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. Moreover, these new PDE10A inhibitors were able to decrease the nitrite production in LPS-stimulated cells, thus demonstrating their potential as anti-inflammatory agents.

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.

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.

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.

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. A 3D-QSAR modelling study (GRIND/ALMOND methodology) performed on the hA(1) data gave further support to the pharmacological results, and it is presented as a useful tool for the future design of ligands with better pharmacological profiles.

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 5.5 nM, respectively) and selective towards hA(2A) (hA(2A)/hA(2B) SI=912, 159 and 630, respectively), hA(3) (hA(3)/hA(2B) SI=>100, 3090 and >180, respectively) and hA(1) (hA(1)/hA(2B) SI=>100, 44 and 120, respectively), SI being the selectivity index. A number of selected ligands tested in functional assays in vitro showed very interesting antagonist activities and efficacies at both A(2A) and A(2B) receptor subtypes, with pA(2) values close to the corresponding pK(i)s. Structure-affinity and structure-selectivity relationships suggested that the binding potency at the hA(2B) receptor may be increased by lipophilic substituents at the N4-position of piperazinamides and that an ortho-methoxy substituent at the 8-phenyl ring and alkyl groups at N1 larger than the ones at N3, in the 9-deazaxanthine ring, may strongly enhance the hA(2A)/hA(2B) SI.

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.

Serotonin-2A homodimers are needed for signalling via both phospholipase A2 and phospholipase C in transfected CHO cells.

Different ligands differentially activate phospholipase A2 (PLA2) and phospholipase C (PLC) signalling pathways that are coupled to the serotonin 2A (5-HT2A) receptor, a class-A G-protein coupled receptor (GPCR). The serotonin 5-HT2A receptor has been shown to be expressed as a homodimer displaying some ligands negative cooperativity between protomers in the PLA2 signalling pathway. We hypothesized that the homodimeric complex is the minimum functional unit required for activation of the PLA2 and PLC pathways by the serotonin 5-HT2A receptor. To investigate this hypothesis, we partially blocked the serotonin 5-HT2A receptors with ritanserin and measured PLA2 and PLC activity simultaneously. We subsequently added the competitive antagonist spiperone to release the inactivator through a crosstalk mechanism and thus allow the dimer to return to a reactive state. Partial inactivation of the homodimer by ritanserin binding decreased the activity of the receptor by 59±13% and 70±4% in the PLA2 and PLC pathways respectively (P<0.001), with no difference in the potency of the serotonin (5-HT) was observed. The subsequent binding of spiperone released ritanserin due to the crosstalk between protomers and recovery of the receptor activity to 74±7% and 72±4%. Negative cooperativity between protomers in the dimer was maintained during arachidonic acid (AA) release after blocking ritanserin, as indicated by the biphasic inhibition curves for clozapine over 1µM serotonin (5-HT) in these conditions. These findings provide evidence that serotonin 5-HT2A receptors must be expressed as homodimers in order to activate both the PLA2 and PLC signalling pathways.

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

The serotonin 2A (5-HT2A) receptor is a G-protein coupled receptor (GPCR) with a conserved disulfide bridge formed by Cys148 (transmembrane helix 3, TM3) and Cys227 (extracellular loop 2, ECL-2). We hypothesized that disulfide bridges may determine serotonin 5-HT2A 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. A DTT-induced decrease in the number of binding sites (1190 ± 63.55 fmol/mg protein for control cells compared with 921.2 ± 60.84 fmol/mg protein for DTT-treated cells) as well as in the efficacy of both signalling pathways characterized was observed, although the affinity and potency were unchanged. Bioluminiscence resonance energy transfer (BRET) assays revealed the DTT treatment did not modify the homodimeric nature of serotonin 5-HT2A receptors. In molecular dynamic simulations, the ECL-2 of the receptor with a broken cysteine bond adopts a wider variety of conformations, some of which protrude deeper into the receptor orthosteric binding pocket leading to collapse of the pocket. A shrunken binding pocket would be incapable of accommodating lysergic acid diethylamide (LSD). Our findings suggest that the decrease of efficacy may be due to disruption of disulfide bridge between TM3 and ECL-2. This reveals the integrity of the ECL-2 epitope, which should be explored in the development of novel ligands acting as allosteric modulators of serotonin 5-HT2A receptors.

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. CONCLUSION: Here, we demonstrate the neuroprotective effect of PDE10A inhibitors in cellular models of PD. [Formula: see text].

Design, synthesis, and structure-activity relationships of 1-,3-,8-, and 9-substituted-9-deazaxanthines at the human A2B adenosine receptor.

Over two hundred 1-, 3-, 8-, and 9-substituted-9-deazaxanthines were prepared and evaluated for their binding affinity at the recombinant human adenosine receptors, in particular at the hA(2B) and hA(2A) subtypes. Several ligands endowed with sub-micromolar to low nanomolar binding affinity at hA(2B) receptors, good selectivity over hA(2A) and hA(3), but a relatively poor selectivity over hA(1) were obtained. Good antagonistic potencies and efficacies, with pA(2) values close to the corresponding pK(i)s, were observed in functional assays in vitro performed on a selected series of compounds. 1,3-Dimethyl-8-phenoxy-(N-p-halogenophenyl)-acetamido-9-deazaxanthine derivatives appeared as the most interesting leads, some of them showing outstanding hA(2B) affinities, high selectivity over hA(2A) and hA(3), but low selectivity over hA(1). Structure-affinity relationships suggested that the binding potency at the hA(2B) receptor was mainly modulated by the steric (lipophilic) properties of the substituents at positions 1 and 3 and by the electronic and lipophilic characteristics of the substituents at position 8. A comparison among affinity and selectivity profiles of 9-deazaxanthines with the corresponding xanthines suggested some possible differences in their binding mode.

