Allosteric modulation of dopamine D2L receptor in complex with Gi1 and Gi2 proteins: the effect of subtle structural and stereochemical ligand modifications.

BACKGROUND: Allosteric modulation of G protein-coupled receptors (GPCRs) is nowadays one of the hot topics in drug discovery. In particular, allosteric modulators of D2 receptor have been proposed as potential modern therapeutics to treat schizophrenia and Parkinson's disease. METHODS: To address some subtle structural and stereochemical aspects of allosteric modulation of D2 receptor, we performed extensive in silico studies of both enantiomers of two compounds (compound 1 and compound 2), and one of them (compound 2) was synthesized as a racemate in-house and studied in vitro. RESULTS: Our molecular dynamics simulations confirmed literature reports that the R enantiomer of compound 1 is a positive allosteric modulator of the D2L receptor, while its S enantiomer is a negative allosteric modulator. Moreover, based on the principal component analysis (PCA), we hypothesized that both enantiomers of compound 2 behave as silent allosteric modulators, in line with our in vitro studies. PCA calculations suggest that the most pronounced modulator-induced receptor rearrangements occur at the transmembrane helix 7 (TM7). In particular, TM7 bending at the conserved P7.50 and G7.42 was observed. The latter resides next to the Y7.43, which is a significant part of the orthosteric binding site. Moreover, the W7.40 conformation seems to be affected by the presence of the positive allosteric modulator. CONCLUSIONS: Our work reveals that allosteric modulation of the D2L receptor can be affected by subtle ligand modifications. A change in configuration of a chiral carbon and/or minor structural modulator modifications are solely responsible for the functional outcome of the allosteric modulator.

N-(3-{4-[3-(trifluoromethyl)phenyl]piperazin-1-yl}propyl)-1H-indazole-3-carboxamide (D2AAK3) as a potential antipsychotic: In vitro, in silico and in vivo evaluation of a multi-target ligand.

Schizophrenia is a mental illness of not adequately understood causes that is not satisfactorily enough treated by current antipsychotics. In search for novel potential antipsychotics we performed structure-based virtual screening aimed to identify new dopamine D2 receptor antagonists. We found compound D2AAK3 with affinity to dopamine D2 receptor of 115 nM. D2AAK3 possesses additional nanomolar or low micromolar affinity to D1, D3, 5-HT1A, 5-HT2A and 5-HT7 receptors, which makes it a good hit for further development as a multifunctional ligand. The compound has also some affinity to M1 and H1 receptors. We used homology modeling, molecular docking and molecular dynamics to study interactions of D2AAK3 with its molecular targets at the molecular level. In behavioral studies D2AAK3 decreases amphetamine-induced hyperactivity (when compared to the amphetamine-treated group) measured as spontaneous locomotor activity in mice. In addition, passive avoidance test demonstrated that D2AAK3 improves memory consolidation after acute treatment in mice. Elevated plus maze tests indicated that D2AAK3 induces anxiogenic activity 30 min after acute treatment, whereas this effect has no longer been observed 60 min after administration of the studied compound in mice.

N-(2-Hydroxyphenyl)-1-[3-(2-oxo-2,3-dihydro-1H- benzimidazol-1-yl)propyl]piperidine-4-Carboxamide (D2AAK4), a Multi-Target Ligand of Aminergic GPCRs, as a Potential Antipsychotic.

N-(2-hydroxyphenyl)-1-[3-(2-oxo-2,3-dihydro-1H-benzimidazol -1-yl)propyl]piperidine-4-carboxamide (D2AAK4) is a multitarget ligand of aminergic G protein-coupled receptors (GPCRs) identified in structure-based virtual screening. Here we present detailed in vitro, in silico and in vivo investigations of this virtual hit. D2AAK4 has an atypical antipsychotic profile and low affinity to off-targets. It interacts with aminergic GPCRs, forming an electrostatic interaction between its protonatable nitrogen atom and the conserved Asp 3.32 of the receptors. At the dose of 100 mg/kg D2AAK4 decreases amphetamine-induced hyperactivity predictive of antipsychotic activity, improves memory consolidation in passive avoidance test and has anxiogenic properties in elevated plus maze test (EPM). Further optimization of the virtual hit D2AAK4 will be aimed to balance its multitarget profile and to obtain analogs with anxiolytic activity.

