Romana Fato

Noise-Induced Hearing Loss (NIHL) as a Target of Oxidative Stress-Mediated Damage: Cochlear and Cortical Responses after an Increase in Antioxidant Defense

This study addresses the relationship between cochlear oxidative damage and auditory cortical injury in a rat model of repeated noise exposure. To test the effect of increased antioxidant defenses, a water-soluble coenzyme Q10 analog (Qter) was used. We analyzed auditory function, cochlear oxidative stress, morphological alterations in auditory cortices and cochlear structures, and levels of coenzymes Q9 and Q10 (CoQ9 and CoQ10, respectively) as indicators of endogenous antioxidant capability. We report three main results. First, hearing loss and damage in hair cells and spiral ganglion was determined by noise-induced oxidative stress. Second, the acoustic trauma altered dendritic morphology and decreased spine number of II–III and V–VI layer pyramidal neurons of auditory cortices. Third, the systemic administration of the water-soluble CoQ10 analog reduced oxidative-induced cochlear damage, hearing loss, and cortical dendritic injury. Furthermore, cochlear levels of CoQ9 and CoQ10 content increased. These findings indicate that antioxidant treatment restores auditory cortical neuronal morphology and hearing function by reducing the noise-induced redox imbalance in the cochlea and the deafferentation effects upstream the acoustic pathway.

Naphthoquinone Derivatives Exert Their Antitrypanosomal Activity via a Multi-Target Mechanism

Background and Methodology Recently, we reported on a new class of naphthoquinone derivatives showing a promising anti-trypanosomatid profile in cell-based experiments. The lead of this series (B6, 2-phenoxy-1,4-naphthoquinone) showed an ED50 of 80 nM against Trypanosoma brucei rhodesiense, and a selectivity index of 74 with respect to mammalian cells. A multitarget profile for this compound is easily conceivable, because quinones, as natural products, serve plants as potent defense chemicals with an intrinsic multifunctional mechanism of action. To disclose such a multitarget profile of B6, we exploited a chemical proteomics approach. Principal Findings A functionalized congener of B6 was immobilized on a solid matrix and used to isolate target proteins from Trypanosoma brucei lysates. Mass analysis delivered two enzymes, i.e. glycosomal glycerol kinase and glycosomal glyceraldehyde-3-phosphate dehydrogenase, as potential molecular targets for B6. Both enzymes were recombinantly expressed and purified, and used for chemical validation. Indeed, B6 was able to inhibit both enzymes with IC50 values in the micromolar range. The multifunctional profile was further characterized in experiments using permeabilized Trypanosoma brucei cells and mitochondrial cell fractions. It turned out that B6 was also able to generate oxygen radicals, a mechanism that may additionally contribute to its observed potent trypanocidal activity. Conclusions and Significance Overall, B6 showed a multitarget mechanism of action, which provides a molecular explanation of its promising anti-trypanosomatid activity. Furthermore, the forward chemical genetics approach here applied may be viable in the molecular characterization of novel multitarget ligands.

Multitarget Drug Design Strategy: Quinone–Tacrine Hybrids Designed To Block Amyloid-β Aggregation and To Exert Anticholinesterase and Antioxidant Effects

We report the identification of multitarget anti-Alzheimer compounds designed by combining a naphthoquinone function and a tacrine fragment. In vitro, 15 compounds displayed excellent acetylcholinesterase (AChE) inhibitory potencies and interesting capabilities to block amyloid-β (Aβ) aggregation. The X-ray analysis of one of those compounds in complex with AChE allowed rationalizing the outstanding activity data (IC50 = 0.72 nM). Two of the compounds showed negligible toxicity in immortalized mouse cortical neurons Neuro2A and primary rat cerebellar granule neurons. However, only one of them was less hepatotoxic than tacrine in HepG2 cells. In T67 cells, both compounds showed antioxidant activity, following NQO1 induction. Furthermore, in Neuro2A, they were able to completely revert the decrease in viability induced by Aβ. Importantly, they crossed the blood-brain barrier, as demonstrated in ex vivo experiments with rats. When ex vivo results were combined with in vitro studies, these two compounds emerged to be promising multitarget lead candidates worthy of further pursuit.

