3D Pharmacophore,hierarchical methods,and 5-HT4 receptor binding data

5-Hydroxytryptamine subtype-4 (5-HT₄) receptors have stimulated considerable interest amongst scientists and clinicians owing to their importance in neurophysiology and potential as therapeutic targets. A comparative analysis of hierarchical methods applied to data from one thousand 5-HT₄ receptor–ligand binding interactions was carried out. The chemical structures were described as chemical and pharmacophore fingerprints. The definitions of indices, related to the quality of the hierarchies in being able to distinguish between active and inactive compounds, revealed two interesting hierarchies with the Unity (1 active cluster) and pharmacophore fingerprints (4 active clusters). The results of this study also showed the importance of correct choice of metrics as well as the effectiveness of a new alternative of the Ward clustering algorithm named Energy (Minimum E-Distance method). In parallel, the relationship between these classifications and a previously defined 3D 5-HT₄ antagonist pharmacophore was established.     

Fragment library screening reveals remarkable similarities between the G protein-coupled receptor histamine H₄ and the ion channel serotonin 5-HT₃A

A fragment library was screened against the G protein-coupled histamine H(4) receptor (H(4)R) and the ligand-gated ion channel serotonin 5-HT(3A) (5-HT(3A)R). Interestingly, significant overlap was found between H(4)R and 5-HT(3A)R hit sets. The data indicates that dual active H(4)R and 5 HT(3A)R fragments have a higher complexity than the selective compounds which has important implications for chemical genomics approaches. The results of our fragment-based library screening study illustrate similarities in ligand recognition between H(4)R and 5-HT(3A)R and have important consequences for selectivity profiling in ongoing drug discovery efforts on H(4)R and 5-HT(3A)R. The affinity profiles of our fragment screening studies furthermore match the chemical properties of the H(4)R and 5-HT(3A)R binding sites and can be used to define molecular interaction fingerprints to guide the in silico prediction of protein-ligand interactions and structure.     

Synthesis and structure–affinity relationships of novel small molecule natural product derivatives capable of discriminating between serotonin 5-HT1A, 5-HT2A, 5-HT2C receptor subtypes

Efforts to develop ligands that distinguish between clinically relevant 5-HT2A and 5-HT2C serotonin receptor subtypes have been challenging, because their sequences have high homology. Previous studies reported that a novel aplysinopsin belonging to a chemical class of natural products isolated from a marine sponge was selective for the 5-HT2C over the 5-HT2A receptor subtype. Our goal was to explore the 5-HT2A/2C receptor structure–affinity relationships of derivatives based on the aplysinopsin natural product pharmacophore. Twenty aplysinopsin derivatives were synthesized, purified and tested for their affinities for cloned human serotonin 5-HT1A, 5-HT2A, and 5-HT2C receptor subtypes. Four compounds in this series had >30-fold selectivity for 5-HT2A or 5-HT2C receptors. The compound (E)-5-((5,6-dichloro-1H-indol-3-yl)methylene)-2-imino-1,3-dimethylimidazolidin-4-one (UNT-TWU-22, 16) had approximately 2100-fold selectivity for the serotonin 5-HT2C receptor subtype: an affinity for 5-HT2C equal to 46 nM and no detectable affinity for the 5-HT1A or 5-HT2A receptor subtypes. The two most important factors controlling 5-HT2A or 5-HT2C receptor subtype selectivity were the combined R1,R3-alkylation of the imidazolidinone ring and the type and number of halogens on the indole ring of the aplysinopsin pharmacophore.     

