Novel Proline Transporter Inhibitor (LQFM215) Presents Antipsychotic Effect in Ketamine Model of Schizophrenia.

The glutamatergic hypothesis of schizophrenia suggests a correlation between NMDA receptor hypofunction and negative psychotic symptoms. It has been observed that the expression of the proline transporter (PROT) in the central nervous system (CNS) is associated with glutamatergic neurotransmission, as L-proline has the capacity to activate and modulate AMPA and NMDA receptors. In this study, we aimed to investigate whether inhibition of proline transporters could enhance glutamatergic neurotransmission and potentially exhibit antipsychotic effects in an experimental schizophrenia model. Using molecular dynamics analysis in silico, we validated an innovative PROT inhibitor, LQFM215. We quantified the cytotoxicity of LQFM215 in the Lund human mesencephalic cell line (LUHMES). Subsequently, we employed the ketamine-induced psychosis model to evaluate the antipsychotic potential of the inhibitor, employing behavioral tests including open-field, three-chamber interaction, and prepulse inhibition (PPI). Our results demonstrate that LQFM215, at pharmacologically active concentrations, exhibited negligible neurotoxicity when astrocytes were co-cultured with neurons. In the ketamine-induced psychosis model, LQFM215 effectively reduced hyperlocomotion and enhanced social interaction in a three-chamber social approach task across all administered doses. Moreover, the compound successfully prevented the ketamine-induced disruption of sensorimotor gating in the PPI test at all tested doses. Overall, these findings suggest that PROT inhibition could serve as a potential therapeutic target for managing symptoms of schizophrenia model.

Stimulation of de novo glutathione synthesis by nitrofurantoin for enhanced resilience of hepatocytes.

Toxicity is not only a function of damage mechanisms, but is also determined by cellular resilience factors. Glutathione has been reported as essential element to counteract negative influences. The present work hence pursued the question how intracellular glutathione can be elevated transiently to render cells more resistant toward harmful conditions. The antibiotic nitrofurantoin (NFT) was identified to stimulate de novo synthesis of glutathione in the human hepatoma cell line, HepG2, and in primary human hepatocytes. In intact cells, activation of NFT yielded a radical anion, which subsequently initiated nuclear-factor-erythroid 2-related-factor-2 (Nrf2)-dependent induction of glutamate cysteine ligase (GCL). Application of siRNA-based intervention approaches confirmed the involvement of the Nrf2-GCL axis in the observed elevation of intracellular glutathione levels. Quantitative activation of Nrf2 by NFT, and the subsequent rise in glutathione, were similar as observed with the potent experimental Nrf2 activator diethyl maleate. The elevation of glutathione levels, observed even 48 h after withdrawal of NFT, rendered cells resistant to different stressors such as the mitochondrial inhibitor rotenone, the redox cycler paraquat, the proteasome inhibitors MG-132 or bortezomib, or high concentrations of NFT. Repurpose of the antibiotic NFT as activator of Nrf2 could thus be a promising strategy for a transient and targeted activation of the endogenous antioxidant machinery. Graphical abstract.

Profiling of Human Neural Crest Chemoattractant Activity as a Replacement of Fetal Bovine Serum for In Vitro Chemotaxis Assays.

Fetal bovine serum (FBS) is the only known stimulus for the migration of human neural crest cells (NCCs). Non-animal chemoattractants are desirable for the optimization of chemotaxis as-says to be incorporated in a test battery for reproductive and developmental toxicity. We con-firmed here in an optimized transwell assay that FBS triggers directed migration along a con-centration gradient. The responsible factor was found to be a protein in the 30-100 kDa size range. In a targeted approach, we tested a large panel of serum constituents known to be chem-otactic for NCCs in animal models (e.g., VEGF, PDGF, FGF, SDF-1/CXCL12, ephrins, endothelin, Wnt, BMPs). None of the corresponding human proteins showed any effect in our chemotaxis assays based on human NCCs. We then examined, whether human cells would produce any fac-tor able to trigger NCC migration in a broad screening approach. We found that HepG2 hepa-toma cells produced chemotaxis-triggering activity (CTA). Using chromatographic methods and by employing the NCC chemotaxis test as bioassay, the responsible protein was enriched by up to 5000-fold. We also explored human serum and platelets as a direct source, independent of any cell culture manipulations. A CTA was enriched from platelet lysates several thousand-fold. Its temperature and protease sensitivity suggested also a protein component. The capacity of this factor to trigger chemotaxis was confirmed by single-cell video-tracking analysis of migrating NCCs. The human CTA characterized here may be employed in the future for the setup of assays testing for the disturbance of directed NCC migration by toxicants.

