Identification of NQO1 and ferrochelatase as interaction partners for neuroprotective N-{[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}-arylamides.

Affinity chromatography was used to identify potential cellular targets that are responsible for neuroprotective activity of N-{[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}-arylamides. Active and inactive representatives of N-{[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}-arylamides bearing an extended linker were synthesized and immobilized on an agarose-based matrix. This was followed by the identification of specifically bound proteins isolated out of the whole rat brain extract. Inducible flavoprotein NAD(P)H:quinone oxidoreductase (NQO1) was identified as candidates for cellular targets.

Synthesis, Biological, and Computational Evaluation of Substituted 1-(2-Methoxyphenyl)-4-(1-phenethylpiperidin-4-yl)piperazines and 1-(2-Methoxyphenyl)-4-[(1-phenethylpiperidin-4-yl)methyl]piperazines as Dopaminergic Ligands.

Sixteen new 1-(2-methoxyphenyl)-4-(1-phenethylpiperidin-4-yl)piperazines and 1-(2-methoxyphenyl)-4-[(1-phenethylpiperidin-4-yl)methyl]piperazines were synthesized to be used as probes for mapping the dopamine D2 receptor (D2 DAR) arylpiperazine binding site. All compounds were evaluated for their affinity toward D2 DAR in an in vitro competitive displacement assay. The most active one was 1-(2-methoxyphenyl)-4-{[1-(3-nitrophenethyl)piperidin-4-yl]methyl}piperazine (25) with an affinity of Ki = 54 nM. Docking analysis was conducted on all herein described compounds, whereas molecular dynamic simulation was performed on ligand 25 to establish its mode of interaction with D2 DAR. Two possible docking orientations are proposed; the one with a salt bridge between the piperidine moiety and Asp114 of D2 DAR is more stable.

Modeling of Dopamine D2 Receptor - Overview of 35-Year Evolution.

Research on dopamine (DA) and its receptors, and in particular the D2 receptor subclass, has been an intriguing and fast developing scientific field in the past 35 years. Methods of medicinal chemistry, molecular and structural biology as well as computational chemistry were used in the studies of DA receptors (DRs). Early attempts to describe DRs were based on a small amount of experimental data available and produced crude models at best. Once crystal structures of bacteriorhodopsin, rhodopsine, various G-protein coupled receptors, and finally D3 DR receptor became available, better and more detailed D2 DR receptor models emerged. These models gave us an insight into the mechanism of ligand-receptor interactions, and paved the way for the synthesis of new dopaminergic compounds, both agonists and antagonists and possible drugs for the treatment of different imbalances of the dopaminergic system. This review covers the key discoveries on the path to the creation of the D2 DR receptor model.

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, TH 1 cytokine IFN-γ, TH 17 cytokine IL-17, as well as the signature transcription factors of TH 1 (T-bet) and TH 17 (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. Arylpiperazine dopaminergic/serotonergic ligands reduce neurological symptoms of acute autoimmune encephalomyelitis in rats without affecting the activation of autoreactive immune response, through mechanisms involving a decrease in CNS immune infiltration, as well as direct protection of CNS from immune-mediated damage. These data indicate potential usefulness of arylpiperazine-based compounds in the treatment of neuroinflammatory disorders such as multiple sclerosis.

Exploration of N-arylpiperazine Binding Sites of D2 Dopaminergic Receptor.

The crystal structures of the D3 dopamine receptor and several other G-protein coupled receptors (GPCRs) were published in recent times. Those 3D structures are used by us and other scientists as a template for the homology modeling and ligand docking analysis of related GPCRs. Our main scientific interest lies in the field of pharmacologically active N-arylpiperazines that exhibit antipsychotic and/or antidepressant properties, and as such are dopaminergic and serotonergic receptor ligands. In this short review article we are presenting synthesis and biological data on the new N-arylpipereazine as well our results on molecular modeling of the interactions of those N-arylpiperazines with the model of D2 dopamine receptors. To obtain that model the crystal structure of the D3 dopamine receptor was used. Our results show that the N-arylpiperazines binding site consists of two pockets: one is the orthosteric binding site where the N-arylpiperazine part of the ligand is docked and the second is a non-canonical accessory binding site for N-arylpipereazine that is formed by a second extracellular loop (ecl2) of the receptor. Until now, the structure of this receptor region was unresolved in crystal structure analyses of the D3 dopamine receptor. To get a more complete picture of the ligand - receptor interaction, DFT quantum mechanical calculations on N-arylpiperazine were performed and the obtained models were used to examine those interactions.

Regenerative nanotechnology in oral and maxillofacial surgery.

Regenerative nanotechnology is at the forefront of medical research, and translational medicine is a challenge to both scientists and clinicians. Although there has been an exponential rise in the volume of research generated about it for both medical and surgical uses, key questions remain about its actual benefits. Nevertheless, some people think that therapeutics based on its principles may form the core of applied research for the future. Here we give an account of its current use in oral and maxillofacial surgery, and implications and challenges for the future.

