Vukic Soskic

Nonenzymatic posttranslational protein modifications in ageing.

One of the most fundamental molecular aspects of aging is accumulating oxidative damage caused by reactive oxygen species (ROS) as proposed by the free radical theory of aging. These unwanted chemical side products of normal metabolism lead to the formation of altered, less active and potentially toxic species of DNA, RNA, proteins, lipids, and small molecules. Due to gradually accumulating small contributions of irreversible reactions during ageing, uncatalyzed chemical side reactions occur with increasing frequencies and repair functions decline. Eventually key biochemical pathways are impaired by increasingly less efficient cellular stress management. In this review, we describe the chemical nature of nonenzymatic age-related modifications of proteins and provide an overview of related analytical challenges and approaches, with a focus on mass spectrometry. We include the description of a strategy to rapidly exploit the wealth of mass spectrometric information from standard MALDI-TOF peptide fingerprints for the characterisation of age-related oxidative amino acid modifications.

Functional proteomics of signal transduction by membrane receptors

Functional proteomic methods have been developed and applied to the investigation of signal transduction systems involving platelet‐derived growth factor (PDGF), endothelin and bradykinin receptors. Mouse fibroblast cells have been stimulated with PDGF or endothelin. Phosphorylation/dephosphorylation of several hundred proteins has been followed as a function of time following stimulation using 2‐D gel electrophoresis and anti‐phosphotyrosine or anti‐phosphoserine antibodies. Up to 100 of these proteins showed strong changes in phosphorylation with minutes of receptor stimulation. Identification of some of these proteins by mass fingerprinting using matrix‐assisted laser desorption/ionization‐time of flight (MALDI‐TOF) mass spectrometry and by partial peptide sequencing with ion trap electrospray mass spectrometry has identified proteins which were previously known to be associated with PDGF signaling, proteins which have been shown to be involved in other signaling pathways, but not PDGF and proteins not previously associated with signal transduction. Parallel to these studies, new methods for rapid, single‐step isolation of peptide receptors using a peptide coupled to a (dA)30 oligonucleotide have been developed and applied to mass spectrometric studies of post‐translational modifications of the endothelin B and bradykinin B2 receptors under in vivo conditions. Both receptors have been shown to undergo extensive phosphorylation as well as palmitoylation. The patterns of post‐translational modifications are more complex than previously recognized and provide new indications of possible roles for these modifications in the regulation and response of these receptors.

Post-translational Modifications of Endothelin Receptor B from Bovine Lungs Analyzed by Mass Spectrometry

A new mild experimental approach for isolation of peptide membrane receptors and subsequent analysis of post-translational modifications is described. Endothelin receptors A and B were isolated on oligo(dT)-cellulose usingN-(ε-maleimidocaproyloxy)succinimide endothelin coupled to a protected (dA)-30-mer. This allowed a one-step isolation of the receptor from oligo(dT)-cellulose via variation solely of salt concentration. The identity of the receptor was confirmed by direct amino acid sequencing of electroblotted samples or by using antibodies against ETA and ETB receptors. The method used here is very fast, requires only very mild elution conditions and, for the first time, gave both ETA and ETB receptors concurrently in very good yield. Following enzymatic in-gel digestion, MALDI, and electrospray ion trap mass spectrometric analysis of the isolated endothelin B receptor showed phosphorylation at Ser-304, -418, -438, -439, -440, and -441. Further phosphorylation at either Ser-434 or -435 was observed. The endothelin B receptor is also palmitoylated at Cys residues 402 and 404. Phosphorylation of Ser304 may play a role in Hirschsprung’s disease.

Arylpiperazine-mediated activation of Akt protects SH-SY5Y neuroblastoma cells from 6-hydroxydopamine-induced apoptotic and autophagic death.

