Probing the Binding Pocket of the Broadly Tuned Human Bitter Taste Receptor TAS2R14 by Chemical Modification of Cognate Agonists

Sensing potentially harmful bitter substances in the oral cavity is achieved by a group of ^?25 receptors, named TAS2Rs, which are expressed in specialized sensory cells and recognize individual but overlapping sets of bitter compounds. The receptors differ in their tuning breadths ranging from narrowly to broadly tuned receptors. One of the most broadly tuned human bitter taste receptors is the TAS2R14 recognizing an enormous variety of chemically diverse synthetic and natural bitter compounds, including numerous medicinal drugs. This suggests that this receptor possesses a large readily accessible ligand binding pocket. To allow probing the accessibility and size of the ligand binding pocket, we chemically modified cognate agonists and tested receptor responses in functional assays. The addition of large functional groups to agonists was usually possible without abolishing agonistic activity. The newly synthesized agonist derivatives were modeled in the binding site of the receptor, providing comparison to the mother substances and rationalization of the in vitro activities of this series of compounds.     

Ligand Binding Sensitivity of the Extracellular Loop Two of the Cannabinoid Receptor 1

The cannabinoid receptor one (CB1) is a class A G-protein-coupled receptor thought to bind ligands primarily within its helical bundle. Evidence suggests, however, that the extracellular domain may also play a role. We have previously shown that the C-terminus of the extracellular loop 2 of CB1 is important in binding some compounds; receptors with mutations in this region (F268W, P269A, H270A, and I271A) bound some agonists with severely reduced affinity relative to the wild-type receptor. In the present work, we examine the impact of these mutations on binding a chemically diverse set of ligands. The receptors, F268W and I271A, exhibited a greater sensitivity to binding the inverse agonists/antagonists SLV319, AVE1625, NESS0327 relative to P269A and H270A, suggesting that the Pro and His are not involved in binding those compounds. In contrast, binding of the agonists, BAY593074 and WIN55212-2, was diminished in all four receptors, suggesting the conformational unit contributed by all four residues is important. A more marked loss in binding was observed for agonists of the nonclassical (CP55940) and classical (HU-210, JWH061, JWH179) cannabinoid classes and for a silent antagonist derivative (O-2050), pointing to the critical nature of this region for binding both the bicyclic/tricyclic core and the alkyl chain of these derivatives. However, moving the location of the alkyl chain on a series of pyrazole analogues shows it can be better accommodated in certain locations (O-1255) than others (O-1302, O-1690) and underscores the involvement of residues F268 and I271.     

Muscarinic Receptor Activity of Some Malaysian Plant Species

Abstract

Muscarinic receptor binding activity was tested on 224 plant extracts obtained from more than 50 plant families found in Malaysia. The plant extracts were evaluated by a 96-well microplate filtration–based radioligand competitive assay, centered on the ability of the plant extracts to competitively displace the radioligand, [³H]N.-methylscopolamine, from binding to the muscarinic membrane receptors. The screening assay was initially carried out at 50 µg/assay point, and those showing inhibition at and above 61% were retested at 10 µg/assay point. The extracts of Ficus septica. Burm. f. (Moraceae) [65.85±3.75% inhibition; mean (n = 3)±SD], Polyalthia microtus. Miq. (Annonaceae) (32.63±1.38% inhibition), and Popowia odoardoi. Diels (Annonaceae) (35.79±7.11% inhibition) at 10 µg/assay point exhibited muscarinic properties, which are worthy of further investigation.     

Effects of the Partial M1 Muscarinic Cholinergic Receptor Agonist CDD-0102A on Stereotyped Motor Behaviors and Reversal Learning in the BTBR Mouse Model of Autism

BackgroundAutism spectrum disorders (ASD) are a set of neurodevelopmental disorders marked by a lack of social interaction, restrictive interests, and repetitive behaviors. There is a paucity of pharmacological treatments to reduce core ASD symptoms. Various lines of evidence indicate that reduced brain muscarinic cholinergic receptor activity may contribute to an ASD phenotype.MethodsThe present experiments examined whether the partial M₁ muscarinic receptor agonist, 5-(3-ethyl-1,2,4-oxadiazol-5-yl)-1,4,5,6-tetrahydropyrimidine hydrochloride (CDD-0102A), alleviates behavioral flexibility deficits and/or stereotyped motor behaviors in the BTBR mouse model of autism. Behavioral flexibility was tested using a reversal learning test. Stereotyped motor behaviors were measured by eliciting digging behavior after removal of nesting material in a home cage and by measuring repetitive grooming.ResultsCDD-0102A (0.2 and 0.6 mg/kg but not 1.2 mg/kg) injected prior to reversal learning attenuated a deficit in BTBR mice but did not affect performance in B6 mice. Acute CDD-0102A treatment (1.2 and 3 mg/kg) reduced self-grooming in BTBR mice and reduced digging behavior in B6 and BTBR mice. The M₁ muscarinic receptor antagonist VU0255035 (3 mg/kg) blocked the effect of CDD-0102A on grooming behavior. Chronic treatment with CDD-0102A (1.2 mg/kg) attenuated self-grooming and digging behavior in BTBR mice. Direct CDD-0102A infusions (1 µg) into the dorsal striatum reduced elevated digging behavior in BTBR mice. In contrast, CDD-0102A injections in the frontal cortex were not effective.ConclusionsThe results suggest that treatment with a partial M₁ muscarinic receptor agonist may reduce repetitive behaviors and restricted interests in autism in part by stimulating striatal M₁ muscarinic receptors.     

