Non-selective Ligands Research Articles

Molecular dynamics approach to probe PKCβII–ligand interactions and influence of crystal water molecules on these interactions

Objective: PKCβII is a potential target for therapeutic intervention against pandemic diabetic complications. Present study probes the molecular interactions of PKCβII with its clinically important ligands, viz. ruboxistaurin, enzastaurin and co-crystallized ligand, 2-methyl-1H-indol-3-yl-BIM-1.Research design and methods: The essentials of PKCβII–ligand interaction, crystal water-induced alterations in these interactions and key interacting flexible residues are analyzed. Computational methodologies, viz. molecular docking and molecular simulation coupled with molecular mechanics-Poisson–Boltzmann surface area and generalized born surface area (MM-PB[GB]SA) are employed.Results: The structural changes in the presence and absence of crystal water molecules in PKCβII ATP binding site residues, and its interaction with bound ligand, are identified. Difference in interaction of selective and nonselective ligand with ATP binding site residues of PKCβII is reported.Conclusions: The study showed that the nonbonding interactions contribute significantly in PKCβII–ligand binding and presence of crystal water molecules affects the interactions. The findings of present work may integrate the new aspects in the drug design process of PKCβII inhibitors.

Sa2050 Corticotropin-Releasing Factor (CRF) Peptides Modulates Rat Colonic Neuronal Tau Phosphorylation: Differential Role of CRF Receptors

BACKGROUND: Corticotropin releasing factor receptor-1 (CRF1)-overexpression enhances experimental stress or CRF-induced tau (TAU) phosphorylation in the mouse brain (PNAS, 2012: 109(16):6277-82). CRF is a key mediator of the gut response to stress. Whether CRF affect enteric TAU and whether enteric neuronal TAU modulation impacts gastrointestinal tract functions are not known. AIM: 1) Determine 1) the effect of selective activation of CRF receptors on rat colon primary myneteric neuron Tau 2) the colonic motor response of Tau transgenic mice to acute stress. METHODS: Rat distal colonic primary myenteric neurons were incubated with non-selective CRF1 and 2 receptor agonists (CRF, urocortin 1 (Ucn1)) and selective CRF2 receptor agonist (Ucn2) for 30 min (10 or 100 nM) and pTAU probed by western blot. Rat colon primary myenteric neurons were processed for immunostaining of Tau. Human TauP301L overexpressing and wild-type (WT) mice were exposed to 1-hour novel environment stress and cumulative Fecal Pellet Output (FPO) at 5, 15, 30, 45 and 60 min was monitored. The overexpression of human Tau in the TauOE mice was confirmed by western blotting. RESULTS: Rat colon primary neurons have constitutively active pTau that is suppressed by selective CRF2 receptor activation but not by the non-selective CRF receptor ligand, CRF. Rat colon primary myenteric neurons have abundant Tau. Compared to WT, Tau overexpressing mice have 2-4X increased total Tau mRNA expression, increased defecation (60.7±6.5% increase in 60 min cumulative FPO) response to mild novel environment stress and display altered ileal and colonic CRF2 receptor expression. CONCLUSION: The differential modulation of colonic primary myenteric TAU phophorylation by CRF peptides and the altered colonic motor response to stress in the mice that express phospho TAU in the intestine and the modulation of colonic CRF2 receptor mRNA profile in these mice, suggest a possible interplay between myenteric TAU and CRFsignaling in the colonic response to stress. Supported by R01DK078676 (MM); Veterans Administration Merit Review (MEH-W)

Bioisosteric Replacement and Related Analogs in the Design, Synthesis and Evaluation of Ligands for Muscarinic Acetylcholine Receptors

Previous structure-activity relationship studies involving a series of lactone-based muscarinic ligands identified a lead compound containing a diphenylmethylpiperazine moiety (4; IC50 = 340 nM). The purpose of the present work is to investigate 1,3-benzodioxoles, 4,4-diethyl substituted tetrahydrofurans, 5-substituted oxazolidinones and chromones as bioisosteric replacements for the lactone ring in a novel series of muscarinic ligands. The approach provided compounds with improved % inhibition values and identified a non-selective muscarinic ligand with an IC50 value of 280 nM. The structure-activity relationship for this new series will be discussed. Selected compounds were evaluated in preliminary assays for subtype selectivity and were found to be non-selective.

