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The recently developed and used molecular modeling approach to search for privileged amino acids for halogen bonding (XB hot spots) through XSAR sets has been applied to 5-HT 7 R. Herein, among all identified 5-HT 7 R XB hot spots, the S5x42 was employed in a virtual screening protocol as a constraint. Through a designed virtual screening protocol, 63 XSAR sets (156 compounds) were selected from more than 8 million commercially available compounds and examined using in vitro assay toward 5-HT 7 R. A 68% accuracy was found in predicting halogenated derivatives with higher affinity for 5-HT 7 R than their unsubstituted analogs. Moreover, it was observed that a halogen bond formed between S5x42 and a chlorine atom at the 3-position of the arylpiperazine fragment caused the most remarkable, 35.4-fold increase in binding affinity for 5-HT 7 R when compared to the nonhalogenated analog. Interestingly, molecular dynamics simulations showed the formation of a bifurcated halogen bond with S5x42.

5-HT 7 R belongs to a family of G protein-coupled receptors and is associated with a variety of physiological processes in the central nervous system via the activation of the neurotransmitter serotonin (5-HT). To develop selective and biased 5-HT 7 R ligands, we designed and synthesized a series of pyrazolyl-diazepanes 2 and pyrazolyl-piperazines 3 , which were evaluated for binding affinities to 5-HTR subtypes and functional selectivity for G protein and β-arrestin signaling pathways of 5-HT 7 R. Among them, 1-(3-(3-chlorophenyl)-1 H -pyrazol-4-yl)-1,4-diazepane 2c showed the best binding affinity for 5-HT 7 R and selectivity over other 5-HTR subtypes. It was also revealed as a G protein-biased antagonist. The self-grooming behavior test was performed with 2c in vivo with Shank3 -/- transgenic (TG) mice, wherein 2c significantly reduced self-grooming duration time to the level of wild-type mice. The results suggest that 5-HT 7 R could be a potential therapeutic target for treating autism spectrum disorder stereotypy.

There has been significant attention concerning the biased agonism of G protein-coupled receptors (GPCRs), and it has resulted in various pharmacological benefits. 5-HT 7 R belongs to a GPCR, and it is a promising pharmaceutical target for the treatment of neurodevelopmental and neuropsychiatric disorders. Based on our previous research, we synthesized a series of 6-chloro-2'-methoxy biphenyl derivatives 1 , 2 , and 3 with a variety of amine scaffolds. These compounds were evaluated for their binding affinities to 5-HTR subtypes and their functional selectivity toward the Gs protein and the β-arrestin signaling pathways of 5-HT 7 R. Among them, 2-(6-chloro-2'-methoxy-[1,1'-biphenyl]-3-yl)- N -ethylethan-1-amine, 2b , was found to be a G-protein-biased ligand of 5-HT 7 R. In an in vivo study with Shank3 transgenic mice, the self-grooming behavior test was performed with 2b , which increased the duration of self-grooming. The experiments further suggested that 5-HT 7 R is associated with autism spectrum disorders (ASDs) and could be a therapeutic target for the treatment of stereotypy in ASDs.

It is well-documented that serotonin (5-HT) exerts its pharmacological effects through a series of 5-HT receptors. The most recently identified member of this family, 5-HT 7 , was first identified in 1993. Over the course of the last 25 years, this receptor has been the subject of intense investigation, and it has been demonstrated that 5-HT 7 plays an important role in a wide range of pharmacological processes. As a result of these findings, modulation of 5-HT 7 activity has been the focus of numerous drug discovery and development programs. This review provides an overview of the roles of 5-HT 7 in normal physiology and the therapeutic potential of this interesting drug target.

A computational approach combining a structure-activity relationship library of halogenated and the corresponding unsubstituted ligands (called XSAR) with QM-based molecular docking and binding free energy calculations was used to search for amino acids frequently targeted by halogen bonding (hot spots) in a 5-HT 7 R as a case study. The procedure identified two sets of hot spots, extracellular (D2.65, T2.64, and E7.35) and transmembrane (C3.36, T5.39, and S5.42), which were further verified by a synthesized library of halogenated arylsulfonamide derivatives of (aryloxy)ethylpiperidines. It was found that a halogen bond formed between T5.39 and a bromine atom at 3-position of the aryloxy fragment caused the most remarkable, 35-fold increase in binding affinity for 5-HT 7 R when compared to the nonhalogenated analog. The proposed paradigm of halogen bonding hot spots was additionally verified on D 4 dopamine receptor showing that it can be used in rational drug design/optimization for any protein target.

Serotonin 5-HT 7 receptor (5-HT 7 R) has been the subject of intense research efforts because of its presence in brain areas such as the hippocampus, hypothalamus, and cortex. Preclinical data link the 5-HT 7 R to a variety of central nervous system processes including the regulation of circadian rhythms, mood, cognition, pain processing, and mechanisms of addiction. 5-HT 7 R blockade has antidepressant effects and may ameliorate cognitive deficits associated with schizophrenia. 5-HT 7 R has been recently shown to modulate neuronal morphology, excitability, and plasticity, thus contributing to shape brain networks during development and to remodel neuronal wiring in the mature brain. Therefore, the activation of 5-HT 7 R has been proposed as a therapeutic approach for neurodevelopmental and neuropsychiatric disorders associated with abnormal neuronal connectivity. This Perspective celebrates the silver jubilee of the discovery of 5-HT 7 R by providing a survey of recent studies on the medicinal chemistry of 5-HT 7 R ligands and on the neuropharmacology of 5-HT 7 R.