A Ligand Selection Strategy Identifies Chemical Probes Targeting the Proteases of SARS-CoV-2.

Activity-based probes are valuable tools for chemical biology. However, finding probes that specifically target the active site of an enzyme remains a challenging task. Herein, we present a ligand selection strategy that allows to rapidly tailor electrophilic probes to a target of choice and showcase its application for the two cysteine proteases of SARS-CoV-2 as proof of concept. The resulting probes were specific for the active site labeling of 3CLpro and PLpro with sufficient selectivity in a live cell model as well as in the background of a native human proteome. Exploiting the probes as tools for competitive profiling of a natural product library identified salvianolic acid derivatives as promising 3CLpro inhibitors. We anticipate that our ligand selection strategy will be useful to rapidly develop customized probes and discover inhibitors for a wide range of target proteins also beyond corona virus proteases.

Design and evaluation of bi-functional iron chelators for protection of dopaminergic neurons from toxicants.

While the etiology of non-familial Parkinson's disease (PD) remains unclear, there is evidence that increased levels of tissue iron may be a contributing factor. Moreover, exposure to some environmental toxicants is considered an additional risk factor. Therefore, brain-targeted iron chelators are of interest as antidotes for poisoning with dopaminergic toxicants, and as potential treatment of PD. We, therefore, designed a series of small molecules with high affinity for ferric iron and containing structural elements to allow their transport to the brain via the neutral amino acid transporter, LAT1 (SLC7A5). Five candidate molecules were synthesized and initially characterized for protection from ferroptosis in human neurons. The promising hydroxypyridinone SK4 was characterized further. Selective iron chelation within the physiological range of pH values and uptake by LAT1 were confirmed. Concentrations of 10-20 µM blocked neurite loss and cell demise triggered by the parkinsonian neurotoxicants, methyl-phenyl-pyridinium (MPP+) and 6-hydroxydopamine (6-OHDA) in human dopaminergic neuronal cultures (LUHMES cells). Rescue was also observed when chelators were given after the toxicant. SK4 derivatives that either lacked LAT1 affinity or had reduced iron chelation potency showed altered activity in our assay panel, as expected. Thus, an iron chelator was developed that revealed neuroprotective properties, as assessed in several models. The data strongly support the role of iron in dopaminergic neurotoxicity and suggests further exploration of the proposed design strategy for improving brain iron chelation.

An adverse outcome pathway for parkinsonian motor deficits associated with mitochondrial complex I inhibition.

Epidemiological studies have observed an association between pesticide exposure and the development of Parkinson's disease, but have not established causality. The concept of an adverse outcome pathway (AOP) has been developed as a framework for the organization of available information linking the modulation of a molecular target [molecular initiating event (MIE)], via a sequence of essential biological key events (KEs), with an adverse outcome (AO). Here, we present an AOP covering the toxicological pathways that link the binding of an inhibitor to mitochondrial complex I (i.e., the MIE) with the onset of parkinsonian motor deficits (i.e., the AO). This AOP was developed according to the Organisation for Economic Co-operation and Development guidelines and uploaded to the AOP database. The KEs linking complex I inhibition to parkinsonian motor deficits are mitochondrial dysfunction, impaired proteostasis, neuroinflammation, and the degeneration of dopaminergic neurons of the substantia nigra. These KEs, by convention, were linearly organized. However, there was also evidence of additional feed-forward connections and shortcuts between the KEs, possibly depending on the intensity of the insult and the model system applied. The present AOP demonstrates mechanistic plausibility for epidemiological observations on a relationship between pesticide exposure and an elevated risk for Parkinson's disease development.