We investigated the ability of 19 recently synthesized arylpiperazine compounds to protect human SH-SY5Y neuroblastoma cells from the neurotoxin 6-hydroxydopamine (6-OHDA). The compound with the most potent neuroprotective action was N-{3-[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}-picolinamide (6b), which reduced 6-OHDA-induced apoptotic death through stabilization of mitochondrial membrane and subsequent prevention of superoxide production, caspase activation and DNA fragmentation. 6-OHDA-triggered autophagic response was also reduced by 6b, which prevented inactivation of the main autophagy repressor mTOR, upregulation of proautophagic beclin-1, conversion of microtubule-associated protein 1 light chain 3 (LC3)-I to autophagosome-associated LC3-II, as well as intracytoplasmic acidification induced by 6-OHDA. The inhibition of autophagy using LC3β gene silencing or pharmacological autophagy blockers 3-methyladenine or bafilomycin A1, mimicked the cytoprotective effect of 6b. While the treatment with 6b had no effect on the phosphorylation of proapoptotic MAP kinases ERK and JNK, it markedly increased the phosphorylation of the prosurvival kinase Akt in 6-OHDA-treated cells. Akt inhibitor DEBC or RNA interference-mediated Akt silencing reduced the ability of 6b to block 6-OHDA-triggered apoptotic and autophagic responses, thus confirming their dependency on Akt activation. The cytoprotective effect of 6b was also observed in 6-OHDA-treated neuronal PC12 cells, but not in SH-SY5Y or PC12 cells exposed to 1-methyl-4-phenylpyridinium, indicating that the observed neuroprotection was dependent on the cytotoxic stimulus. Because of the ability to prevent 6-OHDA induced apoptotic/autophagic cell death through activation of Akt, the investigated arylpiperazines could be potential candidates for treatment of neurodegenerative diseases.

Synthesis of Several Substituted Phenylpiperazines Behaving as Mixed D2/5HT1A Ligands

Twenty‐two different compounds have been synthesized with the aim of creating new, mixed D2/5HT1A ligands. For this purpose 1‐substituted phenylpiperazines attached by the N‐4 nitrogen to dopaminergic pharmacophores of the 2‐(5‐benzimidazole)ethyl‐, 2‐(5‐benztriazole)ethyl‐, 2‐[5‐(benzimidazole‐2‐thione)]ethyl‐ and 2‐[6‐(1,4‐dihydroquinoxaline‐2,3‐dione)]ethyl‐type were selected according to known structure‐affinity requirements of 1‐arylpiperazines.

Stem cells in head and neck squamous cell carcinoma

The existence of a small subpopulation of tumourigenic cancer stem cells in the bulk of human head and neck squamous cancers (SCC) has been recognised in recent reports. This subpopulation has self-renewal properties and is responsible for the production of differentiated daughter cells that form the bulk of the tumour. Stem cells in head and neck SCC can be identified functionally using their self-renewal properties, or by their characteristic surface markers. As their resistance to contemporary cancer treatments may eventually lead to the failure of treatment there is an urgent need to better understand their biology with the ultimate goal of developing new diagnostic markers and curative cancer treatments.

Neuroprotective arylpiperazine dopaminergic/serotonergic ligands suppress experimental autoimmune encephalomyelitis in rats.

Arylpiperazine‐based dopaminergic/serotonergic ligands exert neuroprotective activity. We examined the effect of arylpiperazine D2/5‐HT1A ligands, N‐{4‐[2‐(4‐phenyl‐piperazin‐1‐yl)‐ethyl}‐phenyl]‐picolinamide (6a) and N‐{3‐[2‐(4‐phenyl‐piperazin‐1‐yl)‐ethyl]‐phenyl}‐picolinamide (6b), in experimental autoimmune encephalomyelitis (EAE), a model of neuroinflammation. Both compounds (10 mg/kg i.p.) reduced EAE clinical signs in spinal cord homogenate‐immunized Dark Agouti rats. Compound 6b was more efficient in delaying the disease onset and reducing the maximal clinical score, which correlated with its higher affinity for D2 and 5‐HT1A receptors. The protection was retained if treatment was limited to the effector (from day 8 onwards), but not the induction phase (day 0–7) of EAE. Compound 6b reduced CNS immune infiltration and expression of mRNA encoding the proinflammatory cytokines tumor necrosis factor, IL‐6, IL‐1, and GM‐CSF, TH1 cytokine IFN‐γ, TH17 cytokine IL‐17, as well as the signature transcription factors of TH1 (T‐bet) and TH17 (RORγt) cells. Arylpiperazine treatment reduced apoptosis and increased the activation of anti‐apoptotic mediators Akt and p70S6 kinase in the CNS of EAE animals. The in vitro treatment with 6b protected oligodendrocyte cell line OLN‐93 and neuronal cell line PC12 from mitogen‐activated normal T cells or myelin basic protein‐activated encephalitogenic T cells. In conclusion, arylpiperazine dopaminergic/serotonergic ligands suppress EAE through a direct neuroprotective action and decrease in CNS inflammation.

