A study on application of impregnated synthetic peptide TLC stationary phases for the screening of 5-HT1A ligands. Part 2

Six extended analogues of the recently described peptides (LDVL, ADVL) were designed and synthesized on a solid support, and then impregnated on TLC stationary phases. The impact of the impregnated peptide sequence modifications on the chromatographic retard, Delta R(f) (difference in the migration of tested compound on control and impregnated plates), of 42 arylpiperazine 5-HT(1A) receptor ligands was studied. None of the new models tested made a better prediction of 5-HT(1A) affinity than that utilizing ADVL tetrapeptide. Further validation of the ADVL model on a set of 22 structurally differentiated 2-methoxy-phenylpiperazine derivatives confirmed its effectiveness in the affinity discrimination of a coherent group of 5-HT(1A) receptor ligands.     

A new class of arylpiperazine derivatives: the library synthesis on SynPhase lanterns and biological evaluation on serotonin 5-HT(1A) and 5-HT(2A) receptors

An efficient solid-supported method for the synthesis of a new class of arylpiperazine derivatives containing amino acid residues has been developed. A 72-membered library was synthesized on SynPhase Lanterns functionalized by a BAL linker. A one-pot cleavage/cyclization step of aspartic and glutamic acid derivatives yielded succinimide- and pyroglutamyl-containing ligands (chemsets 9 and 10). The library representatives under study showed different levels of affinity for 5-HT(1A) and 5-HT(2A) receptors (estimated K(i) = 24-4000 and 1-2130 nM, respectively). Several dual 5-HT(1A)/5-HT(2A) ligands were found, of which two (9(3,3) and 9(3,5)) displayed high 5-HT(2A) affinity comparable to that of the reference drug ritanserin. A set of individual fragment contributions for the prediction of 5-HT(1A) and 5-HT(2A) affinity of all the library members were defined on the basis of the Free-Wilson analysis of 26 compounds. An alkylarylpiperazine fragment had essentially the same impact on the affinity for both receptors, whereas different terminal amide fragments were preferred by 5-HT(1A) (chemset 17, R(2) = adamantyl) and 5-HT(2A) (chemset 9, R(2) = norborn-2-ylmethyl) binding sites.     

G-protein coupled receptor assays: to measure affinity or efficacy that is the question

Cell-based assays have always played an important role in the pharmaceutical industry, providing information about the functional effects of compounds. These functional assays have traditionally accompanied facile biochemical high throughput screening programmes, being applied as secondary assays in the later stages of lead development. However, with the disappointing reality that there is not likely to be a plethora of novel, druggable targets in the post-genomic era, the role of cell-based assays in drug discovery is beginning to change. Competition to develop the "best" agents for well established targets and find more effective ways of identifying "novel" agents is driving the industry towards a "quality" versus "quantity" approach. Advances in genetic engineering, automation compatible functional assay technologies and the introduction of more sophisticated robotic systems, have facilitated the application of cell-based assays to primary screening. However, despite some apparent success to move these assays into the routine "toolbox" for high throughput screening, certain preconceptions and concerns about cell-based assays persist and the subject remains a topic of much debate. Here we use examples from the screening portfolio at Pfizer, Sandwich, to discuss the practical and theoretical considerations of employing cell-based assays in HTS with a focus on G-protein coupled receptors.     

Synthesis and structure-activity relationships of 4-amino-5-chloro-N-(1,4-dialkylhexahydro-1,4-diazepin-6-yl)-2-methoxybenzamide derivatives,novel and potent serotonin 5-HT3 and dopamine D2 receptors dual antagonist