Simultaneous IR-Spectroscopic Observation of α-Synuclein, Lipids, and Solvent Reveals an Alternative Membrane-Induced Oligomerization Pathway.

The intrinsically disordered protein α-synuclein (αS), a known pathogenic factor for Parkinson's disease, can adopt defined secondary structures when interacting with membranes or during fibrillation. The αS-lipid interaction and the implications of this process for aggregation and damage to membranes are still poorly understood. Therefore, we established a label-free infrared (IR) spectroscopic approach to allow simultaneous monitoring of αS conformation and membrane integrity. IR showed its unique sensitivity for identifying distinct ß-structured aggregates. A comparative study of wild-type αS and the naturally occurring splicing variant αS Δexon3 yielded new insights into the membrane's capability for altering aggregation pathways.

Stem Cell Transcriptome Responses and Corresponding Biomarkers That Indicate the Transition from Adaptive Responses to Cytotoxicity.

Analysis of transcriptome changes has become an established method to characterize the reaction of cells to toxicants. Such experiments are mostly performed at compound concentrations close to the cytotoxicity threshold. At present, little information is available on concentration-dependent features of transcriptome changes, in particular, at the transition from noncytotoxic concentrations to conditions that are associated with cell death. Thus, it is unclear in how far cell death confounds the results of transcriptome studies. To explore this gap of knowledge, we treated pluripotent stem cells differentiating to human neuroepithelial cells (UKN1 assay) for short periods (48 h) with increasing concentrations of valproic acid (VPA) and methyl mercury (MeHg), two compounds with vastly different modes of action. We developed various visualization tools to describe cellular responses, and the overall response was classified as "tolerance" (minor transcriptome changes), "functional adaptation" (moderate/strong transcriptome responses, but no cytotoxicity), and "degeneration". The latter two conditions were compared, using various statistical approaches. We identified (i) genes regulated at cytotoxic, but not at noncytotoxic, concentrations and (ii) KEGG pathways, gene ontology term groups, and superordinate biological processes that were only regulated at cytotoxic concentrations. The consensus markers and processes found after 48 h treatment were then overlaid with those found after prolonged (6 days) treatment. The study highlights the importance of careful concentration selection and of controlling viability for transcriptome studies. Moreover, it allowed identification of 39 candidate "biomarkers of cytotoxicity". These could serve to provide alerts that data sets of interest may have been affected by cell death in the model system studied.

Switching from astrocytic neuroprotection to neurodegeneration by cytokine stimulation.

Astrocytes, the largest cell population in the human brain, are powerful inflammatory effectors. Several studies have examined the interaction of activated astrocytes with neurons, but little is known yet about human neurotoxicity under such situations and about strategies of neuronal rescue. To address this question, immortalized murine astrocytes (IMA) were combined with human LUHMES neurons and stimulated with an inflammatory (TNF, IL-1) cytokine mix (CM). Neurotoxicity was studied both in co-cultures and in monocultures after transfer of conditioned medium from activated IMA. Interventions with >20 drugs were used to profile the model system. Control IMA supported neurons and protected them from neurotoxicants. Inflammatory activation reduced this protection, and prolonged exposure of co-cultures to CM triggered neurotoxicity. Neither the added cytokines nor the release of NO from astrocytes were involved in this neurodegeneration. The neurotoxicity-mediating effect of IMA was faithfully reproduced by human astrocytes. Moreover, glia-dependent toxicity was also observed, when IMA cultures were stimulated with CM, and the culture medium was transferred to neurons. Such neurotoxicity was prevented when astrocytes were treated by p38 kinase inhibitors or dexamethasone, whereas such compounds had no effect when added to neurons. Conversely, treatment of neurons with five different drugs, including resveratrol and CEP1347, prevented toxicity of astrocyte supernatants. Thus, the sequential IMA-LUHMES neuroinflammation model is suitable for separate profiling of both glial-directed and directly neuroprotective strategies. Moreover, direct evaluation in co-cultures of the same cells allows for testing of therapeutic effectiveness in more complex settings, in which astrocytes affect pharmacological properties of neurons.

Multiparameter toxicity assessment of novel DOPO-derived organophosphorus flame retardants.

