Show Subtype Selectivity Research Articles

Development of Tetrachlorophthalimides as Liver X Receptor β (LXRβ)-Selective Agonists.

Liver X receptor (LXR) agonists are candidates for the treatment of atherosclerosis via induction of ABCA1 (ATP-binding cassette A1) gene expression, which contributes to reverse cholesterol transport (RCT) and to cholesterol efflux from the liver and intestine. However, LXR agonists also induce genes involved in lipogenesis, such as SREBP-1c (sterol regulatory binding element protein 1c) and FAS (fatty acid synthase), thereby causing an undesirable increase in plasma and hepatic triglyceride (TG) levels. Recent studies indicate that LXRα contributes to lipogenesis in liver, and selective LXRβ activation improves RCT in mice. Therefore, LXRβ-selective agonists are promising candidates to improve atherosclerosis without increasing plasma or hepatic TG levels. However, the ligand-binding domains in the two LXR isoforms α/β share high sequence identity, and few LXR ligands show subtype selectivity. In this study we identified a tetrachlorophthalimide analogue as an LXRβ-selective agonist. Structural development led to (E)-4,5,6,7-tetrachloro-2-(2-styrylphenyl)isoindoline-1,3-dione (24 a), which shows potent and selective LXRβ agonistic activity in reporter gene assays. In binding assays, compound 24 a bound to LXRβ preferentially over LXRα. It also induced the expression of ABCA1 mRNA but not SREBP-1c mRNA in cells. Compound 24 a appears to be a promising lead compound for therapeutic agents to treat atherosclerosis without the side effects induced by LXRα/β dual agonists.

Accelerated structure-based design of chemically diverse allosteric modulators of a muscarinic G protein-coupled receptor

Design of ligands that provide receptor selectivity has emerged as a new paradigm for drug discovery of G protein-coupled receptors, and may, for certain families of receptors, only be achieved via identification of chemically diverse allosteric modulators. Here, the extracellular vestibule of the M2 muscarinic acetylcholine receptor (mAChR) is targeted for structure-based design of allosteric modulators. Accelerated molecular dynamics (aMD) simulations were performed to construct structural ensembles that account for the receptor flexibility. Compounds obtained from the National Cancer Institute (NCI) were docked to the receptor ensembles. Retrospective docking of known ligands showed that combining aMD simulations with Glide induced fit docking (IFD) provided much-improved enrichment factors, compared with the Glide virtual screening workflow. Glide IFD was thus applied in receptor ensemble docking, and 38 top-ranked NCI compounds were selected for experimental testing. In [(3)H]N-methylscopolamine radioligand dissociation assays, approximately half of the 38 lead compounds altered the radioligand dissociation rate, a hallmark of allosteric behavior. In further competition binding experiments, we identified 12 compounds with affinity of ≤30 μM. With final functional experiments on six selected compounds, we confirmed four of them as new negative allosteric modulators (NAMs) and one as positive allosteric modulator of agonist-mediated response at the M2 mAChR. Two of the NAMs showed subtype selectivity without significant effect at the M1 and M3 mAChRs. This study demonstrates an unprecedented successful structure-based approach to identify chemically diverse and selective GPCR allosteric modulators with outstanding potential for further structure-activity relationship studies.

Development and characterization of the α3β4α5 nicotinic receptor cellular membrane affinity chromatography column and its application for on line screening of plant extracts

The α3β4α5 nAChR has been recently shown to be a useful target for smoking cessation pharmacotherapies. Herein, we report on the development and characterization of the α3β4α5 nicotinic receptor column by frontal displacement chromatography. The binding affinity of the nicotine and minor alkaloids found in tobacco smoke condensates were determined for both the α3β4 and α3β4α5 nicotinic receptors. It was demonstrated that while no subtype selectivity was observed for nicotine and nornicotine, anabasine was selective for the α3β4α5 nicotinic receptor. The non-competitive inhibitor binding site was also studied and it was demonstrated while mecamylamine was not selective between subtypes, buproprion showed subtype selectivity for the α3β4 nicotinic receptor. The application of this methodology to complex mixtures was then carried out by screening aqueous-alcoholic solutions of targeted plant extracts, including Lycopodium clavatum L. (Lycopodiaceae) and Trigonella foenum graecum L. (Fabaceae) against both the α3β4 and α3β4α5 nAChRs.