Biotransformation of beta-endorphin and possible therapeutic implications

Endogenous opioid peptides have been aligned with a diverse array of effects. Their activity is not only attributable to action the three main opioid receptors, mu (MOR), delta (DOR), and kappa (KOR) opioid receptors but their impacts appear to extend to activities at sodium channels, cytokine receptors (Finley et al., 2008), calcium channels and non-specific and partially undefined pharmacological effects inconsistent with G-protein coupled opioid receptor activity. Of the family of opioid peptides beta-endorphin (BE 1-31) is one of the most prominent and is the prototypical endogenous peptide for the MOR class of opioid receptors and is found within the CNS and the immune system (Cabot et al., 1997). BE 1-31 is derived from pro-opiomelanocortin (POMC) in the cytosol of cell bodies. BE has been shown to possess peripheral and central analgesic activity (Van Den Burg et al., 2001), producing a morphine-like effect by inhibiting the signals of C- and Aδ-fiber activation (Duggan and Fleetwood-Walker, 1993). In addition, BE 1-31 is a non-selective endogenous peptide with the highest affinities for MOR and DOR (Binder et al., 2004), suggesting that the endogenous system is not modulated by specific and selective opioid agonists in isolation. This concept touches on a new theme evolving in novel therapeutic strategies in the pain field, i.e. the targeting of multiple channels with either one non-selective ligand or a combination of selective ligands to produce effects that are either synergistic or, at a minimum, differential in terms of side effects. This could seemingly point to a multitude of combinations of drugs of both G-protein receptor targeting ligands or extend to those targeting other receptor classes including sodium channels (Su et al., 2002), potassium channels (Welch and Dunlow, 1993) and calcium channels (Smart et al., 1995). The scope of the possible therapeutic targets is immense and, potentially of even greater complexity, is the dose determination for such combinations. Perhaps the answer in part lies in the endogenous opioid system, which is, in essence, the system designed to mediate noxious stimuli as well as interact with the immune system in disease (Figure ​(Figure11). Figure 1 Biotransformation of beta-endorphin 1–31 within inflamed tissue and fragment actions. Immune cells containing beta-endorphin migrate to inflamed tissue in a site directed manner. Beta-endorphin is released within the inflammatory mileu and biotransformed ...

Nanoporous Bicontinuous Structures via Addition of Thermally-Stable Amphiphilic Nanoparticles within Block Copolymer Templates

Herein, we fabricated the bicontinuous structures from nanocomposites of poly(styrene-b-methyl methacrylate) (PS-b-PMMA) block copolymer and the shell-cross-linked, thermally stable gold nanoparticles (Au NPs). The surface property of Au NPs was controlled with ligands containing various compositions of PS and PMMA so that the resulting Au NPs were selective to PS or PMMA block or nonselective (i.e., neutral) to both blocks. The amphiphilic Au NPs were also prepared by coating the surface of Au NPs with equimolar mixtures of PS and PMMA selective ligands. Consequently, it was found that the morphological behaviors of thermally annealed nanocomposites containing amphiphilic Au NPs and PS-b-PMMA were dramatically different from the case of neutral Au NPs that were coated with nonselective ligands. With increasing the amount of amphiphilic Au NPs, a transition from lamellar to bicontinuous structures was observed, whereas the neutral Au NPs were aggregated within the PS-b-PMMA lamellae. Furthermore, the nanoporous bicontinuous thin films were fabricated on the silicon substrates and the morphological behaviors were quantitatively investigated by grazing-incidence small-angle X-ray scattering (GISAXS) analysis.