Modulators of the serotonin 5-HT6 receptor (5-HT6R) offer a promising strategy for the treatment of the cognitive deficits that are associated with dementia and Alzheimer's disease. Herein, we report the design, synthesis, and characterization of a novel class of 5-HT6R antagonists that is based on the 1H-pyrrolo[3,2-c]quinoline core. The most active compounds exhibited comparable binding affinity to the reference compound, SB-742457, and markedly improved selectivity. Lead optimization led to the identification of (S)-1-[(3-chlorophenyl)sulfonyl]-4-(pyrrolidine-3-yl-amino)-1H-pyrrolo[3,2-c]quinoline (14) (Ki = 3 nM and Kb = 0.41 nM). Pharmacological characterization of the 5-HT6R's constitutive activity at Gs signaling revealed that 14 behaved as a neutral antagonist, while SB-742457 was classified as an inverse agonist. Both compounds 14 and SB-742457 reversed phencyclidine-induced memory deficits and displayed distinct procognitive properties in cognitively unimpaired animals (3 mg/kg) in NOR tasks. Compounds 14 and SB-742457 were also active in the Vogel test, yet the anxiolytic effect of 14 was 2-fold higher (MED = 3 mg/kg). Moreover, 14 produced, in a 3-fold higher dose (MED = 10 mg/kg), antidepressant-like effects that were similar to those produced by SB-742457 (MED = 3 mg/kg). Together, these data suggest that the 4-(pyrrolidine-3-yl-amino)-1H-pyrrolo[3,2-c]quinoline scaffold is an attractive molecular framework for the development of procognitive agents. The results are promising enough to warrant further detailed mechanistic studies on the therapeutic potential of 5-HT6R antagonists and inverse agonists for the treatment of cognitive decline and depression/anxiety symptoms that are comorbidities of Alzheimer's disease.

Within the past decade several novel targets have been indicated as key players in Alzheimer-type dementia and associated conditions, including a "frightening" memory loss as well as severe cognitive impairments. These proteins are deeply implicated in crucial cell processes, e.g., autophagy, growth and progression, apoptosis, and metabolic equilibrium. Since recently, 5-HT6R has been considered as one of the most prominent biological targets in AD drug therapy. Therefore, we investigated the potential procognitive and neuroprotective effects of our novel selective 5-HT6R antagonist, AVN-211. During an extensive preclinical evaluation the lead compound demonstrated a relatively high therapeutic potential and improved selectivity toward 5-HT6R as compared to reference drug candidates. It was thoroughly examined in different in vivo behavioral models directly related to AD and showed evident improvements in cognition and learning. In many cases, the observed effect was considerably greater than that determined for the reported drugs and drug candidates, including memantine, SB-742457, and Lu AE58054, evaluated under the same conditions. In addition, AVN-211 showed a similar or better anxiolytic efficacy than fenobam, rufinamide, lorazepam, and buspirone in an elevated plus-maze model, elevated platform, and open field tests. The compound demonstrated low toxicity and no side effects in vivo, an appropriate pharmacokinetic profile, and stability. In conclusion, AVN-211 significantly delayed or partially halted the progressive decline in memory function associated with AD, which makes it an interesting drug candidate for the treatment of neurodegenerative and psychiatric disorders. Advanced clinical trials are currently under active discussion and in high priority.

Molecular docking, despite its undeniable usefulness in computer-aided drug design protocols and the increasing sophistication of tools used in the prediction of ligand-protein interaction energies, is still connected with a problem of effective results analysis. In this study, a novel protocol for the automatic evaluation of numerous docking results is presented, being a combination of Structural Interaction Fingerprints and Spectrophores descriptors, machine-learning techniques, and multi-step results analysis. Such an approach takes into consideration the performance of a particular learning algorithm (five machine learning methods were applied), the performance of the docking algorithm itself, the variety of conformations returned from the docking experiment, and the receptor structure (homology models were constructed on five different templates). Evaluation using compounds active toward 5-HT6 and 5-HT7 receptors, as well as additional analysis carried out for beta-2 adrenergic receptor ligands, proved that the methodology is a viable tool for supporting virtual screening protocols, enabling proper discrimination between active and inactive compounds.

Molecular imaging methods such as positron emission tomography (PET) are increasingly involved in the development of new drugs. Using radioactive tracers as imaging probes, PET allows the determination of the pharmacokinetic and pharmacodynamic properties of a drug candidate, via recording target engagement, the pattern of distribution, and metabolism. Because of the noninvasive nature and quantitative end point obtainable by molecular imaging, it seems inherently suited for the examination of a pharmaceutical's behavior in the brain. Molecular imaging, most especially PET, can therefore be a valuable tool in CNS drug research. In this Perspective, we present the basic principles of PET, the importance of appropriate tracer selection, the impact of improved radiopharmaceutical chemistry in radiotracer development, and the different roles that PET can fulfill in CNS drug research.

Alzheimer's disease (AD) is one of the most frequent causes of death and disability worldwide and has a significant clinical and socioeconomic impact. In the search for novel therapeutic strategies, serotonin 5-HT6 receptor (5-HT6R) has been proposed as a promising drug target for cognition enhancement in AD. This manuscript reviews the compelling evidence for the implication of this receptor in learning and memory processes. We have summarized the current status of the medicinal chemistry of 5-HT6R antagonists and the encouraging preclinical findings that demonstrate their significant procognitive behavioral effects in a number of learning paradigms, probably acting through modulation of multiple neurotransmitter systems and signaling pathways. The results of the ongoing clinical trials are eagerly awaited to shed some light on the validation of 5-HT6R antagonists as a new drug class for the treatment of symptomatic cognitive impairment in AD, either as stand-alone therapy or in combination with established agents.

The recent increase in the number of atomic-resolution structures of G protein-coupled receptors (GPCRs) has contributed to a deeper understanding of ligand binding to several important drug targets. However, reliable modeling of GPCR-ligand complexes for the vast majority of receptors with unknown structure remains to be one of the most challenging goals for computer-aided drug design. The GPCR Dock 2013 assessment, in which researchers were challenged to predict the crystallographic structures of serotonin 5-HT(1B) and 5-HT(2B) receptors bound to ergotamine, provided an excellent opportunity to benchmark the current state of this field. Our contributions to GPCR Dock 2013 accurately predicted the binding mode of ergotamine with RMSDs below 1.8 Å for both receptors, which included the best submissions for the 5-HT(1B) complex. Our models also had the most accurate description of the binding sites and receptor-ligand contacts. These results were obtained using a ligand-guided homology modeling approach, which combines extensive molecular docking screening with incorporation of information from multiple crystal structures and experimentally derived restraints. In this work, we retrospectively analyzed thousands of structures that were generated during the assessment to evaluate our modeling strategies. Major contributors to accuracy were found to be improved modeling of extracellular loop two in combination with the use of molecular docking to optimize the binding site for ligand recognition. Our results suggest that modeling of GPCR-drug complexes has reached a level of accuracy at which structure-based drug design could be applied to a large number of pharmaceutically relevant targets.

