Novel fused arylpyrimidinone based allosteric modulators of the M1 muscarinic acetylcholine receptor

Benzoquinazolinone 1 is a positive allosteric modulator (PAM) of the M1 muscarinic acetylcholine receptor (mAChR), which is significantly more potent than the prototypical PAM, 1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline- 3-carboxylic acid (BQCA). In this study, we explored the structural determinants that underlie the activity of 1 as a PAM of the M1 mAChR. We paid particular attention to the importance of the tricyclic scaffold of compound 1, for the activity of the molecule. Complete deletion of the peripheral fused benzene ring caused a significant decrease in affinity and binding cooperativity with acetylcholine (ACh). This loss of affinity was rescued with the addition of either one or two methyl groups in the 7- and/or 8-position of the quinazolin-4(3H)-one core. These results demonstrate that the tricyclic benzo[h]quinazolin-4(3H)-one core could be replaced with a quinazolin-4(3H)-one core and maintain functional affinity. As such, the quinazolin-4(3H)-one core represents a novel scaffold to further explore M1 mAChR PAMs with improved physicochemical properties

Probe dependence of allosteric enhancers on the binding affinity of adenosine A1‐receptor agonists at rat and human A1‐receptors measured using NanoBRET

Background and Purpose: Adenosine is a local mediator that regulates a number of physiological and pathological processes via activation of adenosine A1‐receptors. The activity of adenosine can be regulated at the level of its target receptor via drugs that bind to an allosteric site on the A1‐receptor. Here, we have investigated the species and probe dependence of two allosteric modulators on the binding characteristics of fluorescent and nonfluorescent A1‐receptor agonists. Experimental Approach: A Nano‐luciferase (Nluc) BRET (NanoBRET) methodology was used. This used N‐terminal Nluc‐tagged A1‐receptors expressed in HEK293T cells in conjunction with both fluorescent A1‐receptor agonists (adenosine and NECA analogues) and a fluorescent antagonist CA200645.Key Results: PD 81,723 and VCP171 elicited positive allosteric effects on the binding affinity of orthosteric agonists at both the rat and human A1‐receptors that showed clear probe dependence. Thus, the allosteric effect on the highly selective partial agonist capadenoson was much less marked than for the full agonists NECA, adenosine, and CCPA in both species. VCP171 and, to a lesser extent, PD 81,723, also increased the specific binding of three fluorescent A1‐receptor agonists in a species‐dependent manner that involved increases in Bmax and pKD.Conclusions and Implications: These results demonstrate the power of the NanoBRET ligand‐binding approach to study the effect of allosteric ligands on the binding of fluorescent agonists to the adenosine A1‐receptor in intact living cells. Furthermore, our studies suggest that VCP171 and PD 81,723 may switch a proportion of A1‐receptors to an active agonist conformation (R*)

Subtle modifications to a thieno[2,3-d]pyrimidine scaffold yield negative allosteric modulators and agonists of the dopamine D2 receptor

We recently described a structurally novel series of negative allosteric modulators (NAMs) of the dopamine D2 receptor (D2R) based on thieno[2,3-d]pyrimidine 1, showing it can be structurally simplified to reveal low molecular weight, fragment-like NAMs that retain robust negative cooperativity, such as 3. Herein, we report the synthesis and functional profiling of analogues of 3, placing specific emphasis on examining secondary and tertiary amino substituents at the 4-position, combined with a range of substituents at the 5/6-positions (e.g. aromatic/aliphatic carbocycles). We identify analogues with diverse pharmacology at the D2R including NAMs (19fc) with sub-?M affinity (9h) and, surprisingly, low efficacy partial agonists (9d and 9i)

Synthesis, biological evaluation, and utility of fluorescent ligands targeting the μ-opioid receptor

Fluorescently labeled ligands are useful pharmacological research tools for studying receptor localization, trafficking, and signaling processes via fluorescence imaging. They are also employed in fluorescent binding assays. This study is centered on the design, synthesis, and pharmacological evaluation of fluorescent probes for the opioid receptors, for which relatively few non-peptidic fluorescent probes currently exist. The known μ-opioid receptor (MOR) partial agonist, buprenorphine, was structurally elaborated to include an amidoalkylamine linker moiety that was coupled with a range of fluorophores to afford new fluorescent probes. All compounds proved to be selective MOR antagonists. Confocal fluorescence microscopy studies revealed that the probe incorporating a sulfonated cyanine-5 fluorophore was the most appropriate for imaging studies. This ligand was subsequently employed in an automated fluorescence-based competition binding assay, allowing the pKi values of several well-known opioid ligands to be determined. Thus, this new probe will prove useful in future studies of MOR receptor pharmacology

