Interaction of different third intracellular loop fragments of human dopamine d2l receptor with a-subunit of gi1 protein - prospective therapeutic application

In order to find the essential structural motif of the D2L dopamine receptor necessary for the interaction with a-subunit of Gi1 protein, four fragments of the third cytoplasmic loop (CPL3) of this receptor were cloned, expressed in E. coli and purified. After that, fusion proteins with glutathione-S-transferase (GST) were prepared and the interactions quantified by a colorimetric assay for GST activity determination. The presence of D2L-CPL3 fragment-Gia1 complexes was detected by SDS-polyacrylamide gel electrophoresis (PAGE). Kd values for the interaction of the three fragments with Gia1 were similar and in nmol/L range of concentrations, while the peptide representing the insert in the long form of the dopamine D2 receptor expressed about 10-fold lower binding affinity. These results could serve to design new therapeutic agents that might act at the level of receptor/G protein interaction rather than at the level of ligand-receptor binding.U cilju pronalaženja bitnih strukturnih motiva potrebnih za interakciju sa a podjedinicom Gi1 proteina klonirana su, eksprimirana i prečišćena 4 fragmenta treće citoplazmatične petlje (CPL3) dopaminskog D2L receptora koji su dalje pripremljeni kao fuzioni proteini sa glutation-S-transferazom (GST). Interakcije su kvantifikovane bojenom reakcijom za određivanje aktivnosti GST. Postojanje kompleksa D2L-CPL3 fragment- Gia1 je dokazano elektroforetskom analizom na SDS-poliakrilamidnom gelu (PAGE). Kd vrednosti za tri fragmenta su bile vrlo slične i u nmol/L opsegu koncentracija, dok je peptid koji predstavlja insert u dugom obliku dopaminskog D2 receptora posedovao oko 10 puta manji afinitet vezivanja za Gia1. Ovi rezultati mogu biti osnova za sintezu novih terapeutskih agenasa koji bi delovali na nivou interakcije receptora i G proteina umesto na nivou vezivanja liganda za receptor

Application of Hybrid Density Functional Theory in Calculation of Edge-to-Face Interactions of Receptor-Ligand System

Our previously described research on docking analysis of a series of isosteric N4-arylpiperazines on a model of 5-HT1A receptor was used earlier to investigate interactions of different ligands with the receptor binding site. Due to the limitations of molecular mechanics (MM) methods, docking analysis failed to give precise results about interactions that influence binding affinity of the ligands, but we presumed that aromatic-aromatic interactions, or edge-to-face, to be more precise, play an important role in the binding process. In order to further elaborate on this hypothesis, ab initio approach was used to calculate possible edge-to-face interactions on a model system and correlate them to ligand affinity. Obtained results indicate that those dispersive interactions can show notable influence on the binding of the ligands to 5-HT1A receptor. Stabilization energies of modeled receptor-ligand complex, calculated using Becke's "half-and-half" hybrid DFT method showed strong correlation with the affinity of investigated ligands towards 5-HT1A receptor

Synthesis and pharmacological evaluation of several N-(2-nitrophenyl) piperazine derivatives

Six newly synthesized heterocyclic (2-nitrophenyl)piperazines, with a specific structure of the heteroaryl group, whichmimics the catechol moiety of dopamine (benzimidazoles and substituted benzimidazoles), were evaluated for their binding affinity to rat dopamine (DA), serotonin (5-HT) and _1 receptors. All compounds with a benzimidazole group had a 5-HT2A/D2 receptors binding ratio characteristic for atypical neuroleptics (>1, pK i values). Compound 7c, 4-bromo-6-{2-_4-(2-nitrophenyl)piperazin- 1-yl_ethyl}-1H-benzimidazole, expressed higher affinities for all receptor classes than clozapine. Also, it exhibited the best characteristic for atypical neuroleptics and presents a compound with the best profile for further in vivo investigations.Sintetisano je šest heterocikličnih (2-nitrofenil)piperazina sa specifičnom heteroaril grupom, koja podražava kateholsku grupu dopamina (benzimidazoli i supstituisani benzimidazoli), i ispitan je njihov afinitet ka dopaminskim, serotoninskim i _1 receptorima. Sva jedinjenja sa benzimidazolskim grupama su pokazala 5-HT 1A/D2 odnos vezivanja karakterističan za atipične neuroleptike (>1, pK i vrednosti). Jedinjenje 7c, 4-bromo-6-{2-_4-(2-nitrofenil)piperazin-1-il_etil}-1H-benzimidazol, pokazalo je izraženiji afinitet ka svim klasama receptora u poređenju sa klozapinom i takođe predstavlja jedinjenje sa najboljim karakteristikama za dalja in vivo istraživanja.nul