Insights into binding modes of 5-HT2c receptor antagonists with ligand-based and receptor-based methods

5-hydroxytryptamine-2c (5-HT2c) receptor antagonists have clinical utility in the management of nervous system. In this work, ligand-based and receptor-based methods were used to investigate the binding mode of h5-HT2c receptor antagonists. First, the pharmacophore modeling of the h5-HT2c receptor antagonists was carried out by CATALYST. Then, the h5-HT2c antagonists were docked to the h5-HT2c receptor model. Subsequently, the comprehensive analysis of the pharmacophore and docking results revealed the structure-activity relationship of 5-HT2c receptor antagonists and the key residues involved in the interactions. For example, three hydrophobic points in the ligands corresponded to the region surrounded by Val135, Val208, Phe214, Ala222, Phe327, Phe328 and Val354 of the h5-HT2c receptor. The carbonyl group of compound 1 formed a hydrogen bond with Asn331. The nitrogen atom in the piperidine of compound 1 corresponding to the positive ionizable position of the best pharmacophore formed the electrostatic interactions with the carbonyl of Asp134, Asn331 and Val354, and with the hydroxyl group of Ser334. In addition, a predictive CoMFA model was developed based on the 24 compounds that were used as the training set in the pharmacophore modeling. Our results were not only useful to explore the detailed mechanism of the interactions between the h5-HT2c receptor and antagonists, but also provided suggestions in the discovery of novel 5-HT2c receptor antagonists.     

Discovery of 2-substituted benzoxazole carboxamides as 5-HT3 receptor antagonists

A new class of 2-substituted benzoxazole carboxamides are presented as potent functional 5-HT(3) receptor antagonists. The chemical series possesses nanomolar in vitro activity against human 5-HT(3)A receptors. A chemistry optimization program was conducted and identified 2-aminobenzoxazoles as orally active 5-HT(3) receptor antagonists with good metabolic stability. These novel analogues possess drug-like characteristics and have potential utility for the treatment of diseases attributable to improper 5-HT(3) receptor function, especially diarrhea predominant irritable bowel syndrome (IBS-D).     

Discovery of 5-HT6 receptor ligands based on virtual HTS

Based on a pharmacophore alignment on a 5-HT(6) ligand applying 4SCan technology, a new lead series was identified and further structurally investigated. K(i)s down to 8 nM were achieved.     

Stepwise development of structure–activity relationship of diverse PARP-1 inhibitors through comparative and validated in silico modeling techniques and molecular dynamics simulation

Inhibitors of poly (ADP-ribose) polymerase-1 (PARP-1) enzyme are useful for the treatment of various diseases including cancer. Comparative in silico studies were performed on different ligand-based (2D-QSAR, Kernel-based partial least square (KPLS) analysis, Pharmacophore Search Engine (PHASE) pharmacophore mapping), and structure-based (molecular docking, MM-GBSA analyses, Gaussian-based 3D-QSAR analyses on docked poses) modeling techniques to explore the structure–activity relationship of a diverse set of PARP-1 inhibitors. Two-dimensional (2D)-QSAR highlighted the importance of charge topological index (JGI7), fractional polar surface area (Jurs_FPSA3), and connectivity index (CIC2) along with different molecular fragments. Favorable and unfavorable fingerprints were demonstrated in KPLS analysis, whereas important pharmacophore features (one acceptor, one donor, and two ring aromatic) along with favorable and unfavorable field effects were demonstrated in PHASE-based pharmacophore model. MM-GBSA analyses revealed significance of different polar, non-polar, and solvation energies. Docking-based alignment of ligands was used to perform Gaussian-based 3D-QSAR study that further demonstrated importance of different field effects. Overall, it was found that polar interactions (hydrogen bonding, bridged hydrogen bonding, and pi–cation) play major roles for higher activity. Steric groups increase the total contact surface area but it should have higher fractional polar surface area to adjust solvation energy. Structure-based pharmacophore mapping spotted the positive ionizable feature of ligands as the most important feature for discriminating highly active compounds from inactives. Molecular dynamics simulation, conducted on highly active ligands, described the dynamic behaviors of the protein complexes and supported the interpretations obtained from other modeling analyses. The current study may be useful for designing PARP-1 inhibitors.     