Different mtDNA mutations modify tumor progression in dependence of the degree of respiratory complex I impairment

Mitochondrial DNA mutations are currently investigated as modifying factors impinging on tumor growth and aggressiveness, having been found in virtually all cancer types and most commonly affecting genes encoding mitochondrial complex I (CI) subunits. However, it is still unclear whether they exert a pro- or anti-tumorigenic effect. We here analyzed the impact of three homoplasmic mtDNA mutations (m.3460G>A/MT-ND1, m.3571insC/MT-ND1 and m.3243A>G/MT-TL1) on osteosarcoma progression, chosen since they induce different degrees of oxidative phosphorylation impairment. In fact, the m.3460G>A/MT-ND1 mutation caused only a reduction in CI activity, whereas the m.3571insC/MT-ND1 and the m.3243A>G/MT-TL1 mutations induced a severe structural and functional CI alteration. As a consequence, this severe CI dysfunction determined an energetic defect associated with a compensatory increase in glycolytic metabolism and AMP-activated protein kinase activation. Osteosarcoma cells carrying such marked CI impairment displayed a reduced tumorigenic potential both in vitro and in vivo, when compared with cells with mild CI dysfunction, suggesting that mtDNA mutations may display diverse impact on tumorigenic potential depending on the type and severity of the resulting oxidative phosphorylation dysfunction. The modulation of tumor growth was independent from reactive oxygen species production but correlated with hypoxia-inducible factor 1α stabilization, indicating that structural and functional integrity of CI and oxidative phosphorylation are required for hypoxic adaptation and tumor progression.

Respiratory complex I is essential to induce a Warburg profile in mitochondria-defective tumor cells.

BackgroundAerobic glycolysis, namely the Warburg effect, is the main hallmark of cancer cells. Mitochondrial respiratory dysfunction has been proposed to be one of the major causes for such glycolytic shift. This hypothesis has been revisited as tumors appear to undergo waves of gene regulation during progression, some of which rely on functional mitochondria. In this framework, the role of mitochondrial complex I is still debated, in particular with respect to the effect of mitochondrial DNA mutations in cancer metabolism. The aim of this work is to provide the proof of concept that functional complex I is necessary to sustain tumor progression.MethodsComplex I-null osteosarcoma cells were complemented with allotopically expressed complex I subunit 1 (MT-ND1). Complex I re-assembly and function recovery, also in terms of NADH consumption, were assessed. Clones were tested for their ability to grow in soft agar and to generate tumor masses in nude mice. Hypoxia levels were evaluated via pimonidazole staining and hypoxia-inducible factor-1α (HIF-1α) immunoblotting and histochemical staining. 454-pyrosequencing was implemented to obtain global transcriptomic profiling of allotopic and non-allotopic xenografts.ResultsComplementation of a truncative mutation in the gene encoding MT-ND1, showed that a functional enzyme was required to perform the glycolytic shift during the hypoxia response and to induce a Warburg profile in vitro and in vivo, fostering cancer progression. Such trigger was mediated by HIF-1α, whose stabilization was regulated after recovery of the balance between α-ketoglutarate and succinate due to a recuperation of NADH consumption that followed complex I rescue.ConclusionRespiratory complex I is essential for the induction of Warburg effect and adaptation to hypoxia of cancer cells, allowing them to sustain tumor growth. Differently from other mitochondrial tumor suppressor genes, therefore, a complex I severe mutation such as the one here reported may confer anti-tumorigenic properties, highlighting the prognostic values of such genetic markers in cancer.

A Water Soluble CoQ10 Formulation Improves Intracellular Distribution and Promotes Mitochondrial Respiration in Cultured Cells

Background Mitochondria are both the cellular powerhouse and the major source of reactive oxygen species. Coenzyme Q10 plays a key role in mitochondrial energy production and is recognized as a powerful antioxidant. For these reasons it can be argued that higher mitochondrial ubiquinone levels may enhance the energy state and protect from oxidative stress. Despite the large number of clinical studies on the effect of CoQ10 supplementation, there are very few experimental data about the mitochondrial ubiquinone content and the cellular bioenergetic state after supplementation. Controversial clinical and in vitro results are mainly due to the high hydrophobicity of this compound, which reduces its bioavailability. Principal Findings We measured the cellular and mitochondrial ubiquinone content in two cell lines (T67 and H9c2) after supplementation with a hydrophilic CoQ10 formulation (Qter®) and native CoQ10. Our results show that the water soluble formulation is more efficient in increasing ubiquinone levels. We have evaluated the bioenergetics effect of ubiquinone treatment, demonstrating that intracellular CoQ10 content after Qter supplementation positively correlates with an improved mitochondrial functionality (increased oxygen consumption rate, transmembrane potential, ATP synthesis) and resistance to oxidative stress. Conclusions The improved cellular energy metabolism related to increased CoQ10 content represents a strong rationale for the clinical use of coenzyme Q10 and highlights the biological effects of Qter®, that make it the eligible CoQ10 formulation for the ubiquinone supplementation.

Antiinflammatory effect of phytosterols in experimental murine colitis model: prevention, induction, remission study.