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. We aimed to develop a multiplex functional assay in 96-well plates for the simultaneous measurement of the PLA2 and PLC pathways coupled to 5-HT2A receptors; this approach enables the detection of either functional selectivity or cooperativity phenomena in early drug screening stages. The suitability of the method for running screening campaigns was tested using the Prestwick Chemical Library, and 22 confirmed hits with activities of more than 90% were identified; 11 of these hits produced statistically significant differences between the two effector pathways. Thus, we have developed a miniaturized multiplex assay in 96-well plates to measure functional selectivity for 5-HT2A receptors in the early stages of the drug discovery process.

Different pharmacological properties of two equipotent antagonists (clozapine and rauwolscine) for 5-HT2B receptors in rat stomach fundus.

On the basis of the previously demonstrated constitutive activity in natural systems and the possibility of specific ligand-induced conformations, the aims of this study were: (i) to characterize the effects of two competitive antagonists (rauwolscine, RAU and clozapine, CLO) with very similar potencies for 5-HT(2B) receptors in a natural system (rat stomach fundus), and (ii) to evaluate a new method for detecting ligand-specific generated conformations through the study of the effects of RAU and CLO in 5-HT efficacy and in the time course of the response to the agonists. RAU and CLO behaved as competitive antagonists and showed similar potencies (pA(2) 7.56+/-0.25 and 7.50+/-0.30, respectively). However, RAU displayed greater efficacy than CLO in relaxing basal tension (10 microM CLO represented 64+/-6% of 10 microM RAU-induced relaxation). CLO partially reverted RAU-induced relaxation and RAU promoted an additional relaxation of maximal CLO-induced relaxation. This may indicate different degrees of inverse agonism. RAU also was more effective in generating insurmountable antagonism after long-term incubation (>3 hr) and modified the time course of the 5-HT(2B) response to 5-HT; conversely, CLO did not affect the time course of this response. This suggests that classical competitive antagonists may generate different specific conformational states and differential effects on receptor system regulation.

8-Substituted-9-deazaxanthines as adenosine receptor ligands: design, synthesis and structure-affinity relationships at A2B.

A number of 8-substituted-9-deazaxanthine derivatives (1,3-dialkyl-6-substituted-1H-pyrrolo[3,2-d]pyrimidine-2,4(3H,5H)-diones) were prepared and tested for their antagonistic activity at the recombinant human adenosine receptors, in particular at the A(2B) and A(2A) receptor subtypes. Compounds endowed with micromolar to nanomolar binding affinities, but with poor A(2B)/A(2A) selectivity, were obtained. Preliminary quantitative structure-affinity relationships suggested that the binding potency at the A(2B) receptor is mainly modulated by the electronic and lipophilic properties of the ligands.

Modulation of cAMP-specific PDE without emetogenic activity: new sulfide-like PDE7 inhibitors.

A forward chemical genetic approach was followed to discover new targets and lead compounds for Parkinson's disease (PD) treatment. By analysis of the cell protection produced by some small molecules, a diphenyl sulfide compound was revealed to be a new phosphodiesterase 7 (PDE7) inhibitor and identified as a new hit. This result allows us to confirm the utility of PDE7 inhibitors as a potential pharmacological treatment of PD. On the basis of these data, a diverse family of diphenyl sulfides has been developed and pharmacologically evaluated in the present work. Moreover, to gain insight into the safety of PDE7 inhibitors for human chronic treatment, we evaluated the new compounds in a surrogate emesis model, showing nonemetic effects.

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. Compounds 18b and 19b, increased [(3)H] NPA binding at concentrations between 10(-12) and 10(-9) M, which is similar to the effect of PLG in this assay and, provides evidences that these compounds are acting as allosteric modulators of dopamine D2 receptors.

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. The compounds elicited long-lasting (at least 24 h) concentration-dependent inhibition of radioligand binding at 5-HT7-binding sites in whole-cell radioligand binding assays, after pretreatment of the cells with the compounds and subsequent compound removal. In cAMP assays, pretreatment of cells with the compounds rendered 5-HT7 receptors unresponsive to 5-CT and also rendered 5-HT7-expressing HEK293 cells unresponsive to forskolin. Compound 1-(2-biphenyl)piperazine (RA-7), a known active metabolite of LP-211 present in vivo, was able to partially inhibit 5-HT7 radioligand binding in a long-lasting irreversible manner. Hence, LP-211 and MEL-9 were identified as high-affinity long-acting inhibitors of human 5-HT7 receptor binding and function in cell lines. The detailed in vitro characterization of these two pharmacological tools targeting 5-HT7 receptors may benefit the study of 5-HT7 receptor function and it may lead to the development of novel selective pharmacological tools with defined functional properties at 5-HT7 receptors.

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. Thanks to these studies we have proved the existence of allosteric sites in PDE7 and we have been able to explain the binding modes of the employed PDE7 inhibitors.

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.

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.