Design and synthesis of dual inhibitors of acetylcholinesterase and serotonin transporter targeting potential agents for Alzheimer's disease

Highly efficient acetylcholinesterase (AChE) and serotonin transporter (SERT) dual inhibitors, (S)-4 and (R)-13 were designed and synthesized on the basis of the hypothetical model of AChE active site. Both compounds showed potent inhibitory activities against AChE and SERT. [structure: see text]     

Mass-spectrometric studies of new 6-nitroquipazines—serotonin transporter inhibitors

Six synthesized 6-nitroquipazine derivatives were examined by electron ionization (EI) and electrospray ionization (ESI) mass spectrometry in positive and negative ion mode. The compounds exhibit high affinity for the serotonin transporter (SERT) and belong to a new class of SERT inhibitors. The EI mass spectra registered in negative ion mode showed prominent molecular ions for all the compounds studied. All EI mass spectra and all ESI mass spectra showed similar fragmentation pathways of molecular ions, but the pathways differed between EI and ESI. The differences were explained with the aid of theoretical evaluation of the stability of the respective radical ions (EI MS) and protonated ions (ESI MS).     

Nanopharmacological Force Sensing to Reveal Allosteric Coupling in Transporter Binding Sites

Controversy regarding the number and function of ligand binding sites in neurotransmitter/sodium symporters arose from conflicting data in crystal structures and molecular pharmacology. Here, we have designed novel tools for atomic force microscopy that directly measure the interaction forces between the serotonin transporter (SERT) and the S‐ and R‐enantiomers of citalopram on the single molecule level. This approach is based on force spectroscopy, which allows for the extraction of dynamic information under physiological conditions thus inaccessible via X‐ray crystallography. Two distinct populations of characteristic binding strengths of citalopram to SERT were revealed in Na⁺‐containing buffer. In contrast, in Li⁺‐containing buffer, SERT showed only low force interactions. Conversely, the vestibular mutant SERT‐G402H merely displayed the high force population. These observations provide physical evidence for the existence of two binding sites in SERT when accessed in a physiological context. Competition experiments revealed that these two sites are allosterically coupled and exert reciprocal modulation.     

A comparative QSAR analysis and molecular docking studies of phenyl piperidine derivatives as potent dual NK1R antagonists/serotonin transporter (SERT) inhibitors

Depression is a critical mood disorder that affects millions of patients. Available therapeutic antidepressant agents are associated with several undesirable side effects. Recently, it has been shown that Neurokinin 1 receptor (NK₁R) antagonists can potentiate the antidepressant effects of serotonin-selective reuptake inhibitors (SSRIs). In this study, a series of phenyl piperidine derivatives as potent dual NK₁R antagonists/serotonin transporter (SERT) inhibitors were applied to quantitative structure–activity relationship (QSAR) analysis. A collection of chemometrics methods such as multiple linear regression (MLR), factor analysis–based multiple linear regression (FA-MLR), principal component regression (PCR), and partial least squared combined with genetic algorithm for variable selection (GA-PLS) were applied to make relations between structural characteristics and NK₁R antagonism/SERT inhibitory of these compounds. The best multiple linear regression equation was obtained from GA-PLS and MLR for NK₁R and SERT, respectively. Based on the resulted model, an in silico-screening study was also conducted and new potent lead compounds based on new structural patterns were designed for both targets. Molecular docking studies of these compounds on both targets were also conducted and encouraging results were acquired. There was a good correlation between QSAR and docking results. The results obtained from validated docking studies indicate that the important amino acids inside the active site of the cavity that are responsible for essential interactions are Glu33, Asp395 and Arg26 for SERT and Ala30, Lys7, Asp31, Phe5 and Tyr82 for NK₁R receptors.     

