In Vitro Evaluation of the Antioxidant Capacity of 3,3-Disubstituted-3H-benzofuran-2-one Derivatives in a Cellular Model of Neurodegeneration.

Oxidative stress represents a hallmark for many degenerative pathologies of the Central Nervous System. Throughout life, the constant pressure of noxious stimuli and/or episodes of traumatic events may expose the brain to a microenvironment where the non-balanced reactive oxygen species inevitably lead to neuronal loss and cognitive decline. HO-1, a 32 kDa heat-shock protein catalyzing the degradation of heme into carbon monoxide (CO), iron and biliverdin/bilirubin is considered one of the main antioxidant defense mechanisms playing pivotal roles in neuroprotection. Restoring the redox homeostasis is the goal of many natural or synthetic antioxidant molecules pursuing beneficial effects on brain functions. Here, we investigated the antioxidant capacity of four selected benzofuran-2-one derivatives in a cellular model of neurodegeneration represented by differentiated SH-SY5Y cells exposed to catechol-induced oxidative stress. Our main results highlight how all the molecules have antioxidant properties, especially compound 9, showing great abilities in reducing intracellular ROS levels and protecting differentiated SH-SY5Y cells from catechol-induced death. This compound above all seems to boost HO-1 mRNA and perinuclear HO-1 protein isoform expression when cells are exposed to the oxidative insult. Our findings open the way to consider benzofuran-2-ones as a novel and promising adjuvant antioxidant strategy for many neurodegenerative disorders.

Trait - driven analysis of the 2p15p16.1 microdeletion syndrome suggests a complex pattern of interactions between candidate genes.

BACKGROUND: Individuals with the 2p15p16.1 microdeletion syndrome share a complex phenotype including neurodevelopmental delay, brain malformations, microcephaly, and autistic behavior. The analysis of the shortest region of overlap (SRO) between deletions in ~ 40 patients has led to the identification of two critical regions and four strongly candidate genes (BCL11A, REL, USP34 and XPO1). However, the delineation of their role in the occurrence of specific traits is hampered by their incomplete penetrance. OBJECTIVE: To better delineate the role of hemizygosity of specific regions in selected traits by leveraging information both from penetrant and non - penetrant deletions. METHODS: Deletions in patients that do not present a specific trait cannot contribute to delineate the SROs. We recently developed a probabilistic model that, by considering also the non - penetrant deletions, allows a more reliable assignment of peculiar traits to specific genomic segments. We apply this method adding two new patients to the published cases. RESULTS: Our results delineate an intricate pattern of genotype - phenotype correlation where BCL11A emerges as the main gene for autistic behavior while USP34 and/or XPO1 haploinsufficiency are mainly associated with microcephaly, hearing loss and IUGR. BCL11A, USP34 and XPO1 genes are broadly related with brain malformations albeit with distinct patterns of brain damage. CONCLUSIONS: The observed penetrance of deletions encompassing different SROs and that predicted when considering each single SRO as acting independently, may reflect a more complex model than the additive one. Our approach may improve the genotype/phenotype correlation and may help to identify specific pathogenic mechanisms in contiguous gene syndromes.

Mutations in ACTL6B, coding for a subunit of the neuron-specific chromatin remodeling complex nBAF, cause early onset severe developmental and epileptic encephalopathy with brain hypomyelination and cerebellar atrophy.

Developmental and epileptic encephalopathies (DEEs) are genetically heterogenous conditions, often characterized by early onset, EEG interictal epileptiform abnormalities, polymorphous and drug-resistant seizures, and neurodevelopmental impairments. In this study, we investigated the genetic defects in two siblings who presented with severe DEE, microcephaly, spastic tetraplegia, diffuse brain hypomyelination, cerebellar atrophy, short stature, and kyphoscoliosis. Whole exome next-generation sequencing (WES) identified in both siblings a homozygous non-sense variant in the ACTL6B gene (NM_016188:c.820C>T;p.Gln274*) coding for a subunit of the neuron-specific chromatin remodeling complex nBAF. To further support these findings, a targeted ACTL6B sequencing assay was performed on a cohort of 85 unrelated DEE individuals, leading to the identification of a homozygous missense variant (NM_016188:c.1045G>A;p.Gly349Ser) in a patient. This variant did not segregate in the unaffected siblings in this family and was classified as deleterious by several prediction softwares. Interestingly, in both families, homozygous patients shared a rather homogeneous phenotype. Very few patients with ACTL6B gene variants have been sporadically reported in WES cohort studies of patients with neurodevelopmental disorders and/or congenital brain malformations. However, the limited number of patients with incomplete clinical information yet reported in the literature did not allow to establish a strong gene-disease association. Here, we provide additional genetic and clinical data on three new cases that support the pathogenic role of ACTL6B gene mutation in a syndromic form of DEE.

TGFß-Activated USP27X Deubiquitinase Regulates Cell Migration and Chemoresistance via Stabilization of Snail1.

In cancer cells, epithelial-to-mesenchymal transition (EMT) is controlled by Snail1, a transcriptional factor also required for the activation of cancer-associated fibroblasts (CAF). Snail1 is short-lived in normal epithelial cells as a consequence of its coordinated and continuous ubiquitination by several F-box-specific E3 ligases, but its degradation is prevented in cancer cells and in activated fibroblasts. Here, we performed an siRNA screen and identified USP27X as a deubiquitinase that increases Snail1 stability. Expression of USP27X in breast and pancreatic cancer cell lines and tumors positively correlated with Snail1 expression levels. Accordingly, downregulation of USP27X decreased Snail1 protein in several tumor cell lines. USP27X depletion impaired Snail1-dependent cell migration and invasion and metastasis formation and increased cellular sensitivity to cisplatin. USP27X was upregulated by TGFß during EMT and was required for TGFß-induced expression of Snail1 and other mesenchymal markers in epithelial cells and CAF. In agreement with this, depletion of USP27X prevented TGFß-induced EMT and fibroblast activation. Collectively, these results indicate that USP27X is an essential protein controlling Snail1 expression and function and may serve as a target for inhibition of Snail1-dependent tumoral invasion and chemoresistance. SIGNIFICANCE: These findings show that inhibition of USP27X destabilizes Snail1 to impair EMT and renders tumor cells sensitive to chemotherapy, thus opening new strategies for the inhibition of Snail1 expression and its protumoral actions.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/1/33/F1.large.jpg.

