Synthetic and Biological Exploration of (+)-Boldine - Identification of potential CNS receptor ligands

(+)-Boldine, an aporphine alkaloid, is reported to be biologically active at various Central Nervous System(CNS) receptors. However, only a few Structure Activity Relationship(SAR) studies have been conducted using boldine’s aporphine scaffold. A library of novel analogs was synthesized from boldine to understand the effect of bisbenzylation at C2 and C9 positions on the affinity and selectivity at the serotonin receptors

G protein-coupled receptor allosterism and complexing

G protein-coupled receptors (GPCRs) represent the largest family of cell-surface receptors. These receptors are natural allosteric proteins because agonist-mediated signaling by GPCRs requires a conformational change in the receptor protein transmitted between two topographically distinct binding sites, one for the agonist and another for the G protein. It is now becoming increasingly recognized, however, that the agonist-bound GPCR can also form ternary complexes with other ligands or "accessory" proteins and display altered binding and/or signaling properties in relation to the binary agonist-receptor complex. Allosteric sites on GPCRs represent novel drug targets because allosteric modulators possess a number of theoretical advantages over classic orthosteric ligands, such as a ceiling level to the allosteric effect and a potential for greater GPCR subtype-selectivity. Because of the noncompetitive nature of allosteric phenomena, the detection and quantification of such effects often relies on a combination of equilibrium binding, nonequilibrium kinetic, and functional signaling assays. This review discusses the development and properties of allosteric receptor models for GPCRs and the detection and quantification of allosteric effects. Moreover, we provide an overview of the current knowledge regarding the location of possible allosteric sites on GPCRs and candidate endogenous allosteric modulators. Finally, we discuss the potential for allosteric effects arising from the formation of GPCR oligomers or GPCRs complexed with accessory cellular proteins. It is proposed that the study of allosteric phenomena will become of progressively greater import to the drug discovery process due to the advent of newer and more sensitive GPCR screening technologies

Design and Synthesis of Novel Serotonin Receptor Ligands

Novel and potent ligands to the serotonin7 (5-HT7) receptor have been synthesized. The synthesized compounds include a set of substituted pyrimidines which show high affinity to the 5-HT7 receptor, synthesized by previously described methods [1,2] in high yield. Comparing the affinities of substituted pyrimidines to previously calculated models [3,4] yielded new hypotheses about the nature of interaction between the pyrimidine ligands and the 5-HT7 binding site. Several new series of compounds were synthesized by various methods to validate these hypotheses, including a conjugate addition to vinylpyrimidines [5]. These compounds include benzofurans, oximes, hydrazones, as well as a group of substituted piperazines. All series of compounds show affinity to the 5-HT7 receptor comparable to previously synthesized 5-HT7 ligands. Several of the synthesized ligands show affinity which exceeds that of currently available ligands. The synthesized compounds were evaluated quantitatively by calculating a three-dimensional quantitative structure-affinity relationship (3D-QSAR) for the 5-HT7 receptor. Evaluation of the calculated model validated qualitative assumptions about the data set as well as described regions of interaction in greater detail than previously available. These observations give further insight on the nature of ligand-binding site interactions with highly potent ligands such as 4-(3-furyl)-2-(N-methylpiperazino)pyrimidine which will lead to more potent 5-HT7 receptor ligands. Additionally, a model was calculated for affinity to the 5-HT2a receptor. Comparing this model to that calculated for affinity to the 5-HT7 receptor identified two regions which may be exploited in future sets of ligands to increase selectivity to the 5HT7 receptor

Molecular Cloning and Pharmacology of Porcine 5-Hydroxytryptamine Receptors Relevant to the Study of Antimigraine Drugs

At the beginning of last century, Brodie (1900) described in an extensive study that injection of blood serum causes vasoconstriction and a vagally-mediated reflex resulting in a reversible bradycardia, hypotension and arrest of the respiration, while injection of blood plasma was devoid of these effects. About 50 years ago, the hormone and neurotransmitter serotonin (5-hydroxytryptamine; 5-HT) was isolated from blood serum and named due to its origin and vascular action (sero=serum and tonin=vasoconstriction) (Rapport et al., 1948). Similarly, another endogenous substance from the enterochromaffin cells (present in gastrointestinal mucosa) was isolated, functionally characterised and named enteramine (Erspamer & Asero, 1952). The functional properties of enteramine were mainly smooth muscle contraction and further investigations revealed that enteramine and serotonin were chemically and pharmacologically similar (Erspamer, 1954)