1-(2'-Bromobenzyl)-6,7-dihydroxy-N-methyl-tetrahydroisoquinoline and 1,2-Demethyl-nuciferine as Agonists in Human D2 Dopamine Receptors.

Certain D2-like dopamine receptor (DR) agonists are useful therapeutically as antiparkinsonian drugs, whereas D2-like DR antagonists or partial agonists are proven effective as antipsychotics. Two isoquinoline derivatives, 1-(2'-bromobenzyl)-6,7-dihydroxy-N-methyl-tetrahydroisoquinoline (Br-BTHIQ, 1) and 1,2-demethyl-nuciferine (aporphine, 2), were herein synthesized, and their dopaminergic affinity in cloned human D2R, D3R, and D4R subtypes and their behavior as agonists/antagonists were evaluated. They showed affinity values (Ki) for hD2, hD3, and hD4 DR within the nanomolar range. The trends in affinity were hD4R ≫ hD3R > hD2R for Br-BTHIQ (1) and hD2R > hD4R > hD3R for 1,2-demethyl-nuciferine (2). The functional assays of cyclic adenosine monophosphate signaling at human D2R showed a partial agonist effect for Br-BTHIQ (1) and full agonist behavior for aporphine (2), with half maximal effective concentration values of 2.95 and 10.2 µM, respectively. Therefore, both isoquinolines 1 and 2 have emerged as lead molecules for the synthesis of new therapeutic drugs that ultimately may be useful to prevent schizophrenia and Parkinson's disease, respectively.

Synthesis, pharmacological and structural studies of 5-substituted-3-(1-arylmethyl-1,2,3,6-tetrahydropyridin-4-yl)-1H-indoles as multi-target ligands of aminergic GPCRs.

Schizophrenia is a complex disease with not fully understood pathomechanism, involving many neurotransmitters and their receptors. This is why it is best treated with multi-target drugs, such as second generation antipsychotics. Here we present 5-substituted-3-(1-arylmethyl-1,2,3,6-tetrahydropyridin-4-yl)-1H-indoles (1-20) which are ligands of dopamine D2 and serotonin 5-HT1A and 5-HT2A receptors and display affinity in the nanomolar range. These compounds were designed as modifications of the virtual hit experimentally confirmed, D2AAK1, and synthesized from indole or 5-alkoxyindoles and N-substituted piperidin-4-ones in methanol in the presence of potassium hydroxide. Compound 9 was subjected to X-ray studies and it crystallizes in the centrosymmetric monoclinic space group P21/c with one molecule in an asymmetric unit. Three most potent compounds (5, 9 and 17) turned out to be antagonists of both D2 and 5-HT2A receptors what is beneficial for their potential application as antipsychotics. Compound 5 was subjected to behavioral studies and exhibited antipsychotic, pro-cognitive and antidepressant activity in appropriate mice models. Structure-activity relationships for compounds 1-20 were rationalized using molecular docking. It was found that, in general, bulky C5-alkoxy substituents at the indole moiety are not favorable as they direct towards aqueous environment of the extracellular vestibule. Keywords: antipsychotics; behavioral studies, G protein-coupled receptors; indole derivatives; multi-target compounds; schizophrenia.

Synthesis, Structural and Thermal Studies of 3-(1-Benzyl-1,2,3,6-tetrahydropyridin-4-yl)-5-ethoxy-1H-indole (D2AAK1_3) as Dopamine D2 Receptor Ligand.

