Learning Impact of a Virtual Brain Electrical Activity Simulator Among Neurophysiology Students: Mixed-Methods Intervention Study.

BACKGROUND: Virtual simulation is the re-creation of reality depicted on a computer screen. It offers the possibility to exercise motor and psychomotor skills. In biomedical and medical education, there is an attempt to find new ways to support students' learning in neurophysiology. Traditionally, recording electroencephalography (EEG) has been learned through practical hands-on exercises. To date, virtual simulations of EEG measurements have not been used. OBJECTIVE: This study aimed to examine the development of students' theoretical knowledge and practical skills in the EEG measurement when using a virtual EEG simulator in biomedical laboratory science in the context of a neurophysiology course. METHODS: A computer-based EEG simulator was created. The simulator allowed virtual electrode placement and EEG graph interpretation. The usefulness of the simulator for learning EEG measurement was tested with 35 participants randomly divided into three equal groups. Group 1 (experimental group 1) used the simulator with fuzzy feedback, group 2 (experimental group 2) used the simulator with exact feedback, and group 3 (control group) did not use a simulator. The study comprised pre- and posttests on theoretical knowledge and practical hands-on evaluation of EEG electrode placement. RESULTS: The Wilcoxon signed-rank test indicated that the two groups that utilized a computer-based electrode placement simulator showed significant improvement in both theoretical knowledge (Z=1.79, P=.074) and observed practical skills compared with the group that studied without a simulator. CONCLUSIONS: Learning electrode placement using a simulator enhances students' ability to place electrodes and, in combination with practical hands-on training, increases their understanding of EEG measurement.

Deciphering Imidazoline Off-targets by Fishing in the Class A of GPCR field.

Based on the finding that a central antihypertensive agent with high affinity for I1-type imidazoline receptors - rilmenidine, shows cytotoxic effects on cultured cancer cell lines, it has been suggested that imidazoline receptors agonists might have a therapeutic potential in the cancer therapy. Nevertheless, potential rilmenidine side effects caused by activation of α-adrenoceptors, or other associated receptors and enzymes, might hinder its therapeutic benefits. Considering that human α-adrenoceptors belong to the rhodopsin-like class A of G-protein-coupled receptors (GPCRs) it is reasonable to assume that imidazolines might have the affinity for other receptors from the same class. Therefore, to investigate possible off-target effects of imidazoline ligands we have prepared a reverse docking protocol on class A GPCRs, using imidazoline ligands and their decoys. To verify our in silico results, three ligands with high scores and three ligands with low scores were tested for antagonistic activity on α2 - adrenoceptors.

A combined ligand- and structure-based approach for the identification of rilmenidine-derived compounds which synergize the antitumor effects of doxorubicin.

The clonidine-like central antihypertensive agent rilmenidine, which has high affinity for I1-type imidazoline receptors (I1-IR) was recently found to have cytotoxic effects on cultured cancer cell lines. However, due to its pharmacological effects resulting also from α2-adrenoceptor activation, rilmenidine cannot be considered a suitable anticancer drug candidate. Here, we report the identification of novel rilmenidine-derived compounds with anticancer potential and devoid of α2-adrenoceptor effects by means of ligand- and structure-based drug design approaches. Starting from a large virtual library, eleven compounds were selected, synthesized and submitted to biological evaluation. The most active compound 5 exhibited a cytotoxic profile similar to that of rilmenidine, but without appreciable affinity to α2-adrenoceptors. In addition, compound 5 significantly enhanced the apoptotic response to doxorubicin, and may thus represent an important tool for the development of better adjuvant chemotherapeutic strategies for doxorubicin-insensitive cancers.

Transfer of SAR information from hypotensive indazole to indole derivatives acting at α-adrenergic receptors: In vitro and in vivo studies.

In a search for novel antihypertensive drugs we applied scaffold hopping from the previously described α1-adrenergic receptor antagonists, 1-[(imidazolin-2-yl)methyl]indazoles. The aim was to investigate whether the α-adrenergic properties of the indazole core were transferable to the indole core. The newly obtained 1-[(imidazolin-2-yl)methyl]indole analogues were screened in vitro for their binding affinities for α1-and α2-adrenoceptors, which allowed the identification of the target-based SAR transfer (T_SAR transfer) as well as structure-based SAR transfer (S_SAR transfer) events. However, when screened in vivo with use of anaesthetized male Wistar rats, the new indole ligands showed a different hemodynamic profile than expected. Instead of the immediate hypotensive effect characteristic of peripheral vasodilatator α1 blockers, a biphasic effect was observed, reminiscent of clonidine-like centrally acting antihypertensive agents. This was supported by subsequent in vitro functional studies in [(35)S]GTPγS binding assay, where the indole analogues displayed partial agonist properties at α2-adrenergic receptors. Since no correlation was found between the in vitro binding to α-adrenoceptors and the in vivo hemodynamic effects of the two series of indazole and indole bioisosteric compounds, in a search for new imidazoline-containing adrenergic drugs, the structure-based SAR transfer information obtained from in vitro binding studies should be treated with caution.