The Relationship between Functional Inhibition and Binding for KCa2 Channel Blockers

Small conductance calcium-activated potassium channels (KCa2.1,2.2,2.3) are blocked with high affinity by both peptide toxins (e.g. apamin) and small molecule blockers (e.g. UCL 1848). In electrophysiological experiments, apamin shows subtype selectivity with IC50s of ∼100 pM and ∼1 nM for block KCa2.2 and KCa2.3 respectively. In binding studies, however, apamin appears not to discriminate between KCa2.2 and 2.3 and is reported to have a significantly higher (∼20–200-fold) affinity (∼5 pM). This discrepancy between binding and block has been suggested to reflect an unusual mode of action of apamin. However, these binding and electrophysiological block experiments have not been conducted in the same ionic conditions, so it is also possible that the discrepancy arises simply because of differences in experimental conditions. We have now examined this latter possibility. Thus, we measured 125I-apamin binding to intact HEK 293 cells expressing KCa2 channels under the same ionic conditions (i.e. normal physiological conditions) that we also used for current block measurements. We find that binding and block experiments agree well if the same ionic conditions are used. Further, the binding of apamin and other blockers showed subtype selectivity when measured in normal physiological solutions (e.g.125I-apamin bound to KCa2.2 with K L 91±40 pM and to KCa2.3 with K L 711±126 pM, while inhibiting KCa2.2 current at IC50 103±2 pM). We also examined KCa2 channel block in Ca2+ and Mg2+ free solutions that mimic conditions reported in the literature for binding experiments. Under these (non-physiological) conditions the IC50 for apamin block of KCa2.2 was reduced to 20±3 pM. Our results therefore suggest that the apparent discrepancy between blocking and binding reported in the literature can be largely accounted for by the use of non-physiological ionic conditions in binding experiments.

Flavonoid modulation of GABAA receptors

There has been a resurgence of interest in synthetic and plant-derived flavonoids as modulators of γ-amino butyric acid-A (GABA(A) ) receptor function influencing inhibition mediated by the major inhibitory neurotransmitter GABA in the brain. Areas of interest include (i) flavonoids that show subtype selectivity in recombinant receptor studies in vitro consistent with their behavioural effects in vivo, (ii) flumazenil-insensitive modulation of GABA(A) receptor function by flavonoids, (iii) the ability of some flavonoids to act as second-order modulators of first-order modulation by benzodiazepines and (iv) the identification of the different sites of action of flavonoids on GABA(A) receptor complexes. An emerging area of interest is the activation of GABA(A) receptors by flavonoids in the absence of GABA. The relatively rigid shape of flavonoids means that they are useful scaffolds for the design of new therapeutic agents. Like steroids, flavonoids have wide-ranging effects on numerous biological targets. The challenge is to understand the structural determinants of flavonoid effects on particular targets and to develop agents specific for these targets.

Induction of Larval Tissue Resorption in Xenopus laevis Tadpoles by the Thyroid Hormone Receptor Agonist GC-1

A major challenge in understanding nuclear hormone receptor function is to determine how the same ligand can cause very different tissue-specific responses. Tissue specificity may result from the presence of more than one receptor subtype arising from multiple receptor genes or alternative splicing. Recently, high affinity analogs of nuclear receptor ligands have been synthesized that show subtype selectivity. These analogs can greatly facilitate the study of receptor subtype-specific functions in organisms where mutational analysis is problematic or where it is desirable for receptors to be expressed in their normal physiological contexts. We describe here the effects of the synthetic thyroid hormone analog GC-1 on the metamorphosis of the frog Xenopus laevis. The most potent natural thyroid hormone, 3,5,3'-triidothyronine or T3, shows similar binding affinity and transactivation dose-response curves for both thyroid hormone receptor isotypes, designated TRalpha and TRbeta. GC-1, however, binds to and activates TRbeta at least an order of magnitude better than it does TRalpha. GC-1 efficiently induces death and resorption of premetamorphic tadpole tissues such as the gills and the tail, two tissues that strongly induce thyroid hormone receptor beta during metamorphosis. GC-1 has less effect on the growth of adult tissues such as the hindlimbs, which express high TRalpha levels. The effectiveness of GC-1 in inducing tail resorption and tail gene expression correlates with increasing TRbeta levels. These results illustrate the utility of subtype selective ligands as probes of nuclear receptor function in vivo.

Characterisation of [formula omitted]-apamin binding sites in rat brain membranes and HEK293 cells transfected with SK channel subtypes