Positron emission tomography imaging of dopamine D2 receptors using a highly selective radiolabeled D2 receptor partial agonist

A series of microPET imaging studies were conducted in anesthetized rhesus monkeys using the dopamine D2-selective partial agonist, [(11)C]SV-III-130. There was a high uptake in regions of brain known to express a high density of D2 receptors under baseline conditions. Rapid displacement in the caudate and putamen, but not in the cerebellum, was observed after injection of the dopamine D2/3 receptor nonselective ligand S(-)-eticlopride at a low dosage (0.025mg/kg/i.v.); no obvious displacement in the caudate, putamen and cerebellum was observed after the treatment with a dopamine D3 receptor selective ligand WC-34 (0.1mg/kg/i.v.). Pretreatment with lorazepam (1mg/kg, i.v. 30min) to reduce endogenous dopamine prior to tracer injection resulted in unchanged binding potential (BP) values, a measure of D2 receptor binding in vivo, in the caudate and putamen. d-Amphetamine challenge studies indicate that there is a significant displacement of [(11)C]SV-III-130 by d-Amphetamine-induced increases in synaptic dopamine levels.

Conversion of a non-selective adenosine receptor antagonist into A3-selective high affinity fluorescent probes using peptide-based linkers

Advances in fluorescence-based imaging technologies have helped propel the study of real-time biological readouts and analysis across many different areas. In particular the use of fluorescent ligands as chemical tools to study proteins such as G protein-coupled receptors (GPCRs) has received ongoing interest. Methods to improve the efficient chemical synthesis of fluorescent ligands remain of paramount importance to ensure this area of bioanalysis continues to advance. Here we report conversion of the non-selective GPCR adenosine receptor antagonist Xanthine Amine Congener into higher affinity and more receptor subtype-selective fluorescent antagonists. This was achieved through insertion and optimisation of a dipeptide linker between the adenosine receptor pharmacophore and the fluorophore. Fluorescent probe 27 containing BODIPY 630/650 (pK(D) = 9.12 ± 0.05 [hA3AR]), and BODIPY FL-containing 28 (pK(D) = 7.96 ± 0.09 [hA3AR]) demonstrated clear, displaceable membrane binding using fluorescent confocal microscopy. From in silico analysis of the docked ligand-receptor complexes of 27, we suggest regions of molecular interaction that could account for the observed selectivity of these peptide-linker based fluorescent conjugates. This general approach of converting a non-selective ligand to a selective biological tool could be applied to other ligands of interest.

A σ1 receptor pharmacophore derived from a series of N-substituted 4-azahexacyclo[5.4.1.02,6.03,10.05,9.08,11]dodecan-3-ols (AHDs)

A library of N-substituted 4-azahexacyclo[5.4.1.02,6.03,10.05,9.08,11]dodecan-3-ols (AHDs) was synthesized and subjected to competition binding assays at σ1 and σ2 receptors, as well as off-target screening of representative members at 44 other common central nervous system (CNS) receptors, transporters, and ion channels. Excluding 3 low affinity analogs, 31 ligands demonstrated nanomolar Ki values for either σ receptor subtype. Several selective σ1 and σ2 ligands were discovered, with selectivities of up to 29.6 times for σ1 and 52.4 times for σ2, as well as several high affinity, subtype non-selective ligands. The diversity of structures and σ1 affinities of the ligands allowed the generation of a σ1 receptor pharmacophore that will enable the rational design of increasingly selective and potent σ1 ligands for probing σ1 receptor function.

Characterisation of the Contribution of the GABA-Benzodiazepine α1 Receptor Subtype to [11C]Ro15-4513 PET Images

This positron emission tomography (PET) study aimed to further define selectivity of [11C]Ro15-4513 binding to the GABARα5 relative to the GABARα1 benzodiazepine receptor subtype. The impact of zolpidem, a GABARα1-selective agonist, on [11C]Ro15-4513, which shows selectivity for GABARα5, and the nonselective benzodiazepine ligand [11C]flumazenil binding was assessed in humans. Compartmental modelling of the kinetics of [11C]Ro15-4513 time-activity curves was used to describe distribution volume (VT) differences in regions populated by different GABA receptor subtypes. Those with low α5 were best fitted by one-tissue compartment models; and those with high α5 required a more complex model. The heterogeneity between brain regions suggested spectral analysis as a more appropriate method to quantify binding as it does not a priori specify compartments. Spectral analysis revealed that zolpidem caused a significant VT decrease (∼10%) in [11C]flumazenil, but no decrease in [11C]Ro15-4513 binding. Further analysis of [11C]Ro15-4513 kinetics revealed additional frequency components present in regions containing both α1 and α5 subtypes compared with those containing only α1. Zolpidem reduced one component (mean±s.d.: 71%±41%), presumed to reflect α1-subtype binding, but not another (13%±22%), presumed to reflect α5. The proposed method for [11C]Ro15-4513 analysis may allow more accurate selective binding assays and estimation of drug occupancy for other nonselective ligands.

