Serotonin 2A receptor disulfide bridge integrity is crucial for ligand binding to different signalling states but not for its homodimerization.

The serotonin 2 (5-HT) receptor is a G-protein coupled receptor (GPCR) with a conserved disulfide bridge formed by Cys (transmembrane helix 3, TM3) and Cys (extracellular loop 2, ECL-2). We hypothesized that disulfide bridges may determine serotonin 5-HT receptor functions such as receptor activation, functional selectivity and ligand recognition. We used the reducing agent dithiothreitol (DTT) to determine how the reduction of disulfide bridges affects radioligand binding, second messenger mobilization and receptor dimerization.

Targeting PDE10A GAF Domain with Small Molecules: A Way for Allosteric Modulation with Anti-Inflammatory Effects.

Phosphodiesterase (PDE) enzymes regulate the levels of cyclic nucleotides, cAMP, and/or cGMP, being attractive therapeutic targets. In order to modulate PDE activity in a selective way, we focused our efforts on the search of allosteric modulators. Based on the crystal structure of the PDE10A GAF-B domain, a virtual screening study allowed the discovery of new hits that were also tested experimentally, showing inhibitory activities in the micromolar range.

Effect of Nitrogen Atom Substitution in A Adenosine Receptor Binding: N-(4,6-Diarylpyridin-2-yl)acetamides as Potent and Selective Antagonists.

We report the first family of 2-acetamidopyridines as potent and selective A adenosine receptor (AR) antagonists. The computer-assisted design was focused on the bioisosteric replacement of the N1 atom by a CH group in a previous series of diarylpyrimidines. Some of the generated 2-acetamidopyridines elicit an antagonistic effect with excellent affinity (K < 10 nM) and outstanding selectivity profiles, providing an alternative and simpler chemical scaffold to the parent series of diarylpyrimidines.

Pharmacological tools based on imidazole scaffold proved the utility of PDE10A inhibitors for Parkinson's disease.

Aim: Since neuroinflammation is partially mediated by cAMP levels and PDE10A enzyme is able to regulate these levels being highly expressed in striatum, its inhibitors emerged as useful drugs to mitigate this inflammatory process and hence the neuronal death associated with Parkinson's disease (PD). Methodology & results: To study the utility of PDE10A as a pharmacological target for PD, in this work we propose the search and development of new PDE10A inhibitors that could be useful as pharmacological tools in models of the disease and presumably as potential drug candidates. By using different medicinal chemistry approaches we have discovered imidazole-like PDE10A inhibitors and showed their neuroprotective actions.

Enantiospecific Recognition at the A Adenosine Receptor by Alkyl 2-Cyanoimino-4-substituted-6-methyl-1,2,3,4-tetrahydropyrimidine-5-carboxylates.

A novel family of structurally simple, potent, and selective nonxanthine AAR ligands was identified, and its antagonistic behavior confirmed through functional experiments. The reported alkyl 2-cyanoimino-4-substituted-6-methyl-1,2,3,4-tetrahy-dropyrimidine-5-carboxylates (16) were designed by bioisosteric replacement of the carbonyl group at position 2 in a series of 3,4-dihydropyrimidin-2-ones. The scaffold (16) documented herein contains a chiral center at the heterocycle.

Serotonin-2A homodimers are needed for signalling via both phospholipase A and phospholipase C in transfected CHO cells.

Different ligands differentially activate phospholipase A (PLA) and phospholipase C (PLC) signalling pathways that are coupled to the serotonin 2 (5-HT) receptor, a class-A G-protein coupled receptor (GPCR). The serotonin 5-HT receptor has been shown to be expressed as a homodimer displaying some ligands negative cooperativity between protomers in the PLA signalling pathway. We hypothesized that the homodimeric complex is the minimum functional unit required for activation of the PLA and PLC pathways by the serotonin 5-HT receptor.

Development of a Multiplex Assay for Studying Functional Selectivity of Human Serotonin 5-HT2A Receptors and Identification of Active Compounds by High-Throughput Screening.

