Ursolic acid ameliorates stress and reactive oxygen species in C. elegans knockout mutants by the dopamine Dop1 and Dop3 receptors.

BACKGROUND: Depression and stress-related disorders are leading causes of death worldwide. Standard treatments elevating serotonin or noradrenaline levels are not sufficiently effective and cause adverse side effects. A connection between dopamine pathways and stress-related disorders has been suggested. Compounds derived from herbal medicine could be a promising alternative. We examined the neuroprotective effects of ursolic acid (UA) by focusing on dopamine signalling. METHODS: Trolox equivalent capacity assay was used to determine the antioxidant activities of UA in vitro. C. elegans N2 wildtype and dopamine receptor-knockout mutants (dop1-deficient RB665 and dop3-deficient LX703 strains) were used as in vivo models. H2DCFDA and acute juglone assays were applied to determine the antioxidant activity in dependency of dopamine pathways in vivo. Stress was assessed by heat and acute osmotic stress assays. The influence of UA on overall survival was analyzed by a life span assay. The dop1 and dop3 mRNA expression was determined by real time RT-PCR. We also examined the binding affinity of UA towards C. elegans Dop1 and Dop3 receptors as well as human dopamine receptors D1 and D3 by molecular docking. RESULTS: Antioxidant activity assays showed that UA exerts strong antioxidant activity. UA increased resistance towards oxidative, osmotic and heat stress. Additionally, UA increased life span of nematodes. Moreover, dop1 and dop3 gene expression was significantly enhanced upon UA treatment. Docking analysis revealed stronger binding affinity of UA to C. elegans and human dopamine receptors than the natural ligand, dopamine. Binding to Dop1 was stronger than to Dop3. CONCLUSION: UA reduced stress-dependent ROS generation and acted through Dop1 and to a lesser extent through Dop3 to reduce stress and prolong life span in C. elegans. These results indicate that UA could be a promising lead compound for the development of new antidepressant medications.

A novel psychoanalytical approach: An electrochemical ligand-binding assay to screen antipsychotics.

Schizophrenia treatment may see a paradigm shift due to development of new atypical antipsychotic drugs (APDs), with better tolerability due to more selective dopamine (DA) receptor blockade. Monitoring of these APD candidates in biological fluids is of great importance to reduce the development cost, to clarify the mechanism of action and ultimately to support the demonstration of efficacy of these molecules. Electrochemical approaches have attracted great attention for monitoring DA and APD levels but none of the methods developed so far aimed to screen APD candidates. Herein, by this work, we propose for the first time an electrochemical ligand-binding approach for antipsychotic drug screening where competitive binding of a novel APD and DA to a dopamine D3 receptor (D3R) was investigated by looking at electrochemical signals of DA and drug before and after D3R interaction. D3R peptide was incubated with DA and/or drug first and then changes in electrochemical oxidation signals of free DA and the drug was measured by cyclic voltammetry (CV) and differential pulse voltammetry (DPV). Circular Dichroism spectroscopy was used to investigate the secondary structure of the peptide upon binding with either drug and/or DA.

Using click chemistry toward novel 1,2,3-triazole-linked dopamine D3 receptor ligands.

The dopamine D3 receptor (D3R) is a target of pharmacotherapeutic interest in a variety of neurological disorders including schizophrenia, Parkinson's disease, restless leg syndrome, and drug addiction. A common molecular template used in the development of D3R-selective antagonists and partial agonists incorporates a butylamide linker between two pharmacophores, a phenylpiperazine moiety and an extended aryl ring system. The series of compounds described herein incorporates a change to that chemical template, replacing the amide functional group in the linker chain with a 1,2,3-triazole group. Although the amide linker in the 4-phenylpiperazine class of D3R ligands has been previously deemed critical for high D3R affinity and selectivity, the 1,2,3-triazole moiety serves as a suitable bioisosteric replacement and maintains desired D3R-binding functionality of the compounds. Additionally, using mouse liver microsomes to evaluate CYP450-mediated phase I metabolism, we determined that novel 1,2,3-triazole-containing compounds modestly improves metabolic stability compared to amide-containing analogues. The 1,2,3-triazole moiety allows for the modular attachment of chemical subunit libraries using copper-catalyzed azide-alkyne cycloaddition click chemistry, increasing the range of chemical entities that can be designed, synthesized, and developed toward D3R-selective therapeutic agents.

Targeting dopamine D3 and serotonin 5-HT1A and 5-HT2A receptors for developing effective antipsychotics: synthesis, biological characterization, and behavioral studies.

Combination of dopamine D3 antagonism, serotonin 5-HT1A partial agonism, and antagonism at 5-HT2A leads to a novel approach to potent atypical antipsychotics. Exploitation of the original structure-activity relationships resulted in the identification of safe and effective antipsychotics devoid of extrapyramidal symptoms liability, sedation, and catalepsy. The potential atypical antipsychotic 5bb was selected for further pharmacological investigation. The distribution of c-fos positive cells in the ventral striatum confirmed the atypical antipsychotic profile of 5bb in agreement with behavioral rodent studies. 5bb administered orally demonstrated a biphasic effect on the MK801-induced hyperactivity at dose levels not able to induce sedation, catalepsy, or learning impairment in passive avoidance. In microdialysis studies, 5bb increased the dopamine efflux in the medial prefrontal cortex. Thus, 5bb represents a valuable lead for the development of atypical antipsychotics endowed with a unique pharmacological profile for addressing negative symptoms and cognitive deficits in schizophrenia.

Homology modeling of dopamine D2 and D3 receptors: molecular dynamics refinement and docking evaluation.

Dopamine (DA) receptors, a class of G-protein coupled receptors (GPCRs), have been targeted for drug development for the treatment of neurological, psychiatric and ocular disorders. The lack of structural information about GPCRs and their ligand complexes has prompted the development of homology models of these proteins aimed at structure-based drug design. Crystal structure of human dopamine D(3) (hD(3)) receptor has been recently solved. Based on the hD(3) receptor crystal structure we generated dopamine D(2) and D(3) receptor models and refined them with molecular dynamics (MD) protocol. Refined structures, obtained from the MD simulations in membrane environment, were subsequently used in molecular docking studies in order to investigate potential sites of interaction. The structure of hD(3) and hD(2L) receptors was differentiated by means of MD simulations and D(3) selective ligands were discriminated, in terms of binding energy, by docking calculation. Robust correlation of computed and experimental K(i) was obtained for hD(3) and hD(2L) receptor ligands. In conclusion, the present computational approach seems suitable to build and refine structure models of homologous dopamine receptors that may be of value for structure-based drug discovery of selective dopaminergic ligands.

Different binding modes of structurally diverse ligands for human D3DAR.

Five different dopamine D3 receptors (D3DARs) models were created considering some suggested binding modes for D3DAR antagonists reported in earlier computational studies. Different hypotheses are justified because of the lack of experimental information about the putative site of interaction and are also due to the variability in scaffolds and size of D3DAR ligands. In this study 114 potent and selective D3DAR antagonists or partial agonists are used as key experimental information to discriminate the most reliable receptor model and to build a docking based 3D quantitative structure-activity relationship model able to indicate the ligand properties and the residues important for activity. The ability of this D3DAR model to discriminate the binding mode of different classes of ligands, showing a good quantitative correlation with their activity, encourages us to use it for screening novel lead compounds.