Halogen-free organophosphorus flame retardants are considered as replacements for the phased-out class of polybrominated diphenyl ethers (PBDEs). However, toxicological information on new flame retardants is still limited. Based on their excellent flame retardation potential, we have selected three novel 9,10-dihydro-9-oxa-10-phosphaphenanthrene-10-oxide (DOPO) derivatives and assessed their toxicological profile using a battery of in vitro test systems in order to provide toxicological information before their large-scale production and use. PBDE-99, applied as a reference compound, exhibited distinct neuro-selective cytotoxicity at concentrations ≥10 µM. 6-(2-((6-oxido-6H-dibenzo[c,e][1,2]oxaphosphinin-6-yl)amino)ethoxy)-6H-dibenzo[c,e][1,2]oxaphosphinine 6-oxide (ETA-DOPO) and 6,6'-(ethane-1,2-diylbis(oxy))bis(6H-dibenzo[c,e][1,2]oxaphosphinine-6-oxide) (EG-DOPO) displayed adverse effects at concentrations >10 µM in test systems reflecting the properties of human central and peripheral nervous system neurons, as well as in a set of non-neuronal cell types. DOPO and its derivative 6,6'-(ethane-1,2-diylbis(azanediyl))bis(6H-dibenzo[c,e][1,2]oxaphosphinine-6-oxide) (EDA-DOPO) were neither neurotoxic, nor did they exhibit an influence on neural crest cell migration, or on the integrity of human skin equivalents. The two compounds furthermore displayed no inflammatory activation potential, nor did they affect algae growth or daphnia viability at concentrations ≤400 µM. Based on the superior flame retardation properties, biophysical features suited for use in polyurethane foams, and low cytotoxicity of EDA-DOPO, our results suggest that it is a candidate for the replacement of currently applied flame retardants.

Definition of transcriptome-based indices for quantitative characterization of chemically disturbed stem cell development: introduction of the STOP-Toxukn and STOP-Toxukk tests.

Stem cell-based in vitro test systems can recapitulate specific phases of human development. In the UKK test system, human pluripotent stem cells (hPSCs) randomly differentiate into cells of the three germ layers and their derivatives. In the UKN1 test system, hPSCs differentiate into early neural precursor cells. During the normal differentiation period (14 days) of the UKK system, 570 genes [849 probe sets (PSs)] were regulated >fivefold; in the UKN1 system (6 days), 879 genes (1238 PSs) were regulated. We refer to these genes as 'developmental genes'. In the present study, we used genome-wide expression data of 12 test substances in the UKK and UKN1 test systems to understand the basic principles of how chemicals interfere with the spontaneous transcriptional development in both test systems. The set of test compounds included six histone deacetylase inhibitors (HDACis), six mercury-containing compounds ('mercurials') and thalidomide. All compounds were tested at the maximum non-cytotoxic concentration, while valproic acid and thalidomide were additionally tested over a wide range of concentrations. In total, 242 genes (252 PSs) in the UKK test system and 793 genes (1092 PSs) in the UKN1 test system were deregulated by the 12 test compounds. We identified sets of 'diagnostic genes' appropriate for the identification of the influence of HDACis or mercurials. Test compounds that interfered with the expression of developmental genes usually antagonized their spontaneous development, meaning that up-regulated developmental genes were suppressed and developmental genes whose expression normally decreases were induced. The fraction of compromised developmental genes varied widely between the test compounds, and it reached up to 60 %. To quantitatively describe disturbed development on a genome-wide basis, we recommend a concept of two indices, 'developmental potency' (D p) and 'developmental index' (D i), whereby D p is the fraction of all developmental genes that are up- or down-regulated by a test compound, and D i is the ratio of overrepresentation of developmental genes among all genes deregulated by a test compound. The use of D i makes hazard identification more sensitive because some compounds compromise the expression of only a relatively small number of genes but have a high propensity to deregulate developmental genes specifically, resulting in a low D p but a high D i. In conclusion, the concept based on the indices D p and D i offers the possibility to quantitatively express the propensity of test compounds to interfere with normal development.

Adverse outcome pathways: opportunities, limitations and open questions.