G protein-coupled receptors (GPCRs) exist as collections of conformations in equilibrium, and the efficacy of drugs has been proposed to be associated with their absolute and relative affinities for these different conformations. The serotonin 2A (5-HT2A) receptor regulates multiple physiological functions, is involved in the pathophysiology of schizophrenia, and serves as an important target of atypical antipsychotic drugs. This receptor was one of the first GPCRs for which the functional selectivity phenomenon was observed, with its various ligands exerting differential effects on the phospholipase A2 (PLA2) and phospholipase C (PLC) signaling pathways.

The arylpiperazine derivatives N-(4-cyanophenylmethyl)-4-(2-diphenyl)-1-piperazinehexanamide and N-benzyl-4-(2-diphenyl)-1-piperazinehexanamide exert a long-lasting inhibition of human serotonin 5-HT7 receptor binding and cAMP signaling.

We performed a detailed in vitro pharmacological characterization of two arylpiperazine derivatives, compound N-(4-cyanophenylmethyl)-4-(2-diphenyl)-1-piperazinehexanamide (LP-211) previously identified as a high-affinity brain penetrant ligand for 5-hydroxytryptamine (serotonin) type 7 (5-HT7) receptors, and its analog N-benzyl-4-(2-diphenyl)-1-piperazinehexanamide (MEL-9). Both ligands exhibited competitive displacement of [(3)H]-(2R)-1-[(3-hydroxyphenyl)sulfonyl]-2-[2-(4-methyl-1-piperidinyl)ethyl]pyrrolidine ([(3)H]-SB-269970) radioligand binding and insurmountable antagonism of 5-carboxamidotryptamine (5-CT)-stimulated cyclic adenosine monophosphate (cAMP) signaling in human embryonic kidney (HEK293) cells stably expressing human 5-HT7 receptors. They also inhibited forskolin-stimulated adenylate cyclase activity in 5-HT7-expressing HEK293 cells but not in the parental cell line.

Discovery of 3,4-Dihydropyrimidin-2(1H)-ones As a Novel Class of Potent and Selective A2B Adenosine Receptor Antagonists.

We describe the discovery and optimization of 3,4-dihydropyrimidin-2(1H)-ones as a novel family of (nonxanthine) A2B receptor antagonists that exhibit an unusually high selectivity profile. The Biginelli-based hit optimization process enabled a thoughtful exploration of the structure-activity and structure-selectivity relationships for this chemotype, enabling the identification of ligands that combine structural simplicity with excellent hA2B AdoR affinity and remarkable selectivity profiles.

Unraveling phosphodiesterase surfaces. Identification of phosphodiesterase 7 allosteric modulation cavities.

The last findings of our group by using chemical genetic approaches have shown that PDE7 is an interesting target in neurodegenerative diseases. The following step in this travel to unravel PDE7 is the design of more selective inhibitors. In this sense we have proposed to perform an analysis of PDE7 surface to identify possible allosteric sites following by a docking study of different PDE7 inhibitors synthesized by our group.

Synthesis and allosteric modulation of the dopamine receptor by peptide analogs of L-prolyl-L-leucyl-glycinamide (PLG) modified in the L-proline or L-proline and L-leucine scaffolds.

Novel analogs of L-prolyl-L-leucylglycinamide (PLG) were synthesized wherein the prolyl residue was replaced with other amino acids based on a 3,5-disubstituted proline scaffold. In some examples, the L-leucyl residue was also replaced by L-valine. These analogs were tested for their ability to enhance the binding of [(3)H]-N-propylnorapomorphine to short isoform of human dopamine D₂ receptors.

Selective and potent adenosine A3 receptor antagonists by methoxyaryl substitution on the N-(2,6-diarylpyrimidin-4-yl)acetamide scaffold.