By introducing distal substituents on a tetracyclic scaffold resembling the ergoline structure, two series of analogues were achieved exhibiting subnanomolar receptor binding affinities for the dopamine D2 and serotonin 5-HT6 receptor subtype, respectively. While the 5-HT6 ligands were antagonists, the D2 ligands displayed intrinsic activities ranging from full agonism to partial agonism with low intrinsic activity. These structures could potentially be interesting for treatment of neurological diseases such as schizophrenia, Parkinson's disease, and cognitive deficits.

Homology modeling is a reliable method of predicting the three-dimensional structures of proteins that lack NMR or X-ray crystallographic data. It employs the assumption that a structural resemblance exists between closely related proteins. Despite the availability of many crystal structures of possible templates, only the closest ones are chosen for homology modeling purposes. To validate the aforementioned approach, we performed homology modeling of four serotonin receptors (5-HT1AR, 5-HT2AR, 5-HT6R, 5-HT7R) for virtual screening purposes, using 10 available G-Protein Coupled Receptors (GPCR) templates with diverse evolutionary distances to the targets, with various approaches to alignment construction and model building. The resulting models were further validated in two steps by means of ligand docking and enrichment calculation, using Glide software. The final quality of the models was determined in virtual screening-like experiments by the AUROC score of the resulting ROC curves. The outcome of this research showed that no correlation between sequence identity and model quality was found, leading to the conclusion that the closest phylogenetic relative is not always the best template for homology modeling.

The 5-HT6 receptors are potent therapeutic targets for psychiatric and neurological diseases (schizophrenia, Alzheimer's disease, etc.). However, with lack of specific radiopharmaceuticals, their pharmacology is still incomplete and their exploration is limited to animal models. In this context, we have designed a fluorinated PET radiotracer, [(18)F]2FNQ1P, that possesses a high affinity and selectivity for 5-HT6. In vitro PET autoradiographies in rat brain sections with this radiotracer were in accordance with the 5-HT6 distribution pattern.

In this paper, a new approach for generating receptor-based 3D pharmacophore models for rapid in silico virtual screening is presented. The method combines information from docking poses of known ligands of different structures and further ligand-receptor complexes analyses using structural interaction fingerprints (SIFts). Next, the best linear combination of three-, four-, and five-feature pharmacophores in terms of selected performance parameter (i.e., recall, F-score, and MCC) is constructed. The resultant queries showed significantly better VS performance and new scaffold recognition when compared with the known ligand- and receptor-based pharmacophore models. The approach was developed and validated on 5-HT₇ receptor homology models created on available crystal structure templates. The efficiency of the obtained linear combinations exhibited only a minor dependence on the template selection.

The formation of ligand-protein complexes requires simultaneous adaptation of the binding partners. In structure based virtual screening, high throughput docking approaches typically consider the ligand flexibility, but the conformational freedom of the protein is usually taken into account in a limited way. The goal of this study is to elaborate a methodology for incorporating protein flexibility to improve the virtual screening enrichments on GPCRs. Explicit-solvated molecular dynamics simulations (MD) were carried out in lipid bilayers to generate an ensemble of protein conformations for the X-ray structures and homology models of both aminergic and peptidergic GPCRs including the chemokine CXCR4, dopamine D3, histamine H4, and serotonin 5HT6 holo receptor complexes. The quality of the receptor models was assessed by enrichment studies to compare X-ray structures, homology models, and snapshots from the MD trajectory. According to our results, selected frames from the MD trajectory can outperform X-ray structures and homology models in terms of enrichment factor and AUC values. Significant changes were observed considering EF1% values: comparing the original CXCR4, D3, and H4 targets and the additional 5HT6 initial models to that of the best MD frame resulted in 0 to 6.7, 0.32 to 3.5 (10×), 13.3 to 26.7 (2×), and 0 to 14.1 improvements, respectively. It is worth noting that rank-average based ensemble evaluation calculated for different ensemble sizes could not improve the results further. We propose here that MD simulation can capture protein conformations representing the key interacting points of the receptor but less biased toward one specific chemotype. These conformations are useful for the identification of a "consensus" binding site with improved performance in virtual screening.

Here we report the design, synthesis, and 5-HT(7) receptor affinity of a set of 1-(3-biphenyl)- and 1-(2-biphenyl)piperazines. The effect on 5-HT(7) affinity of various substituents on the second (distal) phenyl ring was analyzed. Several compounds showed 5-HT(7) affinities in the nanomolar range and >100-fold selectivity over 5-HT(1A) and adrenergic α(1) receptors. 1-[2-(4-Methoxyphenyl)phenyl]piperazine (9a) showed 5-HT(7) agonist properties in a guinea pig ileum assay but blocked 5-HT-mediated cAMP accumulation in 5-HT(7)-expressing HeLa cells.

Of great interest in recent years has been computationally predicting the novel polypharmacology of drug molecules. Here, we applied an "induced-fit" protocol to improve the homology models of 5-HT(2A) receptor, and we assessed the quality of these models in retrospective virtual screening. Subsequently, we computationally screened the FDA approved drug molecules against the best induced-fit 5-HT(2A) models and chose six top scoring hits for experimental assays. Surprisingly, one well-known kinase inhibitor, sorafenib, has shown unexpected promiscuous 5-HTRs binding affinities, K(i) = 1959, 56, and 417 nM against 5-HT(2A), 5-HT(2B), and 5-HT(2C), respectively. Our preliminary SAR exploration supports the predicted binding mode and further suggests sorafenib to be a novel lead compound for 5HTR ligand discovery. Although it has been well-known that sorafenib produces anticancer effects through targeting multiple kinases, carefully designed experimental studies are desirable to fully understand whether its "off-target" 5-HTR binding activities contribute to its therapeutic efficacy or otherwise undesirable side effects.

A graphical method is introduced for compound data mining and structure-activity relationship (SAR) data analysis that is based upon a canonical structural organization scheme and captures a compound-scaffold-skeleton hierarchy. The graph representation has a constant layout, integrates compound activity data, and provides direct access to SAR information. Characteristic SAR patterns that emerge from the graph are easily identified. The molecular hierarchy enables "forward-backward" analysis of compound data and reveals both global and local SAR patterns. For example, in heterogeneous data sets, compound series are immediately identified that convey interpretable SAR information in isolation or in the structural context of related series, which often define SAR pathways through data sets.

