Patrick M. Sexton

CRITICAL ROLE FOR THE SECOND EXTRACELLULAR LOOP IN THE BINDING OF BOTH ORTHOSTERIC AND ALLOSTERIC G PROTEIN-COUPLED RECEPTOR LIGANDS

The second extracellular (E2) loop of G protein-coupled receptors (GPCRs) plays an essential but poorly understood role in the binding of non-peptidic small molecules. We have utilized both orthosteric ligands and allosteric modulators of the M2 muscarinic acetylcholine receptor, a prototypical Family A GPCR, to probe possible E2 loop binding dynamics. We developed a homology model based on the crystal structure of bovine rhodopsin and predicted novel cysteine substitutions that should dramatically reduce E2 loop flexibility via disulfide bond formation and significantly inhibit the binding of both types of ligands. This prediction was validated experimentally using radioligand binding, dissociation kinetics, and cell-based functional assays. The results argue for a flexible “gatekeeper” role of the E2 loop in the binding of both allosteric and orthosteric GPCR ligands.

RNA editing of the serotonin 5HT2C receptor and its effects on cell signalling, pharmacology and brain function

The process of RNA editing involves the modification of mRNA at specific sites by adenosine deaminases that act on RNA (ADAR) enzymes. By catalyzing the conversion of adenosine to inosine, these enzymes alter the way in which the mRNA is translated, and consequently alter the primary structure of the resultant proteins. The serotonin (5HT) 2C receptor (5HT2CR) is currently the only known member of the superfamily of seven transmembrane domain receptors (7TMRs) to undergo this modification, and provides a fascinating case study in the effects of such changes. Here we review the current state of knowledge surrounding the editing of the 5HT2CR, the stark differences in signalling arising due to this process, and the potential for (and difficulties in) exploiting the phenomenon for improved therapeutic intervention in various neurological disorders.

Discovery of antiandrogen activity of nonsteroidal scaffolds of marketed drugs.

Finding good drug leads de novo from large chemical libraries, real or virtual, is not an easy task. High-throughput screening is often plagued by low hit rates and many leads that are toxic or exhibit poor bioavailability. Exploiting the secondary activity of marketed drugs, on the other hand, may help in generating drug leads that can be optimized for the observed side-effect target, while maintaining acceptable bioavailability and toxicity profiles. Here, we describe an efficient computational methodology to discover leads to a protein target from safe marketed drugs. We applied an in silico “drug repurposing” procedure for identification of nonsteroidal antagonists against the human androgen receptor (AR), using multiple predicted models of an antagonist-bound receptor. The library of marketed oral drugs was then docked into the best-performing models, and the 11 selected compounds with the highest docking score were tested in vitro for AR binding and antagonism of dihydrotestosterone-induced AR transactivation. The phenothiazine derivatives acetophenazine, fluphenazine, and periciazine, used clinically as antipsychotic drugs, were identified as weak AR antagonists. This in vitro biological activity correlated well with endocrine side effects observed in individuals taking these medications. Further computational optimization of phenothiazines, combined with in vitro screening, led to the identification of a nonsteroidal antiandrogen with improved AR antagonism and marked reduction in affinity for dopaminergic and serotonergic receptors that are the primary target of phenothiazine antipsychotics.

Structure-function studies of allosteric agonism at M2 muscarinic acetylcholine receptors

The M2 muscarinic acetylcholine receptor (mAChR) possesses at least one binding site for allosteric modulators that is dependent on the residues 172EDGE175, Tyr177, and Thr423. However, the contribution of these residues to actions of allosteric agonists, as opposed to modulators, is unknown. We created mutant M2 mAChRs in which the charge of the 172EDGE175 sequence had been neutralized and each Tyr177 and Thr423 was substituted with alanine. Radioligand binding experiments revealed that these mutations had a profound inhibitory effect on the prototypical modulators gallamine, alcuronium, and heptane-1,7-bis-[dimethyl-3′-phthalimidopropyl]-ammonium bromide (C7/3-phth) but minimal effects on the orthosteric antagonist [3H]N-methyl scopolamine. In contrast, the allosteric agonists 4-I-[3-chlorophenyl]carbamoyloxy)-2-butynyltrimethylammnonium chloride (McN-A-343), 4-n-butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl] piperidine hydrogen chloride (AC-42), and the novel AC-42 derivative 1-[3-(4-butyl-1-piperidinyl)propyl]-3,4-dihydro-2(1H)-quinolinone (77-LH-28-1) demonstrated an increased affinity or proportion of high-affinity sites at the combined EDGE-YT mutation, indicating a different mode of binding to the prototypical modulators. Subsequent functional assays of extracellular signal-regulated kinase (ERK)1/2 phosphorylation and guanosine 5′-(γ-[35S]thio)triphosphate ([35S]GTPγS) binding revealed minimal effects of the mutations on the orthosteric agonists acetylcholine (ACh) and pilocarpine but a significant increase in the efficacy of McN-A-343 and potency of 77-LH-28-1. Additional mutagenesis experiments found that these effects were predominantly mediated by Tyr177 and Thr423, rather than the 172EDGE175 sequence. The functional interaction between each of the allosteric agonists and ACh was characterized by high negative cooperativity but was consistent with an increased allosteric agonist affinity at the combined EDGE-YT mutant M2 mAChR. This study has thus revealed a differential role of critical allosteric site residues on the binding and function of allosteric agonists versus allosteric modulators of M2 mAChRs.