The influence of diverse methoxyphenyl substitution patterns on the N-(2,6-diarylpyrimidin-4-yl)acetamide scaffold is herein explored in order to modulate the A(3) adenosine receptor antagonistic profile. As a result, novel ligands exhibiting excellent potency (K(i) on A(3) AR < 20 nM) and selectivity profiles (above 100-fold within the adenosine receptors family) are reported. Moreover, our joint theoretical and experimental approach allows the identification of novel pharmacophoric elements conferring A(3)AR selectivity, first established by a robust computational model and thereafter characterizing the most salient features of the structure-activity and structure-selectivity relationships in this series.

Identification in silico and experimental validation of novel phosphodiesterase 7 inhibitors with efficacy in experimental autoimmune encephalomyelitis mice.

A neural network model has been developed to predict the inhibitory capacity of any chemical structure to be a phosphodiesterase 7 (PDE7) inhibitor, a new promising kind of drugs for the treatment of neurological disorders. The numerical definition of the structures was achieved using CODES program. Through the validation of this neural network model, a novel family of 5-imino-1,2,4-thiadiazoles (ITDZs) has been identified as inhibitors of PDE7.

Neuroprotective efficacy of quinazoline type phosphodiesterase 7 inhibitors in cellular cultures and experimental stroke model.

A simple and efficient synthetic method for the preparation of quinazoline type phosphodiesterase 7 (PDE7) inhibitors, based on microwave irradiation, has been developed. The use of this methodology improved yields and reaction times, providing a scalable procedure. These compounds are pharmacologically interesting because of their in vivo efficacy both in spinal cord injury and Parkinson's disease models, as shown in previous studies from our group.

Discovery of LAS101057: A Potent, Selective, and Orally Efficacious A2B Adenosine Receptor Antagonist.

The structure-activity relationships for a series of pyrazine-based A2B adenosine receptor antagonists are described. From this work, LAS101057 (17), a potent, selective, and orally efficacious A2B receptor antagonist, was identified as a clinical development candidate. LAS101057 inhibits agonist-induced IL-6 production in human fibroblasts and is active in an ovalbumin (OVA)-sensitized mouse model after oral administration, reducing airway hyperresponsiveness to methacholine, Th2 cytokine production, and OVA-specific IgE levels.

Pyrimidine derivatives as potent and selective A3 adenosine receptor antagonists.

Two regioisomeric series of diaryl 2- or 4-amidopyrimidines have been synthesized and their adenosine receptor affinities were determined in radioligand binding assays at the four human adenosine receptors (hARs). Some of the ligands prepared herein exhibit remarkable affinities (K(i) < 10 nm) and, most noticeably, the absence of activity at the A(1), A(2A), and A(2B) receptors. The structural determinants that support the affinity and selectivity profiles of the series were highlighted through an integrated computational approach, combining a 3D-QSAR model built on the second generation of GRid INdependent Descriptors (GRIND2) with a novel homology model of the hA(3) receptor.

Pyrazolo[1',5':1,6]pyrimido[4,5-d]pyridazin-4(3H)-ones as selective human A(1) adenosine receptor ligands.

A series of pyrazolo[1',5':1,6]pyrimido[4,5-d]pyridazin-4(3H)-ones was synthesized and tested in radioligand binding assays to determine their affinities for the human adenosine A(1), A(2A), A(2B) and A(3) receptors. Results indicated that this scaffold is appropriate for adenosine receptor subtype A(1) ligands and that the best arranged groups around this scaffold are 3- and 4-pyridinyl at position 1, benzyl at position 3, hydrogen at position 6 and 3-thienyl or phenyl at position 9. The most interesting compounds showed K(i) for A1 in the nanomolar range and an appreciable selectivity for other receptor subtypes.

Synthesis of novel 1-alkyl-8-substituted-3-(3-methoxypropyl) xanthines as putative A(2B) receptor antagonists.

