The specific NQO2 inhibitor, S29434, only marginally improves the survival of dopamine neurons in MPTP-intoxicated mice.

Over the years, evidence has accumulated on a possible contributive role of the cytosolic quinone reductase NQO2 in models of dopamine neuron degeneration induced by parkinsonian toxin, but most of the data have been obtained in vitro. For this reason, we asked the question whether NQO2 is involved in the in vivo toxicity of MPTP, a neurotoxin classically used to model Parkinson disease-induced neurodegeneration. First, we show that NQO2 is expressed in mouse substantia nigra dopaminergic cell bodies and in human dopaminergic SH-SY5Y cells as well. A highly specific NQO2 inhibitor, S29434, was able to reduce MPTP-induced cell death in a co-culture system of SH-SY5Y cells with astrocytoma U373 cells but was inactive in SH-SY5Y monocultures. We found that S29434 only marginally prevents substantia nigra tyrosine hydroxylase+ cell loss after MPTP intoxication in vivo. The compound produced a slight increase of dopaminergic cell survival at day 7 and 21 following MPTP treatment, especially with 1.5 and 3 mg/kg dosage regimen. The rescue effect did not reach statistical significance (except for one experiment at day 7) and tended to decrease with the 4.5 mg/kg dose, at the latest time point. Despite the lack of robust protective activity of the inhibitor of NQO2 in the mouse MPTP model, we cannot rule out a possible role of the enzyme in parkinsonian degeneration, particularly because it is substantially expressed in dopaminergic neurons.

Lowering Mutant Huntingtin Using Tricyclo-DNA Antisense Oligonucleotides As a Therapeutic Approach for Huntington's Disease.

Huntington's disease is a neurodegenerative disorder caused by a CAG repeat expansion in the first exon of huntingtin gene (HTT) encoding for a toxic polyglutamine protein. This disease is characterized by motor, psychiatric, and cognitive impairments. Currently, there is no disease modifying treatment. However, reducing the expression of the huntingtin protein (HTT) using antisense oligonucleotides (ASOs) has been shown as a promising therapeutic strategy. In this study, we explore the therapeutic potential of ASO made of tricyclo-DNA (tcDNA), a conformationally constrained DNA analog, to silence HTT. We used a gapmer ASO, containing central DNA nucleotides flanked by tcDNA modifications on 5' and 3' ends, allowing the recruitment of RNAse H and subsequent degradation of the messenger RNA. After transfection of tcDNA-ASO in patient-derived fibroblast cell lines, we show a strong decrease of HTT mRNA and protein levels. As a control, 2'O-methyl-RNA targeting the same region of HTT was also tested and did not induce a significant effect. tcDNA-ASO were also evaluated in vivo in the YAC128 mice, containing the full-length human HTT gene with 128 CAG repeat expansion. Single intracerebroventricular (ICV) injections of tcDNA induce a significant decrease of HTT messenger and protein levels in the cortex, hippocampus, striatum, and cerebellum of treated mice. tcDNA-ASO were found well distributed in the central nervous system (CNS) and show long lasting effect with protein levels still low, 12 weeks after a single ICV injection. This proof of concept study suggests the therapeutic potential of gapmer tcDNA ASO to downregulate huntingtin in vitro and in vivo.

Evaluating the Impact of Variable Phosphorothioate Content in Tricyclo-DNA Antisense Oligonucleotides in a Duchenne Muscular Dystrophy Mouse Model.

Antisense oligonucleotides (ASOs) hold promise for therapeutic splice switching correction for genetic diseases, in particular for Duchenne muscular dystrophy (DMD), for which ASO-exon skipping represents one of the most advanced therapeutic strategies. We have previously reported the therapeutic potential of tricyclo-DNA (tcDNA) in mouse models of DMD, highlighting the unique pharmaceutical properties and unprecedented uptake in many tissues after systemic delivery, including the heart and central nervous system. TcDNA-ASOs demonstrate an encouraging safety profile and no particular class-related toxicity, however, when administered in high doses for several months, mild renal toxicity is observed secondary to predictable phosphorothioate (PS)-ASO accumulation in kidneys. In this study, we investigate the influence of the relative content of PS linkages in tcDNA-ASOs on exon skipping efficacy. Mdx mice were injected intravenously once weekly for 4 weeks with tcDNA carrying various amounts of PS linkages (0%, 25%, 33%, 50%, 67%, 83%, and 100%). The results indicate that levels of exon-23 skipping and dystrophin rescue increase with the number of PS linkages in most skeletal muscles except in the heart. As expected, plasma coagulation times are shortened with decreasing PS content, and tcDNA-protein binding in serum directly correlates with the number of PS linkages on the tcDNA backbone. Altogether, these data contribute in establishing the appropriate sulfur content within the tcDNA backbone for maximal efficacy and minimal toxicity of the oligonucleotide.

Interest of colchicine in the treatment of acute myocardial infarct responsible for heart failure in a mouse model.