In Vitro and in Vivo Molecular Imaging of Estrogen Receptor α and β Homo- and Heterodimerization: Exploration of New Modes of Receptor Regulation

Estrogen receptor (ER) biology reflects the actions of estrogens through the two receptors, ERα and ERβ, although little is known regarding the preference for formation of ER homo- vs. heterodimers, and how this is affected by the level of ligand occupancy and preferential ligand affinity for one of the ER subtypes. In this report, we use a split optical reporter-protein complementation system to demonstrate the physical interaction between ERα and ERβ in response to different ER ligands in cells and, for the first time, by in vivo imaging in living animals. The genetically encoded reporter vectors constructed with the ligand-binding domains of ERα and ERβ, fused to split firefly or Renilla luciferase (Fluc or hRluc) fragments, were used for this study. This molecular proteomic technique was used to detect ERα/ERα or ERβ/ERβ homodimerization, or ERα/ERβ heterodimerization induced by ER subtype-selective and nonselective ligands, and selective ER modulators (SERM), as well as in dimers in which one mutant monomer was unable to bind estradiol. The SERM-bound ERα and ERβ form the strongest dimers, and subtype-preferential homodimerization was seen with ERα-selective ligands (methyl piperidino pyrazole/propyl pyrazole triol) and the ERβ-selective ligands (diarylpropionitrile/tetrahydrochrysene/genistein). We also demonstrated that a single ligand-bound monomer can form homo- or heterodimers with an apo-monomer. Xenografts of human embryonic kidney 293T cells imaged in living mice by bioluminescence showed real-time ligand induction of ERα/ERβ heterodimerization and reversal of dimerization upon ligand withdrawal. The results from this study demonstrate the value of the split luciferase-based complementation system for studying ER-subtype interactions in cells and for evaluating them in living animals by noninvasive imaging. They also probe what combinations of ERα and ERβ dimers might be the mediators of the effects of different types of ER ligands given at different doses.

Discovery and optimisation of a selective non-steroidal glucocorticoid receptor antagonist

High-throughput screening of 3.87million compounds delivered a novel series of non-steroidal GR antagonists. Subsequent rounds of optimisation allowed progression from a non-selective ligand with a poor ADMET profile to an orally bioavailable, selective, stable, glucocorticoid receptor antagonist.

An investigation of non-selective and selective GABAA ligands using the beam walking assay in rats and mice

An investigation of non-selective and selective GABAA ligands using the beam walking assay in rats and mice

Exploring the structure of opioid receptors with homology modeling based on single and multiple templates and subsequent docking: A comparative study

Opioid receptors are the principal targets for opioids, which have been used as analgesics for centuries. Opioid receptors belong to the rhodopsin family of G-protein coupled receptors (GPCRs). In the absence of crystal structures of opioid receptors, 3D homology models have been reported with bovine rhodopsin as a template, though the sequence homology is low. Recently, it has been reported that use of multiple templates results in a better model for a target having low sequence identity with a single template. With the objective of carrying out a comparative study on the structural quality of the 3D models based on single and multiple templates, the homology models for opioid receptors (mu, delta and kappa) were generated using bovine rhodopsin as single template and the recently deposited crystal structures of squid rhodopsin, turkey β-1 and human β-2 adrenoreceptors along with bovine rhodopsin as multiple templates. In this paper we report the results of comparison between the refined 3D models based on multiple sequence alignment (MSA) and models built with bovine rhodopsin as template, using validation programs PROCHECK, PROSA, Verify 3D, Molprobity and docking studies. The results indicate that homology models of mu and kappa with multiple templates are better than those built with only bovine rhodopsin as template, whereas, in many aspects, the homology model of delta opioid receptor with single template is better with respect to the model based on multiple templates. Three nonselective ligands were docked to both the models of mu, delta and kappa opioid receptors using GOLD 3.1. The results of docking complied well with the pharamacophore, reported for nonspecific opioid ligands. The comparison of docking results for models with multiple templates and those with single template have been discussed in detail. Three selective ligands for each receptor were also docked. As the crystallographic structures are not yet known, this comparison will help in choosing better homology models of opioid receptors for studying ligand receptor interactions to design new potent opioid antagonists.