Interaction of different third intracellular loop fragments of human dopamine d2l receptor with a-subunit of gi1 protein - prospective therapeutic application

In order to find the essential structural motif of the D2L dopamine receptor necessary for the interaction with a-subunit of Gi1 protein, four fragments of the third cytoplasmic loop (CPL3) of this receptor were cloned, expressed in E. coli and purified. After that, fusion proteins with glutathione-S-transferase (GST) were prepared and the interactions quantified by a colorimetric assay for GST activity determination. The presence of D2L-CPL3 fragment-Gia1 complexes was detected by SDS-polyacrylamide gel electrophoresis (PAGE). Kd values for the interaction of the three fragments with Gia1 were similar and in nmol/L range of concentrations, while the peptide representing the insert in the long form of the dopamine D2 receptor expressed about 10-fold lower binding affinity. These results could serve to design new therapeutic agents that might act at the level of receptor/G protein interaction rather than at the level of ligand-receptor binding.U cilju pronalaženja bitnih strukturnih motiva potrebnih za interakciju sa a podjedinicom Gi1 proteina klonirana su, eksprimirana i prečišćena 4 fragmenta treće citoplazmatične petlje (CPL3) dopaminskog D2L receptora koji su dalje pripremljeni kao fuzioni proteini sa glutation-S-transferazom (GST). Interakcije su kvantifikovane bojenom reakcijom za određivanje aktivnosti GST. Postojanje kompleksa D2L-CPL3 fragment- Gia1 je dokazano elektroforetskom analizom na SDS-poliakrilamidnom gelu (PAGE). Kd vrednosti za tri fragmenta su bile vrlo slične i u nmol/L opsegu koncentracija, dok je peptid koji predstavlja insert u dugom obliku dopaminskog D2 receptora posedovao oko 10 puta manji afinitet vezivanja za Gia1. Ovi rezultati mogu biti osnova za sintezu novih terapeutskih agenasa koji bi delovali na nivou interakcije receptora i G proteina umesto na nivou vezivanja liganda za receptor

Synthesis of novel 5-HT1A arylpiperazine ligands: Binding data and computer-aided analysis of pharmacological potency

Serotonin receptors modulate numerous behavioral and neuropsychological processes. Therefore, they are the target for the action of many drugs, such as antipsychotics, antidepressants, antiemetics, migraine remedies, and many others. The 5-HT1A receptors have been involved in the pathogenesis and treatment of anxiety and depression and represent a promising target for new drugs with reduced extrapyramidal side effects. In most antidepressants, a piperazine-based structural motif can be identified as a common moiety. Here we describe the synthesis, pharmacological, and in silico characterization of a novel arylpiperazines series with excellent 5-HT1A affinity. The final compounds, 4a, 8a, and 8b, were selected according to predictions of in silico pharmacokinetics, docking analysis, and molecular dynamics in conjunction with physical properties, and metabolic stability. The accentuated molecules could serve as a lead compound for developing 5-HT1A drug-like molecules for depression treatment

New substituted 2-methylthiomethyl- and 2-methylsulphinylmethylenebenz- imidazoles with D 2 /5-HT(1A) activity

Several 2-methylthiomethylbenzimidazoles (3a-e) and the corresponding sulphinyl derivatives 4a-e were synthesized and evaluated measuring their in vitro binding affinity at the D 1 and D 2 dopamine (source: synaptosomal membranes of the bovine nucleus caudatus) and 5-HT(1A) serotonin (source: synaptosomal membranes of the bovine hippocampus) receptors. [ 3 H]SCH 23390, [ 3 H]spiperone, and [ 3 H]-8-OH-DPAT were employed as specific radioligands for the D 1 , D 2 and 5-HT(1A) receptors, respectively. None of the compounds except for 3b acting as a moderate [ 3 H]SCH 23390, competitor, expressed binding affinity at the D 1 receptor. Compounds 4a and 4e were inactive displacers of both [ 3 H]spiperone and [ 3 H]-8-OH-DPAT. Ligands 4b, 3d and 4d acted as weak to moderate [ 3 H]spiperone competitors and 3a was a weak [ 3 H]- 8-OH-DPAT displacer. The remaining ligands expressed binding affinity at the corresponding receptors in a nanomolar concentration range. Among them, compound 3b with K(i) of 14.2 nM and 8.4 nM in [ 3 H]spiperone and [ 3 H]-8- OH-DPAT binding assay, respectively, was the most potent mixed dopaminergic/serotonergic ligand. Although sterically similar, the two classes of ligands differ with regard to electronic properties of substituents in position 2 of the benzimidazole ring. Oxidation of 2- (methylthiomethyl)benzimidazoles afforded ligands devoid of binding affinity at the 5-HT(1A) receptor and significantly reduced binding affinity at the D 2 receptor. This points to the importance of electronic properties of substituents in position 2 of benzimidazole ring for the D 2 /5-HT(1A) affinity ratio of this type of ligands