In search of a dopamine D2 and serotonin 5-HT3 receptors dual antagonist as a potential broad antiemetic agent, a number of benzamides were prepared from 4-amino-5-chloro-2-methoxybenzoic acid derivatives and 6-amino-1,4-dialkylhexahydro-1,4-diazepines and evaluated for their binding affinity for the dopamine D2 and the serotonin 5-HT3 receptors using rat brain synaptic and rat cortical membranes, respectively. From the results of both in vitro receptor binding and in vivo biological assays for the dopamine D2 receptor, 1-ethyl-4-methylhexahydro-1,4-diazepine ring was selected as an optimum amine moiety. Introduction of one methyl group on the nitrogen atom at the 4-position and/or modification of the substituent at the 5-position of the 4-amino-5-chloro-2-methoxybenzoyl moiety caused a marked increase in the dopamine D2 receptor binding affinity along with a potent 5-HT3 receptor binding affinity. Among the compounds, 5-chloro-N-(1-ethyl-4-methylhexahydro-1,4-diazepin-6-yl)-2-methoxy-4-methylaminobenzamide (82), 5-bromo (110), and 5-iodo (112) analogues exhibited a much higher affinity for the dopamine D2 receptor than that of metoclopramide (IC50=17.5-61.0 nM vs. 483 nM). In particular, 82 showed a potent antagonistic activity for both receptors in vivo tests. Optical resolution of the racemate 82 brought about a dramatic change in the pharmacological profile with the (R)-enantiomer exhibiting a strong affinity for both the dopamine D2 and the 5-HT3 receptors, while the corresponding (S)-enantiomer had a potent and selective serotonin 5-HT3 receptor binding affinity.     

Quantitative structure-affinity relationship of 5-HT1A receptor ligands by the classification tree method

The influence of molecular structure of 346 ligands on their affinity for 5-HT1A receptors was investigated. It was shown that the effectiveness of the proposed novel approach for interpretation of decision tree models compared favourably with the PLS method. In the context of the proposed approach, molecular fragments and their values of the relative influence on the affinity for 5-HT1A receptors were defined.     

A first step towards the understanding of the 5-HT(3) receptor subunit heterogeneity from a computational point of view

The functional serotonin type-3 receptor (5-HT(3)-R), which is the target of many neuroactive drugs, is known to be a homopentamer made of five identical subunits A (5-HT(3A)-R) or a binary heteropentamer made of subunits A and B (5-HT(3A/B)-R) with a still debated arrangement and stoichiometry. This complex picture has been recently further complicated by the discovery of additional 5-HT(3)-R subunits, C, D, and E, which, similarly to the B subunit, are apparently able to form functional receptors only if co-expressed with subunit A. Being the binding site for both serotonin and antagonists (i.e. drugs) located at the extracellular interface between two adjacent subunits, the large variability of the 5-HT(3)-R composition becomes a crucial issue, since it can originate many different interfaces providing non-equivalent ligand binding sites and complicating the pharmacological modulation. Here, the different 5-HT(3)-R interfaces are analysed, on the bases of the structural conformations of previously built 3D homology models and of the known subunit sequences, by addressing their physicochemical characterization. The results confirm the presence of an aromatic cluster located in the core of the A-A interface as a key determinant for having an interface both stable and functional. This is used as a discriminant to make hypotheses about the capability of all the other possible interfaces constituted by the known 5-HT(3)-R sequences A, B, C, D, and E to build active receptors.     

A miniaturized glucocorticoid receptor translocation assay using enzymatic fragment complementation evaluated with qHTS

Nuclear translocation is an important step in glucocorticoid receptor (GR) signaling and assays that measure this process allow the identification of nuclear receptor ligands independent of subsequent functional effects. To facilitate the identification of GR-translocation agonists, an enzyme fragment complementation (EFC) cell-based assay was scaled to a 1536-well plate format to evaluate 9,920 compounds using a quantitative high throughput screening (qHTS) strategy where compounds are assayed at multiple concentrations. In contrast to conventional assays of nuclear translocation the qHTS assay described here was enabled on a standard luminescence microplate reader precluding the requirement for imaging methods. The assay uses beta-galactosidase alpha complementation to indirectly detect GR-translocation in CHO-K1 cells. 1536-well assay miniaturization included the elimination of a media aspiration step, and the optimized assay displayed a Z' of 0.55. qHTS yielded EC(50) values for all 9,920 compounds and allowed us to retrospectively examine the dataset as a single concentration-based screen to estimate the number of false positives and negatives at typical activity thresholds. For example, at a 9 microM screening concentration, the assay showed an accuracy that is comparable to typical cell-based assays as judged by the occurrence of false positives that we determined to be 1.3% or 0.3%, for a 3sigma or 6sigma threshold, respectively. This corresponds to a confirmation rate of approximately 30% or approximately 50%, respectively. The assay was consistent with glucocorticoid pharmacology as scaffolds with close similarity to dexamethasone were identified as active, while, for example, steroids that act as ligands to other nuclear receptors such as the estrogen receptor were found to be inactive.     

