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Depto. de Química en Ciencias Farmacéuticas, Fac. de Farmacia, pub

Depto. de Química en Ciencias Farmacéuticas, Fac. de Farmacia, pub

Hybrids based on an aza-analogue of CGP37157, a mitochondrial Na+/Ca2+ exchanger antagonist, and lipoic acid were obtained in order to combine in a single molecule the antioxidant and NRF2 induction properties of lipoic acid and the neuroprotective activity of CGP37157. The four possible enantiomers of the hybrid structure were synthesized by using as the key step a fully diastereoselective reduction induced by Ellman’s chiral auxiliary. After computational druggability studies that predicted good ADME profiles and blood–brain permeation for all compounds, the DPPH assay showed moderate oxidant scavenger capacity. Following a cytotoxicity evaluation that proved the compounds to be non-neurotoxic at the concentrations tested, they were assayed for NRF2 induction capacity and for anti-inflammatory properties and measured by their ability to inhibit nitrite production in the lipopolysaccharide-stimulated BV2 microglial cell model. Moreover, the compounds were studied for their neuroprotective effect in a model of oxidative stress achieved by treatment of SH-SY5Y neuroblastoma cells with the rotenone–oligomycin combination and also in a model of hyperphosphorylation induced by treatment with okadaic acid. The stereocenter configuration showed a critical influence in NRF2 induction properties, and also in the neuroprotection against oxidative stress experiment, leading to the identification of the compound with S and R configuration as an interesting hit with a good neuroprotective profile against oxidative stress and hyperphosphorylation, together with a relevant anti-neuroinflammatory activity. This interesting multitarget profile will be further characterized in future work.

Hybrids based on an aza-analogue of CGP37157, a mitochondrial Na+/Ca2+ exchanger antagonist, and lipoic acid were obtained in order to combine in a single molecule the antioxidant and NRF2 induction properties of lipoic acid and the neuroprotective activity of CGP37157. The four possible enantiomers of the hybrid structure were synthesized by using as the key step a fully diastereoselective reduction induced by Ellman’s chiral auxiliary. After computational druggability studies that predicted good ADME profiles and blood–brain permeation for all compounds, the DPPH assay showed moderate oxidant scavenger capacity. Following a cytotoxicity evaluation that proved the compounds to be non-neurotoxic at the concentrations tested, they were assayed for NRF2 induction capacity and for anti-inflammatory properties and measured by their ability to inhibit nitrite production in the lipopolysaccharide-stimulated BV2 microglial cell model. Moreover, the compounds were studied for their neuroprotective effect in a model of oxidative stress achieved by treatment of SH-SY5Y neuroblastoma cells with the rotenone–oligomycin combination and also in a model of hyperphosphorylation induced by treatment with okadaic acid. The stereocenter config- uration showed a critical influence in NRF2 induction properties, and also in the neuroprotection against oxidative stress experiment, leading to the identification of the compound with S and R configuration as an interesting hit with a good neuroprotective profile against oxidative stress and hyperphosphorylation, together with a relevant anti-neuroinflammatory activity. This interesting multitarget profile will be further characterized in future work.

Parkinson’s disease (PD) is the second most prevalent neurodegenerative disease. It is characterized by a complex network of physiopathological events where oxidative stress plays a central role among other factors such as neuroinflammation and protein homeostasis. Nuclear factor-erythroid 2 p45-related factor 2 (NRF2) has a multitarget profile itself as it controls a plethora of cellular processes involved in the progression of the disease. In this line, we designed a novel family of 2-(1H-indol-3-yl)ethan-1-amine derivatives as NRF2 inducers with complementary activities. Novel compounds are based on melatonin scaffold and include, among other properties, selective monoamine oxidase B (MAO-B) inhibition activity. Novel multitarget compounds exhibited NRF2 induction activity and MAO-B selective inhibition, combined with anti-inflammatory, antioxidant, and blood–brain barrier permeation properties. Furthermore, they exert neuroprotective properties against oxidative stress toxicity in PD-related in vitro. Hit compound 14 reduced oxidative stress markers and exerted neuroprotection in rat striatal slices exposed to 6-hydroxydopamine or rotenone. In conclusion, we developed a promising family of dual NRF2 inducers and selective MAO-B inhibitors that could serve as a novel therapeutic strategy for PD treatmen