Receptor Activity-Modifying Proteins Differentially Modulate the G Protein-Coupling Efficiency of Amylin Receptors

Receptor activity-modifying proteins (RAMPs) 1, 2, and 3 are prototypic G protein-coupled receptor accessory proteins that can alter not only receptor trafficking but also receptor phenotype. Specific RAMP interaction with the calcitonin receptor (CTR) generates novel and distinct receptors for the peptide amylin; however, the role of RAMPs in receptor signaling is not understood. The current study demonstrates that RAMP interaction with the CTRa in COS-7 or HEK-293 cells leads to selective modulation of signaling pathways activated by the receptor complex. There was a 20- to 30-fold induction in amylin potency at CTR/RAMP1 (AMY1) and CTR/RAMP3 (AMY3) receptors, compared with CTR alone, for formation of the second-messenger cAMP that parallels an increase in amylin binding affinity. In contrast, only 2- to 5-fold induction of amylin potency was seen for mobilization of intracellular Ca++ or activation of ERK1/2. In addition, in COS-7 cells, the increase in amylin potency for Ca++ mobilization was 2-fold greater for AMY3 receptors, compared with AMY1 receptors and this paralleled the relative capacity of overexpression of Galphaq proteins to augment induction of high affinity 125I-amylin binding. These data demonstrate that RAMP-complexed receptors have a different signaling profile to CTRs expressed in the absence of RAMPs, and this is likely due to direct effects of the RAMP on G protein-coupling efficiency.

Molecular approximations between residues 21 and 23 of secretin and its receptor : Development of a model for peptide docking with the amino terminus of the secretin receptor

The structurally unique amino-terminal domain of class II G protein-coupled receptors is critically important for ligand binding and receptor activation. Understanding the precise role it plays requires detailed insights into the molecular basis of its ligand interactions and the conformation of the ligand-receptor complex. In this work, we used two high-affinity, full-agonist, secretin-like photolabile probes having sites for covalent attachment in positions 21 and 23 and used sequential proteolysis and sequencing of the labeled region of the receptor to identify two new spatial approximation constraints. The position 21 probe labeled receptor residue Arg15, whereas the position 23 probe labeled receptor residue Arg21. A homology model of the amino-terminal domain of the secretin receptor was developed using the NMR structure of the analogous domain of the corticotropin-releasing factor receptor. This was attached to a homology model of the secretin receptor transmembrane bundle, with the two domains oriented relative to each other based on continuity of the peptide backbone and by imposing a distance restraint recently identified between the amino-terminal WDN sequence and the region of the helical bundle above transmembrane segment six. Secretin was docked to this model using seven sets of spatial approximation constraints identified in previous photoaffinity labeling studies. This model was found to fully accommodate all existing constraints, as well as the two new approximations identified in this work.

3- and 6-Substituted 2-amino-4,5,6,7-tetrahydrothieno[2,3-c]pyridines as A1 adenosine receptor allosteric modulators and antagonists

A series of 2-amino-4,5,6,7-tetrahydrothieno[2,3-c]pyridines were prepared and evaluated as potential allosteric modulators at the A(1) adenosine receptor. The structure-activity relationships of the 3- and 6-positions of a series of 2-amino-4,5,6,7-tetrahydrothieno[2,3-c]pyridines were explored. Despite finding that 3- and 6-substituted 2-amino-4,5,6,7-tetrahydrothieno[2,3-c]pyridines possess the ability to recognize an allosteric site on the agonist-occupied A(1)AR at relatively high concentrations, the structural modifications we have performed on this scaffold favor the expression of orthosteric antagonist properties over allosteric properties. This research has identified 2-amino-4,5,6,7-tetrahydrothieno[2,3-c]pyridines as novel class of orthosteric antagonist of the A(1)AR and highlighted the close relationship between structural elements governing allosteric modulation and orthosteric antagonism of agonist function at the A(1)AR.