High throughput screening (HTS) in identification new ligands and drugable targets of G protein-coupled receptors (GPCRs)

G protein-coupled receptors (GPCRs) which constitute one of the largest and most versatile families of cell surface receptors are involved in a wide spectrum of physiological functions, such as, neuronal transmission, chemotaxis, pacemaker activity and embryonic development. Therefore, in the past a few years GPCR families have become very important targets in pharmaceutical design. However, according to the human genome project, there are approximately 1000 genes encoding GPCRs, only about 200 of GPCRs have known ligands and functions. Searching for ligands of the unknown GPCRs and better modulators of known GPCRs are currently attracting lots of interest. High throughput screening (HTS), which is commonly defined as an automatic process of testing potential drug candidates efficiently, is widely used in drug discovery. In this review, the use of high throughput screening (HTS) in studying GPCRs and the choice of screening technology in different G-protein signaling pathways were summarized.     

A ligand-based approach to mining the chemogenomic space of drugs

The practical implementation and validation of a ligand-based approach to mining the chemogenomic space of drugs is presented and applied to the in silico target profiling of 767 drugs against 684 targets of therapeutic relevance. The results reveal that drugs targeting aminergic G protein-coupled receptors (GPCRs) show the most promiscuous pharmacological profiles. The detection of cross-pharmacologies between aminergic GPCRs and the opioid, sigma, NMDA, and 5-HT3 receptors aggravate the potential promiscuity of those drugs, predominantly including analgesics, antidepressants, and antipsychotics.     

Opioid ligands with mixed properties from substituted enantiomeric N-phenethyl-5-phenylmorphans. Synthesis of a micro-agonist delta-antagonist and delta-inverse agonists

Enantiomeric N-phenethyl-m-hydroxyphenylmorphans with various substituents in the ortho, meta or para positions of the aromatic ring in the phenethylamine side-chain (chloro, hydroxy, methoxy, nitro, methyl), as well as a pyridylethyl and a indolylethyl moiety on the nitrogen atom, were synthesized and their binding affinity to the mu-, delta-, and kappa-opioid receptors was examined. The higher affinity ligands were further examined in the [(35)S]GTPgammaS assay to study their function and efficacy. 3-((1R,5S)-(-)-2-(4-Nitrophenethyl)-2-aza-bicyclo[3.3.1]nonan-5-yl)phenol ((-)-) was found to be a mu-agonist and delta-antagonist in that functional assay and was about 50 fold more potent than morphine in vivo. 3-((1R,5S)-(-)-2-(4-Chlorophenethyl)-2-aza-bicyclo[3.3.1]nonan-5-yl)phenol ((-)-) and several other ligands displayed inverse agonist activity at the delta-opioid receptor. The absolute configuration of all of the reported compounds was established by chemical conversion of (-)- to 1R,5S-(-)-.HBr.     

Measuring intracellular calcium fluxes in high throughput mode

The measurement of intracellular calcium fluxes in real time is widely applied within the pharmaceutical industry to measure the activation of G-protein coupled receptors (GPCRhyp;s), either for pharmacological characterisation or to screen for new surrogate ligands. Initially restricted to G(q) coupled GPCRs, the introduction of promiscuous and chimeric G-proteins has further widened the application of these assays. The development of new calcium sensitive dyes and assays has provided sensitive, homogeneous assays which can be readily applied to high throughput screening (HTS). In this paper we describe the full automation of this assay type using a fluorometric imaging plate reader (FLIPR ) integrated into a Beckman/Sagian system to establish a simple robotic system that is well suited for the current medium throughput screening in this area of lead discovery. Using a recently completed HTS we discuss important determinants for FLIPR based screening, highlight some limitations of the current approach, and look at the requirements for future automated systems capable of keeping up with expanding compound files.     