The pharmacology of [ 125 I] -apamin binding sites was examined in rat cortical and hippocampal tissue and compared with membranes prepared from human embryonic kidney (HEK293) cells transfected with SK channel subtypes hSK1, rSK2 and rSK3. The K D of [ 125 I] -apamin in rat cortex and hippocampus was similar to the apamin-sensitive subtypes, rSK2 and rSK3 ( K D (pM): 6.4, 7.08, 6.56 and 8.94, respectively). In addition, [ 125 I] -apamin had a K D=270.4 pM for the putatively ‘apamin-insensitive’ hSK1. Apamin had about a three-fold higher affinity than [ 125 I] -apamin in brain tissue and in the cells expressing the different SK channel subtypes. Pancuronium, bicuculline and d-tubocurarine displayed micromolar affinity for all five-membrane preparations, whereas dequalinium and gallamine appear to show some subtype selectivity. Tetraethylammonium (TEA) and 4-aminopyridine (4-AP) had millimolar affinity and linopirdine had no effect. In conclusion, the pharmacology of [ 125 I] -apamin binding in the cortex and hippocampus was similar to that in the apamin-sensitive clones, rSK2 and rSK3. In addition, we demonstrated low affinity [ 125 I] -apamin binding for hSK1 and identified compounds that show subtype selectivity. These data cast further doubt on the identification of SK1 as encoding for the K + channel responsible for the apamin-insensitive sAHP.

Characterisation of Neuropeptide Y Receptor Subtypes by Synthetic NPY Analogues and by Anti-receptor Antibodies

Neuropeptide Y (NPY), a 36-mer neuromodulator, binds to the receptors Y1, Y2, Y4 and Y5 with nanomolar affinity. They all belong to the rhodopsin-like G-protein coupled, seven transmembrane helix spanning receptors. In this study, Ala-substituted and centrally truncated NPY analogues were compared with respect to affinity to the Y-receptors. Furthermore, antibodies against the second (E2) and the third (E3) extracellular loop of NPY Y1-, Y2- and Y5-receptor subtypes were raised and affinity to intact cells was tested by immunofluorescence assays. Both methods were applied in order to receive subtype selective tools and to characterise ligand binding. The analogues [A13]-pNPY and [A27]-pNPY showed subtype selectivity for the Y2-receptor. Sera against the E2 loop of the Y1-receptor and against the E2 loop of the Y2-receptor were subtype selective. Two antibodies against the Y5 E2 and E3 loop recognised the Y5- and Y2-receptor subtypes. In combination, these sera are able to distinguish between the Y1-, Y2-, and Y5-receptor subtypes. The analogues and antibodies represent valuable tools to distinguish NPY receptors on membranes and intact cells.

Pharmacological characterization of [ 3H]-JTH-601, a novel α 1-adrenoceptor antagonist : Binding to recombinant human α 1-adrenoceptors and human prostates

Several α 1-adrenoceptor (AR) selective antagonists are now widely used to improve lower urinary tract symptoms in benign prostatic hyperplasia patients. However, these drugs often result in orthostatic hypotension, because of their poor uroselectivity; the blockade of α 1-AR not only in prostate but also in vasculature. Here we have investigated uroselectivity of JTH-601, a newly developed antagonist, in radioligand binding experiment using recombinant human α 1-AR subtypes and human prostate. In saturation experiments, [ 3H]-JTH-601 showed subtype selectivity: high affinity to α 1a-AR (pKd; 9.88±0.09), lower affinity to α 1b-AR (pKd; 8.96±0.17) and no specific binding at concentrations up to 3000 pM to α 1d-AR. In competition experiments, JTH-601 and its metabolic compound (JTH-601-G1) also showed α 1a-AR selectivity, exhibiting approximately 5 times higher affinity for α 1a-AR than for α 1b-AR, 10 to 20 times higher affinity than for α 1d-AR, respectively. [ 3H]-JTH-601 also bound to human prostate membranes in monophasic manner with high affinity constant (pKd; 9.89±0.12, Bmax=123.6±16 fmol/mg protein). JTH-601 is a unique α 1-AR antagonist that shows high affinity and selectivity for human recombinant α 1a- and human prostate. This new compound is useful for understanding α 1-AR pharmacology and may have a therapeutic value.

Transduction is a major factor influencing receptor characterization.

Two state (agonist-antagonist) receptor systems may explain many discrepancies in receptor classification, but the role of transduction (G protein coupling) may be critical. We propose that in some instances synthetic agonists and antagonists may interact with the receptor in such a way as to modify coupling compared with endogenous agonists, and that the transduction system together with the local environment, may contribute more to the rank order of potency of agonists and antagonists than the receptor subtype as defined by structure. Allosteric interactions at ion channels and receptors require a modification of concepts of coupling. Imidazoline ligands have different efficacy in coupling alpha 2-adrenoceptors to G proteins, compared with adrenaline and noradrenaline, and do not show a marked sodium shift, implying that the sodium site, and by implication the arginine switch, is implicated in the differential coupling. The alpha 2-adrenoceptor labeled with a natural agonist does not show subtype selectivity whereas antagonist-labeled alpha 2-adrenoceptors show subtype selectivity. In the 5-HT1A receptor, palmitoylation (of receptor or G proteins) allows the expression of different agonist states. Thus transduction and G protein coupling must be taken into account in receptor classification, even if the primary classification may be structural.