1-Aryl-4-(4-succinimidobutyl)piperazines and their conformationally constrained analogues: synthesis, binding to serotonin (5-HT1A, 5-HT2A, 5-HT7), alpha1-adrenergic, and dopaminergic D2 receptors, and in vivo 5-HT1A functional characteristics

Starting with the structure of potent 5-HT(1A) ligands, that is, MM77 [1-(2-methoxyphenyl)-4-(4-succinimidobutyl)piperazine, 4] and its constrained version 5 (MP349), previously obtained in our laboratory, a series of their direct analogues with differently substituted aromatic ring (R=H, m-Cl, m-CF(3), m-OCH(3), p-OCH(3)) were synthesized. The flexible and the corresponding 1e,4e-disubstituted cyclohexane derivatives were designed in order to investigate the influence of rigidification on 5-HT(1A) affinity, selectivity for 5-HT(2A), 5-HT(7), D(1), and D(2) binding sites and functional profile at pre- and postsynaptic 5-HT(1A) receptors. The new compounds 19-25 were found to be highly active 5-HT(1A) receptor ligands (K(i)=4-44 nM) whereas their affinity for other receptors was: either significantly decreased after rigidification (5-HT(7)), or controlled by substituents in the aromatic ring (alpha(1)), or influenced by both those structural modifications (5-HT(2A)), or very low (D(2), K(i)=5.3-31 microM). Since a distinct disfavor towards rigid compounds was observed for 5-HT(7) receptors only, it seems that the bioactive conformation of chain derivatives at those sites should differ from the extended one. Several in vivo models were used to asses functional activity of 19-25 at pre- (hypothermia in mice) and postsynaptic 5-HT(1A) receptors (lower lip retraction in rats and serotonin syndrome in reserpinized rats). Unlike the parent antagonists 4 and 5, all the new derivatives tested were classified as partial agonists with different potency, however, similar effects were observed within pairs (flexible and rigid) of the analogues. The obtained results indicated that substitution in the aromatic ring, but not spacer rigidification, controls the 5-HT(1A) functional activity of the investigated compounds. Moreover, an o-methoxy substituent in the structure of 5 seems to be necessary for its full antagonistic properties. Of all the new compounds studied, trans-4-(4-succinimidocyclohexyl)-1-(3-trifluoromethylphenyl)piperazine 24 was the most potent 5-HT(1A) receptor ligand in vitro (K(i)=4 nM) and in vivo, with at least 100-fold selectivity for the other receptors tested.     

Discovery of potential pancreatic cholesterol esterase inhibitors using pharmacophore modelling, virtual screening, and optimization studies

Pancreatic cholesterol esterase (CEase) is a serine hydrolase involved in the hydrolysis of variety of lipids and transport of free cholesterol. In this study, pharmacophore hypotheses based on known inhibitors were generated using common feature pharmacophore generation protocol available in Discovery Studio program. The best pharmacophore model containing two hydrogen bond acceptor and three hydrophobic features was selected and validated. It was further used in screening three diverse chemical databases. Hit compounds were subjected to drug-likeness and molecular docking studies. Four hits, namely SEW00846, NCI0040784, GK03167, and CD10645, were selected based on the GOLD fitness score and interaction with active site amino acids. All hit compounds were further optimized to improve their binding in the active site. The optimized compounds were found to have improved binding at the active site. Strongly binding optimized hits at the active site can act as virtual leads in potent CEase inhibitor designing.     

Nature of pharmacophore influences active site specificity of proteasome inhibitors

Proteasomes degrade most proteins in mammalian cells and are established targets of anti-cancer drugs. The majority of proteasome inhibitors are composed of short peptides with an electrophilic functionality (pharmacophore) at the C terminus. All eukaryotic proteasomes have three types of active sites as follows: chymotrypsin-like, trypsin-like, and caspase-like. It is widely believed that active site specificity of inhibitors is determined primarily by the peptide sequence and not the pharmacophore. Here, we report that active site specificity of inhibitors can also be tuned by the chemical nature of the pharmacophore. Specifically, replacement of the epoxyketone by vinyl sulfone moieties further improves the selectivity of β5-specific inhibitors NC-005, YU-101, and PR-171 (carfilzomib). This increase in specificity is likely the basis of the decreased cytotoxicity of vinyl sulfone-based inhibitors to HeLa cells as compared with that of epoxyketone-based inhibitors.     