5-羟色胺转运蛋白显像剂的研究进展

中枢神经系统5-羟色胺神经元功能异常,特别是突触前膜的5-羟色胺转运蛋白（SERT）密度的变化常导致复杂的精神紊乱疾病。SERT的正电子发射断层（PET）和单光子发射断层（SPECT）活体显像剂有助于研究该系统的变化与精神紊乱疾病的关系,以及精神紊乱病人疗效的监测。本文综述了近年来SERT显像剂的最新研究进展,并指出了今后该类显像剂的发展趋势。     

In vitro continuous amperometric monitoring of 5-hydroxytryptamine release from enterochromaffin cells of the guinea pig ileum

A diamond microelectrode was used to sensitively, reproducibly and stably record overflow of 5-hydroxytryptamine (5-HT, serotonin) from enterochromaffin cells (EC) of the intenstinal mucosal layer. 5-HT is an important neurotransmitter and paracrine signalling molecule in the gastrointestinal tract. The diamond microelectrode was formed by overcoating a sharpened 76 microm diameter Pt wire with a thin layer of conducting diamond. After insulation with polypropylene, the conically-shaped microelectrode had a diameter of about 10 microm at the tip and 80 microm at the cylindrical portion. The exposed length was 100-200 microm. Continuous amperometry with the microelectrode poised at a detection potential of 700 mV vs. Ag|AgCl was used to measure 5-HT overflow as an oxidation current. 5-HT overflow was elicited by both mechanical and electrical stimulation. Some minor electrode fouling, a common problem with the oxidative detection of 5-HT, was seen for diamond but the response stabilized enabling recording in vitro. Both 5-HT and the paracrine hormone, melatonin, were detected in the extracellular solution. The 5-HT oxidation current increased in the presence of the serotonin transporter (SERT) inhibitor, fluoxetine (1 microM), providing evidence that the oxidation current was associated with 5-HT.     

Synthesis and <Emphasis Type="Italic">in vitro</Emphasis> evaluation of new diphenyl ether derivatives as serotonin transporter ligands

For the development of new ligands as potential imaging agents for the serotonin transporter (SERT), a series of diphenyl ether derivatives have been synthesized, characterized, and evaluated for their in vitro binding affinities to the SERT. Among the above compounds, 2-(2-((dimethylamino)methyl)-4-fluorophenoxy)-5-bromobenzenamine (15) and 2-(2-((dimethylamino)methyl)-4-fluorophenoxy)-5-iodobenzene amine (16) show high binding affinities for the SERT with K _i values of 0.28 and 0.20 nmol·L⁻¹, respectively. They can be further labeled with carbon-11, fluorine-18, iodine-123 or bromine-76, and evaluated as useful imaging agents for the SERT. Moreover, the study of the structure-activity relationship (SAR) provides some useful information for the future design of new ligands. Supported by the National Natural Science Foundation of China (Grant No. 20471011)     

Three-dimensional quantitative structure-activity relationship (3D QSAR) and pharmacophore elucidation of tetrahydropyran derivatives as serotonin and norepinephrine transporter inhibitors

Three-dimensional quantitative structure-activity relationship (3D QSAR) using comparative molecular field analysis (CoMFA) was performed on a series of substituted tetrahydropyran (THP) derivatives possessing serotonin (SERT) and norepinephrine (NET) transporter inhibitory activities. The study aimed to rationalize the potency of these inhibitors for SERT and NET as well as the observed selectivity differences for NET over SERT. The dataset consisted of 29 molecules, of which 23 molecules were used as the training set for deriving CoMFA models for SERT and NET uptake inhibitory activities. Superimpositions were performed using atom-based fitting and 3-point pharmacophore-based alignment. Two charge calculation methods, Gasteiger-Hückel and semiempirical PM3, were tried. Both alignment methods were analyzed in terms of their predictive abilities and produced comparable results with high internal and external predictivities. The models obtained using the 3-point pharmacophore-based alignment outperformed the models with atom-based fitting in terms of relevant statistics and interpretability of the generated contour maps. Steric fields dominated electrostatic fields in terms of contribution. The selectivity analysis (NET over SERT), though yielded models with good internal predictivity, showed very poor external test set predictions. The analysis was repeated with 24 molecules after systematically excluding so-called outliers (5 out of 29) from the model derivation process. The resulting CoMFA model using the atom-based fitting exhibited good statistics and was able to explain most of the selectivity (NET over SERT)-discriminating factors. The presence of −OH substituent on the THP ring was found to be one of the most important factors governing the NET selectivity over SERT. Thus, a 4-point NET-selective pharmacophore, after introducing this newly found H-bond donor/acceptor feature in addition to the initial 3-point pharmacophore, was proposed.     