Targeting Serotonin 2A and Adrenergic α1 Receptors for Ocular Antihypertensive Agents: Discovery of 3,4-Dihydropyrazino[1,2-b]indazol-1(2H)-one Derivatives.

Glaucoma affects millions of people worldwide and causes optic nerve damage and blindness. The elevation of the intraocular pressure (IOP) is the main risk factor associated with this pathology, and decreasing IOP is the key therapeutic target of current pharmacological treatments. As potential ocular hypotensive agents, we studied compounds that act on two receptors (serotonin 2A and adrenergic α1 ) linked to the regulation of aqueous humour dynamics. Herein we describe the design, synthesis, and pharmacological profiling of a series of novel bicyclic and tricyclic N2-alkyl-indazole-amide derivatives. This study identified a 3,4-dihydropyrazino[1,2-b]indazol-1(2H)-one derivative with potent serotonin 2A receptor antagonism, >100-fold selectivity over other serotonin subtype receptors, and high affinity for the α1 receptor. Moreover, upon local administration, this compound showed superior ocular hypotensive action in vivo relative to the clinically used reference compound timolol.

Synthesis of Benzofuran-2-One Derivatives and Evaluation of Their Antioxidant Capacity by Comparing DPPH Assay and Cyclic Voltammetry.

The present work aimed to synthesise promising antioxidant compounds as a valuable alternative to the currently expensive and easily degradable molecules that are employed as stabilizers in industrial preparation. Taking into account our experience concerning domino Friedel-Crafts/lactonization reactions, we successfully improved and extended the previously reported methodology toward the synthesis of 3,3-disubstituted-3H-benzofuran-2-one derivatives 9-20 starting from polyphenols 1-6 as substrates and either diethylketomalonate (7) or 3,3,3-trifluoromethyl pyruvate (8) as electrophilic counterpart. The antioxidant capacity of the most stable compounds (9-11 and 15-20) was evaluated by both DPPH assay and Cyclic Voltammetry analyses performed in alcoholic media (methanol) as well as in aprotic solvent (acetonitrile). By comparing the recorded experimental data, a remarkable activity can be attributed to few of the tested lactones.

Asymmetric Synthesis of Spirooxindoles via Nucleophilic Epoxidation Promoted by Bifunctional Organocatalysts.

Taking into account the postulated reaction mechanism for the organocatalytic epoxidation of electron-poor olefins developed by our laboratory, we have investigated the key factors able to positively influence the H-bond network installed inside the substrate/catalyst/oxidizing agent. With this aim, we have: (i) tested a few catalysts displaying various effects that noticeably differ in terms of steric hindrance and electron demand; (ii) employed α-alkylidene oxindoles decorated with different substituents on the aromatic ring (11a-g), the exocylic double bond (11h-l), and the amide moiety (11m-v). The observed results suggest that the modification of the electron-withdrawing group (EWG) weakly conditions the overall outcomes, and conversely a strong influence is unambiguously ascribable to either the N-protected or N-unprotected lactam framework. Specifically, when the NH free substrates (11m-u) are employed, an inversion of the stereochemical control is observed, while the introduction of a Boc protecting group affords the desired product 12v in excellent enantioselectivity (97:3 er).

Non-Covalent Organocatalyzed Domino Reactions Involving Oxindoles: Recent Advances.

The ubiquitous presence of spirooxindole architectures with several functionalities and stereogenic centers in bioactive molecules has been appealing for the development of novel methodologies seeking their preparation in high yields and selectivities. Expansion and refinement in the field of asymmetric organocatalysis have made possible the development of straightforward strategies that address these two requisites. In this review, we illustrate the current state-of-the-art in the field of spirooxindole synthesis through the use of non-covalent organocatalysis. We aim to provide a concise overview of very recent methods that allow to the isolation of unique, densely and diversified spirocyclic oxindole derivatives with high structural diversity via the use of cascade, tandem and domino processes.

Synthesis and in vitro, ex-vivo and in vivo activity of hybrid compounds linking a potent ROS and RNS scavenger activity with diverse substrates addressed to pass across the blood-brain barrier.

The synthesis of a small library of CR-6 (a potent ROS and RNS scavenger agent) derivatives bearing covalent linkage with different endogen nutrients that have specific transport through the blood-brain barrier (BBB) is reported. The synthetic sequence involved the preparation of a common precursor ester 6 derived from CR-6, which was easily converted into the carboxylic acid 7a or the amino derivative 11, for its further coupling with the required substrates through amide bonds. Antioxidant in vitro (DPPH) and cellular assays (CAA) with the SH-S5SY cell line performed on these library members revealed that the couplings did not affect the antioxidant activity elicited by CR-6 itself. More interestingly, results from the intraperitoneal administration of selected library components in rats showed that compounds 2b, 2c and 2d were able to pass across the BBB. In particular, the amino acid compound 2d was the most penetrating derivative (15.8 ± 1.7 nmol/g brain with respect to the 4.0 ± 1.2 nmol/g brain found for the parent CR-6).