Phytosterols, besides hypocholesterolemic effect, present anti-inflammatory properties. Little information is available about their efficacy in Inflammatory Bowel Disease (IBD). Therefore, we have evaluated the effect of a mixture of phytosterols on prevention/induction/remission in a murine experimental model of colitis. Phytosterols were administered x os before, during and after colitis induction with Dextran Sodium Sulfate (DSS) in mice. Disease Activity Index (DAI), colon length, histopathology score, 18F-FDG microPET, oxidative stress in the intestinal tissue (ileum and colon) and gallbladder ileum and colon spontaneous and carbachol (CCh) induced motility, plasma lipids and plasma, liver and biliary bile acids (BA) were evaluated. A similar longitudinal study was performed in a DSS colitis control group. Mice treated with DSS developed severe colitis as shown by DAI, colon length, histopathology score, 18F-FDG microPET, oxidative stress. Both spontaneous and induced ileal and colonic motility were severely disturbed. The same was observed with gallbladder. DSS colitis resulted in an increase in plasma cholesterol, and a modification of the BA pattern. Phytosterols feeding did not prevent colitis onset but significantly reduced the severity of the disease and improved clinical and histological remission. It had strong antioxidant effects, almost restored colon, ileal and gallbladder motility. Plasmatic levels of cholesterol were also reduced. DSS induced a modification in the BA pattern consistent with an increase in the intestinal BA deconjugating bacteria, prevented by phytosterols. Phytosterols seem a potential nutraceutical tool for gastrointestinal inflammatory diseases, combining metabolic systematic and local anti-inflammatory effects.

Versatility of the Curcumin Scaffold: Discovery of Potent and Balanced Dual BACE-1 and GSK-3β Inhibitors

The multitarget approach has gained increasing acceptance as a useful tool to address complex and multifactorial maladies such as Alzheimers disease (AD). The concurrent inhibition of the validated AD targets β-secretase (BACE-1) and glycogen synthase kinase-3β (GSK-3β) by attacking both β-amyloid and tau protein cascades has been identified as a promising AD therapeutic strategy. In our study, curcumin was identified as a lead compound for the simultaneous inhibition of both targets; therefore, synthetic efforts were dedicated to obtaining a small library of novel curcumin-based analogues, and a number of potent and balanced dual-target inhibitors were obtained. In particular, 2, 6, and 7 emerged as promising drug candidates endowed with neuroprotective potential and brain permeability. Notably, for some new compounds the symmetrical diketo and the β-keto-enol tautomeric forms were purposely isolated and tested in vitro, allowing us to gain insight into the key requirements for BACE-1 and GSK-3β inhibition.

Novel 8-Hydroxyquinoline Derivatives as Multitarget Compounds for the Treatment of Alzheimer′s Disease

We discovered a small series of hit compounds that show multitargeting activities against key targets in Alzheimer′s disease (AD). The compounds were designed by combining the structural features of the anti‐AD drug donepezil with clioquinol, which is able to chelate redox‐active metals, thus decreasing metal‐driven oxidative phenomena and β‐amyloid (Aβ)‐mediated neurotoxicity. The majority of the new hybrid compounds selectively target human butyrylcholinesterase at micromolar concentrations and effectively inhibit Aβ self‐aggregation. In addition, compounds 5‐chloro‐7‐((4‐(2‐methoxybenzyl)piperazin‐1‐yl)methyl)‐8‐hydroxyquinoline (1u2009b), 7‐((4‐(2‐methoxybenzyl)piperazin‐1‐yl)methyl)‐8‐hydroxyquinoline (2u2009b), and 7‐(((1‐benzylpiperidin‐4‐yl)amino)methyl)‐5‐chloro‐8‐hydroxyquinoline (3u2009a) are able to chelate copper(II) and zinc(II) and exert antioxidant activity inu2005vitro. Importantly, in the case of 2u2009b, the multitarget profile is accompanied by high predicted blood–brain barrier permeability, low cytotoxicity in T67 cells, and acceptable toxicity in HUVEC primary cells.

2-Phenoxy-1,4-naphthoquinones: From a Multitarget Antitrypanosomal to a Potential Antitumor Profile

A small library of 2-phenoxy-1,4-naphthoquinone and 2-phenoxy-1,4-anthraquinone derivatives was initially developed to optimize the antitrypanosomatid profile of the multitarget hit compound B6 (1). The whole series was evaluated against the three most important human trypanosomatid pathogens (Trypanosoma brucei rhodesiense, Trypanosoma cruzi, and Leishmania donovani), and two compounds (14 and 21) showed good activity, despite a concomitant mammalian cytotoxicity. Furthermore, a subset also inhibited the glycolytic TbGAPDH enzyme in vitro. In light of these results and aware of the antitumor properties of quinones, the anticancer potential of some selected derivatives was investigated. Intriguingly, the tested compounds displayed antitumor activity, while being less toxic against noncancerous cells. The observed cytotoxic potency was ascribed to a multitarget mechanism of action accounting for hGAPDH inhibition and mitochondrial toxicity. Overall, the development of further derivatives, able to finely modulate multiple pathways of cancer or parasite cell metabolism, might lead to more effective treatments against these devastating diseases.