No abstract

Parkinson's disease (PD) is a progressive neurodegenerative movement disorder characterized by the loss of nigrostriatal dopaminergic neurons. Mounting evidence suggests that Nrf2 is a promising target for neuroprotective interventions in PD. However, electrophilic chemical properties of the canonical Nrf2-based drugs cause irreversible alkylation of cysteine residues on cellular proteins resulting in side effects. Bach1 is a known transcriptional repressor of the Nrf2 pathway. We report that Bach1 levels are up-regulated in PD postmortem brains and preclinical models. Bach1 knockout (KO) mice were protected against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced dopaminergic neurotoxicity and associated oxidative damage and neuroinflammation. Functional genomic analysis demonstrated that the neuroprotective effects in Bach1 KO mice was due to up-regulation of Bach1-targeted pathways that are associated with both Nrf2-dependent antioxidant response element (ARE) and Nrf2-independent non-ARE genes. Using a proprietary translational technology platform, a drug library screen identified a substituted benzimidazole as a Bach1 inhibitor that was validated as a nonelectrophile. Oral administration of the Bach1 inhibitor attenuated MPTP neurotoxicity in pre- and posttreatment paradigms. Bach1 inhibitor–induced neuroprotection was associated with the up-regulation of Bach1-targeted pathways in concurrence with the results from Bach1 KO mice. Our results suggest that genetic deletion as well as pharmacologic inhibition of Bach1 by a nonelectrophilic inhibitor is a promising therapeutic approach for PD.

Most common drug development failures originate from either bioavailability problems, or unexpected toxic effects. The culprit is often the liver, which is responsible for biotransformation of a majority of xenobiotics. Liver may be modeled using "liver on a chip" devices, which may include established cell lines, primary human cells, and stem cell-derived hepatocyte-like cells. The choice of biological material along with its processing and maintenance greatly influence both the device performance and the resultant toxicity predictions. Impediments to the development of "liver on a chip" technology include the problems with standardization of cells, limitations imposed by culturing and the necessity to develop more complicated fluidic contours. Fortunately, recent breakthroughs in the development of cell-based reporters, including ones with fluorescent label, permits monitoring of the behavior of the cells embed into the "liver on a chip" devices. Finally, a set of computational approaches has been developed to model both particular toxic response and the homeostasis of human liver as a whole; these approaches pave a way to enhance the in silico stage of assessment for a potential toxicity.

Activation of HIF-1α and Nrf2 is a primary component of cellular response to oxidative stress, and activation of HIF-1α and Nrf2 provides neuroprotection in models of neurodegenerative disorders, including ischemic stroke, Alzheimer's and Parkinson's diseases. Screening a library of CNS-targeted drugs using novel reporters for HIF-1α and Nrf2 elevation in neuronal cells revealed histone deacetylase (HDAC) inhibitors as potential activators of these pathways. We report the identification of phenylhydroxamates as single agents exhibiting tripartite inhibition of HDAC6, inhibition of HIF-1 prolyl hydroxylase (PHD), and activation of Nrf2. Two superior tripartite agents, ING-6 and ING-66, showed neuroprotection against various cellular insults, associated with stabilization of both Nrf2 and HIF-1, and expression of their respective target genes in vitro and in vivo. Discovery of the innate ability of phenylhydroxamate HDAC inhibitors to activate Nrf2 and HIF provides a novel route to multifunctional neuroprotective agents and cautions against HDAC6 selective inhibitors as chemical probes of specific HDAC isoform function.

No abstract

L-Ascorbate (L-Asc), but not D-isoascorbate (D-Asc) and N-acetylcysteine (NAC) suppress HIF1 ODD-luc reporter activation induced by various inhibitors of HIF prolyl hydroxylase (PHD). The efficiency of suppression by L-Asc was sensitive to the nature of HIF PHD inhibitor chosen for reporter activation. In particular, the inhibitors developed to compete with alpha-ketoglutarate (αKG), were less sensitive to suppression by the physiological range of L-Asc (40-100 μM) than those having a strong iron chelation motif. Challenging those HIF activators in the reporter system with D-Asc demonstrated that the D-isomer, despite exhibiting the same reducing potency with respect to ferric iron, had almost no effect compared to L-Asc. Similarly, no effect on reporter activation was observed with cell-permeable reducing agent NAC up to 1 mM. Docking of L-Asc and D-Asc acid into the HIF PHD2 crystal structure showed interference of Tyr310 with respect to D-Asc. This suggests that L-Asc is not merely a reducing agent preventing enzyme inactivation. Rather, the overall results identify L-Asc as a co-substrate of HIF PHD that may compete for the binding site of αKG in the enzyme active center. This conclusion is in agreement with the results obtained recently in cell-based systems for TET enzymes and jumonji histone demethylases, where L-Asc has been proposed to act as a co-substrate and not as a reducing agent preventing enzyme inactivation.

Specificity of RNAi to selected target is challenged by off-target effects, both canonical and non-canonical. Notably, more than half of all human microRNAs are co-expressed with hosting them proteincoding genes. Here we dissect regulatory subnetwork centered on IGFBP6 gene, which is associated with low proliferative state and high migratory activity of basal-like breast cancer. We inhibited expression of IGFBP6 gene in a model cell line for basal-like breast carcinoma MDA-MB-231, then traced secondary and tertiary effects of this knockdown to LAMA4, a laminin encoding gene that contributes to the phenotype of triple-negative breast cancer. LAMA4-regulating miRNA miR-4274 and its host gene SORCS2 were highlighted as intermediate regulators of the expression levels of LAMA4, which correlated in a basal-like breast carcinoma sample subset of TCGA to the levels of SORCS2 negatively. Overall, our study points that the secondary and tertiary layers of regulatory interactions are certainly underappreciated. As these types of molecular event may significantly contribute to the formation of the cell phenotypes after RNA interference based knockdowns, further studies of multilayered molecular networks affected by RNAi are warranted.

The effect of the operational potential on the stability of electrochemical biosensors is particularly relevant in the case of peroxidase biosensors, because these enzymes can catalyze the reduction of hydrogen peroxide via either a high-potential redox cycle [involving Compound I, Compound II, and Fe(III)] or a low-potential redox cycle [involving Fe(III) and Fe(II)]. Herein, it is shown that recombinant tobacco peroxidase immobilized on a graphite surface displays two well-separated electrocatalytic waves, associated with each of these two catalytic cycles. While continuous scanning in the highpotential region does not alter significantly the electrocatalytic current, it is shown that just modest incursions into the low-potential region cause an irreversible loss of the electrocatalytic response. A quantitative analysis of the extent of inactivation as a function of time, potential, and hydrogen peroxide concentration is shown to be consistent with a fast inactivation caused by hydroxyl radicals generated by a Fenton-like mechanism. Accordingly, the inactivation process is shown to slow via the addition of radical scavengers to the solution. Preliminary results indicate that the same inactivation process may also be present in horseradish peroxidase-modified electrodes.