Fluorinated analogues of marsanidine, a highly α2-AR/imidazoline I1 binding site-selective hypotensive agent. Synthesis and biological activities.

The aim of these studies was to establish the influence of fluorination of the indazole ring on the pharmacological properties of two selective α2-adrenoceptor (α2-AR) agonists: 1-[(imidazolidin-2-yl)imino]-1H-indazole (marsanidine, A) and its methylene analogue 1-[(4,5-dihydro-1H-imidazol-2-yl)methyl]-1H-indazole (B). Introduction of fluorine into the indazole ring of A and B reduced both binding affinity and α2-AR/I1 imidazoline binding site selectivity. The most α2-AR-selective ligands were 6-fluoro-1-[(imidazolidin-2-yl)imino]-1H-indazole (6c) and 7-fluoro-1-[(imidazolidin-2-yl)imino]-1H-indazole (6d). The in vivo cardiovascular properties of fluorinated derivatives of A and B revealed that in both cases the C-7 fluorination leads to compounds with the highest hypotensive and bradycardic activities. The α2-AR partial agonist 6c was prepared as a potential lead compound for development of a radiotracer for PET imaging of brain α2-ARs.

Synthesis and biological activities of 2-[(heteroaryl)methyl]imidazolines.

A series of 2-[(heteroaryl)methyl]imidazolines was synthesized and tested for their activities at α(1)- and α(2)-adrenoceptors and imidazoline I(1) and I(2) receptors. The most active 2-[(indazol-1-yl)methyl]imidazolines showed high or moderate affinities for α(1)- and α(2)-adrenoceptors. However, their intrinsic activities at α(2A)-adrenoceptors proved to be negligible. A selected 7-chloro derivative behaved as a potent α(1)-adrenoceptor antagonist and exhibited peripherally mediated hypotensive effects in rats.

3-[(Imidazolidin-2-yl)imino]indazole ligands with selectivity for the α(2)-adrenoceptor compared to the imidazoline I(1) receptor.

A series of 3-[(4,5-dihydroimidazolidin-2-yl)imino]indazoles has been synthesized as positional analogues of marsanidine, a highly selective α(2)-adrenoceptor ligand. Parent compound 4a and its 4-chloro (4c) and 4-methyl (4d) derivatives display α(2)-adrenoceptor affinity at nanomolar concentrations (K(i)=39.4, 15.9 and 22.6nM, respectively) and relatively high α(2)/I(1) selectivity ratios of 82, 115 and 690, respectively. Evidence was obtained that these compounds act as partial agonists at α(2A)-adrenoceptors. Compound 4d with intrinsic activity comparable with that of marsanidine, but lower than that of clonidine, elicited pronounced cardiovascular effects in anesthetized rats at doses as low as 0.01mg/kg iv.

Octopamine receptors from the barnacle balanus improvisus are activated by the alpha2-adrenoceptor agonist medetomidine.

G protein-coupled octopamine receptors of insects and other invertebrates represent counterparts of adrenoceptors in vertebrate animals. The alpha(2)-adrenoceptor agonist medetomidine, which is in clinical use as a veterinary sedative agent, was discovered to inhibit the settling process of barnacles, an important step in the ontogeny of this crustacean species. Settling of barnacles onto ship hulls leads to biofouling that has many harmful practical consequences, and medetomidine is currently under development as a novel type of antifouling agent. We now report that medetomidine induces hyperactivity in the barnacle larvae to disrupt the settling process. To identify the molecular targets of medetomidine, we cloned five octopamine receptors from the barnacle Balanus improvisus. We show by phylogenetic analyses that one receptor (BiOctalpha) belongs to the alpha-adrenoceptor-like subfamily, and the other four (BiOctbeta-R1, BiOctbeta-R2, BiOctbeta-R3, and BiOctbeta-R4) belong to the beta-adrenoceptor-like octopamine receptor subfamily. Phylogenetic analyses also indicated that B. improvisus has a different repertoire of beta-adrenoceptor-like octopamine receptors than insects. When expressed in CHO cells, the cloned receptors were activated by both octopamine and medetomidine, resulting in increased intracellular cAMP or calcium levels. Tyramine activated the receptors but with much lesser potency than octopamine. A hypothesis for receptor discrimination between tyramine and octopamine was generated from a homology three-dimensional model. The characterization of B. improvisus octopamine receptors is important for a better functional understanding of these receptors in crustaceans as well as for practical applications in development of environmentally sustainable antifouling agents.

Homogeneous GTP binding assay employing QRET technology.