Relaxin Receptors - New Drug Targets for Multiple Disease States

Relaxin was discovered more than 75 years prior to the identification of the receptors that mediate its actions. There has been a slow emergence in understanding the role of relaxin, with it being denoted initially as a hormone of pregnancy due to its observed effects to relax pubic ligaments and soften the cervix of guinea pigs to facilitate parturition. However, many other physiological roles have been identified for relaxin, including cardiovascular and neuropeptide functions and an ability to induce the matrix metalloproteinases, so it is clear that relaxin is not exclusively a hormone of pregnancy but has a much wider role in vivo. The recent de-orphanisation of four receptors LGR7, LGR8, GPCR135 (SALPR) and GPCR142 (GPR100) that respond to and bind at least one of the three forms of relaxin identified to date, allows dissection of this system to determine the precise role of each receptor and enable the identification of new targets for treatment of numerous disease states. Relaxin has the potential to be useful for the treatment of scleroderma, fibrosis, in orthodontics and to facilitate embryo implantation in humans. Relaxin antagonists may act as contraceptives or prevent the development of breast cancer metastases. Recent research has added considerable knowledge to the signalling pathways activated by relaxin, which will aid our understanding of how relaxin produces its effects. The focus of this review is to bring together recent developments in the relaxin receptor field and to highlight their potential as drug targets.

Benzodiazepine ligands can act as allosteric modulators of the Type 1 cholecystokinin receptor

The cholecystokinin (CCK(1)) receptor is a G protein-coupled receptor important for nutrient homeostasis. The molecular basis of CCK-receptor binding has been debated, with one prominent model suggesting occupation of the same region of the intramembranous helical bundle as benzodiazepines. Here, we used a specific assay of allosteric ligand interaction to probe the mode of binding of devazepide, a prototypic benzodiazepine ligand. Devazepide elicited marked slowing of dissociation of pre-bound CCK, only possible through binding to a topographically distinct allosteric site. This effect was disrupted by chemical modification of a cysteine in the benzodiazepine-binding pocket. Application of an allosteric model to the equilibrium interaction between a series of benzodiazepine ligands and CCK yielded quantitative estimates of each modulators affinity for the allosteric site, as well as the degree of negative cooperativity for the interaction between occupied orthosteric and allosteric sites. The allosteric nature of benzodiazepine binding to the CCK(1) receptor provides new opportunities for small molecule drug development.

Pharmacology of 5HT2C receptor-mediated ERK1/2 phosphorylation: Agonist-specific activation pathways and the impact of RNA editing

We have previously characterized a mechanism of 5HT-stimulated extracellular signal-regulated kinases 1 and 2 (ERK1/2) activation via the non-RNA-edited isoform of the serotonin 5HT(2C) receptor (5HT(2C)R-INI) in a CHO cell line. We have now used CV1 cells, which endogenously express epidermal growth factor receptors (EGFRs), to investigate whether the mechanisms underlying ERK1/2 activation by the 5HT(2C)R change in a time-, agonist-, and cell background-dependent manner. Interrogation of the CV1 5HT(2C)R-INI ERK1/2 signaling pathway, using a variety of pathway-selective inhibitors, revealed a clear time-dependence in the involvement of specific pathway components such as phosphatidylinositol 3-kinase, EGFR, matrix metalloproteases and protein kinase C. The contribution of these components to the overall response also varied with the agonist used to stimulate the receptor, providing further evidence for the ability of 5HT(2C)R-INI to signal in an agonist-specific manner. We also investigated the impact of 5HT(2C)R RNA editing on this phenomenon. Although we found no alteration in antagonist pharmacology, the partially edited VSV and fully edited VGV isoforms of the 5HT(2C)R exhibited altered temporal and pharmacological characteristics, including the degree of dependence on specific effectors, in signaling to ERK1/2 in comparison to the 5HT(2C)R-INI. In conclusion, we provide evidence for remarkable flexibility in 5HT(2C)R-mediated ERK1/2 signaling that can be pharmacologically and mechanistically distinct depending on the agonist or edited isoform involved and on the duration of receptor activation.