Characterization of Novel Diphenylamine Compounds as Ferroptosis Inhibitors.

Ferroptosis is a form of oxidative cell death that is increasingly recognized as a key mechanism not only in neurodegeneration but also in regulated cell death, causing disease in other tissues. In neurons, major hallmarks of ferroptosis involve the accumulation of lipid reactive oxygen species (ROS) and impairment of mitochondrial morphology and function. Compounds that interfere with ferroptosis could provide novel treatment options for neurodegenerative disorders and other diseases involving ferroptosis. In the present study, we developed new compounds by refining structural elements of the BH3 interacting-domain death agonist inhibitor BI-6c9, which was previously demonstrated to block ferroptosis signaling at the level of mitochondria. Here, we inserted an antioxidative diphenylamine (DPA) structure to the BI-6c9 structure. These DPA compounds were then tested in models of erastin, and Ras-selective lethal small molecule 3 induced ferroptosis in neuronal HT22 cells. The DPA compounds showed an increased protective potency against ferroptotic cell death compared with the scaffold molecule BI-6c9. Moreover, hallmarks of ferroptosis such as lipid, cytosolic, and mitochondrial ROS formation were abrogated in a concentration- and time-dependent manner. Additionally, mitochondrial parameters such as mitochondrial morphology, mitochondrial membrane potential, and mitochondrial respiration were preserved by the DPA compounds, supporting the conclusion that lipid ROS toxicity and mitochondrial impairment are closely related in ferroptosis. Our findings confirm that the DPA compounds are very effective agents in preventing ferroptotic cell death by blocking ROS production and, in particular, via mitochondrial protection. SIGNIFICANCE STATEMENT: Preventing neuronal cells from different forms of oxidative cell death was previously described as a promising strategy for treatment against several neurodegenerative diseases. This study reports novel compounds based on a diphenylamine structure that strongly protects neuronal HT22 cells from ferroptotic cell death upon erastin and Ras-selective lethal small molecule 3 induction by preventing the development of different reactive oxygen species and by protecting mitochondria from ferroptotic impairments.

Production of a beam of tensor-polarized deuterons using a carbon target.

An initially unpolarized beam of deuterons is found to acquire tensor polarization after traversing a foil of spin-zero target nuclei. The effect, called nuclear spin dichroism, has been predicted theoretically, albeit resulting in small values of p(zz) of the order of 0.01 for energies around 10 MeV. The experiment was carried out at the Köln tandem accelerator using carbon targets bombarded by deuterons. The observed polarization is as large as p(zz)=-0.28±0.03 for a beam of 14.8 MeV and a 129 mg/cm2 target. The results allow one to produce tensor-polarized deuterons with p(zz) around -0.30 (or +0.25) from an initially unpolarized beam using a carbon target of appropriate thickness.

An imperfect inverted repeat is critical for DNA binding of the response regulator RegR of Bradyrhizobium japonicum.

RegR is the response regulator of the RegSR two-component regulatory system in Bradyrhizobium japonicum. The only target known so far is the fixR-nifA operon, encoding the redox-responsive transcription factor NifA, which activates many genes required for symbiotic nitrogen fixation in soybean nodules. In previous in vivo studies, we identified a 32 bp upstream activating sequence located around position -68, which is essential for RegR-dependent expression of the fixR-nifA operon. Here, we used an in vitro binding-site selection assay (SELEX) to more precisely define the DNA-binding specificity of RegR. The selected sequences comprised an imperfect inverted repeat (GCGGC-N(5)-GTCGC) which is highly similar to an imperfect inverted repeat in the fixR UAS (GCGAC-N(5)-GACGC). In a parallel approach, band-shift experiments were performed with oligonucleotides comprising defined point or deletion mutations in the fixR UAS. This led to the identification of 11 critical nucleotides within a 17 bp minimal RegR binding site centered at position -64 upstream of the fixR-nifA transcription start site. Notably, all 11 critical nucleotides were located either within the half sites of the inverted repeat (four nucleotides in each half site) or in the 5 bp spacer that separates the half sites (three nucleotides). Based on these results, we defined a DNA motif comprising those nucleotides that are critical for RegR binding (RegR box; 5'-GNG(A)(G)C(A)(G)TTNNGNCGC-3'). A comparison of the RegR box with functional binding sites of the RegR-like regulator RegA of Rhodobacter capsulatus revealed considerable similarities. Thus, the RegR box may assist in the identification of new RegR target genes not only in B.japonicum but also in other alpha-proteobacteria possessing RegR-like response regulators.