Functional cell signaling assays have become important tools for measuring ligand-induced receptor activation in cell-based biomolecular screening. Guanosine-5'-triphosphate (GTP) is a generic signaling marker responsible for the first intracellular signaling event of the G-protein-coupled receptors (GPCRs). [(35)S]GTPgammaS binding assay is the classical well-established method for measuring agonist-induced G-protein activation requiring a separation of free and bound fractions prior to measurement. Here a novel, separation-free, time-resolved fluorescence GTP binding assay has been developed based on a non-fluorescence resonance energy transfer (FRET) single-label approach and quenching of a nonbound europium-labeled, nonhydrolyzable GTP analog (Eu-GTP). The quenching resonance energy transfer (QRET) method relies on the use of Eu-GTP, providing a time-resolved fluorescent detection as an alternative to the radiolabel [(35)S]GTPgammaS assay. Upon activation of recombinant human alpha(2A)-adrenoceptors (alpha(2A)-AR) expressed in Chinese hamster ovary cells, guanosine-5'-diphosphate is released from the alpha-subunit of Gi-proteins, enabling the subsequent binding of Eu-GTP. Activation of alpha(2A)-AR with 5 different alpha(2)-AR agonists was measured quantitatively using the developed QRET GTP assay and compared to [(35)S]GTPgammaS and heterogeneous Eu-GTP filtration assays. Equal potencies and efficacy rank orders were observed in all 3 assays but with a lower signal-to-background ratio and increased assay variation in the QRET assay compared to the Eu-GTP filtration and the nonhomogeneous [(35)S]GTPgammaS binding assays.

Alpha2-adrenoreceptors profile modulation. 4. From antagonist to agonist behavior.

The goal of the present study was to modulate the receptor interaction properties of known alpha 2-adrenoreceptor (AR) antagonists to obtain novel alpha 2-AR agonists with desirable subtype selectivity. Therefore, a phenyl group or one of its bioisosteres or aliphatic moieties with similar steric hindrance were introduced into the aromatic ring of the antagonist lead basic structure. The functional properties of the novel compounds allowed our previous observations to be confirmed. The high efficacy of 7, 12, and 13 as alpha 2-AR agonists and the significant alpha 2C-AR subtype selective activation displayed by 11 and 15 demonstrated that favorable interactions to induce alpha 2-AR activation were formed between the pendant groups of the ligands and the aromatic cluster present in transmembrane domain 6 of the binding site cavity of the receptors.

Model structures of alpha-2 adrenoceptors in complex with automatically docked antagonist ligands raise the possibility of interactions dissimilar from agonist ligands.

Antagonist binding to alpha-2 adrenoceptors (alpha2-ARs) is not well understood. Structural models were constructed for the three human alpha2-AR subtypes based on the bovine rhodopsin X-ray structure. Twelve antagonist ligands (including covalently binding phenoxybenzamine) were automatically docked to the models. A hallmark of agonist binding is the electrostatic interaction between a positive charge on the agonist and the negatively charged side chain of D3.32. For antagonist binding, ion-pair formation would require deviations of the models from the rhodopsin structural template, e.g., a rotation of TM3 to relocate D3.32 more centrally within the binding cavity, and/or creation of new space near TM2/TM7 such that antagonists would be shifted away from TM5. Thus, except for the quinazolines, antagonist ligands automatically docked to the model structures did not form ion-pairs with D3.32. This binding mode represents a valid alternative, whereby the positive charge on the antagonists is stabilized by cation-pi interactions with aromatic residues (e.g., F6.51) and antagonists interact with D3.32 via carboxylate-aromatic interactions. This binding mode is in good agreement with maps derived from a molecular interaction library that predicts favorable atomic contacts; similar interaction environments are seen for unrelated proteins in complex with ligands sharing similarities with the alpha2-AR antagonists.

Conserved structural, pharmacological and functional properties among the three human and five zebrafish alpha 2-adrenoceptors.

1. Zebrafish has five distinct alpha(2)-adrenoceptors. Two of these, alpha(2Da) and alpha(2Db), represent a duplicated, fourth alpha(2)-adrenoceptor subtype, while the others are orthologue of the human alpha(2A)-, alpha(2B)- and alpha(2C)-adrenoceptors. Here, we have compared the pharmacological properties of these receptors to infer structural determinants of ligand interactions. 2. The zebrafish alpha(2)-adrenoceptors were expressed in Chinese hamster ovary cells and tested in competitive ligand binding assays and in a functional assay (agonist-stimulated [(35)S]GTPgammaS binding). The affinity results were used to cluster the receptors and, separately, the ligands using both principal component analysis and binary trees. 3. The overall ligand binding characteristics, the order of potency and efficacy of the tested agonists and the G-protein coupling of the zebrafish and human alpha(2)-adrenoceptors, separated by approximately 350 million years of evolution, were found to be highly conserved. The binding affinities of the 20 tested ligands towards the zebrafish alpha(2)-adrenoceptors are generally comparable to those of their human counterparts, with a few compounds showing up to 40-fold affinity differences. 4. The alpha(2A) orthologues and the zebrafish alpha(2D) duplicates clustered as close pairs, but the relationships between the orthologues of alpha(2B) and alpha(2C) were not clearly defined. Applied to the ligands, our clustering methods segregated the ligands based on their chemical structures and functional properties. As the ligand binding pockets formed by the transmembrane helices show only minor differences among the alpha(2)-adrenoceptors, we suggest that the second extracellular loop--where significant sequence variability is located --might contribute significantly to the observed affinity differences.