Here we present the solution phase parallel synthesis of a combinatorial library consisting of 776 new substituted 3-phenylsulfonyl-[1,2,3]triazolo[1,5-a]quinazolines and a study of the relation of their structure with a 5-HT(6) receptor antagonistic activity in a functional cell (HEK 293) analysis and radioligand competitive binding. We have found highly active and selective 5-HT(6)R antagonists. The most active 5-HT(6)R antagonists have IC(50) <100 nM in a functional assay, and K(i) <10 nM in a binding assay, which is 100 times higher than the activity with respect to other serotonin receptors.

Novel 5-cyclic amine-3-arylsulfonylindazoles were prepared, and several analogues within this class have been identified as high-affinity 5-HT(6) receptor ligands with improved pharmacokinetic and pharmacological properties. One selected example, 18b, showed good brain penetrability and a generally favorable pharmacokinetic profile with procognitive efficacy in the rat novel object recognition assay. The synthesis and structure-activity relationship of this potent class are discussed.

On the basis of our previously described pharmacophore model for serotonin 5-HT(6) receptor (5-HT(6)R) antagonists, we have designed, synthesized, and pharmacologically characterized a series of benzimidazole derivatives 1-20 that represent a new family of potent antagonists at the human 5-HT(6)R. Site-directed mutagenesis and a beta(2)-adrenoceptor-based homology model of the 5-HT(6)R were used to predict the mode of binding of antagonist SB-258585 and the new synthesized ligands. Substitution of W6.48, F6.52, or N6.55 by Ala fully impedes compound 4 to block 5-HT-induced activation. Thus, we propose that D3.32 in TM 3 anchors the protonated piperazine ring, the benzimidazole ring expands parallel to EL 2 to hydrogen bond N6.55 in TM 6, and the aromatic ring is placed between TMs 3 and 5 in CH(2)-containing compounds and between TMs 3 and 6 in CO-containing compounds. This combined experimental and computational study has permitted to propose the molecular mechanisms by which the new benzimidazole derivatives act as 5-HT(6)R antagonists.

Homo- and heterodimerization is becoming an assessed concept in G-protein coupled receptor (GPCR) pharmacology, and the notion that GPCRs may dimerize or oligomerize is allowing for a reinterpretation of some inconsistencies or anomalies and is providing medicinal chemists with potentially relevant novel molecular targets for a variety of therapeutic conditions. Recently, it has been reported that two unrelated GPCRs, namely class C metabotropic glutamate receptor type-2 (mGluR2) and class A 5HT(2A) serotoninergic receptor, can heterodimerize at the transmembrane domain level. We performed a 40 ns molecular dynamics simulation of the mGluR2/5HT(2A) heterocomplex constructed around a TM4/TM5 interface and embedded in an explicit phospholipidic bilayer surrounded by water molecules. In a separate experiment, the monomeric 5HT(2A) receptor was simulated for additional 40 ns under the same conditions. The analysis and the comparison of the two simulations allowed us to clearly identify a cross-talk between the two protomers and to put forward an effect of the heterodimerization on the shape of the binding pocket of 5HT(2A). This result provides the first molecular explanation for the reported allosteric effect of mGluR2 on 5HT(2A)-mediated response and suggests that the heterocomplex can be a more suitable target for in silico screening than the monomeric protomers.

A population of 100 graphics models of the human 5-HT6 serotonin receptor was constructed based on the structure of bovine rhodopsin. The endogenous tryptamine-based agonist serotonin (5-HT; 1) and the benzenesulfonyl-containing tryptamine-derived 5-HT6 receptor antagonist MS-245 (4a) were automatically docked with each of the 100 receptor models using a genetic algorithm approach. Similar studies were conducted with the more selective 5-HT6 receptor agonist EMDT (5) and optical isomers of EMDT-related analog 8, as well as with optical isomers of MS-245 (4a)-related and benzenesulfonyl-containing pyrrolidine 6 and aminotetralin 7. Although associated with the same general aromatic/hydrophobic binding cluster, 5-HT (1) and MS-245 (4a) were found to preferentially bind with distinct receptor conformations, and did so with different binding orientations (i.e., poses). A 5-HT pose/model was found to be common to EMDT (5) and its analogs, whereas that identified for MS-245 (4a) was found common to benzenesulfonyl-containing compounds. Specific amino acid residues were identified that can participate in binding, and evaluation of a sulfenamide analog of MS-245 indicates for the first time that the presence of the sulfonyl oxygen atoms enhances receptor affinity. The results indicate that the presence or absence of an N1-benzenesulfonyl group is a major determinant of the manner in which tryptamine-related agents bind at 5-HT6 serotonin receptors.

A set of 31 diversified 5-HT7 receptor antagonists was automatically docked to a conformational ensemble of rhodopsin-based 5-HT7R models (flexible docking). It was found that ergolines, aporphines, and tricyclic psychotropic agents were always docked in a pocket formed by TMHs 4-6, and besides the main ionic bond with Asp3.32, they had specific interactions with Phe6.52, Phe6.51, Trp6.48, and Ser5.42. The arylpiperidine, arylpiperazine, or beta-carboline fragment of the complex ligands occupied the same pocket, whereas the terminal amide/imide part of those compounds reached the second cavity formed by TMHs 7-3 and interacted with Phe3.28, Arg7.36, and Tyr7.43. A similar orientation of selective antagonists of the group of arylsulfonamidoalkylamines was observed, that is, the sulfonamide part was located in the second pocket. Coherent docking results allowed the generation of two receptor-based pharmacophores: first containing features necessary for high 5-HT7R affinity and the other defining selectivity for this receptor subtype. The latter model indicated the importance of specific interactions with the residues of the TMHs 7-3 pocket (especially nonconserved Arg7.36) for selectivity over other monoamine GPCRs.

Various sulfonyl-containing compounds (e.g. sulfonamides, sulfones) bind at human 5-HT6 serotonin receptors, but it has been difficult relating the binding mode(s) of such agents to one another, even though many possess a common SO2 moiety, to identify a common pharmacophore model(s). On the basis of the hypothesis that an ergoline-type conformation might be important for the binding of some sulfonamide-containing arylalkylamines, we prepared for examination at h5-HT6 receptors a series of compounds, including phenylethylamines 6, pyrroloethylamine 7, and phenylpiperazines 9. The results (with Ki values ranging from about 1 nM to >1000 nM) suggest that many of these agents likely bind in a related fashion, and structure-affinity studies indicate that the benzenesulfonamide portion of the phenylethylamine and phenylpiperazine analogues can be "reversed", abbreviated to a sulfone, and moved to an adjacent position with relatively little impact on affinity. Although a benzenesulfonamide (or related arylsulfonamide) group might be common to various 5-HT6 ligands, there appears to be some latitude with regard to the specific constitution and location of the sulfonamide moiety even within the same arylalkylamine structural framework. A pharmacophore model is presented to account for some of the current findings.