Adverse outcome pathways (AOPs) are a recent toxicological construct that connects, in a formalized, transparent and quality-controlled way, mechanistic information to apical endpoints for regulatory purposes. AOP links a molecular initiating event (MIE) to the adverse outcome (AO) via key events (KE), in a way specified by key event relationships (KER). Although this approach to formalize mechanistic toxicological information only started in 2010, over 200 AOPs have already been established. At this stage, new requirements arise, such as the need for harmonization and re-assessment, for continuous updating, as well as for alerting about pitfalls, misuses and limits of applicability. In this review, the history of the AOP concept and its most prominent strengths are discussed, including the advantages of a formalized approach, the systematic collection of weight of evidence, the linkage of mechanisms to apical end points, the examination of the plausibility of epidemiological data, the identification of critical knowledge gaps and the design of mechanistic test methods. To prepare the ground for a broadened and appropriate use of AOPs, some widespread misconceptions are explained. Moreover, potential weaknesses and shortcomings of the current AOP rule set are addressed (1) to facilitate the discussion on its further evolution and (2) to better define appropriate vs. less suitable application areas. Exemplary toxicological studies are presented to discuss the linearity assumptions of AOP, the management of event modifiers and compensatory mechanisms, and whether a separation of toxicodynamics from toxicokinetics including metabolism is possible in the framework of pathway plasticity. Suggestions on how to compromise between different needs of AOP stakeholders have been added. A clear definition of open questions and limitations is provided to encourage further progress in the field.

A transcriptome-based classifier to identify developmental toxicants by stem cell testing: design, validation and optimization for histone deacetylase inhibitors.

Test systems to identify developmental toxicants are urgently needed. A combination of human stem cell technology and transcriptome analysis was to provide a proof of concept that toxicants with a related mode of action can be identified and grouped for read-across. We chose a test system of developmental toxicity, related to the generation of neuroectoderm from pluripotent stem cells (UKN1), and exposed cells for 6 days to the histone deacetylase inhibitors (HDACi) valproic acid, trichostatin A, vorinostat, belinostat, panobinostat and entinostat. To provide insight into their toxic action, we identified HDACi consensus genes, assigned them to superordinate biological processes and mapped them to a human transcription factor network constructed from hundreds of transcriptome data sets. We also tested a heterogeneous group of 'mercurials' (methylmercury, thimerosal, mercury(II)chloride, mercury(II)bromide, 4-chloromercuribenzoic acid, phenylmercuric acid). Microarray data were compared at the highest non-cytotoxic concentration for all 12 toxicants. A support vector machine (SVM)-based classifier predicted all HDACi correctly. For validation, the classifier was applied to legacy data sets of HDACi, and for each exposure situation, the SVM predictions correlated with the developmental toxicity. Finally, optimization of the classifier based on 100 probe sets showed that eight genes (F2RL2, TFAP2B, EDNRA, FOXD3, SIX3, MT1E, ETS1 and LHX2) are sufficient to separate HDACi from mercurials. Our data demonstrate how human stem cells and transcriptome analysis can be combined for mechanistic grouping and prediction of toxicants. Extension of this concept to mechanisms beyond HDACi would allow prediction of human developmental toxicity hazard of unknown compounds with the UKN1 test system.

Alpha-synuclein binds to the inner membrane of mitochondria in an α-helical conformation.

The human alpha-Synuclein (αS) protein is of significant interest because of its association with Parkinson's disease and related neurodegenerative disorders. The intrinsically disordered protein (140 amino acids) is characterized by the absence of a well-defined structure in solution. It displays remarkable conformational flexibility upon macromolecular interactions, and can associate with mitochondrial membranes. Site-directed spin-labeling in combination with electron paramagnetic resonance spectroscopy enabled us to study the local binding properties of αS on artificial membranes (mimicking the inner and outer mitochondrial membranes), and to evaluate the importance of cardiolipin in this interaction. With pulsed, two-frequency, double-electron electron paramagnetic resonance (DEER) approaches, we examined, to the best of our knowledge for the first time, the conformation of αS bound to isolated mitochondria.

Modeling ferroptosis in human dopaminergic neurons: Pitfalls and opportunities for neurodegeneration research.

