with the protein at different states of oxidation. Figure 8B highlights that when the carrier was incubated during the transport activity with 1 mM dithioerythritol (DTE), BSc5371 10 M DAN was able to reduce a major aliquot of oxidized protein (70% recovery of activity compared to the control). When the carrier was incubated with 50 mM DTE, recovery was 25%, demonstrating that the action of DAN is exerted on thiol groups. To identify the cysteines responsible for the activation of the CAC carrier, the proteoliposomes of the WT protein and the mutants C136S, C155S, C136/155S, and C-lessV (C23V/C58V/C89S/C136V/C155V/C283S) were incubated with 10 M DAN. The increase in transport activity observed on WT (180%) was significantly lost when cysteines C136 and C155 were alternately or together mutated (C136S, C155S, and C136/155S) or all cysteine residues were replaced (C-less V), as seen in Figure 8C, indicating that these amino acids are the specific redox targets for DAN. Further evidence of the reducing action of DAN on CAC was obtained by oxidation of the reconstituted WT protein under controlled conditions, i.e., in the presence or absence of hydrogen peroxide and its subsequent degradation by the enzyme catalase [45]. Figure 8D shows that the transport activity decreased to about 50% of the untreated control by adding 1 mM H2O2 to proteoliposomes for 10 min at 37 C. The addition of 20 M DAN led to a significant recovery (about 80%) of the transport function, after treatment of the carrier with H2O2. These data suggest that DAN could reverse the oxidizing action of hydrogen peroxide on the CAC, re-establishing the entry of fatty acids, the activation of the -oxidation, and therefore the production of ATP. Although ALC is able to cross the bloodCbrain barrier [48] and provide neuroprotection in the therapy of neurodegenerative diseases [49], even in various conditions of metabolic stress [50], the bioavailability of ALC in brain after oral administration is very low. The acetyl group of ALC is subjected to fast renal clearance, while in the BSc5371 gut and liver, it is converted in acetyl-CoA to allow acetylation reactions [51]. Since treatment with DAN may reactivate the function of CAC under OS conditions, the enhanced export of endogenous ALC produced during -oxidation [27] could represent an important source of acetyl moieties, conferring effective cell neuroprotection. 3. Materials and Methods All the chemicals, enzymes, solvents, and reagents were purchased from Sigma-Aldrich Europe (Milan, Italy) unless specified, and used without further purification. 3.1. Buffer Stability Assay The analytical HPLC measurements were performed on a Waters 1525 HPLC System (Waters, Milan, Italy) equipped with a Waters 2587 variable-wavelength UV-Vis detector and a Waters 717 plus autosampler. The chromatographic data were acquired using the Waters Breeze software (version 3.20). Analyses were performed on a Phenomenex C18 column (150 4.6 mm i.d., 3 m particle size; Phenomenex srl, Castel Maggiore, Italy) using a mobile phase of methanol/water (75:25 C0214 (given as a gift). Wild-type and mutants rat CAC inclusion body fractions were isolated from 0.05 were considered statistically significant. Data points were derived Ywhaz from the mean of three different experiments, as specified in the figure legends. 4. Conclusions Repurposing of existing drugs is considered a valid shortcut to alternative therapies or, as in the case of neurodegenerative diseases, to give chances to unmet therapeutic needs [61]. The main advantage of this approach is in avoiding the preclinical phase of drug development, thus minimizing the costs required for ADMET profiling and the business risks for companies and investors. On the other hand, the main challenge of drug BSc5371 repurposing is to achieve the desired off-label activities regardless of the original therapeutic effect or balancing it into a risk/benefit evaluation for a new therapeutic use. In this context, we disclosed new biological activities exerted by dantrolene, a drug specifically used in the management of malignant hyperthermia, which has been proposed for repurposing in several pathologies, including MDMA intoxication [8] and Duchenne muscular dystrophy [62]. In this work, we demonstrated for the first time that DAN competitively inhibits MAO B with a micromolar Ki value for the human enzyme, showing, in addition, activity as an AChE inhibitor and hampering the aggregation of A40 and PHF6, i.e., two probes of amyloid aggregation in AD brain. Regarding the antioxidant properties [28], we showed that DAN, ineffective as a radical scavenger, decreases ROS production in a cell-based assay of neuroprotection from OS, this activity being apparently related to its MAO inhibitory activity. A significant.