Assembly of the azepine ring of xantheno[9,1-cd]azepines by electrophilic cyclization of sulfonamide acetals provides access to clavizepine analogues in the form of 2,12b-dihydro- or 4-hydroxy-2,3,4,12b-tetrahydro-1H-xantheno[9,1-cd]azepines, in the latter case producing the trans derivative stereoselectively. Binding assays for clavizepine and analogues at adrenergic, dopaminergic, and serotonergic receptors are reported.

Similarity-based methods for virtual screening are widely used. However, conventional searching using 2D chemical fingerprints or 2D graphs may retrieve only compounds which are structurally very similar to the original target molecule. Of particular current interest then is scaffold hopping, that is, the ability to identify molecules that belong to different chemical series but which could form the same interactions with a receptor. Reduced graphs provide summary representations of chemical structures and, therefore, offer the potential to retrieve compounds that are similar in terms of their gross features rather than at the atom-bond level. Using only a fingerprint representation of such graphs, we have previously shown that actives retrieved were more diverse than those found using Daylight fingerprints. Maximum common substructures give an intuitively reasonable view of the similarity between two molecules. However, their calculation using graph-matching techniques is too time-consuming for use in practical similarity searching in larger data sets. In this work, we exploit the low cardinality of the reduced graph in graph-based similarity searching. We reinterpret the reduced graph as a fully connected graph using the bond-distance information of the original graph. We describe searches, using both the maximum common induced subgraph and maximum common edge subgraph formulations, on the fully connected reduced graphs and compare the results with those obtained using both conventional chemical and reduced graph fingerprints. We show that graph matching using fully connected reduced graphs is an effective retrieval method and that the actives retrieved are likely to be topologically different from those retrieved using conventional 2D methods.

The present study discusses the well-known 5-HT7/5-HT1A selectivity issue through a new series of phenylpyrrole derivatives. The first hits emerged from a virtual screening performed on a chemolibrary. Further study led to an optimization of a preliminary 5-HT7 pharmacophore model. The importance of each pharmacophoric feature is confirmed, but these characteristics have to be coupled to geometric constraints in order to achieve a 5-HT7 selectivity. Indeed, 5-HT1A affinity probably arises from extended conformations, whereas a bent one appears to be best suited for 5-HT7 selectivity.

Herein a general concept for the design of targeted libraries for proteins with binding sites that are divided into subsites is laid out, including several practical aspects and their solutions. The design is based on a chemogenomic classification of the subsites followed by collection of bioactive molecular fragments and virtual library generation. The general process is outlined and applied to the assembly of a library of 500 molecules targeting the serotonin type 7 (5-HT7) receptor, a class A G-Protein Coupled Receptor (GPCR). Utilizing commercially available building blocks of similar size and composition, a reference library was created. Control sets of known ligands for the 5-HT7 receptor, other GPCRs, and nuclear receptors were collected from literature sources. Principal component analysis of molecular descriptors for the two libraries and the literature sets, displayed a focusing of the targeted library to the region in the chemical space defined by the literature actives, suggesting a denser coverage of the bioactive region than for the more diverse reference library. Additional computational validations, including PCA class predictions, 3D pharmacophore modeling, and docking calculations all indicated an enrichment factor of 5-HT7 ligand-like molecules in the range of 2-4 for the targeted library compared to the reference library.

A series of arylpiperazine- and 1,2,3,4-tetrahydroisoquinoline-based arylsulfonamides was synthesized and evaluated for their interactions with the constitutively active 5-HT7 receptor. Effects on basal adenylate cyclase activity were measured using HEK-293 cells expressing the rat 5-HT7. All ligands produced a decrease of adenylate cyclase activity, indicative of their inverse agonism. Additionally, computational studies with a set of 22 inverse agonists, including these novel inverse agonists and inverse agonists known from literature, resulted in a pharmacophore model and a CoMFA model (R2 = 0.97, SE = 0.18). Docking of inverse agonists at the binding site of a model of the helical parts of the 5-HT7 receptor, based on the alpha carbon template for 7-TM GPCRs, revealed interesting molecular interactions and a possible explanation for observed structure-activity relationships.

A definition of a pharmacophore for the 5-HT7 antagonists was carried out by searching the common chemical features of selective antagonists from the literature. A molecular design is described by analyzing the differences between this new pharmacophore and three other 3D serotonin pharmacophores previously described. This comparison led to the synthesis of a new series of potent 5-HT7 antagonists.

Privileged structures are ligand substructures that are widely used to generate high-affinity ligands for more than one type of receptor. To explain this, we surmised that there must be some common feature in the target proteins. For a set of class A GPCRs, we found a good correlation between conservation patterns of residues in the ligand binding pocket and the privileged structure fragments in class A GPCR ligands. A major part of interior surface of the common ligand binding pocket of class A receptors, identified in many GPCRs, is lined with variable residues that are responsible for selectivity in ligand recognition, while other regions, typically located deeper into the binding pocket, are more conserved and retain a predominantly hydrophobic and aromatic character. The latter is reflected in the chemical nature of most GPCR privileged structures and is proposed to be the common feature that is recognized by the privileged structures. Further, we find that this subpocket is conserved even in distant orthologs within the class A family. Three pairs of ligands recognizing widely different receptor types were docked into receptor models of their target receptors utilizing available structure- activity relationships and mutagenesis data. For each pair of ligands, the ligand-receptor complexes reveal that the nature of the privileged structure binding pocket is conserved between the two complexes, in support of our hypothesis. Only part of the privileged structures can be accommodated within the conserved subpocket. Some contacts are established between the privileged structure and the nonconserved parts of the binding pocket. This implies that any one particular privileged structure can target only a subset of receptors, those complementary to the full privileged structure. Our hypothesis leads to a valuable novelty in that ligand libraries can be designed without any foreknowledge of the structure of the endogenous ligand, which in turn means that even orphan receptors can in principle now be addressed as potential drug targets.