Electrostatic surface potential calculation on several new halogenated benzimidazole-like dopaminergic ligands

We examined the effects of the electron density distribution (electrostatic surface potential; ESP) of several new benzimidazole-type ligands on their binding affinity for the D 1 and D 2 dopamine receptors (DAR). Receptors were prepared from synaptosomal membranes of bovine caudate nuclei. [ 3 H]SCH 23390 and [ 3 H]spiperone were used as specific radiolabels for the D 1 and D 2 receptors, respectively. The ESP of these compounds was calculated using Gaussian 98 W software. Calculations performed with known dopaminergic ligands showed that the electron density charge in the aromatic ring of these compounds favors a higher binding affinity for the D 2 DAR. This was confirmed by the synthesis of halogenated analogues of several known dopaminergic ligands. Halogenation resulted in an increase in the positive charge of the aromatic part of the molecule. None of the newly synthesized compounds was efficient in displacing [ 3 H]SCH 23390 from the D 1 DAR. The introduction of chlorine into the molecule led to a higher binding affinity for the D 2 DAR of the new ligands in comparison to both parent compounds and brominated ligands. This difference probably originates from the difference in the sizes of chlorine and bromine atoms, which could influence the interaction of a ligand with the receptor binding site. However, among the new ligands with bromine as a substituent, two compounds (8b and 10b) expressed a higher binding affinity and two of them (9b and 11b) a lower binding affinity for the D 2 DAR, when compared to unsubstituted parent compounds. These results indicate that the electrostatic surface potential of a ligand is an important factor in its interaction with the D 2 DAR and that this should be taken into account during design and synthesis of dopaminergic compounds

Investigation of mixed D(2)/5-HT(1A) activity of N-heteroarylmethyl-N-phenylpiperazines, N-heteroarylethyl-N-phenylpiperazines and N-heteroarylpropyl-N-phenylpiperazines

Eight novel N-heteroarylalkyl-N-phenylpiperazines have been synthesized, chemically characterized and evaluated for in vitro binding affinity at the dopamine and serotonin receptors. Synaptosomal membranes of fresh bovine caudate nuclei (D(1) and D(2)), the membranes of COS-7 cells (D(4.4)) and those prepared from fresh bovine hippocampi (5-HT(1A)) were used as a source of the corresponding receptor subtypes. [(3)H]SCH 23390 (D(1)-selective), [(3)H]spiperone (D(2)- and D(4.4)- selective) and [(3)H]-8-OH-DPAT (5-HT(1A)-selective) served as radioligands. None of the compounds expressed the affinity for the binding at the D(1) subtype receptor. Compounds 7-9 containing a single methylene group serving as a bridge between heteroaryl and N-phenylpiperazine part of the molecule were inactive [(3)H]spiperone and [(3)H]-8-OH-DPAT competitors. Ligands 15-19 (three methylene groups connecting heteroaryl- and N-phenylpiperazine part of the molecule) acted as moderate competitors of [(3)H]spiperone binding at the D(2) receptor subtype, with the exception of 15 (a thione) which expressed a high binding affinity at the D(2) receptor subtype. Compounds 15-19 behaved as moderate displacers of 8-OH-[(3)H]DPAT. Among all eight novel ligands only compound 15 expressed a moderate binding affinity at the D(4.4) receptor subtype

The influence of dispersive interactions on the binding affinities of ligands with an arylpiperazine moiety to the dopamine D2 receptor

Several isosteric 1,3-dihydro-5-[2-(4-aryl-1-piperazinyl)ethyl]-2H-benzimidazole-2-thiones were used to investigate the interactions of different ligands with the binding site of the D2 receptor. Due to limitations of the simulation methods, docking analysis failed to show precisely the interactions that influence the binding affinity of the ligands. It is presumed that dispersive forces or more precisely edge-to-face interactions play an important role in the binding process, especially for the lipophilic part of the ligands. In order to confirm this hypothesis, ab initio calculations were applied on a model system in order to find the stabilization energies of potential edge-to-face interactions and then to correlate them with the ligand affinity. The obtained results indicate that there is a significant correlation between the strength of dispersive inter-actions and ligand affinity. It was shown that for the calculation of stabilization energies of modeled receptor–ligand complexes the Becke “half-and-half” hybrid DFT method can be used, thus speeding up the usually long calculation time and reducing the required computer strength