Pyridazinone derivatives: design, synthesis, and in vitro vasorelaxant activity

In an attempt to identify potential vasodilator-cardiotonic lead compounds, three series of pyridazinones were designed using three-dimensional pharmacophore developed with CATALYST software from a set of potent cyclic nucleotide phosphodiesterase III, cAMP PDEIII inhibitors. The features of the target compounds were based on the structures of many biologically active lead compounds with cAMP phosphodiesterase III inhibiting activity such as Milrinone and others. Compounds with higher fit scores to the developed pharmacophore were synthesized namely; 6-(3-ethoxycarbonyl-4-oxo-1,4-dihydroquinolin-6-yl)-4,5-dihydro-3(2H)-pyridazinones (3a and 3b), 6-[4-(2,6-disubstituted-quinolin-4-ylamino)phenyl]-4,5-dihydropyridazin-3(2H)-ones (5a-f), and 6-[3-(5-cyano-6-oxo-4-aryl-1,6-dihydro-2-pyridyl)phenylamino]-3(2H)pyridazinone (8a and 8b). The vasodilator activity of the newly synthesized compounds was examined on the isolated main pulmonary artery of the rabbit. Some of the tested compounds showed moderate vasorelaxant activity compared with standard drug, Milrinone.     

Capturing Nature's Diversity

Natural products are universally recognized to contribute valuable chemical diversity to the design of molecular screening libraries. The analysis undertaken in this work, provides a foundation for the generation of fragment screening libraries that capture the diverse range of molecular recognition building blocks embedded within natural products. Physicochemical properties were used to select fragment-sized natural products from a database of known natural products (Dictionary of Natural Products). PCA analysis was used to illustrate the positioning of the fragment subset within the property space of the non-fragment sized natural products in the dataset. Structural diversity was analysed by three distinct methods: atom function analysis, using pharmacophore fingerprints, atom type analysis, using radial fingerprints, and scaffold analysis. Small pharmacophore triplets, representing the range of chemical features present in natural products that are capable of engaging in molecular interactions with small, contiguous areas of protein binding surfaces, were analysed. We demonstrate that fragment-sized natural products capture more than half of the small pharmacophore triplet diversity observed in non fragment-sized natural product datasets. Atom type analysis using radial fingerprints was represented by a self-organizing map. We examined the structural diversity of non-flat fragment-sized natural product scaffolds, rich in sp3 configured centres. From these results we demonstrate that 2-ring fragment-sized natural products effectively balance the opposing characteristics of minimal complexity and broad structural diversity when compared to the larger, more complex fragment-like natural products. These naturally-derived fragments could be used as the starting point for the generation of a highly diverse library with the scope for further medicinal chemistry elaboration due to their minimal structural complexity. This study highlights the possibility to capture a high proportion of the individual molecular interaction motifs embedded within natural products using a fragment screening library spanning 422 structural clusters and comprised of approximately 2800 natural products.     

Ergoline Derivatives as a Probe for Featuring the 5-HT1A Receptor Pharmacophore

A 5-HT_1A pharmacophore has been obtained employing a set of rigid templates encompassing the 5-HT1A structure. The use of rigid templates allowed us to overcome the discrepancy found when flexible structures where the energy of the active conformers are sometimes higher than the global minimum energy are used. On the basis of the results herein reported the three-dimensional requirements necessary for the binding interaction have been defined within this set of molecules. In this study forbidden zones of the receptor have been characterised. The pharmacophore model derived places some agonist/antagonist pharmacophore models appeared in the literature.     