On the basis of a set of 20 diverse 5-HT(7) receptor agonists, the pharmacophore for 5-HT(7) receptor agonism was determined. Additionally two CoMFA models were developed, based on different alignments of the agonists. Both models show good correlations between experimental and predictive pK(i) values and show a high degree of similarity. The CoMFA fields were subsequently used to map the agonist binding site of the model of the 5-HT(7) receptor. Important roles in ligand binding are attributed to Asp162 of TM3 (interaction with a protonated nitrogen), and Thr244 of TM5 (interaction with a substituent at an aromatic moiety). Amino acid residues of the aromatic cluster of TM6 are hypothesized to play an important role in ligand binding as pi-pi stacking moieties. Agonists missing a hydrogen-bond-accepting moiety, but possessing an aromatic substituent instead, seem to bind the receptor with high affinity as well by occupying a lipophilic pocket hosted by residues of TM5 and TM6.

5-HT(2A) serotonin receptors represent the principal molecular targets for LSD-like hallucinogens and atypical antipsychotic drugs. It has been proposed that a dysregulation of 5-HT(2A) receptor-mediated signaling may contribute to the pathogenesis of schizophrenia and related diseases. A major mechanism for the attenuation of GPCR signaling following agonist activation typically involves the phosphorylation of serine and/or threonine residues by various kinases. Ser/Thr phosphorylation leads to the binding of accessory proteins and the uncoupling of the G proteins, thereby preventing further signaling. The molecular mechanisms by which 5-HT(2A) receptors are desensitized are unknown, and to date, no residues essential for agonist-mediated desensitization have been identified. Thus, we mutated, individually or in groups, all of the 37 serines and threonines in the cytoplasmic domains of the 5-HT(2A) receptor and assessed the effects of these mutations on agonist-mediated desensitization. We discovered that mutation of two residues, S421 in the C-terminal tail and S188 in the second intracellular loop, to alanine resulted in a significant block of agonist-induced desensitization. Intriguingly, a single-nucleotide polymorphism, of unreported frequency, at the S421 locus has been reported (S421F); the S421F mutation, like the S421A mutation, significantly attenuated agonist-mediated desensitization. Taken together, these findings indicate that the process of agonist-mediated desensitization of 5-HT(2A) receptors requires the presence of two nonconserved serine residues located in distinct intracellular loops.

Using unnatural amino acid mutagenesis, the binding site for serotonin at the novel Caenorhabditis elegans receptor MOD-1 has been probed. As with the closely related serotonin receptor 5-HT3, MOD-1 makes use of a strong cation-pi interaction between the ammonium of serotonin and the indole side chain of a tryptophan. However, the specific Trp used by MOD-1 is different from that used for 5-HT3 (and the nAChR), aligning with a residue more than 40 amino acids distant in sequence space and on a different "loop" of the agonist binding site. This suggests a significant rearrangement of the ligand on binding these two closely related receptors. It is suggested that, unlike enzymes, receptors and other signaling molecules may need only to deliver an agonist to a general binding region, rather than establishing precise drug-receptor interactions.

It has been proposed that 5-HT(1A) receptor antagonists augment the antidepressant efficacy of selective serotonin (5-HT) reuptake inhibitors. In a search toward new and efficient antidepressants, 1-(aryl)-3-[4-arylpiperazin-1-yl]-1-propane molecular hybrids were designed, synthesized, and evaluated for 5-HT reuptake inhibition and 5-HT(1A) receptor affinity. The design was based in coupling structural moieties related to inhibition of serotonin reuptake, such as benzo[b]thiophene derivatives to arylpiperazines, typical 5-HT(1A) receptor ligands. In binding studies, several compounds showed affinity at the 5-HT transporter and at 5-HT(1A) receptors. Molecular modeling studies predicted the pharmacophore elements required for high affinity binding and the features that enable to discriminate between agonist, partial agonist, or antagonist action at 5-HT(1A) receptors and 5-HT transporter inhibition. Solvent interactions in desolvation prior to the binding step along with enthalpy and enthropy compensations might be responsible to explain agonist, partial agonist, and antagonist character. Hydrogen-bonding capability seems to be important to break hydrogen interhelical hydrogen bonds or alternatively to form other bonds upon ligand binding. Partial agonists and antagonists are unable to do this as the full agonist, which interacts closely by long-range forces or directly. The compounds showing the higher affinity at both the 5-HT transporter (K(i) < 50 nM) and the 5-HT(1A) receptors (K(i) < 20 nM) were further explored for their ability to stimulate [(35)S]GTPgammaS binding or to antagonize 8-hydroxy-2-di-n-propylamino-tetralin (8-OH-DPAT)-stimulated [(35)]GTPgammaS binding to rat hippocampal membranes, an index of agonist/antagonist action at 5-HT(1A) receptors, respectively. Compound 8g exhibited agonist activity (EC(50) = 30 nM) in this assay, whereas compounds 7g and 8h,i behaved as weak partial agonists and 7h-j and 8j,l antagonized the R(+)-8-OH-DPAT-stimulated GTPgammaS binding. Functional characterization was performed by measuring the antagonism to 8-OH-DPAT-induced hypothermia in mice.

A series of tryptophan analogues has been introduced into the binding site regions of two ion channels, the ligand-gated nicotinic acetylcholine and serotonin 5-HT(3A) receptors, using unnatural amino acid mutagenesis and heterologous expression in Xenopus oocytes. A cation-pi interaction between serotonin and Trp183 of the serotonin channel 5-HT(3A)R is identified for the first time, precisely locating the ligand-binding site of this receptor. The energetic contribution of the observed cation-pi interaction between a tryptophan and the primary ammonium ion of serotonin is estimated to be approximately 4 kcal/mol, while the comparable interaction with the quaternary ammonium of acetylcholine is approximately 2 kcal/mol. The binding mode of nicotine to the nicotinic receptor of mouse muscle is examined by the same technique and found to differ significantly from that of the natural agonist, acetylcholine.

We have measured fluorescence resonance energy transfer (FRET) between a fluorescent antagonist, bound to the purified detergent-solubilized serotonin type 3 receptor, and a lipophilic acceptor probe partitioned into the micelle surrounding the detergent-solubilized receptor. The experimentally observed FRET efficiency was evaluated on the basis of the characteristic dimensions of the receptor-micelle complex and the average number of acceptor molecules in such micelles. The binding site was determined to be 5.4 +/- 0.9 nm above the center of the detergent micelle. The experiments were performed below the critical micellar concentration of the detergent (C(12)E(9)) used to solubilize the receptor, under which conditions it was demonstrated that the ligand binding activity was fully preserved. This reduces considerably the fluorescence background arising from probes not associated with the receptor, allowing a precise determination of the transfer efficiency.