The activation of ferroptosis is being pursued in cancer research as a strategy to target apoptosis-resistant cells. By contrast, in various diseases that affect the cardiovascular system, kidneys, liver, and central and peripheral nervous systems, attention is directed toward interventions that prevent ferroptotic cell death. Mechanistic insights into both research areas stem largely from studies using cellular in vitro models. However, intervention strategies that show promise in cellular test systems often fail in clinical trials, which raises concerns regarding the predictive validity of the utilized in vitro models. In this study, the human LUHMES cell line, which serves as a model for human dopaminergic neurons, was used to characterize factors influencing the activation of ferroptosis. Erastin and RSL-3 induced cell death that was distinct from apoptosis. Parameters such as the differentiation state of LUHMES cells, cell density, and the number and timing of medium changes were identified as determinants of sensitivity to ferroptosis activation. In differentiated LUHMES cells, interventions at mechanistically divergent sites (iron chelation, coenzyme Q10, peroxidase mimics, or inhibition of 12/15-lipoxygenase) provide almost complete protection from ferroptosis. LUHMES cells allowed the experimental modulation of intracellular iron concentrations and demonstrated a correlation between intracellular iron levels, the rate of lipid peroxidation, as well as the sensitivity of the cells to ferroptotic cell death. These findings underscore the importance of understanding the various factors that influence ferroptosis activation and highlight the need for well-characterized in vitro models to enhance the reliability and predictive value of observations in ferroptosis research, particularly when translating findings into in vivo contexts.

Oxidative and nitrative alpha-synuclein modifications and proteostatic stress: implications for disease mechanisms and interventions in synucleinopathies.

Alpha-synuclein (ASYN) is a major constituent of the typical protein aggregates observed in several neurodegenerative diseases that are collectively referred to as synucleinopathies. A causal involvement of ASYN in the initiation and progression of neurological diseases is suggested by observations indicating that single-point (e.g., A30P, A53T) or multiplication mutations of the gene encoding for ASYN cause early onset forms of Parkinson's disease (PD). The relative regional specificity of ASYN pathology is still a riddle that cannot be simply explained by its expression pattern. Also, transgenic over-expression of ASYN in mice does not recapitulate the typical dopaminergic neuronal death observed in PD. Thus, additional factors must contribute to ASYN-related toxicity. For instance, synucleinopathies are usually associated with inflammation and elevated levels of oxidative stress in affected brain areas. In turn, these conditions favor oxidative modifications of ASYN. Among these modifications, nitration of tyrosine residues, formation of covalent ASYN dimers, as well as methionine sulfoxidations are prominent examples that are observed in post-mortem PD brain sections. Oxidative modifications can affect ASYN aggregation, as well as its binding to biological membranes. This would affect neurotransmitter recycling, mitochondrial function and dynamics (fission/fusion), ASYN's degradation within a cell and, possibly, the transfer of modified ASYN to adjacent cells. Here, we propose a model on how covalent modifications of ASYN link energy stress, altered proteostasis, and oxidative stress, three major pathogenic processes involved in PD progression. Moreover, we hypothesize that ASYN may act physiologically as a catalytically regenerated scavenger of oxidants in healthy cells, thus performing an important protective role prior to the onset of disease or during aging.

A 3-dimensional human embryonic stem cell (hESC)-derived model to detect developmental neurotoxicity of nanoparticles.

Nanoparticles (NPs) have been shown to accumulate in organs, cross the blood-brain barrier and placenta, and have the potential to elicit developmental neurotoxicity (DNT). Here, we developed a human embryonic stem cell (hESC)-derived 3-dimensional (3-D) in vitro model that allows for testing of potential developmental neurotoxicants. Early central nervous system PAX6(+) precursor cells were generated from hESCs and differentiated further within 3-D structures. The 3-D model was characterized for neural marker expression revealing robust differentiation toward neuronal precursor cells, and gene expression profiling suggested a predominantly forebrain-like development. Altered neural gene expression due to exposure to non-cytotoxic concentrations of the known developmental neurotoxicant, methylmercury, indicated that the 3-D model could detect DNT. To test for specific toxicity of NPs, chemically inert polyethylene NPs (PE-NPs) were chosen. They penetrated deep into the 3-D structures and impacted gene expression at non-cytotoxic concentrations. NOTCH pathway genes such as HES5 and NOTCH1 were reduced in expression, as well as downstream neuronal precursor genes such as NEUROD1 and ASCL1. FOXG1, a patterning marker, was also reduced. As loss of function of these genes results in severe nervous system impairments in mice, our data suggest that the 3-D hESC-derived model could be used to test for Nano-DNT.