Pharmacology of capsaicin-, anandamide-, and N-arachidonoyl-dopamine-evoked cell death in a homogeneous transient receptor potential vanilloid subtype 1 receptor population

Transient receptor potential vanilloid subtype 1 (TRPV1) receptor is a primary pain-sensing relay at peripheral sensory nerve endings and is also widespread in the brain, where it is implicated in neurodegeneration. Previous studies of TRPV1 neurotoxicity have utilized heterogeneous receptor populations, non-selective ligands, or non-neuronal cell types. Here, we explored the pharmacology of TRPV1-induced cytotoxicity in a homogeneous, neurone-like cellular environment. Cell death was examined in a human neurone-like cell line, stably expressing recombinant human TRPV1. Cytotoxicity was quantified in terms of nuclear morphology and mitochondrial complex II activity. Immunocytochemical markers of apoptotic cell death were also examined. The TRPV1-selective agonist capsaicin, and the endovanilloids anandamide and N-arachidonoyl-dopamine (NADA), induced TRPV1-dependent delayed cell death in a concentration- and time-dependent manner. Capsaicin exposure time was significantly correlated with potency (r(2)=0.91, P=0.01). Release of cytochrome c from mitochondria, activation of caspase-3, and condensed nuclear chromatin were evident 6 h after capsaicin exposure, but cytotoxicity was unaffected by a pan-caspase inhibitor (zVAD-fmk, 50 microM). We conclude that capsaicin, anandamide, and NADA can initiate TRPV1-dependent delayed cell death in neurone-like cells. This is an apoptosis-like process, but independent of caspase activity.

Modifications to five-substituted 3,3-diethyl-4,5-dihydro-2(3H)-furanones en route to novel muscarinic receptor ligands

Lead lactone-based ligands with modest affinity for muscarinic receptors were modified based on structure–activity relationship data in the literature to provide a new series of 5-substituted 4,5-dihydro-2(3H)-furanones. The modifications included the addition of various nitrogen-containing heterocycles attached to substituted and unsubstituted aromatic rings. The target compounds were synthesized in modest yields and evaluated in preliminary muscarinic binding assays. A lactone-based ligand containing a diphenylmethylpiperazine moiety was identified as a nonselective muscarinic ligand with IC50 of 340 nM. The design of future ligands will be based, in part, on structure–activity data reported herein.

Structure–activity relationships of N-substituted ligands for the α7 nicotinic acetylcholine receptor

A series of α7 neuronal nicotinic acetylcholine receptor ligands were designed based on a structural combination of a potent, but non-selective ligand, epibatidine, with a selective lead structure, 2. Three series of compounds in which aryl moieties were attached via a linker to different positions on the core structure were studied. A potent and functionally efficacious analog, (3a R,6a S)-2-(6-phenylpyridazin-3-yl)-5-(pyridin-3-ylmethyl)octahydropyrrolo[3,4- c]pyrrole ( 3a), was identified.

Cannabinoid receptor ligands as potential anticancer agents — high hopes for new therapies?