The effect of two selective A1-receptor agonists and the bitopic ligand VCP746 on heart rate and regional vascular conductance in conscious rats

Background and purposeAdenosine is a local mediator that regulates physiological and pathological processes via activation of four G protein‐coupled receptors (A1, A2A, A2B, A3). We have investigated the effect of two A1‐receptor selective agonists and the novel A1‐receptor bitopic ligand VCP746 on the rat cardiovascular system.Experimental ApproachTo investigate the effect of these A1‐agonists on the cardiovascular system, we evaluated their regional haemodynamic responses in conscious rats. Male Sprague Dawley rats (350–450g) were chronically implanted with pulsed Doppler flow probes (positioned around the renal and mesenteric arteries, and the descending abdominal aorta) and catheters (jugular vein and caudal artery). Cardiovascular responses were measured following i.v. infusion (3 min each dose) of CCPA (120, 400, 1200 ng.kg‐1.min‐1), capadenoson or adenosine (30, 100, 300 μg.kg‐1.min‐1) or VCP746 (6, 20, 60 μg.kg‐1.min‐1) following pre‐dosing with DPCPX (0.1 mg.kg‐1 i.v.) or vehicle.Key ResultsCCPA produced a significant A1‐receptor‐mediated decrease (p less than 0.05) in heart rate that was accompanied by vasoconstrictions in the renal and mesenteric vascular beds but an increase in hindquarters vascular conductance. The partial agonist capadenoson also produced an A1‐receptor‐mediated bradycardia. In contrast, VCP746 produced increases in heart rate and renal and mesenteric vascular conductance that were not mediated by A1‐receptors. In vitro studies confirmed that VCP746 had potent agonist activity at both A2A and A2B receptors.Conclusions and ImplicationsThese results suggest VCP746 mediates its cardiovascular effects via activation of A2 rather than A1 adenosine receptors. This has implications for the design of future bitopic ligands that incorporate A1 allosteric ligand moieties

Biased allosteric modulation at the CaS receptor engendered by structurally diverse calcimimetics

Background and Purpose Clinical use of cinacalcet in hyperparathyroidism is complicated by its tendency to induce hypocalcaemia, arising partly from activation of calcium-sensing receptors (CaS receptors) in the thyroid and stimulation of calcitonin release. CaS receptor allosteric modulators that selectively bias signalling towards pathways that mediate desired effects [e.g. parathyroid hormone (PTH) suppression] rather than those mediating undesirable effects (e.g. elevated serum calcitonin), may offer better therapies. Experimental Approach We characterized the ligand-biased profile of novel calcimimetics in HEK293 cells stably expressing human CaS receptors, by monitoring intracellular calcium (Ca2+i) mobilization, inositol phosphate (IP)1 accumulation, ERK1/2 phosphorylation (pERK1/2) and receptor expression. Key Results Phenylalkylamine calcimimetics were biased towards allosteric modulation of Ca2+i mobilization and IP1 accumulation. S,R-calcimimetic B was biased only towards IP1 accumulation. R,R-calcimimetic B and AC-265347 were biased towards IP1 accumulation and pERK1/2. Nor-calcimimetic B was unbiased. In contrast to phenylalkylamines and calcimimetic B analogues, AC-265347 did not promote trafficking of a loss-of-expression, naturally occurring, CaS receptor mutation (G670E). Conclusions and Implications The ability of R,R-calcimimetic B and AC-265347 to bias signalling towards pERK1/2 and IP1 accumulation may explain their suppression of PTH levels in vivo at concentrations that have no effect on serum calcitonin levels. The demonstration that AC-265347 promotes CaS receptor receptor signalling, but not trafficking reveals a novel profile of ligand-biased modulation at CaS receptors The identification of allosteric modulators that bias CaS receptor signalling towards distinct intracellular pathways provides an opportunity to develop desirable biased signalling profiles in vivo for mediating selective physiological responses

Structure-Kinetic Profiling of Haloperidol Analogues at the Human Dopamine D2 Receptor