Protein tyrosine nitration and thiol oxidation by peroxynitrite-strategies to prevent these oxidative modifications.

The reaction product of nitric oxide and superoxide, peroxynitrite, is a potent biological oxidant. The most important oxidative protein modifications described for peroxynitrite are cysteine-thiol oxidation and tyrosine nitration. We have previously demonstrated that intrinsic heme-thiolate (P450)-dependent enzymatic catalysis increases the nitration of tyrosine 430 in prostacyclin synthase and results in loss of activity which contributes to endothelial dysfunction. We here report the sensitive peroxynitrite-dependent nitration of an over-expressed and partially purified human prostacyclin synthase (3.3 µM) with an EC50 value of 5 µM. Microsomal thiols in these preparations effectively compete for peroxynitrite and block the nitration of other proteins up to 50 µM peroxynitrite. Purified, recombinant PGIS showed a half-maximal nitration by 10 µM 3-morpholino sydnonimine (Sin-1) which increased in the presence of bicarbonate, and was only marginally induced by freely diffusing NO2-radicals generated by a peroxidase/nitrite/hydrogen peroxide system. Based on these observations, we would like to emphasize that prostacyclin synthase is among the most efficiently and sensitively nitrated proteins investigated by us so far. In the second part of the study, we identified two classes of peroxynitrite scavengers, blocking either peroxynitrite anion-mediated thiol oxidations or phenol/tyrosine nitrations by free radical mechanisms. Dithiopurines and dithiopyrimidines were highly effective in inhibiting both reaction types which could make this class of compounds interesting therapeutic tools. In the present work, we highlighted the impact of experimental conditions on the outcome of peroxynitrite-mediated nitrations. The limitations identified in this work need to be considered in the assessment of experimental data involving peroxynitrite.

Locally resolved membrane binding affinity of the N-terminus of α-synuclein.

α-Synuclein is abundantly present in Lewy bodies, characteristic of Parkinson's disease. Its exact physiological role has yet to be determined, but mitochondrial membrane binding is suspected to be a key aspect of its function. Electron paramagnetic resonance spectroscopy in combination with site-directed spin labeling allowed for a locally resolved analysis of the protein-membrane binding affinity for artificial phospholipid membranes, supported by a study of binding to isolated mitochondria. The data reveal that the binding affinity of the N-terminus is nonuniform.

Neuroprotection by minocycline caused by direct and specific scavenging of peroxynitrite.

Minocycline prevents oxidative protein modifications and damage in disease models associated with inflammatory glial activation and oxidative stress. Although the drug has been assumed to act by preventing the up-regulation of proinflammatory enzymes, we probed here its direct chemical interaction with reactive oxygen species. The antibiotic did not react with superoxide or (•)NO radicals, but peroxynitrite (PON) was scavenged in the range of ∼1 µm minocycline and below. The interaction of pharmacologically relevant minocycline concentrations with PON was corroborated in several assay systems and significantly exceeded the efficacy of other antibiotics. Minocycline was degraded during the reaction with PON, and the resultant products lacked antioxidant properties. The antioxidant activity of minocycline extended to cellular systems, because it prevented neuronal mitochondrial DNA damage and glutathione depletion. Maintenance of neuronal viability under PON stress was shown to be solely dependent on direct chemical scavenging by minocycline. We chose α-synuclein (ASYN), known from Parkinsonian pathology as a biologically relevant target in chemical and cellular nitration reactions. Submicromolar concentrations of minocycline prevented tyrosine nitration of ASYN by PON. Mass spectrometric analysis revealed that minocycline impeded nitrations more effectively than methionine oxidations and dimerizations of ASYN, which are secondary reactions under PON stress. Thus, PON scavenging at low concentrations is a novel feature of minocycline and may help to explain its pharmacological activity.