NIR-mbc94, a fluorescent ligand that binds to endogenous CB(2) receptors and is amenable to high-throughput screening

High-throughput screening (HTS) of chemical libraries is often used for the unbiased identification of compounds interacting with G protein-coupled receptors (GPCRs), the largest family of therapeutic targets. However, current HTS methods require removing GPCRs from their native environment, which modifies their pharmacodynamic properties and biases the screen toward false positive hits. Here, we developed and validated a molecular imaging (MI) agent, NIR-mbc94, which emits near infrared (NIR) light and selectively binds to endogenously expressed cannabinoid CB(2) receptors,?a recognized target for treating autoimmune diseases, chronic pain and cancer. The precision and ease of this assay allows for the HTS of compounds interacting with CB(2) receptors expressed in their native environment.     

Subunit rotation models activation of serotonin 5-HT<Subscript>3AB</Subscript> receptors by agonists

The N-terminal extracellular regions of heterooligomeric 3AB-type human 5-hydroxytryptamine receptors (5-HT3ABR) were modelled based on the crystal structure of snail acetylcholine binding protein AChBP. Stepwise rotation of subunit A by 5 degrees was performed between -10 degrees and 15 degrees to mimic agonist binding and receptor activation. Anticlockwise rotation reduced the size of the binding cavity in interface AB and reorganised the network of hydrogen bonds along the interface. AB subunit dimers with different rotations were applied for docking of ligands with different efficacies: 5-HT, m-chlorophenylbiguanide, SR 57227, quinolinyl piperazine and lerisetron derivatives. All ligands were docked into the dimer with -10 degrees rotation representing ligand-free, open binding cavities similarly, without pharmacological discrimination. Their ammonium ions were in hydrogen bonding distance to the backbone carbonyl of W183. Anticlockwise rotation and contraction of the binding cavity led to distinctive docking interactions of agonists with E129 and cation-pi interactions of their ammonium ions. Side chains of several further amino acids participating in docking (Y143, Y153, Y234 and E236) are in agreement with the effects of point mutations in the binding loops. Our model postulates that 5-HT binds to W183 in a hydrophobic cleft as well as to E236 in a hydrophilic vestibule. Then it elicits anticlockwise rotation to draw in loop C via pi-cation-pi interactions of its ammonium ion with W183 and Y234. Finally, closure of the binding cavity might end in rebinding of 5-HT to E129 in the hydrophilic vestibule.     

Predicted structures and dynamics for agonists and antagonists bound to serotonin 5-HT2B and 5-HT2C receptors

Subtype 2 serotonin (5-hydroxytryptamine, 5-HT) receptors are major drug targets for schizophrenia, feeding disorders, perception, depression, migraines, hypertension, anxiety, hallucinogens, and gastrointestinal dysfunctions. (1) We report here the predicted structure of 5-HT2B and 5-HT2C receptors bound to highly potent and selective 5-HT2B antagonist PRX-08066 3, (pKi: 30 nM), including the key binding residues [V103 (2.53), L132 (3.29), V190 (4.60), and L347 (6.58)] determining the selectivity of binding to 5-HT2B over 5-HT2A. We also report structures of the endogenous agonist (5-HT) and a HT2B selective antagonist 2 (1-methyl-1-1,6,7,8-tetrahydro-pyrrolo[2,3-g]quinoline-5-carboxylic acid pyridine-3-ylamide). We examine the dynamics for the agonist- and antagonist-bound HT2B receptors in explicit membrane and water finding dramatically different patterns of water migration into the NPxxY motif and the binding site that correlates with the stability of ionic locks in the D(E)RY region.     