Structure of the human serotonin 5-HT4 receptor gene and cloning of a novel 5-HT4 splice variant

Several variants of the serotonin 5-HT4 receptor are known to be produced by alternative splicing. To survey the existence and usage of exons in humans, we cloned the human 5-HT4 gene. Based on sequence analysis seven C-terminal variants (a-g) and one internal splice variant (h) were found. We concentrated in this study on the functional characterization of the novel splice variant h, which leads to the insertion of 14 amino acids into the second extracellular loop of the receptor. The h variant was cloned as a splice combination with the C-terminal b variant; therefore, we call this receptor 5-HT4(hb). This novel receptor variant was expressed transiently in COS-7 cells, and its pharmacological profile was compared with those of the previously cloned 5-HT4(a) and 5-HT4(b) isoforms, with the latter being the primary reference for the h variant. In competition binding experiments using reference 5-HT4 ligands, no significant differences were detected. However, the broadly used 5-HT4 antagonist GR113808 discriminated functionally among the receptor variants investigated. As expected, it was an antagonist on the 5-HT4(a) and 5-HT4(b) variant but showed partial agonistic activity on the 5-HT4(hb) variant. These data emphasize the importance of variations introduced by splicing for receptor pharmacology and may help in the understanding of conflicting results seen with 5-HT4 ligands in different model systems.     

Plasma serotonin monitoring by blood microdialysis coupled to high-performance liquid chromatography with electrochemical detection in humans

Plasma serotonin (5-HT) active pool was monitored in male volunteers by intravenous microdialysis coupled to HPLC–EC with 98.6% efficient probes. 5-HT was monitored from 60 min before to 360 min after an oral dose of fluoxetine, a 5-HT uptake inhibitor, or vehicle. The basal values were within nanomolar range (0.55 to 4.6 ng/ml). After administration of fluoxetine, there was a significant increment of 5-HT with respect to controls. These results showed that intravenous microdialysis is an alternative efficient technique to monitor endogenous unbound 5-HT changes in plasma without extracting blood or sample pretreatment procedures before the chemical analysis.     

Identification of novel CDK2 inhibitors by a multistage virtual screening method based on SVM,pharmacophore and docking model

Abstract

Cyclin-dependent kinase 2 (CDK2) is the family of Ser/Thr protein kinases that has emerged as a highly selective with low toxic cancer therapy target. A multistage virtual screening method combined by SVM, protein-ligand interaction fingerprints (PLIF) pharmacophore and docking was utilised for screening the CDK2 inhibitors. The evaluation of the validation set indicated that this method can be used to screen large chemical databases because it has a high hit-rate and enrichment factor (80.1% and 332.83 respectively). Six compounds were screened out from NCI, Enamine and Pubchem database. After molecular dynamics and binding free energy calculation, two compounds had great potential as novel CDK2 inhibitors and they also showed selective inhibition against CDK2 in the kinase activity assay.     

CHEMICAL, ENZYMATIC AND ULTRASTRUCTURAL CHARACTERIZATION OF 5-HYDROXYTRYPTAMINE-CONTAINING NEURONS FROM THE GANGLIA OF APLYSIA CALIFORNICA AND TRITONIA DIOMEDIA

Abstract— Several identified neurons in Aplysia and Tritonia ganglia were shown to contain measurable quantities (4–6 pmol/cell body) of 5-hydroxytryptamine (5-HT). A metabolic correlate for the limited distribution of 5-HT among the neurons of Tritonia is provided by the finding that the enzyme, aromatic acid decarboxylase (AAD), is 500 times more active in nerve cells containing 5-HT than in neurons devoid of the amine. Although all Aplysia neurons have some AAD activity, 5-HT cell bodies in this species are 10-fold more active than cell bodies which do not contain 5-HT. The cytoplasm of 5-HT cell bodies in Aplysia and Tritonia characteristically contains granules that have minimum diameters of approx. 1000 Å and eccentric opaque cores. This type of granule was not found in somata which did not contain measurable 5-HT. These data illustrate the metabolic and morphological specialization in 5-HT-containing neurons of molluscs.     