A fluorescence displacement assay for antidepressant drug discovery based on ligand-conjugated quantum dots

The serotonin (5-hydroxytryptamine, 5-HT) transporter (SERT) protein plays a central role in terminating 5-HT neurotransmission and is the most important therapeutic target for the treatment of major depression and anxiety disorders. We report an innovative, versatile, and target-selective quantum dot (QD) labeling approach for SERT in single Xenopus oocytes that can be adopted as a drug-screening platform. Our labeling approach employs a custom-made, QD-tagged indoleamine derivative ligand, IDT318, that is structurally similar to 5-HT and accesses the primary binding site with enhanced human SERT selectivity. Incubating QD-labeled oocytes with paroxetine (Paxil), a high-affinity SERT-specific inhibitor, showed a concentration- and time-dependent decrease in QD fluorescence, demonstrating the utility of our approach for the identification of SERT modulators. Furthermore, with the development of ligands aimed at other pharmacologically relevant targets, our approach may potentially form the basis for a multitarget drug discovery platform.     

Continuous amperometric detection of co-released serotonin and melatonin from the mucosa in the ileum

Serotonin (5-HT) and melatonin (MEL) are well known neurotransmitters and paracrine signalling molecules. Both compounds are present in enterochromaffin (EC) cells in the mucosa of the gastrointestinal tract and are thought to play a role in controlling gut motility. To date there are no real-time analytical methods for the detection of these two molecules and it is not clear if MEL is actually released from the EC cells. In this paper, I used boron-doped diamond (BDD) microelectrodes to record 5-HT and MEL overflow from EC cells in the mucosa of rabbit ileum. The BDD microelectrode was extremely stable and sensitive for measurements of both compounds when assessed using differential pulse voltammetry (DPV) and flow injection analysis (FIA) using amperometric detection. MEL release was detected in the mucosa, where it is most likely from the EC cells. Mechanical stimulation of individual villi increased 5-HT but not MEL overflow. Application of the serotonin transporter (SERT) inhibitor, fluoxetine, elevated the 5-HT but not the MEL signal. Differences in the amounts of the two gastrointestinal compounds released and the mechanism of which they are released will provide insights to the physiology of the EC cell and disease states.     

QSAR based modeling on a series of lactam fused chroman derivatives as selective 5-HT transporters

One of the most prevalent disorders in the present society is depression. The development of treatments for this disorder, beginning with the tricyclic antidepressants and leading to the development of selective serotonin re-uptake inhibitors, has focused on compounds that block the function of the serotonin transporter (SERT). Quantitative structure–activity relationship has been performed on a series of lactam fused chroman derivatives as selective 5-HT transporters, using different physicochemical parameters along with appropriate indicator variables. It has been found that physicochemical parameters, such as connectivity indices ⁰χ and ²χ, molecular weight (MW), Parachor (Pc), Balaban index (J), Wiener index (W) along with indicator variables are significantly correlated with activity. In this paper best results were obtained by using multiple regression analysis. Different models were generated with high values of R² and low values of PRESS/SSY ratio. The significant equations were statistically tested by using leave one out (LOO) technique and cross-validation methods.     

Targeting cell surface receptors with ligand-conjugated nanocrystals

To explore the potential for use of ligand-conjugated nanocrystals to target cell surface receptors, ion channels, and transporters, we explored the ability of serotonin-labeled CdSe nanocrystals (SNACs) to interact with antidepressant-sensitive, human and Drosophila serotonin transporters (hSERT, dSERT) expressed in HeLa and HEK-293 cells. Unlike unconjugated nanocrystals, SNACs were found to dose-dependently inhibit transport of radiolabeled serotonin by hSERT and dSERT, with an estimated half-maximal activity (EC(50)) of 33 (dSERT) and 99 microM (hSERT). When serotonin was conjugated to the nanocrystal through a linker arm (LSNACs), the EC(50) for hSERT was determined to be 115 microM. Electrophysiology measurements indicated that LSNACs did not elicit currents from the serotonin-3 (5HT(3)) receptor but did produce currents when exposed to the transporter, which are similar to those elicited by antagonists. Moreover, fluorescent LSNACs were found to label SERT-transfected cells but did not label either nontransfected cells or transfected cells coincubated with the high-affinity SERT antagonist paroxetine. These findings support further consideration of ligand-conjugated nanocrystals as versatile probes of membrane proteins in living cells.     