In order to identify a high-affinity, selective antagonist for the A(2B) subtype adenosine receptor, more than 40 1,8-disubstituted-3-(3-methoxypropyl) xanthines were prepared and evaluated for their binding affinity at recombinant human adenosine receptors, mainly of the A(2A) and A(2B) subtypes. Some of the 1-ethyl-3-(3-methoxypropyl)-8-aryl substituted derivatives 15(a-m) showed moderate-to-high affinity at human A(2B) receptors, with compound 15d showing A(2B) selectivity over the other A receptors assayed (A(1), A(2A), A(3)) of 34-fold or over.

Evidence for distinct antagonist-revealed functional states of 5-hydroxytryptamine(2A) receptor homodimers.

The serotonin (5-hydroxytryptamine; 5-HT) 2A receptor is a cell surface class A G protein-coupled receptor that regulates a multitude of physiological functions of the body and is a target for antipsychotic drugs. Here we found by means of fluorescence resonance energy transfer and immunoprecipitation studies that the 5-HT(2A)-receptor homodimerized in live cells, which we linked with its antagonist-dependent fingerprint in both binding and receptor signaling. Some antagonists, like the atypical antipsychotics clozapine and risperidone, differentiate themselves from others, like the typical antipsychotic haloperidol, antagonizing these 5-HT(2A) receptor-mediated functions in a pathway-specific manner, explained here by a new model of multiple active interconvertible conformations at dimeric receptors.

A new chemical tool (C0036E08) supports the role of adenosine A(2B) receptors in mediating human mast cell activation.

Asthma is a chronic inflammatory disease of the airways that involves many cell types, amongst which mast cells are known to be important. Adenosine, a potent bronchoconstricting agent, exerts its ability to modulate adenosine receptors of mast cells thereby potentiating derived mediator release, histamine being one of the first mediators to be released. The heterogeneity of sources of mast cells and the lack of highly potent ligands selective for the different adenosine receptor subtypes have been important hurdles in this area of research.

Synthesis, adenosine receptor binding and 3D-QSAR of 4-substituted 2-(2'-furyl)-1,2,4-triazolo[1,5-a]quinoxalines.

A collection of 25 2-(2'-furyl)-1,2,4-triazolo[1,5-a]quinoxalines incorporating different substitution patterns at position 4 have been synthesized and their binding affinity towards human adenosine receptors (hA(1), hA(2A), hA(2B) and hA(3)) was determined. The biological data show that several potent at hA(1), but lightly selective, adenosine ligands were identified. Moreover, these results confirmed the hypothesis that the structural modifications carried out on the 4-position of the tricyclic system produces a remarkable modification of the adenosine receptorial profile.

1-, 3- and 8-substituted-9-deazaxanthines as potent and selective antagonists at the human A2B adenosine receptor.

A large series of piperazin-, piperidin- and tetrahydroisoquinolinamides of 4-(1,3-dialkyl-9-deazaxanthin-8-yl)phenoxyacetic acid were prepared through conventional or multiple parallel syntheses and evaluated for their binding affinity at the recombinant human adenosine receptors, chiefly at the hA(2B) and hA(2A) receptor subtypes. Several ligands endowed with high binding affinity at hA(2B) receptors, excellent selectivity over hA(2A) and hA(3) and a significant, but lower, selectivity over hA(1) were identified. Among them, piperazinamide derivatives 23 and 52, and piperidinamide derivative 69 proved highly potent at hA(2B) (K(i)=11, 2 and 5.

Discovery and characterization of 4'-(2-furyl)-N-pyridin-3-yl-4,5'-bipyrimidin-2'-amine (LAS38096), a potent, selective, and efficacious A2B adenosine receptor antagonist.

A novel series of N-heteroaryl 4'-(2-furyl)-4,5'-bipyrimidin-2'-amines has been identified as potent and selective A(2B) adenosine receptor antagonists. In particular, compound 5 showed high affinity for the A(2B) receptor (Ki = 17 nM), good selectivity (IC(50): A(1) > 1000 nM, A(2A) > 2500 nM, A3 > 1000 nM), displayed a favorable pharmacokinetic profile in preclinical species, and showed efficacy in functional in vitro models.