A classical quantitative structure-activity relationship (Hansch) study and artificial neural networks (ANNs) have been applied to a training set of 32 substituted phenylpiperazines with affinity for 5-HT(1A) and alpha(1)-adrenergic receptors, to evaluate the structural requirements that are responsible for 5-HT(1A)/alpha(1) selectivity. The resulting models provide a significant correlation of electronic, steric, and hydrophobic parameters with the biological affinities. Although the derived linear Hansch correlations give good statistics and acceptable predictions, the introduction of nonlinear relationships in the analysis gives more solid models and more accurate predictions. In the ANN models on the basis of the obtained 3D plots, the 5-HT(1A) affinity has a nonlinear dependence on F, V(o), V(m), and pi(o), although the nonlinear relationship is not far from a planar one. The alpha(1)-adrenergic receptor affinity has a clear nonlinear dependence on F, V(o), V(m), pi(o), and pi(m). A comparison of both analyses gives an additional understanding for 5-HT(1A)/alpha(1) selectivity: (a) high F values increase the binding affinity for 5-HT(1A) receptors and decrease the affinity for alpha(1) sites; (b) the hydrophobicity at the meta-position has only influence for the alpha(1)-adrenergic receptor; (c) the meta-position seems to be implicated in the 5-HT(1A)/alpha(1) selectivity. While the 5-HT(1A) receptor is able to accommodate bulky substituents in the region of its active site, the steric requirements of the alpha(1)-adrenergic receptor at this position are more restricted. This information was used for the design of the new ligand EF-7412 (33) (5-HT(1A): K(i exptl) = 27 nM, alpha(1): K(i exptl) > 1000 nM; 5-HT(1A): K(i pred) (ANN) = 36 nM, alpha(1): K(i pred ANN) = 2745 nM) which was characterized as an antagonist in vivo in pre- and postsynaptic 5-HT(1A)R sites. Computational simulations of the complex between EF-7412 (33) and a 3D model of the transmembrane domain of the 5-HT(1A) receptor allowed us to define the molecular details of the ligand-receptor interaction that includes: (i) the ionic interaction between the protonated amine of the ligand and Asp 3.32; (ii) the hydrogen bonds between the m-NHSO(2)Et group of the ligand and Asn 7.39; and the hydrogen bonds between the hydantoin moiety of the ligand and (iii) Thr 3.37, (iv) Ser 5.42, and (v) Thr 5.43. These QSAR and ANN results in combination with computational simulations of ligand recognition will be useful for the design of potent selective 5-HT(1A) ligands.

Traditional methods for performing 3D-QSAR rely upon an alignment step that is often time-consuming and can introduce user bias, the resultant model being dependent upon and sensitive to the alignment used. There are several methods which overcome this problem, but in general the necessary transformations prevent a simple interpretation of the resultant models in the original descriptor space (i.e. 3D molecular coordinates). Here we present a novel class of molecular descriptors which we have termed GRid-INdependent Descriptors (GRIND). They are derived in such a way as to be highly relevant for describing biological properties of compounds while being alignment-independent, chemically interpretable, and easy to compute. GRIND are obtained starting from a set of molecular interaction fields, computed by the program GRID or by other programs. The procedure for computing the descriptors involves a first step, in which the fields are simplified, and a second step, in which the results are encoded into alignment-independent variables using a particular type of autocorrelation transform. The molecular descriptors so obtained can be used to obtain graphical diagrams called "correlograms" and can be used in different chemometric analyses, such as principal component analysis or partial least-squares. An important feature of GRIND is that, with the use of appropriate software, the original descriptors (molecular interaction fields) can be regenerated from the autocorrelation transform and, thus, the results of the analysis represented graphically, together with the original molecular structures, in 3D plots. In this respect, the article introduces the program ALMOND, a software package developed in our group for the computation, analysis, and interpretation of GRIND. The use of the methodology is illustrated using some examples from the field of 3D-QSAR. Highly predictive and interpretable models are obtained showing the promising potential of the novel descriptors in drug design.

A series of photolabile o-nitrobenzyl derivatives of serotonin (caged serotonin) were synthesized: the amine-linked serotonin derivatives N-(2-nitrobenzyl) serotonin (Bz-5HT) and N-(alpha-carboxy-2-nitrobenzyl) serotonin (N-CNB-5HT), and O-alpha-carboxy-2-nitrobenzyl) serotonin (O-CNB-5HT), which has the caging group attached to the phenolic OH group. All the derivatives released free serotonin when excited by 308-nm or 337-nm laser pulses. The time constant of serotonin release from N-CNB-5HT was 1. 2 ms, with a quantum yield of 0.08. This is too slow for rapid chemical kinetic measurements. O-CNB-5HT is suitable for transient kinetic investigations of the serotonin 5-HT(3) receptor. It released serotonin with a time constant of 16 micros and a quantum yield of 0.03. The biological properties of O-CNB-5HT were evaluated, and the applicability of the compound for kinetic studies of the 5-HT(3) receptor was demonstrated. O-CNB-5HT does not activate the 5-HT(3) receptor by itself, nor does it modulate the response of a cell when co-applied with serotonin. When irradiated with a 337-nm laser pulse, O-CNB-5HT released free serotonin that evoked 5-HT(3) receptor-mediated whole-cell currents in NIE-115 mouse neuroblastoma cells.

Hydroxyl radical-effected protium/deuterium ((1)H/(2)H) exchange into the C-H bonds present in peptides has been used to identify the site of hydrogen atom abstraction by hydroxyl radical. Radiolysis of anaerobic, N(2)O-saturated D(2)O solutions containing peptide and dithiothreitol generates a hydroxyl radical that mediates (1)H/(2)H exchange into the side chains of peptides of up to 66 atom % excess (2)H. The (1)H/(2)H exchange is determined by measuring the isotope ratio, [M + H + 1](+)/[M + H](+), of the peptide using electrospray ionization-mass spectrometry. The (1)H/(2)H exchange within each residue of the peptide was determined by measuring the isotope ratio of each isolated dansyl amino acid following hydrolysis and derivatization. Generation of 0.40 mM hydroxyl radical effected (1)H/(2)H exchange into each of the five different residues of (Ala(2))-leucine enkephalin (YAGFL). The propensity of the residues to undergo exchange was L > Y > A congruent with F > G, independent of whether they were radiolyzed separately or as the peptide. The minimal exchange into glycine suggests that reaction of hydroxyl radical with the side chain hydrogens predominates over reaction with the polypeptide alpha-hydrogens. The ability of radiolysis to effect (1)H/(2)H exchange into a larger peptide, SNEQKACKVLGI, was also demonstrated.