Haloperidol is a typical antipsychotic drug (APD) associated with an increased risk of extrapyramidal side-effects (EPS) and hyperprolactinemia relative to atypical APDs such as clozapine. Both drugs are dopamine D2 receptor (D2R) antagonists, with contrasting kinetic profiles. Haloperidol displays fast association/slow dissociation at the D2R whereas clozapine exhibits relatively slow association/fast dissociation. Recently, we have provided evidence that slow dissociation from the D2R predicts hyperprolactinemia, whereas fast association predicts EPS. Unfortunately, clozapine can cause severe side-effects independent of its D2R action. Our results suggest an optimal kinetic profile for D2R antagonist APDs that avoids EPS. To begin exploring this hypothesis, we conducted a structure-kinetic relationship study of haloperidol and reveal that subtle structural modifications dramatically change binding kinetic rate constants, affording compounds with a clozapine-like kinetic profile. Thus, optimisation of these kinetic parameters may allow development of novel APDs based on the haloperidol scaffold with improved side-effect profiles

4-Phenylpyridin-2-one derivatives: a novel class of positive allosteric modulator of the M1 muscarinic acetylcholine receptor

Positive allosteric modulators (PAMs) of the M1 muscarinic acetylcholine receptor (M1 mAChR) are a promising strategy for the treatment of the cognitive deficits associated with diseases including Alzheimer’s and schizophrenia. Herein, we report the design, synthesis, and characterization of a novel family of M1 mAChR PAMs. The most active compounds of the 4-phenylpyridin-2-one series exhibited comparable binding affinity to the reference compound, 1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (BQCA) (1), but markedly improved positive cooperativity with acetylcholine, and retained exquisite selectivity for the M1 mAChR. Furthermore, our pharmacological characterization revealed ligands with a diverse range of activities, including modulators that displayed both high intrinsic efficacy and PAM activity, those that showed no detectable agonism but robust PAM activity and ligands that displayed robust allosteric agonism but little modulatory activity. Thus, the 4-phenylpyridin-2-one scaffold offers an attractive starting point for further lead optimization

Delineating the Mode of Action of Adenosine A 1 Receptor Allosteric Modulators □ S

ABSTRACT Despite the identification of 2-amino-3-benzoylthiophenes (2A3BTs) as the first example of small-molecule allosteric potentiators of agonist function at a G protein-coupled receptor (GPCR)-the adenosine A 1 receptor-their mechanism of action is still not fully understood. We now report the mechanistic basis for the complex behaviors noted for 2A3BTs at A 1 receptors. Using a combination of membranebased and intact-cell radioligand binding, multiple signaling assays, and a native tissue bioassay, we found that the allosteric interaction between 2A3BTs and the agonists 2-chloro- 3 H]8-cyclopentyl-1,3-dipropylxanthine is consistent with a ternary complex model involving recognition of a single extracellular allosteric site. However, when allowed access to the intracellular milieu, 2A3BTs have a secondary action as direct G protein inhibitors; this latter property is receptor-independent as it is observed in nontransfected cells and also after stimulation of another GPCR. In addition, we found that 2A3BTs can signal as allosteric agonists in their own right but show bias toward certain pathways relative to the orthosteric agonist, R-PIA. These results indicate that 2A3BTs have a dual mode of action when interacting with the A 1 receptor and that they can engender novel functional selectivity in A 1 signaling. These mechanisms need to be factored into allosteric ligand structure-activity studies

Molecular Determinants of the Intrinsic Efficacy of the Antipsychotic Aripiprazole

Partial agonists of the dopamine D2 receptor (D2R) have been developed to treat the symptoms of schizophrenia without causing the side effects elicited by antagonists. The receptor-ligand interactions that determine the intrinsic efficacy of such drugs, however, are poorly understood. Aripiprazole has an extended structure comprising a phenylpiperazine primary pharmacophore and a 1,2,3,4-tetrahydroquinolin-2-one secondary pharmacophore. We combined site-directed mutagenesis, analytical pharmacology, ligand fragments and molecular dynamics simulations to identify the D2R-aripiprazole interactions that contribute to affinity and efficacy. We reveal that an interaction between the secondary pharmacophore of aripiprazole and a secondary binding pocket defined by residues at the extracellular portions of transmembrane segments 1, 2 and 7 determine the intrinsic efficacy of aripiprazole. Our findings reveal a hitherto unappreciated mechanism through which to fine-tune the intrinsic efficacy of D2R agonists