Evidence for a functional similarity between the two-component regulatory systems RegSR, ActSR, and RegBA (PrrBA) in alpha-Proteobacteria.

The symbiotic bacteria Bradyrhizobium japonicum and Sinorhizobium meliloti, and the purple photosynthetic bacteria Rhodobacter capsulatus, Rhodovulum sulfidophilum, Roseobacter denitrificans and Rhodobacter sphaeroides possess homologous two-component regulatory systems, namely RegSR, ActSR, RegBA and PrrBA. The respective response regulators of these bacteria control expression of different regulons that are involved in N2 fixation, CO2 fixation, photosynthesis or acid tolerance. We therefore asked whether the regulators are functionally exchangeable or whether they have disparate functions in the different species, despite the amino acid sequence similarity. In this study, we showed that purified B. japonicum RegR bound in vitro to genuine DNA targets for Rba. capsulatus RegA, and that RegA was phosphorylated in vitro when RegSc (a soluble variant of the sensor kinase RegS) was added to an Escherichia coli extract containing overexpressed RegA. In vivo, RegA and S. meliloti ActR activated transcription of the B. japonicum fixR-nifA operon, normally a target for RegR. The genes for both regulators, regA and actR, were able to complement a B. japonicum regR mutant with respect to the formation of a nitrogen-fixing symbiosis with soybean. Vice versa, RegR activated in Rba. capsulatus the expression of the photosynthesis operon puc, normally a target for RegA. In conclusion, the results show that B. japonicum RegR, Rba. capsulatus RegA, and S. meliloti ActR are functionally similar.

Phosphorylation, dephosphorylation and DNA-binding of the Bradyrhizobium japonicum RegSR two-component regulatory proteins.

Under low oxygen conditions, induction of many genes required for nitrogen fixation in Bradyrhizobium japonicum depends on the redox-responsive transcriptional activator NifA which is encoded in the fixR-nifA operon. Basal expression of this operon depends on the response regulator RegR and a DNA element located around position -68 in the fixR-nifA promoter region. To investigate the functional properties of RegR and the interaction with its putative cognate kinase, RegS, we overproduced and affinity-purified RegR and a truncated soluble variant of RegS (RegS(C)), both as N-terminally His(6)-tagged proteins. RegS(C) autophosphorylated when incubated with [gamma-(32)P]ATP, and it catalyzed the transfer of the phosphoryl label to RegR. The phosphorylated form of RegS(C) exhibited phosphatase activity on RegR-phosphate. Chemical stability tests and site-specific mutagenesis identified amino acids H219 and D63 of RegS and RegR, respectively, as the phosphorylated residues. Competition experiments with isolated domains demonstrated that the N-terminal but not the C-terminal domain of RegR interacts with RegS(C). Band-shift experiments revealed that phosphorylated RegR had at least eightfold enhanced DNA-binding activity compared with dephosphorylated RegR or the mutant protein RegR-D63N, which cannot be phosphorylated. In conclusion, the RegSR proteins of B. japonicum exhibit functional properties in vitro that are typical of two-component regulatory systems.

Mapping of the constitutive lysyl-tRNA synthetase gene of Escherichia coli K-12.

The constitutive lysyl-tRNA synthetase gene (lysS) was mapped at 62.1 min on the Escherichia coli chromosome by a combination of conjugation and transduction, with physical confirmation by two-dimensional gel electrophoresis. Revertant analysis suggests that the altered isoelectric point and the low amount of the mutant LysS protein may be due to a single mutational event.