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Neurodegenerative diseases are characterized by a progressive loss of neurons in the brain or spinal cord that leads to a loss of function of the aected areas. The lack of eective treatments and the ever-increasing life expectancy is raising the number of individuals aected, having a tremendous social and economic impact. The brain is particularly vulnerable to oxidative damage given the high energy demand, low levels of antioxidant defenses, and high levels of metal ions. Driven by age-related changes, neurodegeneration is characterized by increased oxidative stress leading to irreversible neuronal damage, followed by cell death. Nevertheless, neurodegenerative diseases are known as complex pathologies where several mechanisms drive neuronal death. Herein we discuss the interplay among oxidative stress, proteinopathy, and neuroinflammation at the early stages of neurodegenerative diseases. Finally, we discuss the use of the Nrf2-ARE pathway as a potential therapeutic strategy based on these molecular mechanisms to develop transformative medicines

Neuroinflammation is increasingly associated to the onset and progression of neurodegenerative diseases. Furthermore, several lines of evidence have demonstrated the capacity of aberrant protein aggregates to activate the immune response, accelerating the advance of the disease. Compound ITH12674 is a melatonin-sulforaphane hybrid designed to exert a dual drug-prodrug mechanism of action that combines potent NRF2 induction and free radical scavenger activity. ITH12674 also showed neuroprotective properties in oxidative stress related models, that were dependant on its NRF2 inducing properties. Given the high impact of neuroinflammation in the pathogenesis of neurodegeneration, we foresaw to study the anti-inflammatory properties of ITH12674. ITH12674 reduced inflammatory markers in glial cell cultures and hippocampal tissue after LPS administration. The anti-inflammatory effect was related to inhibition of TLR4 receptors due to a direct interaction with the TLR4/MD2 complex at the hydrophobic cavity of MD2. ITH12674 is endowed with anti-inflammatory properties, that are complementary to the NRF2 inducing activity and neuroprotective properties. Thus, ITH12674 could be of potential interest for the treatment of diseases with chronic neuroinflammation.

New multi-target indole and naphthalene derivatives containing the oxadiazolone scaffold as a bioisostere of the melatonin acetamido group have been developed. The novel compounds were characterized at melatonin receptors MTR and MTR, quinone reductase 2 (QR2), lipoxygenase-5 (LOX-5), and monoamine oxidases (MAO-A and MAO-B), and also as radical scavengers. We found that selectivity within the oxadiazolone series can be modulated by modifying the side chain functionality and co-planarity with the indole or naphthalene ring. In phenotypic assays, several oxadiazolone-based derivatives induced signalling mediated by the transcription factor NRF2 and promoted the maturation of neural stem-cells into a neuronal phenotype. Activation of NRF2 could be due to the binding of indole derivatives to KEAP1, as deduced from surface plasmon resonance (SPR) experiments. Molecular modelling studies using the crystal structures of QR2 and the KEAP1 Kelch-domain, as well as the recently described X-ray free-electron laser (XFEL) structures of chimeric MTR and MTR, provided a rationale for the experimental data and afforded valuable insights for future drug design endeavours.