An investigation into the effects of allosteric modulators of the human 5-Ht3_3A_A receptor

The 5-Ht$_3$$_A$ receptor is a cys-loop ligand gated ion channel re-emerging as an attractive target in irritable bowel syndrome (IBS), which currently affects 5-10% of the global population. At present, IBS therapies involve the complete blockade of the 5-Ht$_3$$_A$ receptor by orthosteric antagonists, which leads to complications such as severe constipation and ischaemic colitis. Allosteric modulation could bypass such side effects, as receptor function relies upon the endogenous neurotransmitter to retain physiological control. Here, we investigate the structure and function of the 5-Ht$_3$$_A$ receptor allosteric binding site by the identification of novel allosteric compounds and the generation of an α7/5-HT$_3$$_A$ chimeric receptor. Intracellular calcium assays and competitive radioligand binding experiments indicated that the halogenated indole derivatives, 5-chloroindole (5-Cl) (Newman et al., 2013) and 5- (trifluoromethyl)indole (5-TFMI) are positive allosteric modulators (PAM) of the 5-Ht$_3$$_A$ receptor; however 5-TFMI also displayed a degree of orthosteric binding. The structural analogues, 5-bromoindazole (5-BI) and 5-bromo-benzimidazole (5-BBI) exhibited contrasting effects, by potentiating and decreasing 5-HT-evoked responses, respectively. Further studies suggested some orthosteric binding by 5-BI and 5-BBI at high concentrations. The allosteric binding site for 5-Cl was previously located in the N-terminus of the mouse 5-Ht$_3$$_A$ receptor. To identify the site in the human receptor, we attempted to construct a human chimeric α7V$_2$$_0$$_1$5-HT$_3$$_A$ receptor to allow further investigation into this question. Our data suggest these halogenated indoles are allosteric compounds and, when studied in combination with the α7V$_2$$_0$$_1$5-HT$_3$$_A$ chimera, could identify the required core structure of high affinity compounds needed for negative allosteric modulation in the treatment of IBS

The pharmacology of the 5-HT2A receptor and the difficulty surrounding functional studies with single target models

Treatment of many disorders can be frequently problematic due to the relatively non selective nature of many drugs available on the market. Symptoms can be complex and expansive, often leading to symptoms representing other disorders in addition to the primary reason for treatment. In particular mental health disorders fall prey to this situation. Targeting treatment can be difficult due to the implication of receptors in more than one disorder, and more than one receptor in a single disorder. In the instance of GPCRs, receptors such as the serotonin receptors (and in particular the 5-HT2A for the interest of this research) belong to a large family of receptors, the GPCR Class A super family. Around 50% of the drugs now commercially available target GPCR receptors (Wise et al 2004, Katugampola & Davenport.,2003) and drugs with action at serotonin receptors are used in the treatment of many disorders, particularly psychotic disorders such as schizophrenia. Inability to target single receptors selectively means that the therapeutic values of the drugs are much lower than desired. In this study, the 5-HT2A receptor was incorporated into a stable, inducible cell line using HEK 293 cells and the Flp-in T-REx system, allowing receptor expression to be under the control of the antibiotic doxycycline and hence allow pharmacology to be explored. There is a variation when looking at the potency of agonists in relation to calcium mobilisation and IP-one accumulation, although following the same order of potency the values differ between each experiment type. The order of potency for the majority of the antagonist ligands is very different when looking at IP-one and Ca2+ experiments, as are the values obtained for affinity. This was surprising due to the both lying downstream of the IP3 pathway. The most closely relating results to the published IUPHAR values stem from binding experiments. Understanding the pharmacology of the single receptor by several methods is essential when screening drugs for effectiveness at the receptor. Here the data exploring the pharmacology of the 5-HT2A receptor demonstrated the difficulty surrounding functional studies using single target models