Protein biomarkers for in vitro testing of toxicology

The vision of the toxicology in the 21st century movement is to overcome the currently used animal tests and identify molecular pathways of toxicity, using human in vitro systems with the aim to provide the most relevant mechanistic information for human risk assessment. It is expected to translate key surrogate biomarkers to novel types of toxicity-related high throughput screening of the many thousands of compounds which need to be tested during development phases of the pharmaceutical industry and with regard to the REACH legislation in Europe. Systems biology, an emerging and increasingly popular field of research, appears to be the discipline of choice to integrate results from transcriptomics, proteomics, epigenomics and metabonomics technologies used to analyze samples from toxicological models. The challenges, however, with respect to data generation, statistical treatment, bioinformatic integration and interpretation or in silico modeling remain formidable. One of the main difficulties is the fact that the sheer number of molecular species is inflated enormously in the course of translation from genes to proteins due to post-translational modifications. Moreover, at the level of proteins, time scales of cellular reactions to toxic insults can be very fast, ranging from milliseconds to seconds. Linear dynamic ranges of concentration differences between conditions can also differ by several orders of magnitude. So, the search for protein biomarkers of toxicity requires sophisticated strategies for time-resolved quantitative differential approaches. The statistical principles, normalization of primary data and principal component and cluster analysis have been well developed for genomics/transcriptomics and partly for proteomics, but have not been widely adapted to technologies like metabonomics. Also, the integration of functional data, in particular data from mass spectrometry, with the aim of modeling pathways of toxicity for human risk assessment, is still at an infant stage.

Arylpiperazine Dopamineric Ligands Protect Neuroblastoma Cells from Nitric Oxide (NO)-Induced Mitochondrial Damage and Apoptosis

The protective ability of novel arylpiperazine‐based dopaminergic ligands against nitric oxide (NO)‐mediated neurotoxicity is investigated. The most potent neuroprotective arylpiperazine identified during the study was N‐{4‐[2‐(4‐phenyl‐piperazin‐1‐yl)ethyl]‐phenyl}picolinamide, which protected SH‐SY5Y human neuron‐like cells from the proapoptotic effect of NO donor sodium nitroprusside (SNP) by decreasing oxidative stress, mitochondrial membrane depolarization, caspase activation and subsequent phosphatydilserine externalization/DNA fragmentation. The protective effect was associated with the inhibition of proapoptotic (JNK, ERK, AMPK) and activation of antiapoptotic (Akt) signaling pathways, in the absence of interference with intracellular NO accumulation. The neuroprotective action of arylpiperazines was shown to be independent of dopamine receptor binding, as it was not affected by the high‐affinity D1/D2 receptor blocker butaclamol. These results reported support the further study of arylpiperazines as potential neuroprotective agents.

Molecular Modeling of 5HT2A Receptor – Arylpiperazine Ligands Interactions

In this paper, we report the molecular modeling of the 5HT2A receptor and the molecular docking of arylpiperazine‐like ligands. The focus of the research was on explaining the effects the ligand structure has on the binding properties of the 5HT2A receptor and on the key interactions between the ligands and the receptor‐binding site. To see what the receptor–ligand interactions were, various substituents were introduced in one part of the ligand, keeping the rest unchanged. In this way, using a docking analysis on the proposed 5HT2A receptor model, we identified key receptor–ligand interactions and determined their properties. Those properties were correlated with experimentally determined binding affinities in order to determine the structure to activity relationship of the examined compounds.