Abstract Objectives The endocannabinoid system is an endogenous lipid signalling network comprising arachidonic-acid-derived ligands, cannabinoid (CB) receptors, transporters and endocannabinoid degrading enzymes. The CB1 receptor is predominantly expressed in neurons but is also co-expressed with the CB2 receptor in peripheral tissues. In recent years, CB receptor ligands, including Δ9-tetrahydrocannabinol, have been proposed as potential anticancer agents. Key findings This review critically discusses the pharmacology of CB receptor activation as a novel therapeutic anticancer strategy in terms of ligand selectivity, tissue specificity and potency. Intriguingly, antitumour effects mediated by cannabinoids are not confined to inhibition of cancer cell proliferation; cannabinoids also reduce angiogenesis, cell migration and metastasis, inhibit carcinogenesis and attenuate inflammatory processes. In the last decade several new selective CB1 and CB2 receptor agents have been described, but most studies in the area of cancer research have used non-selective CB ligands. Moreover, many of these ligands exert prominent CB receptor-independent pharmacological effects, such as activation of the G-protein-coupled receptor GPR55, peroxisome proliferator-activated receptor gamma and the transient receptor potential vanilloid channels. Summary The role of the endocannabinoid system in tumourigenesis is still poorly understood and the molecular mechanisms of cannabinoid anticancer action need to be elucidated. The development of CB2-selective anticancer agents could be advantageous in light of the unwanted central effects exerted by CB1 receptor ligands. Probably the most interesting question is whether cannabinoids could be useful in chemoprevention or in combination with established chemotherapeutic agents.

Histamine H4 receptor ligands and their potential therapeutic applications

Background: The histamine H4 receptor (H4R) was first cloned in 2000. As a new member of the histamine receptor family it was of great interest and a promising drug target for the treatment of inflammatory diseases. Recent studies suggest that H4 antagonists have therapeutic potential, even in several pain conditions. Initially, pharmacological processes mediated by H4R were analyzed primarily by non-selective H4 ligands. Since then, some highly potent and selective H4 antagonists have been reported by different groups. Several companies are working on the development of potent H4 antagonists, although the number of published patent applications is still low. Objective: This review aims to give an overview of the ligands acting on H4R, this is drawn from published in papers and patent applications. Methods: Analysis of scaffolds possessing significant H4 activity and review of their therapeutic potential. Results/conclusion: The pharmacology of currently available selective H4 ligands suggests therapeutic utility for H4 antagonists in inflammatory conditions as well as in chronic pain.

Vitamin D receptor ligand therapy in chronic kidney disease

Chronic kidney disease is a growing public health concern, and secondary hyperparathyroidism is one of the most serious associated comorbidities. In healthy individuals, precisely controlled feedback loops dynamically modulate parathyroid hormone (PTH) levels. As kidney function declines, phosphate retention, decreased 1a,25-dihydroxyvitamin D3, and a tendency to hypocalcemia leads to overstimulation of PTH production and parathyroid hyperplasia. Vitamin D receptor (VDR) ligands are essential tools for controlling parathyroid activity. This review highlights the current clinical and biochemical VDR ligand studies, focusing on the differences between selective and nonselective VDR ligands. It is apparent that VDR ligands have important roles in the management of secondary hyperparathyroidism. Selective VDR ligands, in particular, may offer additional benefits in the treatment of bone disease, and may potentially reduce adverse effects related to cardiovascular disease.

Endothelin Receptor Dimers Evaluated by FRET, Ligand Binding, and Calcium Mobilization

Endothelin-1 (ET-1) mediates physiological responses via endothelin A (ETA) and B (ETB) receptors, which may form homo- and heterodimers with unknown function. Here, we investigated ET-receptor dimerization using fluorescence resonance energy transfer (FRET) between receptors tagged with CFP (donor) and receptors tagged with tetracysteine-FlAsH (fluorescein arsenical hairpin) (acceptor) expressed in HEK293 cells. FRET efficiencies were 15%, 22%, and 27% for ETA/ETA, ETB/ETB and ETA/ETB, respectively, and dimerization was further supported by coimmunoprecipitation. For all dimer pairs, the natural but nonselective ligand ET-1 rapidly (≤30s) reduced FRET by >50%, but did not detectably reduce coimmunoprecipitation. ET-1 stimulated a transient increase in intracellular Ca2+ ([Ca2+]i) lasting 1–2min for both homodimer pairs, and these ET-1 actions on FRET and [Ca2+]i elevation were blocked by the appropriate subtype-selective antagonist. In contrast, ETA/ETB heterodimers mediated a sustained [Ca2+]i increase lasting >10min, and required a combination of ETA and ETB antagonists to block the observed FRET and [Ca2+]i responses. The sensitive CFP/FlAsH FRET assay used here provides new insights into endothelin-receptor dimer function, and represents a unique approach to characterize G-protein-coupled receptor oligomers, including their pharmacology.