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 receptor­ligand 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 receptor­ligand 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.

N-{[2-(4-Phenyl-piperazin-1-yl)-ethyl]-phenyl}-arylamides with dopamine D2 and 5-Hydroxytryptamine 5HT(1A) activity: synthesis, testing, and molecular modeling.

The ratio of affinities toward the dopamine D2 and the 5-hydroxytryptamine 5-HT(1A) receptors is one of the important parameters that determine the efficiency of antipsychotic drugs. Here, we present the synthesis of ortho-, meta-, and para-N-{[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}-arylamides and their structure-activity relationship studies on dopamine D2 and 5-hydroxytryptamine 5-HT(1A) receptors. It was shown that the biological activity of the described ligands strongly depends on their topology as well as on the nature of the heteroaryl group in the head of the molecules. Docking simulations together with conformational analysis revealed a rational explanation for the ligands' behavior. The molecular model of receptor-ligand interactions described herein provided us with a tool for the rational design of new compounds with a favorable D2/5-HT(1A) profile.

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 LC3ß 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.

Determination of key receptor-ligand interactions of dopaminergic arylpiperazines and the dopamine D2 receptor homology model.

Interest in structure-based G-protein-coupled receptor (GPCR) ligand discovery is huge, given that almost 30 % of all approved drugs belong to this category of active compounds. The GPCR family includes the dopamine receptor subtype D2 (D2DR), but unfortunately--as is true of most GPCRs--no experimental structures are available for these receptors. In this publication, we present the molecular model of D2DR based on the previously published crystal structure of the dopamine D3 receptor (D3DR). A molecular modeling study using homology modeling and docking simulation provided a rational explanation for the behavior of the arylpiperazine ligand. The observed binding modes and receptor-ligand interactions provided us with fresh clues about how to optimize selectivity for D2DR receptors.

Interactions of N-{[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}-2-aryl-2-yl-acetamides and 1-{[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}-3-aryl-2-yl-ureas with dopamine D2 and 5-hydroxytryptamine 5HT(1A) receptors.

It is suggested that the ratio of dopamine D(2) to 5-hydroxytryptamine 5-HT(1A) activity is an important parameter that determines the efficiency of antipsychotic drugs. Here we present the synthesis of N-{[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}-2-aryl-2-yl-acetamides and 1-{[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}-3-aryl-2-yl-ureas and their structure-activity relationship studies on dopamine D(2) and 5-hydrohytryptamine 5-HT(1A) receptors. It was shown that ligand selectivity and affinity strongly depends on their topology and the presence of a pyridyl group in the head of molecules. Molecular modeling studies using homology modeling and docking simulation revealed a rational explanation for the ligand behavior. The observed binding modes and receptor-ligand interactions provided us with a clue for optimizing the optimal selectivity towards 5-HT(1A) receptors.

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.

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.

Systems biology approaches and tools for analysis of interactomes and multi-target drugs.

Systems biology is essentially a proteomic and epigenetic exercise because the relatively condensed information of genomes unfolds on the level of proteins. The flexibility of cellular architectures is not only mediated by a dazzling number of proteinaceous species but moreover by the kinetics of their molecular changes: The time scales of posttranslational modifications range from milliseconds to years. The genetic framework of an organism only provides the blue print of protein embodiments which are constantly shaped by external input. Indeed, posttranslational modifications of proteins represent the scope and velocity of these inputs and fulfil the requirements of integration of external spatiotemporal signal transduction inside an organism. The optimization of biochemical networks for this type of information processing and storage results in chemically extremely fine tuned molecular entities. The huge dynamic range of concentrations, the chemical diversity and the necessity of synchronisation of complex protein expression patterns pose the major challenge of systemic analysis of biological models. One further message is that many of the key reactions in living systems are essentially based on interactions of moderate affinities and moderate selectivities. This principle is responsible for the enormous flexibility and redundancy of cellular circuitries. In complex disorders such as cancer or neurodegenerative diseases, which initially appear to be rooted in relatively subtle dysfunctions of multimodal physiologic pathways, drug discovery programs based on the concept of high affinity/high specificity compounds ("one-target, one-disease"), which has been dominating the pharmaceutical industry for a long time, increasingly turn out to be unsuccessful. Despite improvements in rational drug design and high throughput screening methods, the number of novel, single-target drugs fell much behind expectations during the past decade, and the treatment of "complex diseases" remains a most pressing medical need. Currently, a change of paradigm can be observed with regard to a new interest in agents that modulate multiple targets simultaneously, essentially "dirty drugs." Targeting cellular function as a system rather than on the level of the single target, significantly increases the size of the drugable proteome and is expected to introduce novel classes of multi-target drugs with fewer adverse effects and toxicity. Multiple target approaches have recently been used to design medications against atherosclerosis, cancer, depression, psychosis and neurodegenerative diseases. A focussed approach towards "systemic" drugs will certainly require the development of novel computational and mathematical concepts for appropriate modelling of complex data. But the key is the extraction of relevant molecular information from biological systems by implementing rigid statistical procedures to differential proteomic analytics.