Allosteric Binding of MDMA to the Human Serotonin Transporter (hSERT) via Ensemble Binding Space Analysis with ΔG Calculations,Induced Fit Docking and Monte Carlo Simulations

Despite the recent promising results of MDMA (3,4-methylenedioxy-methamphetamine) as a psychotherapeutic agent and its history of misuse, little is known about its molecular mode of action. MDMA enhances monoaminergic neurotransmission in the brain and its valuable psychoactive effects are associated to a dual action on the 5-HT transporter (SERT). This drug inhibits the reuptake of 5-HT (serotonin) and reverses its flow, acting as a substrate for the SERT, which possesses a central binding site (S1) for antidepressants as well as an allosteric (S2) one. Previously, we characterized the spatial binding requirements for MDMA at S1. Here, we propose a structure-based mechanistic model of MDMA occupation and translocation across both binding sites, applying ensemble binding space analyses, electrostatic complementarity, and Monte Carlo energy perturbation theory. Computed results were correlated with experimental data (r = 0.93 and 0.86 for S1 and S2, respectively). Simulations on all hSERT available structures with Gibbs free energy estimations (ΔG) revealed a favourable and pervasive dual binding mode for MDMA at S2, i.e., adopting either a 5-HT or an escitalopram-like orientation. Intermediate ligand conformations were identified within the allosteric site and between the two sites, outlining an internalization pathway for MDMA. Among the strongest and more frequent interactions were salt bridges with Glu494 and Asp328, a H-bond with Thr497, a π-π with Phe556, and a cation-π with Arg104. Similitudes and differences with the allosteric binding of 5-HT and antidepressants suggest that MDMA may have a distinctive chemotype. Thus, our models may provide a framework for future virtual screening studies and pharmaceutical design and to develop hSERT allosteric compounds with a unique psychoactive MDMA-like profile.     

Binding of serotonin to the human serotonin transporter. Molecular modeling and experimental validation

Molecular modeling and structure-activity relationship studies were performed to propose a model for binding of the neurotransmitter serotonin (5-HT) to the human serotonin transporter (hSERT). Homology models were constructed using the crystal structure of a bacterial homologue, the leucine transporter from Aquifex aeolicus, as the template and three slightly different sequence alignments. Induced fit docking of 5-HT into hSERT homology models resulted in two different binding modes. Both show a salt bridge between Asp98 and the charged primary amine of 5-HT, and both have the 5-HT C6 position of the indole ring pointing toward Ala173. The difference between the two orientations of 5-HT is an enantiofacial discrimination of the indole ring, resulting in the 5-hydroxyl group of 5-HT being vicinal to either Ser438/Thr439 or Ala169/Ile172/Ala173. To assess the binding experimentally, binding affinities for 5-HT and 17 analogues toward wild type and 13 single point mutants of hSERT were measured using an approach termed paired mutant-ligand analogue complementation (PaMLAC). The proposed ligand-protein interaction was systematically examined by disrupting it through site-directed mutagenesis and re-establishing another interaction via a ligand analogue matching the mutated residue, thereby minimizing the risk of identifying indirect effects. The interactions between Asp98 and the primary amine of 5-HT and the interaction between the C6-position of 5-HT and hSERT position 173 was confirmed using PaMLAC. The measured binding affinities of various mutants and 5-HT analogues allowed for a distinction between the two proposed binding modes of 5-HT and biochemically support the model for 5-HT binding in hSERT where the 5-hydroxyl group is in close proximity to Thr439.     