N-Skatyltryptamines—Dual 5-HT6R/D2R Ligands with Antipsychotic and Procognitive Potential

A series of N-skatyltryptamines was synthesized and their affinities for serotonin and dopamine receptors were determined. Compounds exhibited activity toward 5-HT_1A, 5-HT_2A, 5-HT₆, and D₂ receptors. Substitution patterns resulting in affinity/activity switches were identified and studied using homology modeling. Chosen hits were screened to determine their metabolism, permeability, hepatotoxicity, and CYP inhibition. Several D₂ receptor antagonists with additional 5-HT₆R antagonist and agonist properties were identified. The former combination resembled known antipsychotic agents, while the latter was particularly interesting due to the fact that it has not been studied before. Selective 5-HT₆R antagonists have been shown previously to produce procognitive and promnesic effects in several rodent models. Administration of 5-HT₆R agonists was more ambiguous—in naive animals, it did not alter memory or produce slight amnesic effects, while in rodent models of memory impairment, they ameliorated the condition just like antagonists. Using the identified hit compounds 15 and 18, we tried to sort out the difference between ligands exhibiting the D₂R antagonist function combined with 5-HT₆R agonism, and mixed D₂/5-HT₆R antagonists in murine models of psychosis.     

N-[1,3-Dialkyl(aryl)-2-oxoimidazolidin-4-ylidene]-aryl(alkyl)sulphonamides as Novel Selective Human Cannabinoid Type 2 Receptor (hCB2R) Ligands; Insights into the Mechanism of Receptor Activation/Deactivation

Cannabinoid type 1 (hCB1) and type 2 (hCB2) receptors are pleiotropic and crucial targets whose signaling contributes to physiological homeostasis and its restoration after injury. Being predominantly expressed in peripheral tissues, hCB2R represents a safer therapeutic target than hCB1R, which is highly expressed in the brain, where it regulates processes related to cognition, memory, and motor control. The development of hCB2R ligands represents a therapeutic opportunity for treating diseases such as pain, inflammation and cancer. Identifying new selective scaffolds for cannabinoids and determining the structural determinants responsible for agonism and antagonism are priorities in drug design. In this work, a series of N-[1,3-dialkyl(aryl)-2-oxoimidazolidin-4-ylidene]-aryl(alkyl)sulfonamides is designed and synthesized and their affinity for human hCB1R and hCB2R is determined. Starting with a scaffold selected from the NIH Psychoactive Drug Screening Program Repository, through a combination of molecular modeling and structure–activity relationship studies, we were able to identify the chemical features leading to finely tuned hCB2R selectivity. In addition, an in silico model capable of predicting the functional activity of hCB2R ligands was proposed and validated. The proposed receptor activation/deactivation model enabled the identification of four pure hCB2R-selective agonists that can be used as a starting point for the development of more potent ligands.     

Synthesis,Docking Studies and Pharmacological Evaluation of Serotoninergic Ligands Containing a 5-Norbornene-2-Carboxamide Nucleus

A new series of 5-norbornene-2-carboxamide derivatives was prepared and their affinities to the 5-HT_1A, 5-HT_2A, and 5-HT_2C receptors were evaluated and compared to a previously synthesized series of derivatives characterized by exo-N-hydroxy-5-norbornene-2,3-dicarboximidenucleus, in order to identify selective ligands for the above-mentioned subtype receptors. Arylpiperazines represents one of the most important classes of 5-HT_1AR ligands, and recent research concerning new derivatives has been focused on the modification of one or more portions of such pharmacophore. The combination of structural elements (heterocyclic nucleus, propyl chain and 4-substituted piperazine), known to be critical to the affinity to 5-HT_1A receptors, and the proper selection of substituents led to compounds with high specificity and affinity towards serotoninergic receptors. The most active compounds were selected for further in vivo assays to determine their functional activity. Finally, to rationalize the obtained results, molecular docking studies were performed. The results of the pharmacological studies showed that Norbo-4 and Norbo-18 were the most active and promising derivatives for the serotonin receptor considered in this study.