Mapping of Allosteric Druggable Sites in Activation‐Associated Conformers of the M2 Muscarinic Receptor

G‐protein‐coupled receptors (GPCRs) are key cellular signaling proteins and have been targeted by approximately 30–40% of marketed drugs for treating many human diseases including cancer and heart failure. Recently, we directly observed activation of the M2 muscarinic receptor through long‐timescale accelerated molecular dynamics (aMD) simulation, which revealed distinct inactive, intermediate and active conformers of the receptor. Here, FTMAP is applied to search for ‘hot spots’ in these activation‐associated conformers using a library of 16 organic probe molecules that represent fragments of potential drugs. Seven allosteric (non‐orthosteric) binding sites are identified in the M2 receptor through the FTMAP analysis. These sites are distributed in the solvent‐exposed extracellular and intracellular mouth regions, as well as the lipid‐exposed pockets formed by the transmembrane α helices TM3‐TM4, TM5‐TM6 and TM7‐TM1/TM2. They serve as promising target sites for designing novel allosteric modulators as receptor‐selective drugs.     

Analogues of Carbacholine: Synthesis and Relationship between Structure and Affinity for Muscarinic and Nicotinic Acetylcholine Receptors

A series of acyclic and heterocyclic analogues of carbacholine ( 1 ) was synthesized using N-methylcarbacholine (MCC, 2 ), N,N-dimethylcarbacholine (DMCC, 3 ), and the corresponding tertiary amine ( 4 ) as leads. Whereas nicotinic acetylcholine receptor affinity was determined using [³H]nicotine as the radioactive ligand, [³H]oxotremorine-M ([³H]Oxo-M) and [³H]quinuclidinyl benzilate ([³H]QNB), in some cases supplemented with [³H]pirenzepine ([³H]PZ), were used as radioligands for muscarinic acetylcholine receptors on rat brain membranes. On the basis of receptor binding data, nicotinic/muscarinic (N/M) selectivity factors were determined, and muscarinic receptor efficacy (M agonist index) and M₁ selectivity (M₂/M₁ index) estimated. In most cases, quaternized analogues showed higher affinity than the corresponding tertiary amines for muscarinic and, in particular, nicotinic receptor sites. Among the new compounds, N,N-diethylcarbacholine ( 9e ) (IC₅₀ = 0.046 μM), (S)-1-methyl-2-(N,N-diethyl-aminocarbonyloxymethyl)pyrrolidine ( 17k ) (IC₅₀ = 0.068 μM), and the corresponding quaternized analogue, 18k (IC₅₀ = 0.018 μM) showed the highest nicotinic receptor affinity. The tertiary amine, 17k showed much higher nicotinic receptor affinity than the acyclic analogue, 4 (IC₅₀ = 5.7 μM), and the N/M selectivity factor determined for 17k (150) is an order of magnitude lower than that of nicotine (1400). The N/M selectivity factors for MCC ( 2 ) and DMCC ( 3 ), previously reported to be highly selective nicotinic receptor ligands, were shown to be 6.5 and 60, respectively, the latter value being comparable with that of 18k (89).     

Calmodulin Binding to Peptides Derived from the i3 Loop of Muscarinic Receptors

Purpose This study was conducted to identify and characterize the structural requirements of a calmodulin-binding motif identified in the third intracellular (i3) loop of muscarinic acetylcholine receptors (M1–M5), a region important for G protein coupling. Methods GST fusion proteins and synthetic peptides derived from the hM1 i3 loop were tested for binding to CaM using a cross-linking gel shift assay and a dansyl-CaM fluorescence assay. Mutagenesis studies further characterized the structural requirements for the interaction and identified critical residues. Results 28-Mer peptides from the C terminus of i3, representing the putative calmodulin domains of M1, M2, and M3, were found capable of interacting with CaM. In addition, smaller peptides defined a 5-amino-acid sequence essential for calmodulin binding. Studies performed with M1 peptides derived from GST fusion proteins, representing larger portions of the i3 C terminus, suggested the presence of a second adjacent CaM binding site. Mutagenesis studies identified two mutants that are unable to bind CaM: a point mutation, E360A, and a deletion mutant, Δ232–358. Conclusion Calmodulin can bind to an M1 region implicated in G protein coupling. This indicates an important role for CaM in the regulation of muscarinic signal transduction.     