Synthesis of 3,7-disubstituted imipramines by palladium-catalysed amination/cyclisation and evaluation of their inhibition of monoamine transporters

We describe a novel approach for the synthesis of a series of 3,7-difunctionalised symmetric and unsymmetrical analogues of the tricyclic antidepressant (TCA) imipramine, which uses a key palladium-catalysed amination/cyclisation of an ester-functionalised dibromide. Of the ester, methyl, hydroxymethyl and methoxymethyl disubstituted compounds prepared, 3,7-dimethyl-imipramine was found to be the most potent against the human serotonin transporter (hSERT). The inhibitory potency of 3,7-dimethyl imipramine was found to be at least as high as the parent imipramine. This novel TCA also exhibits an increased selectivity (relative to imipramine) in binding to hSERT versus the human norepinephrine transporter (hNET). Even higher selectivity could be obtained with 3,7-dihydroxymethyl imipramine, which was found to be 167-fold more selective for hSERT over hNET, representative of a 120-fold gain in selectivity relative to the parent imipramine. These results further validate our previous model for the binding of imipramine and high-affinity analogues of imipramine to the central binding site of hSERT.     

Synthesis of diphenylmethyl analogues and their affinity for the melanocortin-4 receptor and the serotonin transporter

While examining antagonists of the melanocortin-4 receptor (MC4 receptor), we found that compound 12b, containing a diphenylmethyl moiety, had a relatively high affinity for the MC4 receptor. When diphenylmethyl analogues were further examined, compounds 12c and 18 were also found to exhibit a high affinity for the MC4 receptor (IC(50)=46.7 nM and 33.2 nM, respectively). Furthermore, compound 12c was also found to show a high affinity for the serotonin transporter (IC(50)=10.7 nM). Here, we describe the synthesis and biological evaluation of various diphenylmethyl analogues in relation to their actions on the MC4 receptor and the serotonin transporter.     

Nested summation symbols and perturbation theory

A new mathematical concept, the nested summation symbol (NSS) has been developed. This concept is attached to a mathematical linear operator directly related to the summation symbols. After a discussion on its properties, we investigate the potential usefulness of this symbol in the developing of sequential and parallel computational algorithms, constituting a powerful link between mathematical formalism and high level languages programming. A NSS is well suited in order to express some kind of mathematical formulae and to implement them in any computational environment. In this sense, NSSs are directly related toartificial intelligence techniques. Nested sums are connected withgeneralized nested do loop (GNDL) structures, a programming concept developed in our laboratory. This paper shows an application of the NSS. The NSS concept has been used to obtain in a compact form the expressions of the general energy and wavefunction corrections associated to the perturbation theory under the Brillouin-Wigner or the Rayleigh-Schrödinger formalisms.     

Virtual screening methods that complement HTS

In this review, we discuss a number of computational methods that have been developed or adapted for molecule classification and virtual screening (VS) of compound databases. In particular, we focus on approaches that are complementary to high-throughput screening (HTS). The discussion is limited to VS methods that operate at the small molecular level, which is often called ligand-based VS (LBVS), and does not take into account docking algorithms or other structure-based screening tools. We describe areas that greatly benefit from combining virtual and biological screening and discuss computational methods that are most suitable to contribute to the integration of screening technologies. Relevant approaches range from established methods such as clustering or similarity searching to techniques that have only recently been introduced for LBVS applications such as statistical methods or support vector machines. Finally, we discuss a number of representative applications at the interface between VS and HTS.     

Synthesis of 3-(4-heteroaryl-phenyl)-8-oxabicyclo[3.2.1]octane-2-carboxylic acid methyl esters

Cross coupling protocols were applied for the synthesis of 3-(4-heteroaryl-phenyl)-8-oxabicyclo[3.2.1]oct-2-ene-2-carboxylic acid methyl esters. Stille conditions produced the corresponding products in reasonable yields. Samarium iodide reduction of the resulting coupling products produced the 2β-carbomethoxy-3α-aryl-8-oxabicyclo[3.2.1]octane diastereoisomers as the major, and the 2β-carbomethoxy-3β-aryl-8-oxabicyclo[3.2.1]octane diastereoisomer as the minor products. Both diastereomers manifested inhibition of the dopamine (DAT) and serotonin (SERT) transporters, with some selectivity for SERT inhibition.