BACKGROUND: Inflammation is deeply involved in the pathophysiology of ischemia-reperfusion (I/R) lesions and ventricular remodeling due to an acute myocardial infarction (AMI). Colchicine as a pleiotropic anti-inflammatory molecule may exert cardioprotective effects under acute ischemia. Here, we aimed to evaluate the impact of colchicine on reperfusion injury in a mouse model. METHOD: Myocardial ischemia/reperfusion (I/R) injury was induced in C57BL/6 male mice, after 45min ligation of the left coronary artery followed by reperfusion. 400µg/kg of colchicine or the vehicle was administrated intraperitoneally (i.p.) 25min before the reperfusion (blinded administration). Mice were sacrificed at 24h after the acute myocardial ischemia (AMI) and the infarct size was determined. Circulating level of troponin and cytokines profile were assessed 4h after the AMI. An echocardiography was performed in a follow-up group mice, 48h and 8weeks after the AMI. RESULTS: The infarct size was reduced in colchicine treated mice (39.8±3.5% versus 52.9±3.2%, p<0.05). Troponin was significantly lower in the colchicine treated mice (7015.7±1423.7pg/mL, n=5 vs 30,723.7±7959.9pg/mL in the placebo group, n=6; p<0.0001). Fibrosis was decreased in the Colchicine group (24.51±3.13% vs 11.38±2.46%, p=0.03). In the follow-up group mice (n=8), there were no differences between mice treated with placebo (n=9) and mice treated with colchicine (n=9) regarding to cardiac remodeling parameters but outflow approximated by the ITV was higher in the colchicine group. CONCLUSION: In conclusion, colchicine allowed a significant reduction of infarct size in mice, improves hemodynamic parameters and decrease cardiac fibrosis.

TNF-α-mediated caspase-8 activation induces ROS production and TRPM2 activation in adult ventricular myocytes.

AIMS: TRPM2 is a Ca(2+)-permeable cationic channel of the transient receptor potential (TRP) superfamily that is linked to apoptotic signalling. Its involvement in cardiac pathophysiology is unknown. The aim of this study was to determine whether the pro-apoptotic cytokine tumour necrosis factor-α (TNF-α) induces a TRPM2-like current in murine ventricular cardiomyocytes. METHODS AND RESULTS: Adult isolated cardiomyocytes from C57BL/6 mice were exposed to TNF-α (10 ng/mL). Western blotting showed TRPM2 expression, which was not changed after TNF-α incubation. Using patch clamp in whole-cell configuration, a non-specific cation current was recorded after exposure to TNF-α (ITNF), which reached maximal steady-state amplitude after 3 h incubation. ITNF was inhibited by the caspase-8 inhibitor z-IETD-fmk, the antioxidant N-acetylcysteine, and the TRPM2 inhibitors clotrimazole, N-(P-amylcinnamoyl) anthranilic acid and flufenamic acid (FFA). TRPM2 has previously been shown to be activated by ADP-ribose, which is produced by poly(ADP-ribose) polymerase 1 (PARP-1). TNF-α exposure resulted in increased poly-ADP-ribosylation of proteins and the PARP-1 inhibitor 3-aminobenzamide inhibited ITNF. TNF-α exposure increased the mitochondrial production of reactive oxygen species (ROS; measured with the fluorescent indicator MitoSOX Red), and this increase was blocked by the caspase-8 inhibitor z-IETD-fmk. Clotrimazole and TRPM2 inhibitory antibody decreased TNF-α-induced cardiomyocyte death. CONCLUSION: These results demonstrate that TNF-α induces a TRPM2 current in adult ventricular cardiomyocytes. TNF-α induces caspase-8 activation leading to ROS production, PARP-1 activation, and ADP-ribose production. TNF-induced TRPM2 activation may contribute to cardiomyocyte cell death.

From ApoA1 upregulation to BET family bromodomain inhibition: discovery of I-BET151.

The discovery, synthesis and biological evaluation of a novel series of 7-isoxazoloquinolines is described. Several analogs are shown to increase ApoA1 expression within the nanomolar range in the human hepatic cell line HepG2.

Discovery and characterization of small molecule inhibitors of the BET family bromodomains.

Epigenetic mechanisms of gene regulation have a profound role in normal development and disease processes. An integral part of this mechanism occurs through lysine acetylation of histone tails which are recognized by bromodomains. While the biological and structural characterization of many bromodomain containing proteins has advanced considerably, the therapeutic tractability of this protein family is only now becoming understood. This paper describes the discovery and molecular characterization of potent (nM) small molecule inhibitors that disrupt the function of the BET family of bromodomains (Brd2, Brd3, and Brd4). By using a combination of phenotypic screening, chemoproteomics, and biophysical studies, we have discovered that the protein-protein interactions between bromodomains and acetylated histones can be antagonized by selective small molecules that bind at the acetylated lysine recognition pocket. X-ray crystal structures of compounds bound into bromodomains of Brd2 and Brd4 elucidate the molecular interactions of binding and explain the precisely defined stereochemistry required for activity.