The 5-hydroxytryptamine receptor of type 3 was investigated by fluorescence correlation spectroscopy (FCS). Binding constants of fluorescently labeled ligands, the stoichiometry, and the mass of the receptor are readily accessible by this technique, while the duration of measurement is on the order of seconds to minutes. The receptor antagonist 1,2,3, 9-tetrahydro-3-[(5-methyl-1H-imidazol-4-yl)methyl]-9-(3-aminopropyl)- 4H-carbazol-4-one (GR-H) was labeled with the fluorophores rhodamine 6G, fluorescein, N-[7-nitrobenz-2-oxa-1,3-diazol-4-yl], and the cyanine dye Cy5. These labels cover a large part of the visible electromagnetic spectrum. It is shown that the photophysical and chemical properties have a direct influence on the measurement quality (duration of measurement, signal-to-noise ratio) and the ligand-receptor interactions (dissociation constants), respectively. This makes it necessary to choose a suitable label or a combination of labels for receptor studies. The affinities of the fluorescently labeled ligands determined by FCS were virtually identical to the values obtained by radioligand binding experiments. Moreover, the dissociation constant of a nonfluorescent receptor ligand was determined successfully by an FCS competition assay. The experimental results showed that only one antagonist binds to the receptor, in agreement with measurements previously published [Tairi et al. (1998) Biochemistry 37, 15850-15864].

The screening of ligands for membrane receptor proteins is central to the discovery of new pharmaceutical drugs. We present a general method to reversibly attach receptor proteins via an affinity tag to a quartz surface and subsequently detect with high sensitivity the real-time binding of ligands by total internal reflection fluorescence. A serotonin-gated ion channel protein was immobilized, and the binding of a fluorescent ligand was investigated. The affinity and the kinetic parameters of binding were measured, and the effect of unlabeled compounds was determined by competition. The pharmacology of the immobilized receptor was identical to that of the native receptor. The affinity of unlabeled ligands was rapidly and effectively determined. The method described here is generally applicable for membrane proteins and opens new ways for the discovery of pharmacologically active compounds.

Synthesis and pharmacological evaluation of a series of condensed quinoline and pyridine derivatives bearing a N-methylpiperazine moiety attached to the 2-position of the quinoline or pyridine nucleus are described. 5-HT receptor binding studies revealed subnanomolar affinity for the 5-HT3 receptor subtype in some of the compounds under study. The most active compound (5b) displayed a Ki value about 1 order of magnitude higher than that of quipazine along with a higher selectivity. The potential 5-HT3 agonist/antagonist activity of four selected compounds was assessed in vitro on 5-HT3 receptor-dependent [14C]guanidinium uptake in NG 108-15 cells. Compound 5j acted as a 5-HT3 agonist in this assay with an EC50 value close to that reported for quipazine, while 5b was a partial agonist with an EC50 value of about 0.25 nM, and compound 5c possessed antagonist properties with an IC50 value (approximately 8 nM) in the same range as those of previously characterized 5-HT3 receptor antagonists. Qualitative and quantitative structure-affinity relationship studies carried out by making use of theoretical molecular descriptors allowed to elucidate the role of the main pharmacophoric components and to develop a model for the interaction of the 5-HT3 ligands related to quipazine with their receptor.

A conformational analysis, performed on muscarinic M1 agonists, identified four structural features characteristic of the muscarinic M1 pharmacophore: (i) a protonable basic or quaternary nitrogen acting as a cationic head; (ii) an electronegative dipole usually part of a planar mesomeric ester, amide, or amidine function which can be replaced by an ether (muscarine) or a dioxolane (AF 30); (iii) an intercharge distance of 5 +/- 0.5 A between the cationic head and the electronegative atom of the dipole; (iv) an elevation of 0.5 +/- 0.03 A of the cationic head over the plane containing the electronegative dipole. During a reinvestigation of the conformational behavior of published structures of 5-HT3 antagonists, similar features were observed for the 5-HT3 pharmacophore. However many 5-HT3 antagonists possess additional aromatic planes not present in the muscarinic M1 agonists. These observations brought us to predict the chemical modifications that would change muscarinic M1 agonists into 5-HT3 antagonists. Four of the predicted aminopyridazines were actually synthesized and submitted to testing. The observed IC50 values for 5-HT3 receptor binding ([3H] BRL 43694) ranged from 10 to 425 nM, whereas the affinities for the muscarinic receptor preparations ([3H] pirenzepine) layed over 10,000 nM. In electrophysiological studies the two most active compounds 10 and 13 produced antagonist-like effects on the 5-HT receptor channel complexes responsible for the generation of the rapidly desensitizing ionic currents, and agonist-like effects on those responsible for the slowly desensitizing components.

This paper describes a new 3D flexible searching system 3DFS which supports two types of query definitions: simple atom-based definition and generalized function-based definition. The simple and practical definitions of hydrogen bond acceptors/donors, charge centers, aromatic ring centers, and a rapid hydrophobe recognition algorithm are described in detail. 3DFS adopts a four-step searching strategy: a rapid ID screening, an exact 2D substructure searching using the GMA algorithm, a rigid 3D searching, and a conformationally flexible 3D searching using POWELL method. The utility of 3DFS is illustrated by several typical searching examples.

Explicit molecular binding pockets were constructed and optimized around sets of superimposed ligands using the minireceptor concept. The resulting binding sites incorporate the properties of the different ligands and were shown to be suitable for the design of molecules presenting novel interaction patterns. Two applications of minireceptor construction and/or optimization, followed by molecular design are described. In the pursuit of new ligands mimicking the action of paclitaxel, a minireceptor was constructed using the primary amino acid sequence of the target protein as a guide. The active site extracted from a homology-based model of the serotonin 5-HT1A receptor was optimized around a set of three ligands using the same approach.