Evidence for M2 and M3 Muscarinic Receptor Involvement in Cholinergic Excitatory Junction Potentials Through Synergistic Activation of Cation Channels in the Longitudinal Muscle of Mouse Ileum

Cholinergic nerve–mediated excitatory junction potentials (EJPs) in the longitudinal muscle of mouse ileum were characterized by using M₂ or M₃ muscarinic receptor–knockout (KO) mice and 1-[β-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole hydrochloride (SK&F 96365) and pertussis toxin (PTX). EJPs evoked by electrical field stimulation (EFS) in wild-type preparations, initially determined to be cholinergic in origin using tetrodotoxin, atropine, and eserine, were profoundly depressed after SK&F 96365 treatment known to block muscarinic receptor–operated cation channels. A similar depression of the EJPs was also observed by PTX treatment, which is predicted to disrupt M₂-mediated pathways linked to cation channel activation. In M₂-KO mouse preparations, cholinergic EJPs were evoked by EFS with their relative amplitude of 20% – 30% to the wild-type EJP and strongly inhibited by SK&F 96365. No cholinergic EJP was seen in M₃-KO as well as M₂/M₃ double-KO preparations. The results suggest that the wild-type cholinergic EJP is not a simple mixture of M₂ and M₃ responses, but due to synergistic activation of cation channels by both M₂ and M₃ receptors in the murine ileal longitudinal muscle.     

Discovery of VU6028418: A Highly Selective and Orally Bioavailable M4 Muscarinic Acetylcholine Receptor Antagonist

Herein, we report the SAR leading to the discovery of VU6028418, a potent M₄ mAChR antagonist with high subtype-selectivity and attractive DMPK properties in vitro and in vivo across multiple species. VU6028418 was subsequently evaluated as a preclinical candidate for the treatment of dystonia and other movement disorders. During the characterization of VU6028418, a novel use of deuterium incorporation as a means to modulate CYP inhibition was also discovered.     

Interaction of Follicle‐Stimulating Hormone (FSH) Receptor Binding Inhibitor‐8: A Novel FSH‐Binding Inhibitor,with FSH and its Receptor

Follicle‐stimulating hormone (FSH) receptor binding inhibitor (FRBI‐8) is a novel octapeptide purified from human ovarian follicular fluid. In vitro, it inhibits the binding of FSH to granulosa cells and in vivo, it induces atresia in developing follicles in rodents. This peptide, when administered to marmosets and bonnet monkeys, altered the circulating progesterone levels. This study was carried out to elucidate structure of the FRBI‐8 and understand its mechanism of inhibiting interaction of FSH to its receptors. Homology modeling predicted that the FRBI‐8 adopts a turn and random coil. This is further confirmed by circular dichroism and NMR. Docking studies of the FRBI‐8 with reported FSH–FSHR hormone binding (FSHR_HB) domain complex using zdock algorithm revealed that the FRBI‐8 binds to FSHβL2–FSHR_HB binding interface which is otherwise known to be crucial for activation of signal transduction cascade. FRBI‐8 analogs were designed by replacing the acidic amino acid residues at positions 2, 5 and 6 with Ala, individually. Docking studies revealed that D6A mutant (FRBI‐8^D6A) had a higher binding affinity than the native FRBI‐8. In vitro radioreceptor assay with FRBI‐8^D6A showed 50% lower IC₅₀ compared with the FRBI‐8, confirming the in silico observations. Thus, the study reveals that both FRBI‐8 and FRBI‐8^D6A interfered with the binding of FSH to its receptor.     

Prediction of the Human EP1 Receptor Binding Site by Homology Modeling and Molecular Dynamics Simulation

The prostanoid receptor EP1 is a G-protein-coupled receptor (GPCR) known to be involved in a variety of pathological disorders such as pain, fever and inflammation. These receptors are important drug targets, but design of subtype specific agonists and antagonists has been partially hampered by the absence of three-dimensional structures for these receptors. To understand the molecular interactions of the PGE2, an endogen ligand, with the EP1 receptor, a homology model of the human EP1 receptor (hEP1R) with all connecting loops was constructed from the 2.6 Å resolution crystal structure (PDB code: 1L9H) of bovine rhodopsin. The initial model generated by MODELLER was subjected to molecular dynamics simulation to assess quality of the model. Also, a step by step ligand-supported model refinement was performed, including initial docking of PGE2 and iloprost in the putative binding site, followed by several rounds of energy minimizations and molecular dynamics simulations. Docking studies were performed for PGE2 and some other related compounds in the active site of the final hEP1 receptor model. The docking enabled us to identify key molecular interactions supported by the mutagenesis data. Also, the correlation of r²=0.81 was observed between the Ki values and the docking scores of 15 prostanoid compounds. The results obtained in this study may provide new insights toward understanding the active site conformation of the hEP1 receptor and can be used for the structure-based design of novel specific ligands.     