Massive intravascular hemolysis is associated with acute kidney injury (AKI). Nuclear factor erythroid-2-related factor 2 (Nrf2) plays a central role in the defense against oxidative stress by activating the expression of antioxidant proteins. We investigated the role of Nrf2 in intravascular hemolysis and whether Nrf2 activation protected against hemoglobin (Hb)/heme-mediated renal damage in vivo and in vitro. We observed renal Nrf2 activation in human hemolysis and in an experimental model of intravascular hemolysis promoted by phenylhydrazine intraperitoneal injection. In wild-type mice, Hb/heme released from intravascular hemolysis promoted AKI, resulting in decreased renal function, enhanced expression of tubular injury markers (KIM-1 and NGAL), oxidative and endoplasmic reticulum stress (ER), and cell death. These features were more severe in Nrf2-deficient mice, which showed decreased expression of Nrf2-related antioxidant enzymes, including heme oxygenase 1 (HO-1) and ferritin. Nrf2 activation with sulforaphane protected against Hb toxicity in mice and cultured tubular epithelial cells, ameliorating renal function and kidney injury and reducing cell stress and death. Nrf2 genotype or sulforaphane treatment did not influence the severity of hemolysis. In conclusion, our study identifies Nrf2 as a key molecule involved in protection against renal damage associated with hemolysis and opens novel therapeutic approaches to prevent renal damage in patients with severe hemolytic crisis. These findings provide new insights into novel aspects of Hb-mediated renal toxicity and may have important therapeutic implications for intravascular hemolysis-related diseases

This study has been developed within a multidisciplinary project designed to search for multitarget compounds with NRF2 inducing capacity, by inhibiting the Keap1-NRF2 interaction, for the treatment of Alzheimer¿s disease. We have focused on melatonin and resveratrol derivatives, which additionally induce NRF2 transcription factor, master regulator of oxidative stress. For this study, two melatonin (CH213 and CH507) and one resveratrol (PL119) derivatives, which were the ones that duplicated NRF2 expression at concentrations below 10 ¿M, were selected. We found that these compounds were not neurotoxic in the human neuroblastoma cell line SH-SY5Y at the concentration of 100 ¿M. Thereafter, neuroprotection in a tau hyperphosphorylation in vitro model induced by okadaic acid (OA) was studied; compounds CH507 and PLC119 presented significant neuroprotection, while CH213 did not. Nevertheless, neuroprotection of compound CH213 against OA was studied in primary neuronal cultures achieving significant results. Additionally, their potential anti neuroinflammatory effects were studied in primary rat glial cultures exposed to LPS; in this model, only compound CH213 reduced nitric oxide production and IL-1b. CH123 was further studied in an acute model of hippocampal slices, where toxicity was induced by incubation with OA for 6 h and in organotypic hippocampal cultures of 14 months old APPTau mice; in these models, CH213 was able to provide neuroprotection and reduce oxidative stress. Finally, since QR2, also known as MT3, is overexpressed in AD patients, we sought of interest to perform a molecular docking study to evaluate how the compounds could interact with this receptor. In conclusion, CH213 is a melatonin derivative that induces NRF2 with antineuroinflammatory, antioxidant and neuroprotective properties; however, future studies in in vivo AD models need to be conducted to validate the in vitro results here obtained.

Alzheimer¿s Disease (AD) is a complex multifactorial illness with no effective cure, characterized by the irreversible memory loss and global cognitive impairment. Given that the marketed drugs act only at one single target, being a symptomatic treatment, exploration of new molecules acting in different pathways is required. In this sense, we have developed a large family of multitarget directed ligands (MTDL), which interacts in different targets related to neurodegeneration, such as the nuclear factor (erythroid-derived 2)-like 2 (Nrf2), lipoxygenase-5 (LOX-5), melatonin receptors (MT1, MT2, and MT3), and monoamine oxidases (MAO-A and MAO-B). The affinity or inhibition constants of these compounds in such targets are in the nanomolar and micromolar ranges. In general, these molecules have potent antioxidant properties (ORAC assay) and are able to penetrate into the central nervous system (CNS) by passive diffusion (PAMPA-BBB assay). Moreover, some of them are able to promote neurogenesis in vitro by inducing the maturation of neural stem cells into a neuronal phenotype. A structure-activity relationship has been stablished, reaching an optimized structure as possible candidate to treat AD, able to cross the blood brain barrier, reaching the CNS where it would act in three targets involved in the regulation of oxidative stress, as Nrf2 inducer, MT3 ligand and selective MAO B inhibitor. Furthermore, this MTDL is an effective neuroprotective and neurogenic agent.