SAR development of a selective 5-HT1D antagonist/serotonin reuptake inhibitor lead using rapid parallel synthesis

Incorporation of an SRI (serotonin reuptake inhibitor) pharmacophore into a selective 5-HT(1D) agonist has led to the discovery of a molecule having both 5-HT(1D) antagonist and SRI activity. RPS methodology was used to develop the SAR and identify potential approaches to reduce unwanted adrenergic alpha 1 and dopamine D(2) cross-reactivities.     

Virtual screening-driven identification of human carbonic anhydrase inhibitors incorporating an original, new pharmacophore

Combinated ligand- and pharmacophore-based virtual screening approaches were used to discover novel potential pharmacophores acting as carbonic anhydrase (CA, EC 4.2.1.1) inhibitors (CAIs). A free database of commercially available compounds was screened through drug-like filters using a four-point pharmacophore, and followed by docking calculation within the active site of an X-ray structure of isoform CA II. One compound, bearing a trifluoro-dihydroxy-propanone moiety, showed an interesting, selective inhibitory activity in low micromolar range against this isoform versus CA I. The chemical originality of this new pharmacophore can represent an important bioisosteric alternative to the sulfonamido-based functionalities, thus leading to the development of a new class of CAIs.     

Molecular modeling of the dopamine D2 and serotonin 5-HT1A receptor binding modes of the enantiomers of 5-OMe-BPAT.

Molecular modeling studies were undertaken in order to elucidate the possible dopamine D2 and serotonin 5-HT1A receptor binding modes of the enantiomers of 5-methoxy-2-[N-(2-benzamidoethyl)-N-n-propylamino]tetralin (5-OMe-BPAT, 1). For this purpose, a combination of indirect molecular modeling and direct construction of the seven transmembrane (7TM) domains of the receptors was employed in a stepwise, objective manner. Pharmacophore models and corresponding receptor maps were identified by superimposing selected sets of receptor agonists in their presumed pharmacologically active conformations, while taking the conformational freedom of the ligands into account. The 7TM models were then constructed around the agonist pharmacophore models, by adding the TM domains one-by-one. Initially, the relative positions of TM3, TM4, and TM5 were determined using the three-dimensional structure of bacteriorhodopsin, but subsequently the orientations of all TM domains were adjusted in order to mimic the topology of the TM domains of rhodopsin. The presumed dopamine D2 receptor binding conformations of (S)- and (R)-1 were determined by using the semirigid dopamine D2 receptor antagonist N-benzylpiquindone as a template for superposition. Similarly, the selective serotonin 5-HT1A receptor agonist flesinoxan was employed for identifying the serotonin 5-HT1A receptor binding conformations of the enantiomers of 1. After docking of the presumed pharmacologically active conformations in the 7TM models and subsequent optimization of the binding sites, specific interactions between the ligands and the surrounding amino acid residues, consistent with the structure-activity relationships, were observed. Thus, both enantiomers of 1 bound to the dopamine D2 receptor model in a similar fashion: a reinforced electrostatic interaction was present between the protonated nitrogen atoms and Asp114 in TM3; their carbonyl groups accepted a H-bond from Ser121 in TM3; their amide NH groups acted as H-bond donor to Tyr416 in TM7; and their benzamide phenyl rings were involved in a hydrophobic edge-to-face interaction with Trp386 in TM6. Differences were observed in the orientations of the 2-aminotetralin moieties, which occupied the agonist binding site. Whereas the (S)-enantiomer could form a H-bond between its 5-methoxy substituent and Ser193 in TM5, the (R)-enantiomer could not, which may account for the differences in their intrinsic efficacies at the dopamine D2 receptor. In the serotonin 5-HT1A receptor model, the benzamide phenyl rings of both enantiomers were involved in hydrophobic face-to-face interactions with Phe112 in TM3, while their protonated nitrogen atoms formed a reinforced electrostatic interaction with Asp116 in TM3. Consistent with the structure-affinity relationships of 1, the amide moieties were not involved in specific interactions. Both enantiomers of 1 could form a hydrogen bond between their 5-methoxy substituent and Thr200 in TM5, which may account for their full serotonin 5-HT1A receptor agonist properties.