Synchronization of posttranslational modifications during aging: Time is a crucial biological dimension.

There is an urgent need for surrogate biomarkers in clinical diagnostics but also preclinical in toxicology of chemicals and efficacy testing of pharmaceuticals. On the background of the emerging fields of systems biology and theranostics, the importance of time scales and the synchronization of complex biological readouts become increasingly obvious. Systemic effects such as responses to stimuli, medical intervention, cellular stress, or toxicity elicit immediate molecular changes on the protein level. Early events include phosphorylation and oxidation of proteins. These posttranslational modifications have a direct impact on enzyme activities and protein-protein interactions. Only at downstream stages is gene transcription activated. Here we outline the analytical and statistical requirements dealing with complex patterns of protein expression and the extraction of protein surrogate biomarkers defining specific early stages of biological responses. We also present successful examples from research on aging and discuss prerequisites and necessary considerations while moving candidates to validation.

What does systems biology mean for drug development?

The complexity and flexibility of cellular architectures is increasingly recognized by impressive progress on the side of molecular analytics, i.e. proteomics, genomics and metabolomics. One of the messages from systems biology is that the number of molecular species in cellular networks is orders of magnitude bigger than anticipated by genomic analysis, in particular by fast posttranslational modifications of proteins. The requirements to manage external signals, integrate spatiotemporal signal transduction inside an organism and at the same time optimizing networks of biochemical and chemical reactions result in chemically extremely fine tuned molecular entities. Chemical side reactions of enzymatic activity, like e.g. random oxidative damage of proteins by free radicals during aging constantly introduce epigenetic alterations of protein targets. These events gradually and on an individual stochastic scale, keep modifying activities of these targets, and their affinities and selectivities towards biological and pharmacological ligands. One further message is that many of the key reactions in living systems are essentially based on interactions of low affinities and even low selectivities. This principle is responsible for the enormous flexibility and redundancy of cellular circuitries. So, in complex disorders like cancer or neurodegenerative diseases, which are rooted in relatively subtle and multimodal dysfunction of important physiologic pathways, drug discovery programs based on the concept of high affinity/high specificity compounds ("one-target, one-disease"), which still dominate the pharmaceutical industry increasingly turn out to be unsuccessful. Despite improvements in rational drug design and high throughput screening methods, the number of novel, single-target drugs fell much behind expectations during the past decade and the treatment of "complex diseases" remains a most pressing medical need. Currently a change of paradigm can be observed with regard to a new focus on agents that modulate multiple targets simultaneously. Targeting cellular function as a system rather than on the level of the single protein molecule significantly increases the size of the drugable proteome and is expected to introduce novel classes of multi-target drugs with fewer adverse effects and toxicity. Multiple target approaches have recently been used to design medications against atherosclerosis, cancer, depression, psychosis and neurodegenerative diseases. A focussed approach towards "systemic" drugs will certainly require the development of novel computational and mathematical concepts for appropriate modelling of complex data and extraction of "screenable" information from biological systems essentially ruled by deterministic chaotic processes on a background of individual stochasticity.

A connection between the mitochondrial permeability transition pore, autophagy, and cerebral amyloidogenesis.

In a drug reprofiling attempt, we explored novel neuroprotective properties of 4-azasteroids by synthesizing chemical affinity tags capturing adenine nucleotide translocator-1, as a potential target. Dutasteride inhibits the mitochondrial transition pore and induces an increase of autophagosomal structures in human cell lines. In vivo, a surprising reduction of the beta-amyloid plaque load in a model for cerebral amyloidosis appears to connect release of neurotoxic peptides, mitochondrial apoptosis and autophagy.

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.

Synthesis, binding properties and receptor docking of 4-halo-6-[2-(4-arylpiperazin-1-yl)ethyl]-1H-benzimidazoles, mixed ligands of D2 and 5-HT1A receptors.

In this publication we are describing synthesis, binding properties, and receptor docking of 4-halo-6-[2-(4-arylpiperazin-1-yl)ethyl]-1H-benzimidazoles, a new compounds with potential antipsychotics properties. Affinity towards the dopamine D(1)-like and D(2)-like, and serotonin 5-HT(1A) receptors was evaluated using the radioligand binding assays. All compounds tested had affinity for the D(2)-like and 5-HT(1A) receptors, but were inactive towards the D(1)-like receptor. Halogenated 6-[2-(4-arylpiperazin-1-yl)ethyl]-1H-benzimidazoles showed higher affinity compared to their nonhalogenated congeners. In silico docking analysis of selected ligands was performed in order to explain the results of binding assays. Our analysis suggests that stabilizing interactions between the halogen atom at the benzimidazole ring and the Ser-122 of the D(2)-like and Trp-358 of the 5-HT(1A) receptor. Energy contributions for these interactions were calculated using the ab initio method.