Binding of Digitoxin to Human Serum Proteins: Influence of pH on the Binding of Digitoxin to Human Albumin

Abstract: The binding of digitoxin to albumin was studied under various conditions with regard to electrolytes and pH. Considerable variations of Na, K, Ca and Mg within the range of clinical relevance did not influence the binding of digitoxin to albumin. Under the influence of excessive concentrations of digitoxin this binding followed the simple law of mass action with an average number of binding sites of about 0.5 independent of pH. The intrinsic association constants were about twice the apparent association constants, calculated in another way and with the assumption of one binding site per molecule of albumin. The binding of digitoxin to albumin was shown to depend on the pH of the medium with a maximum association constant at pH = 4.8 (intrinsic association constant, K = 5 x 10⁵ l/mol). The binding of digitoxin to human serum proteins was shown to be equal to that of the binding to albumin. Under conditions relevant for clinical interpretations the binding of digitoxin to other proteins was insignificant.     

Cholinergic Innervation of the Human Urethra and Urinary Bladder: A histochemical study and review of methodology.

Abstract The present work gives a survey of the various methods for the histochemical demonstration of cholinergic nervous structures. The theoretical background and the specificity and sensitivity of the various methods are compared. It is concluded that the histochemical localization of the cholinergic transmitter metabolizing enzyme acetylcholine esterase still probably is the best method, giving a fairly good reflection of the distribution of cholinergic nerves. In comparison with the Falck and Hillarp technique for the demonstration of adrenergic nerves this method is much less specific and sensitive. The present work describes a rich supply of cholinergic nerves in the human urethra and urinary bladder in comparison with the scanty adrenergic innervation. Various functional aspects of the cholinergic innervation are discussed.     

Discovery of New Human A(2A) Adenosine Receptor Agonists: Design, Synthesis, and Binding Mode of Truncated 2-Hexynyl-4'-thioadenosine

The truncated C2- and C8-substituted-4'-thioadenosine derivatives 4a-d were synthesized from D-mannose, using palladium-catalyzed cross coupling reactions as key steps. In this study, an A(3) adenosine receptor (AR) antagonist, truncated 4'-thioadenosine derivative 3 was successfully converted into a potent A(2A)AR agonist 4a (K(i) = 7.19 ± 0.6 nM) by appending a 2-hexynyl group at the C2-position of a derivative of 3 that was N(6)-substituted. However, C8-substitution greatly reduced binding affinity at the human A(2A)AR. All synthesized compounds 4a-d maintained their affinity at the human A(3)AR, but 4a was found to be a competitive A(3)AR antagonist/A(2A)AR agonist in cyclic AMP assays. This study indicates that the truncated C2-substituted-4'-thioadenosine derivatives 4a and 4b can serve as a novel template for the development of new A(2A)AR ligands.     

Demonstration of α-Adrenoceptors in the Rabbit Bladder Base and Urethra with 3H-Dihydroergocryptine Ligand Binding

Abstract: The purpose of this work was to demonstrate the presence of α-adrenoceptors in a crude membrane preparation made from rabbit bladder base and urethra. This was achieved by radioligand binding studies, using ³H-dihydro-α-ergocryptine (³H-DHE) as the radioligand. The specific binding, i.e. the binding that could be inhibited by 10?⁵ M phentolamine, was saturable with 73 fmol ³H-DHE bound per mg membrane protein. Binding was at steady state after 60 min., and reversible. Rate constants for association and dissociation were 3 × 10⁷ M^?1 min.^?1, and 2× 10^?2 min.^?1, respectively. A number of compounds were tested for their abilities to compete with ³H-DHE for the binding sites. The relative affinity of some adrenoceptor agonists was: (-)-adrenaline>(-)-noradrenaline(±)-isoprenaline. Stereoselectivity was shown, since (-)-noradrenaline had 42 times higher affinity than (+)-noradrenaline. Adrenoceptor antagonists inhibited ³H-DHE binding in the following order of potency: DHE>phentolamine(±)-propranolol. The dissociation constant, K_D, for DHE to the binding sites was estimated in three different ways. The constants were derived from saturation, competition, and kinetic studies, and gave K_D values of 1.1,1.4 and 0.7 nM, respectively. The results suggest that α-adrenoceptors were labelled by ³H-DHE in the tissue homogenates.     

Probing Binding Modes of Small Molecule Inhibitors to the Polo-Box Domain of Human Polo-like Kinase 1

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