Compound D2AAK1_3 was designed as a modification of the lead structure D2AAK1 (an in vivo active multi-target compound with nanomolar affinity to a number of aminergic GPCRs) and synthesized in the reaction of 5-ethoxyindole and 1-benzyl-4-piperidone. This compound has an affinity to the human dopamine D2 receptor with Ki of 151 nM. The aim of these studies was the structural and thermal characterization of the compound D2AAK1_3. In particular; X-ray studies; molecular docking and molecular dynamics as well as thermal analysis were performed. The studied compound crystallizes in orthorhombic system; in chiral space group P212121. The compound has a non-planar conformation. The studied compound was docked to the novel X-ray structure of the human dopamine D2 receptor in the inactive state (PDB ID: 6CM4) and established the main contact between its protonatable nitrogen atom and Asp (3.32) of the receptor. The obtained binding pose was stable in molecular dynamics simulations. Thermal stability of the compound was investigated using the TG-DSC technique in the air atmosphere, while TG-FTIR analyses in air and nitrogen atmospheres were also performed. The studied compound is characterized by good thermal stability. The main volatile products of combustion are the following gases: CO2; H2O toluene and CO while in the case of pyrolysis process in the FTIR spectra; the characteristic bands of NH3; piperidine and indole are additionally observed.

Structure-Based Virtual Screening for Dopamine D2 Receptor Ligands as Potential Antipsychotics.

Structure-based virtual screening using a D2 receptor homology model was performed to identify dopamine D2 receptor ligands as potential antipsychotics. From screening a library of 6.5 million compounds, 21 were selected and were subjected to experimental validation. From these 21 compounds tested, ten D2 ligands were identified (47.6% success rate, among them D2 receptor antagonists, as expected) that have additional affinity for other receptors tested, in particular 5-HT2A receptors. The affinity (Ki values) of the compounds ranged from 58 nm to about 24 µM. Similarity and fragment analysis indicated a significant degree of structural novelty among the identified compounds. We found one D2 receptor antagonist that did not have a protonatable nitrogen atom, which is a key structural element of the classical D2 pharmacophore model necessary for interaction with the conserved Asp(3.32) residue. This compound exhibited greater than 20-fold binding selectivity for the D2 receptor over the D3 receptor. We provide additional evidence that the amide hydrogen atom of this compound forms a hydrogen bond with Asp(3.32), as determined by tests of its derivatives that cannot maintain this interaction.

In vitro, molecular modeling and behavioral studies of 3-{[4-(5-methoxy-1H-indol-3-yl)-1,2,3,6-tetrahydropyridin-1-yl]methyl}-1,2-dihydroquinolin-2-one (D2AAK1) as a potential antipsychotic.

Antipsychotics currently available to treat schizophrenia suffer several limitations: (1) they are efficient against positive but not negative and cognitive symptoms of the disease; (2) they help only a half of patients; (3) they have severe side effects including neurological and metabolic side effects. Thus, novel drugs to treat schizophrenia are highly demanded. We identified a novel dopamine D2 receptor antagonist, D2AAK1, with Ki of 58 nM using structure-based virtual screening. D2AAK1 possesses additional nanomolar or low micromolar affinity to D1, D3, 5-HT1A and 5-HT2A receptors, making it an ideal candidate for a multi-target drug. Here we present homology modeling, molecular docking and molecular dynamics of D2AAK1 and its molecular targets and animal studies of D2AAK1 as a potential antipsychotic. The main contact of D2AAK1 and all the receptors studied is the electrostatic interaction between the protonable nitrogen atom of the ligand and the conserved Asp(3.32) as typical for orthosteric ligands of aminergic GPCRs. We confirmed antagonistic/partial agonistic properties of D2AAK1 towards all the receptors in in vitro essays and in in silico studies as the ligand stabilizes the ionic lock interaction. We also demonstrated neuroleptic, anxiolytic and, importantly, procognitive properties of D2AAK1 in mouse models.